U.S. patent application number 15/366924 was filed with the patent office on 2017-05-18 for cell carrier and cell carrier containment devices containing regenerative cells.
The applicant listed for this patent is Cytori Therapeutics, Inc.. Invention is credited to John K. Fraser, Marc H. Hedrick, Susan Lynn Riley, Joseph W. Tai.
Application Number | 20170136069 15/366924 |
Document ID | / |
Family ID | 46302281 |
Filed Date | 2017-05-18 |
United States Patent
Application |
20170136069 |
Kind Code |
A1 |
Hedrick; Marc H. ; et
al. |
May 18, 2017 |
CELL CARRIER AND CELL CARRIER CONTAINMENT DEVICES CONTAINING
REGENERATIVE CELLS
Abstract
The present invention relates to a device comprising a cell
carrier portion containing regenerative cells, e.g., stem and
progenitor cells, and a cell carrier containment portion. The
device is useful for the treatment of bone related disorders,
including spinal fusion related disorders and long bone or flat
bone related defects. The device may be used in conjunction with
disclosed automated systems and methods for separating and
concentrating regenerative cells.
Inventors: |
Hedrick; Marc H.; (Solana
Beach, CA) ; Fraser; John K.; (San Diego, CA)
; Riley; Susan Lynn; (San Diego, CA) ; Tai; Joseph
W.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cytori Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
46302281 |
Appl. No.: |
15/366924 |
Filed: |
December 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13598203 |
Aug 29, 2012 |
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15366924 |
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12568533 |
Sep 28, 2009 |
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13598203 |
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10885293 |
Jul 1, 2004 |
7595043 |
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12568533 |
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10316127 |
Dec 9, 2002 |
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10885293 |
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60554455 |
Mar 19, 2004 |
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60338856 |
Dec 7, 2001 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61F 2/4455 20130101;
A61F 2210/0061 20130101; A61K 35/32 20130101; C12N 5/0667 20130101;
A61F 2/447 20130101; A61F 2002/30604 20130101; A61F 2230/0015
20130101; B33Y 80/00 20141201; A61F 2/442 20130101; A61F 2002/30879
20130101; A61F 2002/30841 20130101; A61F 2002/30075 20130101; A61F
2/4465 20130101; A61K 9/0019 20130101; A61F 2002/30593 20130101;
A61K 35/28 20130101; A61F 2002/30062 20130101; A61F 2002/30133
20130101; C12N 5/069 20130101; A61F 2220/005 20130101; C12N
2506/1384 20130101; A61F 2002/4445 20130101; A61F 2002/445
20130101; A61F 2002/30448 20130101; A61F 2210/0004 20130101 |
International
Class: |
A61K 35/32 20060101
A61K035/32; A61F 2/44 20060101 A61F002/44 |
Claims
1. A method of treating a patient, comprising: (a) providing a
tissue removal system; (b) removing adipose tissue from a patient
using the tissue removal system, the adipose tissue having a
concentration of stem cells; (c) processing at least a part of the
adipose tissue to obtain a concentration of stem cells other than
the concentration of stem cells of the adipose tissue before
processing; and (d) administering the stem cells to a patient
without removing the stem cells from the tissue removal system
before being administered to the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/598,203, filed Aug. 29, 2012, entitled CELL
CARRIER AND CELL CARRIER CONTAINMENT DEVICES CONTAINING
REGENERATIVE CELLS, which is a continuation of U.S. patent
application Ser. No. 12/568,533, filed Sep. 28, 2009 entitled CELL
CARRIER AND CELL CARRIER CONTAINMENT DEVICES CONTAINING
REGENERATIVE CELLS, now abandoned, which is a divisional of U.S.
patent application Ser. No. 10/885,293, now U.S. Pat. No.
7,595,043, filed on Jul. 1, 2004, entitled METHOD FOR PROCESSING
AND USING ADIPOSE-DERIVED STEM CELLS, which is a
continuation-in-part application of U.S. patent application Ser.
No. 10/316,127, filed on Dec. 9, 2002, entitled SYSTEMS AND METHODS
FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATE CELLS, now
abandoned, which claims the benefit of U.S. Provisional Application
No. 60/338,856, filed Dec. 7, 2001. U.S. patent application Ser.
No. 10/885,293, now U.S. Pat. No. 7,595,043, also claims priority
to U.S. Provisional Application No. 60/554,455, entitled CELL
CARRIER AND CELL CARRIER CONTAINMENT DEVICES CONTAINING ADIPOSE
DERIVED CELLS, filed Mar. 19, 2004. The contents of each of the
aforementioned applications are expressly incorporated herein by
this reference.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The present invention relates to methods of making and using
a device, comprised of a cell carrier portion and a cell carrier
containment portion, in combination with regenerative cells, e.g.,
adipose derived regenerative cells, for the treatment of bone
related disorders. In particular, the novel combination disclosed
herein is useful for promoting bone and/or cartilage formation. The
present invention is especially useful for promoting bone formation
in normally non-osteoconductive areas, e.g., for disorders
requiring therapy in the form of interbody spinal fusion
surgery.
[0004] Description of the Related Art
[0005] Back pain caused by disc degeneration and pressure on nerves
near the spine affects nearly 65 million Americans. Common early
treatment options include stretching exercises, pain medications or
steroid injections into back muscles. If these treatments fail,
spinal fusion surgery becomes an option. According to the American
Academy of Orthopedic Surgeons, about 250,000 spinal fusion
surgeries are performed every year, mostly on adults between the
ages of 45 to 64.
[0006] Spinal fusion is a process by which two or more of the
vertebrae that make up the spinal column are fused together with
bone grafts and internal devices (such as rods) that heal into a
single solid bone. Spinal fusion can eliminate unnatural motion
between the vertebrae and, in turn, reduce pressure on nerve
endings. In addition, spinal fusion can be used to treat, for
example, injuries to spinal vertebrae caused by trauma; protrusion
and degeneration of the cushioning disc between vertebrae
(sometimes called slipped disc or herniated disc); abnormal
curvatures (such as scoliosis or kyphosis); and weak or unstable
spine caused by infections or tumors.
[0007] Autogenous bone (bone from the patient) or allograft bone
(bone from another individual) are the most commonly used materials
to induce bone formation. Generally, small pieces of bone are
placed into the space between the vertebrae to be fused. Sometimes
larger solid pieces of bone are used to provide immediate
structural support. Autogenous bone is generally considered
superior at promoting fusion. However, this procedure requires
extra surgery to remove bone from another area of the patient's
body such as the pelvis or fibula. Thus, it has been reported that
about 30 percent of patients have significant pain and tenderness
at the graft harvest site, which may be prolonged, and in some
cases outlast the back pain the procedure intended to correct.
Similarly, allograft bone and other bone graft substitutes,
although eliminating the need for a second surgery, have drawbacks
in that they have yet to be proven as cost effective and
efficacious substitutes for autogenous bone fusion.
[0008] An alternative to autogenous or allograft bone is the use of
growth factors that promote bone formation. For example, studies
have shown that the use of bone morphogenic proteins ("BMPs")
results in better overall fusion, less time in the operating room
and, more importantly, fewer complications for patients because it
eliminates the need for the second surgery. However, use of BMPs,
although efficacious in promoting bone growth, can be prohibitively
expensive.
[0009] Another alternative is the use of a genetically engineered
version of a naturally occurring bone growth factor. This approach
also has limitations. Specifically, surgeons have expressed
concerns that genetically engineered BMPs can dramatically speed
the growth of cancerous cells or cause non-cancerous cells to
become more sinister. Another concern is unwanted bone creation.
There is a chance that bone generated by genetically engineered
BMPs could form over the delicate nerve endings in the spine or,
worse, somewhere else in the body.
[0010] Regenerative medicine, which harnesses the ability of
regenerative cells, e.g., stem cells (i.e., the unspecialized
master cells of the body) to renew themselves indefinitely and
develop into mature specialized cells, may be a means of
circumventing the limitations of the prior-art techniques. Stem
cells, i.e., both embryonic and adult stem cells, have been shown
to possess the nascent capacity to become many, if not all, of the
200+ cell and tissue types of the body, including bone. Recently,
adipose tissue has been shown to be a source of adult stem cells
(Zuk et al., 2001; Zuk et al., 2002). Adipose tissue (unlike
marrow, skin, muscle, liver and brain) is comparably easy to
harvest in relatively large amounts with low morbidity (Commons et
al., 2001; Katz et al., 2001b). Accordingly, given the limitations
of the prior art spinal fusion techniques, there exists a need for
a device that incorporates regenerative cells, e.g., stem cells
that posses the ability to induce bone formation.
SUMMARY OF THE INVENTION
[0011] The present invention relates to methods of making and using
a device comprised of a cell carrier portion and a cell carrier
containment portion, and a therapeutically effective dose of
regenerative cells, the combination of which promotes bone and/or
cartilage formation in an intended area for bone formation in the
recipient, i.e., a target area. The device and cells of the present
invention are especially useful for treating disorders that benefit
from bone formation in normally non-osteoconductive areas, e.g.,
for disorders requiring therapy in the form of interbody spinal
fusion surgery. The device and cells of the present invention are
also useful for treating other disorders or defects that cannot
heal by osteoconduction, e.g., defects in long bones as a result of
trauma. In a preferred embodiment, one or more portions of the
device is resorbable.
[0012] The present invention further relates to systems and methods
for preparing regenerative cells, e.g., adult stem and progenitor
cells. The present invention is yet further based on the discovery
of methods and compositions of regenerative cells, e.g., adult stem
and progenitor cells, that promote bone and/or cartilage formation.
Accordingly, in one embodiment, the present invention is directed
to compositions, methods, and systems for using cells derived from
adipose tissue that are placed into the cell carrier portion of the
device along with such additives necessary to promote, engender, or
support bone and/or cartilage formation in a recipient.
[0013] In one embodiment, the entire procedure from tissue
extraction through processing and placement of the device into the
intended bone and/or cartilage formation site of the recipient
would all be performed in the same facility, indeed, even within
the same room of the patient undergoing the procedure. In a
particular embodiment, an effective dose of the regenerative cells,
e.g., stem and progenitor cells, are placed onto the cell carrier
portion of the device prior to insertion in the target area. In
another embodiment, an effective dose of the regenerative cells,
e.g., stem and progenitor cells, are placed onto the cell carrier
subsequent to placement of the device into the intended bone and/or
cartilage formation site of the recipient, e.g., via a syringe. In
yet other embodiments, the cells may be placed into the cell
carrier portion of the device in combination with other cells,
tissue, tissue fragments, or other stimulators of cell growth
and/or differentiation. In a preferred embodiment, the cells, with
any of the above mentioned additives, are placed into the person
(via the device) from whom they were obtained. In a preferred
embodiment, the cell carrier portion and/or the cell carrier
containment portion is resorbable.
[0014] In a certain embodiment, promoting bone and/or cartilage
formation in a patient includes steps of: a) providing a tissue
removal system; b) removing adipose tissue from a patient using the
tissue removal system, the adipose tissue having a concentration of
stem cells; c) processing at least a part of the adipose tissue to
obtain a concentration of stem cells sufficient to constitute an
effective dose for promoting bone and/or cartilage formation; d)
adding the regenerative cells, e.g., stem and progenitor cells,
obtained from the processing step to a cell carrier portion of the
device of the invention; and e) inserting the device containing the
cells into an intended bone and/or cartilage formation site of the
recipient. In a preferred embodiment, the cell carrier portion
and/or the cell carrier containment portion is resorbable. In
another preferred embodiment, the tissue processing system is
automated.
[0015] In another embodiment, promoting bone and/or cartilage
formation in a patient includes steps of: a) providing a tissue
removal system; b) removing adipose tissue from a patient using the
tissue removal system, the adipose tissue having a concentration of
stem cells; c) processing at least a part of the adipose tissue to
obtain a concentration of stem cells sufficient to constitute an
effective dose for promoting bone and/or cartilage formation; d)
inserting the device of the invention into an intended bone and/or
cartilage formation site of the recipient; and e) adding the
regenerative cells obtained from the processing step to the cell
carrier portion of the device inserted into the recipient. In a
preferred embodiment, the cell carrier portion and/or the cell
carrier containment portion is resorbable. In another preferred
embodiment, the tissue processing system is automated.
[0016] Any feature or combination of features described herein are
included within the scope of the present invention provided that
the features included in any such combination are not mutually
inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art. Additional advantages and aspects of the present invention are
apparent in the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1. is an illustration of a system for separating and
concentrating regenerative cells from tissue which includes one
filter assembly.
[0018] FIG. 2 is an illustration of a system similar to FIG. 1
having a plurality of filter assemblies in a serial
configuration.
[0019] FIG. 3 is an illustration of a system similar to FIG. 1
having a plurality of filter assemblies in a parallel
configuration.
[0020] FIG. 4 is an illustration of a system for separating and
concentrating regenerative cells from tissue which includes a
centrifuge chamber.
[0021] FIG. 5 is a sectional view of a collection chamber including
a prefixed filter utilized in a system for separating and
concentrating regenerative cells from tissue.
[0022] FIG. 6 is a sectional view of a processing chamber of a
system for separating and concentrating regenerative cells from
tissue utilizing a percolative filtration system.
[0023] FIG. 7 is a sectional view of a processing chamber of a
system for separating and concentrating regenerative cells
utilizing a centrifuge device for concentrating the regenerative
cells.
[0024] FIG. 8 is another sectional view of the processing chamber
of FIG. 7.
[0025] FIGS. 9A, 9B and 9C illustrate an elutriation component in
use with the system of the invention.
[0026] FIG. 10 is an illustration of a system for separating and
concentrating regenerative cells from tissue utilizing vacuum
pressure to move fluids through the system. A vacuum system can be
constructed by applying a vacuum pump or vacuum source to the
outlet of the system, controlled at a predetermined rate to pull
tissue and fluid through, using a system of stopcocks, vents, and
clamps to control the direction and timing of the flow.
[0027] FIG. 11 is an illustration of a system for separating and
concentrating regenerative cells from tissue utilizing positive
pressure to move fluids through the system. A positive pressure
system uses a mechanical means such as a peristaltic pump to push
or propel the fluid and tissue through the system at a determined
rate, using valves, stopcocks, vents, and clamps to control the
direction and timing of the flow.
[0028] FIG. 12A illustrates a filtration process in which the feed
stream of fluid flows tangentially to the pores of the filter. FIG.
12B illustrates a filtration process in which the feed stream of
fluid flows perpendicular to the pores of the filter.
[0029] FIG. 13 is an illustration of an exemplary disposable set
for a system of the invention.
[0030] FIGS. 14-1 and 14-2 are illustrations of an exemplary
re-usable component for a system of the invention.
[0031] FIGS. 15A-1 and 15A-2 are illustrations of an exemplary
device of the invention assembled using the disposable set of FIG.
13 and a re-usable component of FIG. 14.
[0032] FIG. 15B is a flowchart depicting exemplary pre-programmed
steps, implemented through a software program, that control
automated embodiments of a system of the present invention. Two
alternative processing parameters are shown indicating the
versatility of the system.
[0033] FIG. 16 depicts a three-dimensional view of an exemplary
cell carrier portion of the device of the invention.
[0034] FIG. 17 depicts the cell carrier of FIG. 16 seeded with the
regenerative cells of the present invention.
[0035] FIG. 18 depicts a three-dimensional view of an exemplary
containment portion of the device of the invention.
[0036] FIG. 19 depicts a three-dimensional view of an exemplary
device of the invention.
[0037] FIG. 20 depicts a three-dimensional view of an exemplary
combination of the device and adipose-derived cells of the
invention.
[0038] FIG. 21 depicts a three-dimensional view of another
exemplary containment portion of the device of the invention.
[0039] FIG. 22 depicts a three-dimensional view of another
exemplary containment portion of the device of the invention.
[0040] FIG. 23 depicts a three-dimensional view of another
exemplary containment portion of the device of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention provides a minimally invasive and
cost-effective device for promoting bone and/or cartilage formation
using a therapeutically effective dose of regenerative cells. The
present invention is based, in part, on the discovery that the
regenerative cells of the invention are a rich source of bone and
cartilage progenitor cells, e.g., osteoprogenitor cells.
Furthermore, the regenerative cells of the invention have been
shown herein to form bone and cartilage in vivo. Accordingly, the
device of the present invention can be used to treat bone related
disorders, e.g., by promoting bone and/or cartilage formation.
Specifically, the present invention provides a novel combination of
1) a device, comprising a porous cell carrier portion, and a cell
carrier containment portion (hereinafter referred to herein as "the
device"), and 2) a therapeutically effective dose of adipose
derived regenerative cells, e.g., adult stem and progenitor cells,
that promote bone and/or cartilage formation. In a particular
embodiment, the device and cells of the present invention are
useful for treating disorders requiring bone formation in normally
non-osteoconductive areas, e.g., for the treatment of disorders
requiring therapy in the form of interbody spinal fusion surgery
and the like.
[0042] The present invention also relates to rapid and reliable
systems and methods for separating and concentrating regenerative
cells, e.g., stem cells and/or progenitor cells, from a wide
variety of tissues, including but not limited to, adipose, bone
marrow, blood, skin, muscle, liver, connective tissue, fascia,
brain and other nervous system tissues, blood vessels, and other
soft or liquid tissues or tissue components or tissue mixtures
(e.g., a mixture of tissues including skin, blood vessels, adipose,
and connective tissue). In a preferred embodiment, the system
separates and concentrates regenerative cells from adipose tissue.
In another preferred embodiment, the system is automated such that
the entire method may be performed with minimal user intervention
or expertise. In a particularly preferred embodiment, the
regenerative cells obtained using the systems and methods of the
present invention are suitable for direct placement into a
recipient suffering from a renal disease or disorder from whom the
tissue was extracted.
[0043] Preferably, the entire procedure from tissue extraction
through separating, concentrating and placement of the regenerative
cells into the recipient would all be performed in the same
facility, indeed, even within the same room of the patient
undergoing the procedure. The regenerative cells may be used in a
relatively short time period after extraction and concentration.
For example, the regenerative cells may be ready for use in about
one hour from the harvesting of tissue from a patient, and in
certain situations, may be ready for use in about 10 to 40 minutes
from the harvesting of the tissue. In a preferred embodiment, the
regenerative cells may be ready to use in about 20 minutes from the
harvesting of tissue. The entire length of the procedure from
extraction through separating and concentrating may vary depending
on a number of factors, including patient profile, type of tissue
being harvested and the amount of regenerative cells required for a
given therapeutic application. The cells may also be placed into
the recipient in combination with other cells, tissue, tissue
fragments, scaffolds or other stimulators of cell growth and/or
differentiation in the context of a single operative procedure with
the intention of deriving a therapeutic, structural, or cosmetic
benefit to the recipient. It is understood that any further
manipulation of the regenerative cells beyond the separating and
concentrating phase of the system will require additional time
commensurate with the manner of such manipulation.
[0044] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0045] As used herein, "regenerative cells" refers to any
heterogeneous or homologous cells obtained using the systems and
methods of the present invention which cause or contribute to
complete or partial regeneration, restoration, or substitution of
structure or function of an organ, tissue, or physiologic unit or
system to thereby provide a therapeutic, structural or cosmetic
benefit. Examples of regenerative cells include: ASCs, endothelial
cells, endothelial precursor cells, endothelial progenitor cells,
macrophages, fibroblasts, pericytes, smooth muscle cells,
preadipocytes, differentiated or de-differentiated adipocytes,
keratinocytes, unipotent and multipotent progenitor and precursor
cells (and their progeny), and lymphocytes.
[0046] One mechanism by which the regenerative cells may provide a
therapeutic, structural or cosmetic benefit is by incorporating
themselves or their progeny into newly generated, existing or
repaired tissues or tissue components. For example, ASCs and/or
their progeny may incorporate into newly generated bone, muscle, or
other structural or functional tissue and thereby cause or
contribute to a therapeutic, structural or cosmetic improvement.
Similarly, endothelial cells or endothelial precursor or progenitor
cells and their progeny may incorporate into existing, newly
generated, repaired, or expanded blood vessels to thereby cause or
contribute to a therapeutic, structural or cosmetic benefit.
Another mechanism by which the regenerative cells may provide a
therapeutic, structural or cosmetic benefit is by expressing and/or
secreting molecules, e.g., growth factors, that promote creation,
retention, restoration, and/or regeneration of structure or
function of a given tissue or tissue component.
[0047] The regenerative cells may be used in their `native` form as
present in or separated and concentrated from the tissue using the
systems and methods of the present invention or they may be
modified by stimulation or priming with growth factors or other
biologic response modifiers, by gene transfer (transient or stable
transfer), by further sub-fractionation of the resultant population
on the basis or physical properties (for example size or density),
differential adherence to a solid phase material, expression of
cell surface or intracellular molecules, cell culture or other ex
vivo or in vivo manipulation, modification, or fractionation as
further described herein. The regenerative cells may also be used
in combination with other cells or devices such as synthetic or
biologic scaffolds, materials or devices that deliver factors,
drugs, chemicals or other agents that modify or enhance the
relevant characteristics of the cells as further described
herein.
[0048] As used herein, "regenerative cell composition" refers to
the composition of cells typically present in a volume of liquid
after a tissue, e.g., adipose tissue, is washed and at least
partially disaggregated. For example, a regenerative cell
composition of the invention comprises multiple different types of
regenerative cells, including ASCs, endothelial cells, endothelial
precursor cells, endothelial progenitor cells, macrophages,
fibroblasts, pericytes, smooth muscle cells, preadipocytes,
differentiated or de-differentiated adipocytes, keratinocytes,
unipotent and multipotent progenitor and precursor cells (and their
progeny), and lymphocytes. The regenerative cell composition may
also contain one or more contaminants, such as collagen, which may
be present in the tissue fragments, or residual collagenase or
other enzyme or agent employed in or resulting from the tissue
disaggregation process described herein.
[0049] As used herein, "regenerative medicine" refers to any
therapeutic, structural or cosmetic benefit that is derived from
the placement, either directly or indirectly, of regenerative cells
into a subject. As used herein, the phrase "bone related disorder"
is intended to include all bone related disorders, including, for
example, disorders requiring spinal fixation, spinal stabilization,
repair of segmental defects in the body (such as in long bones and
flat bones) and growth of bone in a normally non-osteoconductive
environment are bone related disorders. In a particular embodiment,
the phrase is intended to include disorders requiring spinal fusion
surgery. In addition, bone related disorders is intended to
encompass disorders of the vertebrae and discs including, but are
not limited to, disruption of the disc annulus such as annular
fissures, chronic inflammation of the disc, localized disc
herniations with contained or escaped extrusions, relative
instability of the vertebrae surrounding the disc and degenerative
disc disease. Treatment of a bone related disease or disorder is
within the ambit of regenerative medicine.
[0050] As used herein, "stem cell" refers to a multipotent
regenerative cell with the potential to differentiate into a
variety of other cell types, which perform one or more specific
functions and have the ability to self-renew. Some of the stem
cells disclosed herein may be multipotent.
[0051] As used herein, "progenitor cell" refers to a multipotent
regenerative cell with the potential to differentiate into more
than one cell type and has limited or no ability to self-renew.
"Progenitor cell", as used herein, also refers to a unipotent cell
with the potential to differentiate into only a single cell type,
which performs one or more specific functions and has limited or no
ability to self-renew. In particular, as used herein, "endothelial
progenitor cell" refers to a multipotent or unipotent cell with the
potential to differentiate into vascular endothelial cells.
[0052] As used herein, "precursor cell" refers to a unipotent
regenerative cell with the potential to differentiate into one cell
type. Precursor cells and their progeny may retain extensive
proliferative capacity, e.g., lymphocytes and endothelial cells,
which can proliferate under appropriate conditions.
[0053] As used herein "stem cell number" or "stem cell frequency"
refers to the number of colonies observed in a clonogenic assay in
which adipose derived cells (ADC) are plated at low cell density
(<10,000 cells/well) and grown in growth medium supporting MSC
growth (for example, DMEM/F12 medium supplemented with 10% fetal
calf serum, 5% horse serum, and antibiotic/antimycotic agents).
Cells are grown for two weeks after which cultures are stained with
hematoxylin and colonies of more than 50 cells are counted as
CFU-F. Stem cell frequency is calculated as the number of CFU-F
observed per 100 nucleated cells plated (for example; 15 colonies
counted in a plate initiated with 1,000 nucleated regenerative
cells gives a stem cell frequency of 1.5%). Stem cell number is
calculated as stem cell frequency multiplied by the total number of
nucleated ADC cells obtained. A high percentage (.about.100%) of
CFU-F grown from regenerative cells express the cell surface
molecule CD105 which is also expressed by marrow-derived stem cells
(Barry et al., 1999). CD105 is also expressed by adipose
tissue-derived stem cells (Zuk et al., 2002).
[0054] As used herein, the term "adipose tissue" refers to fat
including the connective tissue that stores fat. Adipose tissue
contains multiple regenerative cell types, including ASCs and
endothelial progenitor and precursor cells.
[0055] As used herein, the term "unit of adipose tissue" refers to
a discrete or measurable amount of adipose tissue. A unit of
adipose tissue may be measured by determining the weight and/or
volume of the unit. Based on the data identified above, a unit of
processed lipoaspirate, as removed from a patient, has a cellular
component in which at least 0.1% of the cellular component is stem
cells; that is, it has a stem cell frequency, determined as
described above, of at least 0.1%. In reference to the disclosure
herein, a unit of adipose tissue may refer to the entire amount of
adipose tissue removed from a patient, or an amount that is less
than the entire amount of adipose tissue removed from a patient.
Thus, a unit of adipose tissue may be combined with another unit of
adipose tissue to form a unit of adipose tissue that has a weight
or volume that is the sum of the individual units.
[0056] As used herein, the term "portion" refers to an amount of a
material that is less than a whole. A minor portion refers to an
amount that is less than 50%, and a major portion refers to an
amount greater than 50%. Thus, a unit of adipose tissue that is
less than the entire amount of adipose tissue removed from a
patient is a portion of the removed adipose tissue.
[0057] As used herein, the term "processed lipoaspirate" refers to
adipose tissue that has been processed to separate the active
cellular component (e.g., the component containing regenerative)
from the mature adipocytes and connective tissue. This fraction is
referred to herein as "adipose-derived cells" or "ADC." Typically,
ADC refers to the pellet of regenerative cells obtained by washing
and separating and concentrating the cells from the adipose tissue.
The pellet is typically obtained by centrifuging a suspension of
cells so that the cells aggregate at the bottom of a centrifuge
chamber or cell concentrator.
[0058] As used herein, the terms "administering," "introducing,"
"delivering," "placement" and "transplanting" are used
interchangeably herein and refer to the placement of the
regenerative cells of the invention into a subject by a method or
route which results in at least partial localization of the
regenerative cells at a desired site. The regenerative cells can be
administered by any appropriate route which results in delivery to
a desired location in the subject where at least a portion of the
cells or components of the cells remain viable. The period of
viability of the cells after administration to a subject can be as
short as a few hours, e.g., twenty-four hours, to a few days, to as
long as several years.
[0059] As used herein, the term "treating" includes reducing or
alleviating at least one adverse effect or symptom of a disease or
disorder
[0060] As used herein, "therapeutically effective dose of
regenerative cells" refers to an amount of regenerative cells that
are sufficient to bring about a beneficial or desired clinical
effect. Said dose could be administered in one or more
administrations. However, the precise determination of what would
be considered an effective dose may be based on factors individual
to each patient, including, but not limited to, the patient's age,
size, type or extent of disease, stage of the disease, route of
administration of the regenerative cells, the type or extent of
supplemental therapy used, ongoing disease process and type of
treatment desired (e.g., aggressive vs. conventional
treatment).
[0061] As used herein, the term "subject" includes warm-blooded
animals, preferably mammals, including humans. In a preferred
embodiment, the subject is a primate. In an even more preferred
embodiment, the subject is a human.
[0062] Reference will now be made in detail to the presently
preferred embodiments of the invention, examples of which are
illustrated in the accompanying figures. Wherever possible, the
same or similar reference numbers are used in the drawings and the
description to refer to the same or like parts. It should be noted
that the drawings are in simplified form and are not to precise
scale. In reference to the disclosure herein, for purposes of
convenience and clarity only, directional terms, such as, top,
bottom, left, right, up, down, over, above, below, beneath, rear,
and front, are used with respect to the accompanying drawings. Such
directional terms should not be construed to limit the scope of the
invention in any manner.
[0063] Although the disclosure herein refers to certain illustrated
embodiments, it is to be understood that these embodiments are
presented by way of example and not by way of limitation. The
intent of the following detailed description, although discussing
exemplary embodiments, is to be construed to cover all
modifications, alternatives, and equivalents of the embodiments as
may fall within the spirit and scope of the invention as defined by
the appended claims. The present invention may be practiced in
conjunction with various cell or tissue separation techniques that
are conventionally used in the art, and only so much of the
commonly practiced process steps are included herein as are
necessary to provide an understanding of the present invention.
I. Methods of the Invention
[0064] 1. Methods of Obtaining Regenerative Cells (ADC)
[0065] As previously set forth herein, regenerative cells, e.g.,
stem and progenitor cells, can be harvested from a wide variety of
tissues. The system of the present invention may be used for all
such tissues. Adipose tissue, however, is an especially rich source
of regenerative cells. Accordingly, the system of the present
invention is illustrated herein using adipose tissue as a source of
regenerative cells by way of example only and not limitation.
[0066] Adipose tissue can be obtained by any method known to a
person of ordinary skill in the art. For example, adipose tissue
may be removed from a patient by liposuction (syringe or power
assisted) or by lipectomy, e.g., suction-assisted lipoplasty,
ultrasound-assisted lipoplasty, and excisional lipectomy or
combinations thereof. The adipose tissue is removed and collected
and may be processed in accordance with any of the embodiments of a
system of the invention described herein. The amount of tissue
collected depends on numerous factors, including the body mass
index and age of the donor, the time available for collection, the
availability of accessible adipose tissue harvest sites,
concomitant and pre-existing medications and conditions (such as
anticoagulant therapy), and the clinical purpose for which the
tissue is being collected. For example, the regenerative cell
percentage of 100 ml of adipose tissue extracted from a lean
individual is greater than that extracted from an obese donor
(Table 1). This likely reflects a dilutive effect of the increased
fat content in the obese individual. Therefore, it may be
desirable, in accordance with one aspect of the invention, to
obtain larger amounts of tissue from overweight donors compared to
the amounts that would be withdrawn from leaner patients. This
observation also indicates that the utility of this invention is
not limited to individuals with large amounts of adipose
tissue.
TABLE-US-00001 TABLE 1 Effect of Body Mass Index on Tissue and Cell
Yield Amount of Tissue Total Regenerative Body Mass Index Status
Obtained (g) Cell Yield (.times.10.sup.7) Normal .sup. 641 .+-. 142
2.1 .+-. 0.4 Obese 1,225 .+-. 173 2.4 .+-. 0.5 p value 0.03 0.6
[0067] After the adipose tissue is processed, the resulting
regenerative cells are substantially free from mature adipocytes
and connective tissue. Accordingly, the system of the present
invention generates a heterogeneous plurality of adipose derived
regenerative cells which may be used for research and/or
therapeutic purposes. In a preferred embodiment, the cells are
suitable for placement or re-infusion within the body of a
recipient. In other embodiments, the cells may be used for
research, e.g., the cells can be used to establish stem or
progenitor cell lines which can survive for extended periods of
time and be used for further study.
[0068] Referring now to the Figures, a system 10 of the present
invention is generally comprised of one or more of a tissue
collection chamber 20, a processing chamber 30, a waste chamber 40,
an output chamber 50 and a sample chamber 60. The various chambers
are coupled together via one or more conduits 12 such that fluids
containing biological material may pass from one chamber to another
while maintaining a closed, sterile fluid/tissue pathway. The
conduits may comprise rigid or flexible bodies referred to
interchangeably herein as lumens and tubing, respectively. In
certain embodiments, the conduits are in the form of flexible
tubing, such as polyethylene tubing conventionally used in clinical
settings, silicone or any other material known in the art. The
conduits 12 can vary in size depending on whether passage of fluid
or tissue is desired. The conduits 12 may also vary in size
depending on the amount of tissue or fluid that is cycled through
the system. For example, for the passage of fluid, the conduits may
have a diameter ranging from about 0.060 to about 0.750 inches and
for the passage of tissue, the conduits may have a diameter ranging
from 0.312 to 0.750 inches. Generally, the size of the conduits is
selected to balance the volume the conduits can accommodate and the
time required to transport the tissue or fluids through said
conduits. In automated embodiments of the system, the foregoing
parameters, i.e., volume and time for transport, must be identified
such that the appropriate signals can be transmitted to the
processing device of the system. This allows the device to move
accurate volumes of liquid and tissue from one chamber to another.
The flexile tubing used should be capable of withstanding negative
pressure to reduce the likelihood of collapse. The flexible tubing
used should also be capable of withstanding positive pressure which
is generated by, for example, a positive displacement pump, which
may be used in the system.
[0069] All the chambers of the system may be comprised of one or
more ports, e.g., outlet 22 or inlet 21 ports, which accept
standard IV, syringe and suction tubing connections. The ports may
be a sealed port such as a rubber septum closed syringe needle
access port 51. The inlet ports may be coupled to one or more
cannulas (not shown) by way of conduits. For example, a tissue
inlet port 21 may be coupled to an integrated single use
liposuction cannula and the conduit may be a flexible tubing. The
conduits are generally positioned to provide fluid passageways from
one chamber of the system to another. Towards this end, the
conduits and ports may be coupled to, for example, a suction device
(not shown) which may be manually or automatically operated. The
suction device may be, e.g., a syringe or an electric pump. The
suction device should be capable of providing sufficient negative
pressure to aspirate tissue from a patient. Generally, any suitable
suction device known to one of ordinary skill in the art, e.g., a
surgeon, may be used.
[0070] The conduits 12 may further comprise one or more clamps (not
shown) to control the flow of material among various components of
the system. The clamps are useful for maintaining the sterility of
the system by effectively sealing different regions of the system.
Alternatively, the conduits 12 may comprise one or more valves 14
that control the flow of material through the system. The valves 14
are identified as open circles in the Figures. In preferred
embodiments, the valves may be electromechanical pinch valves. In
another embodiment, the valves may be pneumatic valves. In yet
other embodiments, the valves may be hydraulic valves or mechanical
valves. Such valves are preferably activated by a control system
which may be coupled to levers. The levers may be manually
manipulated such that the levers are activated. In automated
embodiments, the control system may be coupled to the levers as
well as to a processing device which may activate the valves at
pre-determined activation conditions. In certain automated
embodiments, activation of the valves may be partially automated
and partially subject to the user's preference such that the
process may be optimized. In yet other embodiments, certain valves
may be activated manually and others automatically through the
processing device. The valves 14 may also be used in conjunction
with one or more pumps, e.g., peristaltic pumps 34 or positive
displacement pumps (not shown). The conduits 12 and/or the valves
14 may also be comprised of sensors 29, e.g., optical sensors,
ultrasonic sensors, pressure sensors or other forms of monitors
known in the art that are capable of distinguishing among the
various fluid components and fluid levels that flow through the
system. In a preferred embodiment, the sensors 29 may be optical
sensors.
[0071] The system may also include a plurality of filters 36. In
certain embodiments, the filters may be within a chamber of the
system 28. Different chambers within the system may be comprised of
different filters. The filters are effective to separate the
regenerative cells, e.g., stem cells and/or progenitor cells, from
undesirable cells and disaggregation agents that may be used in
accordance with the system. In one embodiment, a filter assembly 36
includes a hollow fiber filtration device. In another embodiment, a
filter assembly 36 includes a percolative filtration device, which
may or may not be used with a sedimentation process. In a further
embodiment, the filter assembly 36 comprises a centrifugation
device, which may or may not be used with an elutriation device and
process. In yet another embodiment, the system comprises a
combination of these filtering devices. The filtration functions of
the present invention can be two-fold, with some filters removing
things from the final concentration such as collagen, free lipid,
free adipocytes and residual collagenase, and with other filters
being used to concentrate the final product. The filters of the
system may be comprised of a plurality of pores ranging in
diameters and/or length from 20 to 800 .mu.m. In a preferred
embodiment, the collection chamber 20 has a prefixed filter 28 with
a plurality of pores ranging from 80 to 400 .mu.m. In another
preferred embodiment, the collection chamber 20 has a prefixed
filter 28 with a plurality of 265 .mu.m pores. In other
embodiments, the filters may be detachable and/or disposable.
[0072] The system may also be comprised of one or more temperature
control devices (not shown) that are positioned to adjust the
temperature of the material contained within one or more chambers
of the system. The temperature control device may be a heater, a
cooler or both, i.e., it may be able to switch between a heater and
a cooler. The temperature device may adjust the temperature of any
of the material passing through the system, including the tissue,
the disaggregation agents, the resuspension agents, the rinsing
agents, the washing agents or the additives. For example, heating
of adipose tissue facilitates disaggregation whereas the cooling of
the regenerative cell output is desirable to maintain viability.
Also, if pre-warmed reagents are needed for optimal tissue
processing, the role of the temperature device would be to maintain
the pre-determined temperature rather than to increase or decrease
the temperature.
[0073] To maintain a closed, sterile fluid/tissue pathway, all
ports and valves may comprise a closure that maintains the sealed
configuration of the system. The closure may be a membrane that is
impermeable to fluid, air and other contaminants or it may be any
other suitable closure known in the art. Furthermore, all ports of
the system may be designed such that they can accommodate syringes,
needles or other devices for withdrawing the materials in the
chambers without compromising the sterility of the system.
[0074] As set forth herein, tissue may be extracted from a patient
via any art recognized method. The aspirated tissue may be
extracted prior to being placed in the system for processing. The
aspirated tissue is typically transferred to the collection chamber
20 through conduits 12 via a sealed entry port, such as a rubber
septum closed syringe needle access port (not shown on collection
chamber). Alternatively, the tissue extraction step may be part of
the system. For example, the collection chamber 20 may be comprised
of a vacuum line 11 which facilitates tissue removal using a
standard cannula inserted into the patient. Thus, in this
embodiment, the entire system is attached to the patient. The
tissue may be introduced into the collection chamber 20 through an
inlet port 21 via a conduit such as 12a which are part of a closed
sterile pathway. The collection chamber 20 may be comprised of a
plurality of flexible or rigid canisters or cylinders or
combinations thereof. For example, the collection chamber 20 may be
comprised of one or more rigid canisters of varying sizes. The
collection chamber 20 may also be comprised of one or more flexible
bags. In such systems, the bag is preferably provided with a
support, such as in internal or external frame, that helps reduce
the likelihood that the bag will collapse upon the application of
suction to the bag. The collection chamber 20 is sized to hold the
requisite amount of saline to appropriately wash and disaggregate
the tissue prior to the wash and concentrate stage of the process
performed in the processing chamber 30. Preferably, the volume of
tissue or fluid present in the collection chamber 20 is easily
ascertainable to the naked eye. For example, to obtain regenerative
cells from adipose tissue, a suitable collection chamber has the
capacity to hold 800 ml of lipoaspirate and 1200 ml of saline.
Accordingly, in one embodiment, the collection chamber 20 has a
capacity of at least 2 liters. In another embodiment, to separate
and concentrate red blood cells from blood, the collection chamber
20 has a capacity of at least 1.5 liters. Generally, the size of
the collection chamber 20 will vary depending on the type and
amount of tissue collected from the patient. The collection chamber
20 may be sized to hold as little as about 5 ml to up to about 2
liters of tissue. For smaller tissue volumes, e.g., 5 mls to 100
mls, the tissue may be gathered in a syringe prior to transfer to
the collection chamber 20.
[0075] The collection chamber 20 may be constructed using any
suitable biocompatible material that can be sterilized. In a
preferred embodiment, the collection chamber 20 is constructed of
disposable material that meets biocompatibility requirements for
intravascular contact as described in the ISO 10993 standard. For
example, polycarbonate acrylic or ABS may be used. The fluid path
of the collection chamber 20 is preferably pyrogen free, i.e.,
suitable for blood use without danger of disease transmittal. In
one embodiment, the collection chamber 20 is constructed of a
material that allows the user to visually determine the approximate
volume of tissue present in the chamber. In other embodiments, the
volume of tissue and/or fluid in the collection chamber 20 is
determined by automated sensors 29. The collection chamber 20 is
preferably designed such that in an automated embodiment, the
system can determine the volume of tissue and/or fluid within the
chamber with a reasonable degree of accuracy. In a preferred
embodiment, the system senses the volume within the collection
chamber with an accuracy of plus or minus fifteen percent.
[0076] In a particular embodiment provided by way of example only,
the collection chamber 20 is in the form of a rigid chamber, for
example, a chamber constructed of a medical grade polycarbonate
containing a roughly conical prefixed filter 28 of medical grade
polyester with a mesh size of 265 .mu.m (see FIG. 5). The rigid
tissue collection container may have a size of approximately eight
inches high and approximately five inches in diameter; the wall
thickness may be about 0.125 inches. The interior of the cylinder
may be accessed through, for example, one or more ports for suction
tubing, one or more ports with tubing for connection through
sterile docking technology, and/or one or more ports for needle
puncture access through a rubber septum. The prefixed filter 28 in
the interior of the collection chamber 20 is preferably structured
to retain adipose tissue and to pass non-adipose tissue as, for
example, the tissues are removed from the patient. More
specifically, the filter 28 may allow passage of free lipid, blood,
and saline, while retaining fragments of adipose tissue during, or
in another embodiment after, the initial harvesting of the adipose
tissue. In that regard, the filter 28 includes a plurality of
pores, of either the same or different sizes, but ranging in size
from about 20 .mu.m to 5 mm. In a preferred embodiment, the filter
28 includes a plurality of 400 .mu.m pores. In a preferred
embodiment, the filter 28 is a medical grade polyester mesh of
around 200 .mu.m thickness with a pore size of around 265 .mu.m and
around 47% open area. This material holds the tissue during rinsing
but allows cells to pass out through the mesh following tissue
disaggregation. Thus, when the tissues are aspirated from the
patient, non-adipose tissue may be separated from adipose tissue.
The same functionality could be achieved with different materials,
mesh size, and the number and type of ports. For example, mesh pore
sizes smaller than 100 .mu.m or as large as several thousand
microns would achieve the same purpose of allowing passage of
saline and blood cells while retaining adipose tissue aggregates
and fragments. Similarly, the same purpose could be achieved by use
of an alternative rigid plastic material, or by many other
modifications that would be known to those skilled in the art
[0077] The system 10 may also be comprised of one or more solution
sources 22. The solution source may comprise a washing solution
source 23, and a tissue disaggregation agent source 24, such as
collagenase. The collection chamber 20 is comprised of closed fluid
pathways that allows for the washing and disaggregating solutions
or agents to be added to the tissue in an aseptic manner.
[0078] The containers for the washing solution 23 and the
disaggregation agents 24 may be any suitable container that can
hold their contents in a sterile manner, e.g., a collapsible bag,
such as an IV bag used in clinical settings. These containers may
have conduits 12, such as conduit 12e, coupled to the collection
chamber 20 so that the washing solution and the disaggregation
agent may be delivered to the interior of the collection chamber
20. The washing solution and the disaggregation agent may be
delivered to the interior of the collection chamber 20 through any
art-recognized manner, including simple gravity pressure applied to
the outside of the containers for the saline 23 and/or the
disaggregation agents 24 or by placement of a positive displacement
pump on the conduits, e.g., conduit 12d in FIG. 4. In automated
embodiments, the processing device of the system calculates various
parameters, e.g., the volume of saline and time or number of cycles
required for washing as well as the concentration or amount of
disaggregation agent and the time required for disaggregation based
on information initially entered by the user (e.g., volume of
tissue being processed). Alternatively, the amounts, times etc. can
be manually manipulated by the user.
[0079] The tissue and/or fluid within the collection chamber should
be maintained at a temperature ranging from 30 degrees Celsius to
40 degrees Celsius. In a preferred embodiment, the temperature of
the suspension inside the collection chamber is maintained at 37
degrees Celsius. In certain embodiments, if the surgical procedure
or therapeutic application needs to be delayed, the selected tissue
may be stored in the collection chamber for later use. The tissue
may be stored at or about room temperature or at about 4 degrees
Celsius for up to 96 hours.
[0080] The washing solution may be any solution known to one of
skill in the art, including saline or any other buffered or
unbuffered electrolyte solution. The types of tissue being
processed will dictate the types or combinations of washing
solutions used. Typically, the washing solution, such as saline,
enters the collection chamber 20 after the adipose tissue has been
removed from the patient and placed in the collection chamber.
However, the washing solution may be delivered to the collection
chamber 20 before the adipose tissue is extracted, or may be
delivered to the collection chamber 20 concurrently with the
adipose tissue. In the collection chamber 20, the washing solution
and the extracted adipose tissue may be mixed by any means
including the methods described below.
[0081] For example, the tissue may be washed by agitation (which
maximizes cell viability and minimizes the amount of free lipid
released). In one embodiment, the tissue is agitated by rotating
the entire collection chamber 20 through an arc of varying degrees
(e.g., through an arc of about 45 degrees to about 90 degrees) at
varying speeds, e.g., about 30 revolutions per minute. In other
embodiments, the tissue is agitated by rotating the entire
collection chamber 20, wherein the collection chamber 20 is
comprised of one or more paddles or protrusions rigidly attached to
an inside surface of the collection chamber, through an arc of
varying degrees (e.g., through an arc of about 45 degrees to about
90 degrees) at varying speeds, e.g., about 30 revolutions per
minute. The rotation of the collection chamber 20 described above
may be accomplished by a drive mechanism attached to or in
proximity with the collection chamber 20. The drive mechanism may
be a simple belt or gear or other drive mechanism known in the art.
The speed of the rotation may be, for example, 30 revolutions per
minute. Generally, higher speeds have been found to generate larger
volumes of free lipids and may not be optimal.
[0082] In other embodiments, the tissue is agitated by placing a
rotatable shaft 25 inside the collection chamber 20, wherein the
rotatable shaft is comprised of one or more paddles 25a or
protrusions rigidly attached to the rotatable shaft 25 which pass
through the mixture as the shaft is being rotated. In certain
embodiments, the rotatable shaft 25 with rigidly attached 25a
paddles may be rested on the bottom of the collection chamber 20.
This may be accomplished, for example, by placing the paddle-like
device into a spinning magnetic field (e.g., magnetic stirrer).
Alternatively, agitating of the tissue may be accomplished using a
simple agitator known in the art, i.e. a device implementing
shaking up and down without rotation. The tissue may also be washed
using any other art-recognized means including rocking, stirring,
inversion, etc.
[0083] After a desired amount of wash cycles, a tissue
disaggregation agent may be delivered to the collection chamber 20
to separate the regenerative cells from the remaining adipose
tissue components. The disaggregation agent may be any
disaggregation agent known to one of skill in the art.
Disaggregation agents that may be used include neutral proteases,
collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease,
members of the Blendzyme enzyme mixture family, e.g., Liberase H1,
pepsin, ultrasonic or other physical energy, lasers, microwaves,
other mechanical devices and/or combinations thereof. A preferred
disaggregation agent of the invention is collagenase. The
disaggregation agents may be added with other solutions. For
example, saline, such as saline delivered from a saline source 23
as described above, may be added to the adipose tissue along with
or immediately followed by addition of collagenase. In one
embodiment, the washed adipose tissue is mixed with a
collagenase-containing enzyme solution at or around 37.degree. C.
for about 20-60 minutes. In other embodiments, a higher
concentration of collagenase or similar agent may be added to
decrease the digestion time. The washed adipose tissue and the
tissue disaggregation agent may then be agitated in manners similar
to the agitation methods described above, until the washed adipose
tissue is disaggregated. For example, the washed adipose tissue and
the tissue disaggregation agent may be agitated by rotating the
entire collection chamber through an arc of approximately 90
degrees, by having a shaft which contains one or more paddles which
pass through the solution as the shaft is being rotated, and/or by
rotating the entire collection chamber which contains paddles or
protrusions on the inside surface of the collection chamber.
[0084] Depending on the purpose for which the adipose derived cells
will be used, the adipose tissue may either be partially
disaggregated, or completely disaggregated. For example, in
embodiments in which the adipose derived cells are to be combined
with a unit of adipose tissue, it may be desirable to partially
disaggregate the harvested adipose tissue, to remove a portion of
the partially disaggregated adipose tissue, and then continue
disaggregating the remaining portion of adipose tissue remaining in
the collection chamber. Alternatively, a portion of washed adipose
tissue may be removed and set aside in a sample container prior to
any digestion. In another embodiment, harvested adipose tissue is
partially disaggregated to concentrate cells before being
reintroduced back into the patient. In one embodiment, the adipose
tissue is mixed with a tissue disaggregation agent for a period of
time generally less than about 20 minutes. A portion of the
partially disaggregated tissue may then be removed from the
collection chamber, and the remaining partially disaggregated
tissue may be further disaggregated by mixing the adipose tissue
with a tissue disaggregation agent for another 40 minutes. When the
adipose derived cells are to be used as an essentially pure
population of regenerative cells, the adipose tissue may be fully
disaggregated.
[0085] After digestion, the tissue and disaggregation agent
solution is allowed to settle for a period of time sufficient to
allow the buoyant and non-buoyant components of the solution to
differentiate within the collection chamber. Typically, the time
ranges from about 15 seconds to several minutes but other times may
be implemented in modified embodiments. The buoyant layer is
comprised of the regenerative cells that require further washing
and concentrating. The non-buoyant layer comprises blood, collagen,
lipids and other non-regenerative cell components of the tissue.
The non-buoyant layer must be removed to the waste chamber.
[0086] Accordingly, the collection chamber 20 is preferably
comprised of an outlet port 22 at the lowest point of the chamber
such that blood and other non-buoyant components of the tissue may
be drained to one or more waste containers 40 via one or more
conduits 12. The collection chamber 20 is generally in (or may be
placed in) an upright position such that the outlet ports 22 are
located at the bottom of the collection chamber. The draining may
be passive or active. For example, the non-buoyant components
described above could be drained using gravity, by applying
positive or negative pressure, by use of pumps 34 or by use of
vents 32. In automated embodiments, the processing device can
signal certain valves and/or pumps to drain the non-buoyant layer
from the collection chamber 20. The automated embodiments may also
be comprised of sensors 29 which can detect when the interface
between the buoyant and non-buoyant liquids has been reached. The
automated embodiments may also be comprised of a sensor 29, e.g.,
an optical sensor, which may be capable of detecting a change in
the light refraction of the effluent which is flowing in the
conduit leading out of the collection chamber. The appropriate
change in the light refraction may signal the presence of the
buoyant layer in the outgoing conduits which indicates that the
non-buoyant layer has been drained. The sensor 29 can then signal
the processing device to proceed with the next step.
[0087] In certain embodiments however, the tissue may be processed
to retrieve the non-regenerative cell component of the tissue. For
example, in certain therapeutic or research applications, collagen,
proteins, matrix or stromal components, lipids, adipocytes or other
components of the tissue may be desired. In such embodiments, it is
the buoyant layer comprising the regenerative cells that must be
removed as described above to the waste chamber. The non-buoyant
layer is then retained in the system for further processing as
needed.
[0088] Once the non-buoyant layer is removed, the buoyant layer
comprising the regenerative cells may be washed one or more times
to remove residual contaminants. Accordingly, the collection
chamber 20 typically includes one or more ports 21 for permitting
the washing solution to be delivered to the interior of the
chamber, and one or more ports 22 for permitting waste and other
materials to be directed out from the collection chamber 20. For
example, the collection chamber may include one or more sealed
entry ports as described herein. The collection chamber 20 may also
include one or more caps (not shown), such as a top cap and a
bottom cap to further ensure that the system remains sterile while
washing solution is delivered into the collection chamber and/or
waste is transported out. The ports 21 may be provided on the caps
of the collection chamber or on a sidewall of the collection
chamber.
[0089] The process of washing with fresh wash solution may be
repeated until the residual content of non-buoyant contaminants in
the solution reaches a pre-determined level. In other words, the
remaining material in the collection chamber 20, which comprises
the buoyant material of the mixture described above, including
adipose tissue fragments, may be washed one or more additional
times until the amount of undesired material is reduced to a
desired pre-determined level. One method of determining the end
point of the washing is to measure the amount of red blood cells in
the tissue solution. This can be accomplished by measuring the
light absorbed on the 540 nm wavelength. In a preferred embodiment,
a range between about 0.546 and about 0.842 is deemed
acceptable.
[0090] During the washing and/or disaggregation, one or more
additives may be added to the various containers as needed to
enhance the results. Some examples of additives include agents that
optimize washing and disaggregation, additives that enhance the
viability of the active cell population during processing,
anti-microbial agents (e.g., antibiotics), additives that lyse
adipocytes and/or red blood cells, or additives that enrich for
cell populations of interest (by differential adherence to solid
phase moieties or to otherwise promote the substantial reduction or
enrichment of cell populations). Other possible additives include
those that promote recovery and viability of regenerative cells
(for example, caspase inhibitors) or which reduce the likelihood of
adverse reaction on infusion or emplacement (for example,
inhibitors of re-aggregation of cells or connective tissue).
[0091] After a sufficient settling time has elapsed, the
non-buoyant fraction of the resulting mixture of washed adipose
tissue fragments and tissue disaggregation agents will contain
regenerative cells, e.g., stem cells and other adipose derived
progenitor cells. As discussed herein, the non-buoyant fraction
containing the regenerative cells will be transferred to the
processing chamber 30 wherein the regenerative cells of interest,
such as the adipose derived stem cells, will be separated from
other cells and materials present in the non-buoyant fraction of
the mixture. This non-buoyant fraction is referred to herein as the
regenerative cell composition and comprises multiple different
types of cells, including stem cells, progenitor cells, endothelial
precursor cells, adipocytes and other regenerative cells described
herein. The regenerative cell composition may also contain one or
more contaminants, such as collagen and other connective tissue
proteins and fragments thereof, which were present in the adipose
tissue fragments, or residual collagenase from the tissue
disaggregation process.
[0092] The processing chamber 30 of the invention is preferably
positioned within the system such that the regenerative cell
composition moves from the collection chamber 20 to the processing
chamber 30 by way of tubing 12, valves 14 and pump 34 in a sterile
manner. The processing chamber is sized to accommodate tissue/fluid
mixtures ranging from 10 mL to 1.2 L. In a preferred embodiment,
the processing chamber is sized to accommodate 800 mLs. In certain
embodiments, the entire regenerative cell composition from the
collection chamber 20 is directed to the processing chamber 30.
However, in other embodiments, a portion of the regenerative cell
composition is directed to the processing chamber 30, and another
portion is directed to a different region of the system, e.g., the
sample chamber 60, to be recombined with cells processed in the
processing chamber 30 at a later time.
[0093] The processing chamber 30 may be constructed using any
suitable biocompatible material that can be sterilized. In a
preferred embodiment, the processing chamber 30 is constructed of
disposable material that meets biocompatibility requirements for
intravascular contact, as described in the ISO 10993 standard. For
example, polycarbonate, acrylic, ABS, ethylene vinyl acetate or
styrene-butadiene copolymers (SBC) may be used. In another
embodiment, the fluid path of the disposable processing chamber is
pyrogen free. The processing chamber may be in the form of a
plastic bag, such as those conventionally used in processing blood
in blood banks; or in other embodiments, it may be structurally
rigid (FIG. 6). In one embodiment, the processing chamber 30 may be
similar to the processing chamber disclosed in commonly owned U.S.
application Ser. No. 10/316,127, filed Dec. 7, 2001 and U.S.
application Ser. No. 10/325,728, filed Dec. 20, 2002, the contents
of which in their entirety are hereby incorporated by
reference.
[0094] The processing chamber 30 may be constructed in any manner
suitable for separating and concentrating cells, including
filtration and centrifugation and/or combinations thereof. In
certain embodiments, the regenerative cell composition from the
collection chamber 20 is introduced into the processing chamber 30
where the composition can be filtered to separate and/or
concentrate a particular regenerative cell population. Cell
filtration is a method of separating particular components and
cells from other different components or types of cells. For
example, the regenerative cell composition of the invention
comprises multiple different types of cells, including stem cells,
progenitor cells and adipocytes, as well as one or more
contaminants, such as collagen, which was present in the adipose
tissue fragments, or residual collagenase from the tissue
disaggregation process. The filters 36 present in the processing
chamber 30 may allow for separation and concentration of a
particular subpopulation of regenerative cells, e.g., stem cells or
endothelial progenitors cells etc.
[0095] Some variables which are associated with filtration of cells
from a liquid include, but are not limited to, pore size of the
filter media, geometry (shape) of the pore, surface area of the
filter, flow direction of the solution being filtered,
trans-membrane pressure, dilution of the particular cell
population, particulate size and shape as well as cell size and
cell viability. In accordance with the disclosure herein, the
particular cells that are desired to be separated or filtered are
typically adipose derived stem cells. However, in certain
embodiments, the particular cells may include adipose derived
progenitor cells, such as endothelial precursor cells, alone or in
combination with the stem cells.
[0096] The regenerative cell composition may be directed through a
filter assembly, such as filter assembly 36. In certain
embodiments, the filter assembly 36 comprises a plurality of
filters which are structured to perform different functions and
separate the regenerative cell composition into distinct parts or
components. For example, one of the filters may be configured to
separate collagen from the regenerative cell composition, one of
the filters may be configured to separate adipocytes and/or lipid
components from the regenerative cell composition, and one of the
filters may be configured to separate residual enzymes, such as the
tissue disaggregation agent, from the regenerative cell
composition. In certain embodiments, one of the filters is capable
of performing two functions, such as separating collagen and the
tissue disaggregation agent from the composition. The plurality of
filters are typically serially arranged; however, at least a
portion of the filters may be arranged in parallel, as well. A
serial arrangement of the filters of the filter assembly 36 is
shown in FIG. 2. A parallel arrangement of the filters of the
filter assembly 36 is shown in FIG. 3.
[0097] In one embodiment, the filter assembly 36 comprises a first
filter, a second filter, and a third filter. The first filter is
configured to remove collagen particles present in the regenerative
cell composition. These collagen particles are typically
approximately 0.1 microns in diameter and can be up to 20 microns
long. The collagen particles may be of varying sizes depending on
the digestion. They also may be fibrils, meaning they have twists
and turns. Any of the filters described herein may be made from
polyethersulfone, polyester, PTFE, polypropylene, PVDF, or possibly
cellulose. There are two possibilities for filtering the collagen.
One is to try to remove the larger particles first, letting the
cells go through, which would require for example a filter probably
in the 10 micron range. The second method is to use a smaller size
filter, such as 4.5 micron, with the intent that the collagen would
be well digested, so as to trap the cells, and let the collagen
pass through. This would require a means to float the cells back
off the filter. There may also be a possibility of implementing a
filter which would attract and hold the collagen fibers.
[0098] The second filter is configured to remove free immature
adipocytes which are not buoyant in the regenerative cell
composition. In one embodiment the second filter can be constructed
of polyester and have a pore size between about 30 and about 50
microns with a preferred pore size being about 40 microns. Although
referred to as a second filter, placement of such a device may be
in a first, rather than second, position to facilitate an initial
removal of larger cells and particles. The third filter is
configured to remove the unused or residual collagenase or other
tissue disaggregation agent present in the composition. In a
preferred implementation, the collagenase may degenerate over time.
In one embodiment, the third filter comprises a plurality of pores
having a diameter, or length less than 1 .mu.m. In certain
embodiments, the pores may have diameters that are smaller than 1
.mu.m. In other embodiments, the pores have diameters between 10 kD
and 5 microns. In certain embodiments, the third filter may be
configured to concentrate the regenerative cell population into a
small volume of saline or other washing solution, as discussed
herein. As presently preferred, only the final filter is the hollow
fiber unit. It is not necessary for any of the filters to be of the
hollow fiber type. The hollow fiber unit is used for the final
filter in a preferred implementation because it is the most
efficient in removing the collagenase with the smallest detrimental
effect to the regenerative cells. In an embodiment wherein the
device is a collection of off the shelf items, the three filters
are in separate housings. It is feasible to have the first and
second filters combined into one housing if a hollow fiber unit is
used for the third filter. If the final filter is not a hollow
fiber set-up then all three filters can be contained in one
housing.
[0099] The filters of the filter assembly 36 may be located in the
processing chamber 30 or may be provided as components separate
from the processing chamber 30. In addition, the filters of the
filter assembly 36 may be provided in multiple processing chambers
or in an inline fashion. In certain embodiments, the conduits or
tubing may act as a processing chamber or chambers. The processing
chamber can be reduced in size such that it becomes the inside
volume of the conduits which connect the filters. This type of
system will function correctly if the volume of tissue solution is
sized appropriately. Thus, the conduits may act as the processing
chamber by containing the fluid with cells as it is being run
through the filters. Care may be taken to minimize the volume of
the conduits so that cells/tissue are not unnecessarily lost in the
process of priming and running the system.
[0100] Referring to the embodiment described above, the
regenerative cell composition, containing the washed cells and
residual collagen, adipocytes, and/or undigested tissue
disaggregation agent, may be directed through the first filter to
remove at least a portion of and preferably substantially all of
the collagen particles from the composition so that fewer, and
preferably no, collagen particles are present in the filtered
solution. The filtered regenerative cell composition containing the
adipocytes and/or undigested tissue disaggregation agent, may then
be directed through the second filter to remove at least a portion
of and preferably substantially all of the free adipocytes from the
filtered regenerative cell composition. Subsequently, the twice
filtered regenerative cell composition, containing the undigested
tissue disaggregation agent, may be directed through the third
filter, such as a hollow fiber filtration device, as discussed
herein, to remove or reduce the undigested tissue disaggregation
agent from the regenerative cell composition.
[0101] The thrice-filtered regenerative cell composition (i.e., the
composition remaining after being passed through the first, second,
and third filters) may then be directed to multiple outlets, which
may include a portion of the processing chamber 30 comprising
multiple outlets. These outlets can serve to maintain the necessary
pressure, as well as to provide connections via conduits to other
containers which may include the collection chamber 20, the output
chamber 50, and/or the waste container 40.
[0102] In one embodiment, a filter of the filter assembly 36
comprises a hollow-fiber filtration member. Or, in other words, the
filter comprises a collection of hollow tubes formed with the
filter media. Examples of filter media which can be used with the
disclosed system 10 include polysulfone, polyethersulfone or a
mixed ester material, and the like. These hollow fibers or hollow
tubes of filter media may be contained in a cylindrical cartridge
of the filter assembly 36. The individual tubes or fibers of filter
media typically have an inside diameter which ranges from about 0.1
mm to about 1 mm with a preferred value being about 0.5 mm. The
diameter and length of a suitable cylindrical cartridge will
determine the number of individual tubes of filter media which can
be placed inside the cartridge. One example of a suitable hollow
fiber filter cartridge is the FiberFlo.RTM. Tangential Flow Filter,
catalog #M-C-050-K (Minntech, Minneapolis, Minn.). Pore sizes of
the filter media can range between about 10 kiloDaltons and about 5
microns with a preferred pore size being about 0.5 microns.
[0103] In the hollow-fiber filter, each hollow tube has a body with
a first end, a second end, and a lumen located in the body and
extending between the first end and second end. The body of each
hollow tube includes a plurality of pores. The pores are generally
oriented in the body so that a regenerative cell composition is
filtered by flowing through the lumen of the body, and the products
to be filtered tangentially pass through the pores, as shown in
FIG. 12A. In other words, the smaller particles in the liquid pass
tangentially through the pores relative the flow of fluid through
the lumen of the body. The composition with the regenerative cells
passes through the lumen of each hollow tube when the composition
is being filtered. Preferably, the flow of the composition is
tangential to the pores of the body of each hollow tube.
[0104] By using a tangential flow of fluid, the efficiency of
filtration of the stem cells may be enhanced relative to other
filtration techniques. For example, in accordance with some
filtration techniques, the pores of the filter media are placed in
such a manner that the filter is orientated perpendicular to the
flow of the fluid so that the Filter media blocks the path of the
fluid being filtered, as illustrated in FIG. 12B. In this type of
filtration, the particles which are being filtered out of the
regenerative cell composition, e.g., the stem cells, tend to build
up on one side of the filter and block the flow of the fluid
through the pores. This blockage can reduce the efficiency of the
filter. In addition, the cells are constantly compressed by the
pressure of the fluid flow as well as the weight of the cells
accumulating on the upstream side of the filter. This can lead to
increased lysis of stem cells. Thus, in such filtration techniques
wherein the flow of fluid is parallel to the orientation of the
pores in the filter, both large cells and small particles can be
undesirably directed against the filter media as the fluid is
passed through the pores. Consequently, larger products in the
liquid such as cells may block the pores, thereby decreasing the
filtering effect and increasing an occurrence of cell rupture or
injury.
[0105] In contrast, in the hollow fiber configuration of the
present system 10, the fluid which is being filtered flows inside
the lumen of the hollow tube. The portion of the fluid which has
the ability to pass through the pores of the body of the filter
does so with the aid of the positive pressure of the fluid on the
inside of the body as well as a negative pressure which is applied
on the outside of the body. In this embodiment, the cells typically
are not subjected to the pressure of the fluid flow or the weight
of other cells, and therefore, the shear forces on the stem cells
are reduced Thus, the efficiency and effectiveness of the
filtration can be enhanced by the reduction in clogging rates and
the reduction in regenerative cell lysis. Due to the size of the
saline and unwanted protein molecules, during filtration, these
molecules and other small components pass through the pores of the
bodies of the hollow tubes to the outside of the hollow tubes and
are directed to the waste container 40. In one embodiment,
filtration is enhanced by generating a vacuum on the outside of the
hollow tube filter media. Due to the size of the regenerative
cells, e.g., stem cells or progenitor cells, these cells typically
cannot pass through the pores of the body and therefore remain on
the inside of the hollow tube filter (e.g., in the lumens of the
tubes) and are directed back to the processing chamber 30 via a
conduit between the filter and the processing chamber, or to the
output chamber 50.
[0106] In one specific embodiment, the hollow fiber filter has
about a 0.05 micron pore size, and contains approximately 550
cm.sup.2 surface area of filter media. An individual media tube
typically has a diameter of about 0.5 mm. In processing 130 ml of
the regenerative cell composition, approximately 120 ml of
additional saline may be added to the composition. The processing
or filter time may be approximately 8 minutes. The differential of
the pressures on either side of the body of the hollow fiber tube
(e.g., the pressure inside the lumen of the body, and outside the
body) is considered the trans-membrane pressure. The trans-membrane
pressure can range from about 1 mmHg to about 500 mmHg with a
preferred pressure being about 200 mmHg. The average nucleated cell
recovery and viability using hollow fiber filtration can be
approximately 80% of viable cells.
[0107] The amount of collagenase which is typically removed in such
a system equates to a three log reduction. For example if the
initial concentration of collagenase in the regenerative cell
composition which is transferred from the collection chamber to the
processing chamber is 0.078 U/ml the collagenase concentration of
the final regenerative cell composition would be 0.00078 U/ml. The
collagenase is removed in the hollow fiber filter, and the hollow
fiber filter corresponds to the third filter discussed above.
[0108] Processing chambers illustrating one or more cell filtration
methods described above are shown in the Figures, particularly
FIGS. 1-3. With reference to FIGS. 1-3, between the processing
chamber 30 and the filtering chamber of the filter assembly 36, a
pump may be provided, such as pump 34. In addition, vent and
pressure sensors, such as vent 32, and pressure sensor 39, may be
provided in line with the processing chamber 30 and the filter
assembly 36. Fittings for the output chamber 50 may also be
provided. These optional components (e.g., the pump 34, the vent
32, the pressure sensor 39, and the fittings for the output chamber
50) may be provided between the processing chamber 30 and the
filter assembly 36 so that liquid contained in the processing
chamber 30 may flow to one or more of these optional components
before flowing through the filter assembly 36. For example, liquid
may flow through the pump 34 before it is passed to the filter
assembly 36. Or, liquid may pass through the pressure sensor 39
before passing through the filter assembly to obtain a pre-filter
liquid pressure in the system. In certain situations, one or more
of these components may also be provided as an element of the
processing chamber 30, such as the vent 32 as illustrated in FIG.
6. In the illustrated embodiment, the pressure sensor 39 is in line
to determine the pressure of the regenerative cell composition
which is generated by the pump 34 as it enters the filtering
chamber of the filter assembly 36. This construction can facilitate
monitoring of the trans-membrane pressure across the filter
membrane. Additional saline or other buffer and washing solution
can be added to the regenerative cell composition to assist in the
removal of unwanted proteins as the composition is being filtered
through the filter assembly 36. This repeated washing can be
performed multiple times to enhance the purity of the regenerative
cells. In certain embodiments, the saline can be added at any step
as deemed necessary to enhance filtration.
[0109] In one specific embodiment, which is provided by way of
example and not limitation, the unwanted proteins and saline or
other washing solution is removed in the following manner. The
composition with the regenerative cells, as well as collagen and
connective tissue particles or fragments, adipocytes, and
collagenase, is cycled through a series of filters until a minimum
volume is reached. The minimum volume is a function of the total
hold up volume of the system and some predetermined constant. The
hold up volume is the volume of liquid which is contained in the
tubing and conduits if all of the processing chambers are empty. In
one embodiment, the minimum volume is 15 ml. When the minimum
volume is reached, a predetermined volume of washing solution is
introduced into the system to be mixed with the regenerative cell
composition. This mixture of washing solution and the regenerative
cell composition is then cycled through the filters until the
minimum volume is reached again. This cycle can be repeated
multiple times to enhance the purity of the regenerative cells, or
in other words, to increase the ratio of regenerative cells in the
composition to the other materials in the composition. See FIGS. 10
and 11.
[0110] After it has been determined that the regenerative cell
composition has been cleansed of unwanted proteins and concentrated
sufficiently (in exemplary embodiments, minimum concentrations
within a range of about 1.times.10.sup.5 to about 1.times.10.sup.7
cells/ml can be used and, in a preferred embodiment the minimum
concentration can be about 1.times.10.sup.7 cells/ml), an output
chamber 50, such as an output bag, may be connected to an outlet
port of the processing chamber 30 and/or the filter assembly 36,
depending on the specific embodiment. A vent, such as the vent 32,
may then be opened to facilitate the output of the concentrated
regenerative cells. In one implementation, this determination of
when a minimum concentration has been reached is made empirically
after experiments have been run and programmed into the electronic
controls of the device. The determination can be an input into the
process of what is desired to yield, i.e., how many stem/progenitor
cells are desired, or range of cell concentration. Based on
scientific data, a predefined amount of adipose tissue needs to be
obtained and placed into the system to achieve the desired output.
With the vent 32 open, a pump, such as the pump 34, can function to
transfer the concentrated regenerative cells into the output bag.
In one embodiment, the output bag 50 is similar to an empty blood
bag which has a tube with a fitting on one end. In a sterile
fashion, the fitting on the output bag may be attached to the
outlet port, and the concentrated regenerative cells may be
transferred to the output bag.
[0111] As illustrated in FIGS. 1-3, a vacuum pump 26 may be
provided in the system 10 to change the pressure in the system,
among other things. For example, the vacuum pump 26 may be coupled
to the collection chamber 20 via a conduit, such as conduit 12b, to
cause a decrease in pressure within the collection chamber 20.
Vacuum pump 26 may also be coupled to the processing chamber 30 by
way of a conduit, such as conduit 12g. Regarding the operation of
vacuum pump 26 in connection with pump 34, two separate vacuum
pumps or sources may be implemented, or a single one may be
implemented by using valves which direct the vacuum pull to the
different conduits that need it at specific points in the process.
In addition, vacuum pump 26 may be coupled to the waste container
40 via a conduit, such as conduit 12f.
[0112] With reference to FIGS. 10 and 11, the pressure generated by
the vacuum pump 26 can be used to direct the flow of fluids,
including the regenerative cells, through the conduits 12. This
pressure can be supplied in multiple directions, for example, by
automatically or manually controlling the position of one or more
valves 14 in the system 10. The system 10 can be made to function
properly with the use of positive pressure or through the use of
negative pressure, or combinations thereof. For instance, the
regenerative cells can be pulled through the first and second
filters described above into a soft sided container which is
connected to the third filter. The soft-sided container can be in
line (serial) connected ahead of the third filter. The final output
chamber may be a soft sided container which is on the other side
(e.g., the downstream side) of the third filter. In this
embodiment, pressure is used to move the regenerative cells from
one soft sided container to a second soft sided container through
the filter.
[0113] In another embodiment of the system 10, the filtration of
the stem cells and/or adipose derived progenitor cells may be
accomplished using a combination of percolative filtration and
sedimentation. For example, such a system uses saline that is
passed through a tissue regenerative cell composition (e.g., the
composition containing the stem cells and/or adipose derived
progenitor cells) and then through a filter. Some of the variables
which are associated with percolative filtration of cells from a
regenerative cell composition include, but are not limited to, pore
size of the filter media, pore geometry or shape, surface area of
the filter, flow direction of the regenerative cell composition
being filtered, flow rate of the infused saline, trans-membrane
pressure, dilution of the cell population, cell size and
viability.
[0114] In one embodiment of the system 10, the processing chamber
30 uses a filter assembly 36 which implements percolative
filtration and sedimentation to separate and concentrate the
regenerative cells. By way of example, and not by way of
limitation, the processing chamber 30 is defined as a generally
cylindrical body having a sidewall 30a, a top surface 30b, and a
bottom surface 30c, as shown in FIG. 6. A sterile vent 32 is
provided in the top surface 30b.
[0115] In the embodiment of FIG. 6, the processing chamber 30 is
illustrated as including a filter assembly 36, which includes two
filters, such as large pore filter 36a, and small pore filter 36b.
The pore sizes of the filters 36a and 36b typically are in a range
between about 0.05 microns and about 10 microns. The large pore
filter 36a may comprise pores with a diameter of about 5 .mu.m, and
the small pore filter 36b may comprise pores with a diameter of
about 1-3 .mu.m. In one embodiment, the filters have a surface area
of about 785 mm.sup.2. Filters 36a and 36b divide an interior of
the processing chamber 30 to include a first chamber 37a, a second
chamber 37b, and a third chamber 37c. As shown in FIG. 6, first
chamber 37a is located between second chamber 37b and third chamber
37c. In addition, first chamber 37a is shown as being the region of
the processing chamber 30 having an inlet port 31a and an outlet
port 31b. The illustrated processing chamber 30 includes a
plurality of ports providing communication paths from an exterior
of the processing chamber 30 to the interior of the processing
chamber 30, such as ports 31a, 31b, and 31c. The ports 31a, 31b,
and 31c, are illustrated as being disposed in the sidewall 30a of a
body of the processing chamber 30. However, the ports 31a, 31b, and
31c could be positioned in other regions, as well. Port 31a is
illustrated as a sample inlet port, which is constructed to be
coupled to a conduit so that a composition containing regenerative
cells can be passed into the interior of the processing chamber 30.
Port 31b is illustrated as an outlet port constructed to be coupled
to a conduit so that the separated and concentrated cells may be
removed from the interior of the processing chamber 30. Port 31c is
illustrated as an inlet port constructed to be coupled to a conduit
for delivery of a fresh washing solution, such as saline into the
interior of the processing chamber 30.
[0116] In use, the regenerative cells may be introduced into the
central chamber 37a via inlet port 31a. Saline or other buffer is
introduced into the bottom chamber 37b through inlet port 31c. The
saline may be directed through the regenerative cell composition in
chamber 37a at a rate of about 10 ml/min. The flow rate of the
saline is such that it counteracts the force of gravity. The flow
of saline gives the cells in the chamber the ability to separate
based on the density of the cells. Typically, as the saline is
forced up through the composition the larger cells in the
composition will settle to the bottom of the central chamber 37a,
and the smaller cells and proteins will be carried away through the
second filter 36b into the top chamber 37c. This filtering is
accomplished by adjusting the flow rate of the saline such that the
larger cells are rolled in place which allows the smaller particles
to be liberated and carried off with the saline. The sterile vent
32 is included in the chamber 30 to ensure that the correct
pressure gradient is maintained in the three chambers within the
processing unit. The upper chamber 37c can comprise an absorbent
media 33. The purpose of the absorbent media is to trap the
unwanted proteins in the solution to ensure that they do not cross
the filter media back into the processing solution, if, for
example, the saline flow rate decreases. An absorbent media can be
a type of filter material that is absorbent, or attracts materials
or components to be filtered out. An outflow port can be added
above the top filter to help draw off the waste. Another embodiment
of this may be to apply a gentle vacuum from the top to help pull
off waste. Absorbent media can be implemented when, as in the
illustrated embodiment, the flow rates are relatively small. Excess
saline and proteins are then carried away to a waste container.
[0117] When the larger cells, (e.g., the adipose derived stem cells
and/or progenitor cells) have been sufficiently separated from
smaller cells and proteins, the composition containing the
separated cells may be concentrated, as discussed herein. The
composition may be further concentrated after it has been removed
from chamber 37a through outlet port 31b, or while it is in the
chamber 37a. In one embodiment, the concentration of cells in the
composition is increased in the following manner. After the cells
have been sufficiently separated the filters, such as filters 36a
and 36b, may be moved towards each other. This movement has the
effect of reducing the volume between the two filters (e.g., the
volume of chamber 37a). A vibrating member may also be provided in
connection with the processing chamber 30 to facilitate
concentrating of the cells in the composition. In one embodiment,
the vibrating member may be coupled to the filter 36b (e.g., the
small pore filter). Vibrating can reduce an incidence of cells
becoming trapped in the filters. The reduction in volume of the
composition allows the excess saline to be removed as waste and the
cells to be concentrated in a smaller volume.
[0118] In another embodiment, the concentration of the regenerative
cells is accomplished in the following manner. After the cells have
been sufficiently separated, the regenerative cell composition can
be transferred to another chamber (not shown) which uses gravity to
filter out the excess saline. In a preferred embodiment, the
sedimentation can occur at the same time as the percolation. This
sedimentation may be accomplished by introducing the composition on
top of a filter which has a pore size ranging from about 10 kD to
about 2 microns. In one embodiment, a suitable filter has a pore
size of about 1 micron. The force of gravity will allow the saline
and smaller particles to be passed through the filter while
preventing the cells in the composition to flow through the filter.
After the desired concentration of cells has been obtained, and
after the filtered smaller particles have been removed from below
the filter, the regenerative cell composition may be agitated to
remove the cells from the filter and, subsequently, the
concentrated regenerative cells may be transferred to the output
bag. The smaller particles can be drawn off as waste through an
outlet.
[0119] In a particular embodiment, the regenerative cell
composition from the collection chamber 20 is transported to the
processing chamber 30 wherein the composition can be centrifuged to
separate and concentrate regenerative cells. Centrifugation
principles are well know in the art and will be not be repeated
herein in the interest of brevity. Standard, art-recognized
centrifugation devices, components and parameters are utilized
herein. An exemplary processing chamber for use as part of a
centrifuge device is shown in FIGS. 7 and 8. Typically, a
centrifuge device causes a centrifuge chamber (such as the one
shown in FIG. 7) to spin around an axis to thereby increasing the
force on the cells in the solution to be greater than gravity. The
denser or heavier materials in the solution typically settle to one
end of the centrifuge chamber, i.e., an output chamber 50 of FIG.
7, to form a regenerative cell pellet. The pellet may then be
re-suspended to obtain a solution with a desired concentration of
cells and/or a desired volume of cells and medium. The processing
chamber shown in FIG. 7 is constructed to separate and concentrate
cells using both centrifugal and gravitational forces.
Specifically, during centrifugation, centrifugal force directs the
denser components of the regenerative cell composition, e.g., the
regenerative cells, towards the outermost ends of the centrifuge
chamber. As the centrifuge chamber slows down and eventually stops,
gravitational force helps the regenerative cells to remain in the
outermost ends of the centrifuge chamber and form a cell pellet.
Accordingly, the unwanted components of the regenerative cell
composition, i.e., the waste, can be removed without disturbing the
cell pellet.
[0120] In yet another embodiment of the invention, the processing
chamber may be comprised of a cell concentrator in the form of a
spinning membrane filter. In a further embodiment of the
centrifugation process, centrifugal elutriation may also be
applied. In this embodiment, the cells may be separated based on
the individual cell sedimentation rate such that the directional
(e.g., outward) force applied by centrifugation causes cells and
solutes to sediment at different rates. In elutriation, the
sedimentation rate of the target cell population is opposed by an
opposite (e.g., inward) flow rate applied by pumping solution in
the opposite direction to the centrifugal force. The counterflow is
adjusted so that the cells and particles within the solution are
separated. Elutriation has been applied in many instances of cell
separation (Inoue, Carsten et al. 1981; Hayner, Braun et al. 1984;
Noga 1999) and the principles and practices used to optimize flow
and centrifugal parameters can be applied herein in light of the
present disclosure by one skilled in the art.
[0121] FIG. 9 illustrates principles associated with an elutriation
implementation in accordance with the present invention. The
elutriation embodiment can be similar to a centrifugation
implementation to the extent that a force is applied to the
solution using a spinning rotor. Some of the variables which are
associated with the presently embodied elutriation separation
include, but are not limited to, the size and shape of the spinning
chamber, the diameter of the rotor, the speed of the rotor, the
diameter of the counter flow tubing, the flow rate of the counter
flow, as well as the size and density of the particles and cells
which are to be removed from solution. As in centrifugation, the
regenerative cells can be separated based on individual cell
densities.
[0122] In one embodiment the regenerative cell composition, e.g.,
the solution containing the regenerative cells and the collagenase,
is introduced into a chamber of a spinning rotor, as shown in FIG.
9A. After the solution is added to the chamber additional saline is
added to the chamber at a predetermined flow rate. The flow rate of
the saline can be predetermined as a function of the speed of the
rotor, the cell diameter, and the chamber constant which has been
established empirically. The flow rate will be controlled for
example with a device similar to an IV pump. A purpose of the
additional saline is to provide a condition inside the rotor
chamber where the larger particles will move to one side of the
chamber and the smaller particles will move to the other, as
illustrated in FIG. 9B. The flow is adjusted so that, in this
application, the smaller particles will exit the chamber and move
to a waste container, as shown in FIG. 9C. This movement results in
the solution in the rotor chamber having a substantially homogenous
population of cells, such as stem cells. After it has been
determined that the stem cells have been separated from the rest of
the items in the solution (with unwanted proteins and free lipids
having been removed from the chamber), the counter flow is stopped.
The cells inside the chamber will then form a concentrated pellet
on the outside wall of the chamber. The counter flow is reversed
and the cell pellet is transferred to the output bag.
[0123] As previously set forth herein, the processing chamber 30 or
the output chamber 50 may include one or more ports, e.g., ports 51
or 52. One or more of these ports may be designed to transport the
regenerative cells obtained using any combination of methods
described above, or a portion thereof, via conduits to other
surgical devices, cell culturing devices, cell marinading devices,
gene therapy devices or purification devices. These ports may also
be designed to transport the regenerative cells via conduits to
additional chambers or containers within the system or as part of
another system for the same purposes described above. The ports and
conduits may be also be used to add one or more additives, e.g.,
growth factors, re-suspension fluids, cell culture reagents, cell
expansion reagents, cell preservation reagents or cell modification
reagents including agents that transfer genes to the cells. The
ports and conduits may also be used to transport the regenerative
cells to other targets such as implant materials (e.g., scaffolds
or bone fragments) as well as other surgical implants and
devices.
[0124] Further processing of the cells may also be initiated by
reconfiguring the interconnections of the disposable sets of the
existing system, re-programming the processing device of the
existing system, by providing different or additional containers
and/or chambers for the existing system, by transporting the cells
to a one or more additional systems or devices and/or any
combinations thereof. For example, the system can be reconfigured
by any of the means described above such that the regenerative
cells obtained using the system may be subject to one or more of
the following: cell expansion (of one or more regenerative cell
types) and cell maintenance (including cell sheet rinsing and media
changing); sub-culturing; cell seeding; transient transfection
(including seeding of transfected cells from bulk supply);
harvesting (including enzymatic, non-enzymatic harvesting and
harvesting by mechanical scraping); measuring cell viability; cell
plating (e.g., on microtiter plates, including picking cells from
individual wells for expansion, expansion of cells into fresh
wells); high throughput screening; cell therapy applications; gene
therapy applications; tissue engineering applications; therapeutic
protein applications; viral vaccine applications; harvest of
regenerative cells or supernatant for banking or screening,
measurement of cell growth, lysis, inoculation, infection or
induction; generation of cells lines (including hybridoma cells);
culture of cells for permeability studies; cells for RNAi and viral
resistance studies; cells for knock-out and transgenic animal
studies; affinity purification studies; structural biology
applications; assay development and protein engineering
applications.
[0125] For example, if expansion of a regenerative cell population
is required for a particular application, an approach using culture
conditions to preferentially expand the population while other
populations are either maintained (and thereby reduced by dilution
with the growing selected cells) or lost due to absence of required
growth conditions could be used. Sekiya et al have described
conditions which might be employed in this regard for bone
marrow-derived stem cells (Sekiya et al., 2002). This approach
(with or without differential adherence to the tissue culture
plastic) could be applied to a further embodiment of this
invention. In this embodiment the final regenerative cell pellet is
removed from the output chamber and placed into a second system
providing the cell culture component. This could be in the form of
a conventional laboratory tissue culture incubator or a
Bioreactor-style device such as that described by Tsao et al., U.S.
Pat. No. 6,001,642, or by Armstrong et al., U.S. Pat. No.
6,238,908. In an alternative embodiment, the cell expansion or cell
culture component could be added to the existing system, e.g., into
the output chamber, allowing for short-term adherence and/or cell
culture of the adipose derived cell populations. This alternate
embodiment would permit integration of the cell culture and/or cell
expansion component to the system and remove the need for removing
the cells from this system and placement within another.
[0126] During the processing, one or more additives may be added to
or provided with the various chambers or containers as needed to
enhance the results. These additives may also be provided as part
of another system associated with the existing system or separate
from the existing system. For example, in certain embodiments, the
additives are added or provided without the need for removing the
regenerative cells from the system. In other embodiments, the
additives are added or provided by connecting a new container or
chamber comprising the additives into an unused port of the system
in a sterile manner. In yet other embodiments, the additives are
added or provided in a second system or device that is not
connected to the system of the present invention. Some examples of
additives include agents that optimize washing and disaggregation,
additives that enhance the viability of the active cell population
during processing, anti-microbial agents (e.g., antibiotics),
additives that lyse adipocytes and/or red blood cells, or additives
that enrich for cell populations of interest (by differential
adherence to solid phase moieties or to otherwise promote the
substantial reduction or enrichment of cell populations) as
described herein.
[0127] For example, to obtain a homogenous regenerative cell
population, any suitable method for separating and concentrating
the particular regenerative cell type may be employed, such as the
use of cell-specific antibodies that recognize and bind antigens
present on, for example, stem cells or progenitor cells, e.g.,
endothelial precursor cells. These include both positive selection
(selecting the target cells), negative selection (selective removal
of unwanted cells), or combinations thereof. Intracellular markers
such as enzymes may also be used in selection using molecules which
fluoresce when acted upon by specific enzymes. In addition, a solid
phase material with adhesive properties selected to allow for
differential adherence and/or elution of a particular population of
regenerative cells within the final cell pellet could be inserted
into the output chamber of the system.
[0128] An alternate embodiment of this differential adherence
approach would include use of antibodies and/or combinations of
antibodies recognizing surface molecules differentially expressed
on target regenerative cells and unwanted cells. Selection on the
basis of expression of specific cell surface markers (or
combinations thereof) is another commonly applied technique in
which antibodies are attached (directly or indirectly) to a solid
phase support structure (Geiselhart et al., 1996; Formanek et al.,
1998; Graepler et al., 1998; Kobari et al., 2001; Mohr et al.,
2001).
[0129] In another embodiment the cell pellet could be re-suspended,
layered over (or under) a fluid material formed into a continuous
or discontinuous density gradient and placed in a centrifuge for
separation of cell populations on the basis of cell density. In a
similar embodiment continuous flow approaches such as apheresis
(Smith, 1997), and elutriation (with or without counter-current)
(Lasch et al., 2000) (Ito and Shinomiya, 2001) may also be
employed.
[0130] Other examples of additives may include additional
biological or structural components, such as cell differentiation
factors, growth promoters, immunosuppressive agents, medical
devices, or any combinations thereof, as discussed herein. For
example, other cells, tissue, tissue fragments, growth factors such
as VEGF and other known angiogenic or arteriogenic growth factors,
biologically active or inert compounds, resorbable scaffolds, or
other additives intended to enhance the delivery, efficacy,
tolerability, or function of the population of regenerative cells
may be added. The regenerative cell population may also be modified
by insertion of DNA or by placement in a cell culture system (as
described herein or known in the art) in such a way as to change,
enhance, or supplement the function of the regenerative cells for
derivation of a structural or therapeutic purpose. For example,
gene transfer techniques for stem cells are known by persons of
ordinary skill in the art, as disclosed in (Morizono et al., 2003;
Mosca et al., 2000), and may include viral transfection techniques,
and more specifically, adeno-associated virus gene transfer
techniques, as disclosed in (Walther and Stein, 2000) and
(Athanasopoulos et al., 2000). Non-viral based techniques may also
be performed as disclosed in (Muramatsu et al., 1998). A gene
encoding one or more cellular differentiating factors, e.g., a
growth factor(s) or a cytokine(s), could also be added. Examples of
various cell differentiation agents are disclosed in (Gimble et
al., 1995; Lennon et al., 1995; Majumdar et al., 1998; Caplan and
Goldberg, 1999; Ohgushi and Caplan, 1999; Pittenger et al., 1999;
Caplan and Bruder, 2001; Fukuda, 2001; Worster et al., 2001; Zuk et
al., 2001).
[0131] Genes encoding anti-apoptotic factors or agents could also
be added. Addition of the gene (or combination of genes) could be
by any technology known in the art including but not limited to
adenoviral transduction, "gene guns," liposome-mediated
transduction, and retrovirus or lentivirus-mediated transduction,
plasmid, adeno-associated virus. These regenerative cells could
then be implanted along with a carrier material bearing gene
delivery vehicle capable of releasing and/or presenting genes to
the cells over time such that transduction can continue or be
initiated in situ.
[0132] When the cells and/or tissue containing the cells are
administered to a patient other than the patient from whom the
cells and/or tissue were obtained, one or more immunosuppressive
agents may be administered to the patient receiving the cells
and/or tissue to reduce, and preferably prevent, rejection of the
transplant. As used herein, the term "immunosuppressive drug or
agent" is intended to include pharmaceutical agents which inhibit
or interfere with normal immune function. Examples of
immunosuppressive agents suitable with the methods disclosed herein
include agents that inhibit T-cell/B-cell costimulation pathways,
such as agents that interfere with the coupling of T-cells and
B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent
Pub. No. 20020182211. A preferred immunosuppressive agent is
cyclosporine A. Other examples include myophenylate mofetil,
rapamicin, and anti-thymocyte globulin. In one embodiment, the
immunosuppressive drug is administered with at least one other
therapeutic agent. The immunosuppressive drug is administered in a
formulation which is compatible with the route of administration
and is administered to a subject at a dosage sufficient to achieve
the desired therapeutic effect. In another embodiment, the
immunosuppressive drug is administered transiently for a sufficient
time to induce tolerance to the regenerative cells of the
invention.
[0133] In these embodiments, the regenerative cells may be
contacted, combined, mixed or added to the additives through any
art recognized manner, including devices such as the agitation
devices and associated methods described herein. For example,
rocking, inversion, compression pulsed or moving rollers may be
used.
[0134] In another aspect, the cell population could be placed into
the recipient and surrounded by a resorbable plastic sheath or
other materials and related components such as those manufactured
by MacroPore Biosurgery, Inc. (see e.g., U.S. Pat. Nos. 6,269,716;
5,919,234; 6,673,362; 6,635,064; 6,653,146; 6,391,059; 6,343,531;
6,280,473).
[0135] In all of the foregoing embodiments, at least a portion of
the separated and concentrated regenerative cells may be
cryopreserved, as described in U.S. patent application Ser. No.
10/242,094, entitled PRESERVATION OF NON EMBRYONIC CELLS FROM NON
HEMATOPOIETIC TISSUES, filed Sep. 12, 2002, which claims the
benefit of U.S. Provisional Patent Application 60/322,070 filed
Sep. 14, 2001, which is commonly assigned, and the contents of
which in their entireties are expressly incorporated herein by
reference.
[0136] At the end of processing, the regenerative cells may be
manually retrieved from the output chamber. The cells may be loaded
into a delivery device, such as a syringe, for placement into the
recipient by either, subcutaneous, intramuscular, or other
technique allowing delivery of the cells to the target site within
the patient. In other words, cells may be placed into the patient
by any means known to persons of ordinary skill in the art.
Preferred embodiments include placement by needle or catheter, or
by direct surgical implantation. In other embodiments, the cells
may be automatically transported to an output chamber which may be
in the form of a container, syringe or catheter etc., which may be
used to place the cells in the patient. The container may also be
used to store the cells for later use or for cryopreservation. All
retrieval methods are performed in a sterile manner. In the
embodiment of surgical implantation, the cells could be applied in
association with additives such as a preformed matrix or scaffold
as described herein.
[0137] In preferred embodiments of the invention (e.g., the
embodiment shown in FIG. 4), the system is automated. In another
embodiment, the system has both automated and manual components.
The system may be comprised of one or more disposable components
connected to or mounted on a re-usable hardware component or
module. The automated systems of the invention provide screen
displays (see FIG. 16) that prompt proper operation of the system.
The automated systems may also provide a screen that provides
status of the procedure and/or the step by step instructions as to
the proper setup of the disposable components of the system. The
screen may also indicate problems or failures in the system if they
occur and provide "troubleshooting" guidance if appropriate. In one
embodiment, the screen is a user interface screen that allows the
user to input parameters into the system through, e.g., a touch
screen.
[0138] The partial and fully automated systems may include a
processing device (e.g., microprocessor or personal computer) and
associated software programs that provide the control logic for the
system to operate and to automate one or more steps of the process
based on user input. In certain embodiments, one or more aspects of
the system may be user-programmable via software residing in the
processing device. The processing device may have one or more
pre-programmed software programs in Read Only Memory (ROM). For
example, the processing device may have pre-programmed software
tailored for processing blood, another program for processing
adipose tissue to obtain small volumes of regenerative cells and
another program for processing adipose tissue to obtain larger
volumes of regenerative cells. The processing device may also have
pre-programmed software which provides the user with appropriate
parameters to optimize the process based on the user's input of
relevant information such as the amount of regenerative cells
required, the type of tissue being processed, the type of
post-processing manipulation required, the type of therapeutic
application, etc.
[0139] The software may also allow automation of steps such as
controlling the ingress and egress of fluids and tissues along
particular tubing paths by controlling pumps and valves of the
system; controlling the proper sequence and/or direction of
activation; detecting blockages with pressure sensors; mixing
mechanisms, measuring the amount of tissue and/or fluid to be moved
along a particular pathway using volumetric mechanisms; maintaining
temperatures of the various components using heat control devices;
and integrating the separation and concentration process with
timing and software mechanisms. The processing device can also
control centrifuge speeds based on the tissue type being processed
and/or the cell population or sub-population being harvested, and
the types of procedures to be performed (e.g., tissue enhancement
using adipose tissue augmented with regenerative cells, or
processing of cells for bone repair applications using regenerative
cell coated bone grafts). The processing device may also include
standard parallel or serial ports or other means of communicating
with other computers or networks. Accordingly, the processing
device can be a stand alone unit or be associated one or more
additional devices for the further processing methods described
herein.
[0140] The software may allow for automated collection of "run
data" including, for example, the lot numbers of disposable
components, temperature and volume measurements, tissue volume and
cell number parameters, dose of enzyme applied, incubation time,
operator identity, date and time, patient identity, etc. In a
preferred embodiment of the device a character recognition system,
such as a bar code reading system would be integrated to permit
data entry of these variables (for example disposable set lot
number and expiration date, lot number and expiration date of the
Collagenase, patient/sample identifiers, etc.) into the processing
device as part of documentation of processing. This would reduce
the opportunity for data entry errors. Such a bar code reading
system could be easily incorporated into the processing device
using a USB or other interface port and system known to the art. In
this way the device would provide integrated control of the data
entry and documentation of the process. A print-out report of these
parameters would be part of the user-defined parameters of a
programmed operation of the system. Naturally this would require
integration of a printer component (hardware and driver) or printer
driver in software plus an interface output connector for a printer
(e.g., a USB port) in the hardware of the device.
[0141] In certain embodiments, the system is a fully automated
system. For example, the user may initially select the amount of
tissue to be processed, attach the system to the patient and the
system may automatically aspirate the required tissue and separate
and concentrate regenerative cells in an uninterrupted sequence
without further user input. The user may also input the amount of
regenerative cells required and allow the system to aspirate the
requisite amount of tissue and process the tissue. A fully
automated system also includes a system which is capable of being
reconfigured based on a number of (e.g., two or more) user input
parameters, e.g., number of wash cycles, speed of centrifugation
etc. The system can also be run in semi-automatic mode during which
the system goes through certain steps without user intervention but
requires user intervention before certain processes can occur. In
other embodiments, the system is a single integrated system that
displays instructions to guide the user to perform predetermined
operations at predetermined times. For example, the processing
device may prompt users through the steps necessary for proper
insertion of tubing, chambers and other components of the system.
Accordingly, the user can ensure that the proper sequence of
operations is being performed. Such a system can additionally
require confirmation of each operational step by the user to
prevent inadvertent activation or termination of steps in the
process. In a further embodiment, the system may initiate automated
testing to confirm correct insertion of tubing, chambers, absence
of blockages etc. In yet another embodiment, the system of the
present invention is capable of being programmed to perform
multiple separation and concentration processes through automated
control of tissue flow through the system. This feature may be
important, for example, during surgery on a patient where tissue
that would otherwise be lost is collected into the system, and
regenerative cells from the tissue are separated and concentrated
and returned to the patient.
[0142] As set forth above, components of the system may be
disposable (referred to herein as "disposable set(s)"), such that
portions of the system can be disposed of after a single use. This
implementation can help ensure that any surface which comes in
contact with the patient's tissue will be disposed of properly
after being used. An exemplary disposable set is illustrated in
FIG. 13. In a preferred embodiment, the disposable components of
the system are pre-sterilized and packaged so as to be usable "off
the shelf" that are easy to use and easy to load and that eliminate
the need for many tubing connections and complex routing of tubing
connections. Such disposable components are relatively inexpensive
to manufacture, and therefore, do not create a substantial expense
due to their disposal. In one embodiment, the disposable system
(referred to interchangeably herein as "disposable set(s)")
comprises, consists essentially of, or consists of, the collection
chamber 20, the processing chamber 30, the waste chamber 40, the
output chamber 50, the filter assemblies 36, the sample bag 60 and
the associated conduits 12 or tubing. In preferred embodiments of
the disposable sets of the system, the collection chamber 20 and
the processing chamber 30 are connected by way of conduits 12 that
are housed in a rigid frame. The rotating seal network (FIGS. 7
& 8) of a processing chamber 30 may also be housed in the same
rigid frame. In another preferred embodiment, the various chambers
and containers of the disposable set are comprised of the necessary
interfaces that are capable of communicating with the processing
device of the system such that the pumps, valves, sensors and other
devices that automate the system are appropriately activated or
de-activated as needed without user intervention. The interfaces
also reduce the time and expertise required to set up the system
and also reduce errors by indicating how to properly set up the
system and alerting the user in the event of an erroneous
setup.
[0143] Most of the disposable sets of the invention will have many
common elements. However, the ordinarily skilled artisan will
recognize that different applications of the system may require
additional components which may be part of the disposable sets.
Accordingly, the disposable sets may further comprise one or more
needles or syringes suitable for obtaining adipose or other tissue
from the patient and returning regenerative cells to the patient.
The type number and variety of the needles and syringes included
will depend on the type and amount of tissue being processed. The
disposable sets may further comprise one or more rigid or flexible
containers to hold washing fluids and other processing reagents
used in the system. For example, the disposable sets may comprise
containers to hold saline, enzymes and any other treatment or
replacement fluids required for the procedure. In addition,
suitable washing solutions, re-suspension fluids, additives, agents
or transplant materials may be provided with the disposable sets
for use in conjunction with the systems and methods of the
invention.
[0144] Any combination of system components, equipment or supplies
described herein or otherwise required to practice the invention
may be provided in the form of a kit. For example, a kit of the
invention may include, e.g., the optimal length and gage needle for
the syringe based liposuction and sterile syringes which contain
the preferred filter media which allows for the processing of small
volumes of tissue. Other exemplary equipment and supplies which may
be used with the invention and may also be included with the kits
of the invention are listed in Tables II and III.
[0145] Table II below identifies examples of supplies that can be
used in to obtain adipose derived regenerative cell in accordance
with the systems and methods of the present invention:
TABLE-US-00002 TABLE II Description Vendor Quantity Note 10 ml
syringe Becton- as req'd Optional, used for Dickinson liposuction
14 GA blunt tip as req'd Optional, used for needle liposuction
Single Blood Baxter 1 Main cell processing bag; Pack (600 ml)
Fenwal bag has spike adaptor on line and two free spike ports
Transfer pack with Baxter 1 Quad bag set coupler (150 ml) Fenwal
Transfer pack with Baxter 1 Waste bag coupler (1 L) Fenwal Sample
Site Coupler Baxter 2 Fenwal 0.9% saline (for Baxter 1 injection)
Fenwal 14 GA sharp needle Monoject as req'd For adding liposuction
tissue to bag 20 GA sharp needle Monoject 3 For adding collagenase
and removing PLA cells 0.2 .mu.m Sterflip Millipore 1 For filtering
collagenase filter Teruflex Aluminium Terumo 4 ME*ACS121 for
temporary sealing clips tube sealing Povidone Iodine Triadine as
req'd 10-3201 prep pad Liberase H1 Roche See Procedure Note 1
Collagenase TSCD wafers Terumo 2 1SC*W017 for use with TSCD Sterile
Tubing Welder
[0146] Table III, below, identifies equipment that may be used with
the systems and methods disclosed herein.
TABLE-US-00003 TABLE III Description Vendor Quantity Note Sorvall
Legend T Fisher 1 75-004-367 Easy Set Centrifuge Scientific Rotor
Kendro/ 1 TTH-750 rotor Sorvall Rotor buckets Kenro/ 4 75006441
round Sorvall buckets Adaptor for 150 ml bags Kendro/ 4 00511
Sorvall Plasma Expressor Baxter 1 4R4414 Fenwal Tube Sealer Sebra 1
Model 1060 TSCD Sterile Tubing Welder Terumo 1 3ME*SC201AD LabLine
Thermal Rocker LabLine 1 4637 `Disposable` plastic Davron 3
hemostat-style clamp Balance Bags Sets 2 Water-filled bags used to
balance centrifuge Biohazard Sharps Chamber 1 Biohazard Waste
Chamber 1
[0147] The re-usable component of the system comprises, consists
essentially of, or consists of the agitation mechanism for the
collection chamber, the pump, and assorted sensors which activate
valves and pump controls, the centrifuge motor, the rotating frame
of the centrifuge motor, the user interface screen and USB ports,
an interlocking or docking device or configuration to connect the
disposable set such that the disposable set is securely attached to
and interface with the re-usable hardware component and other
associated devices. An exemplary re-usable component is illustrated
in FIG. 14. In preferred embodiments, the re-usable component
includes a means for separating and concentrating the regenerative
cells from the regenerative cell composition, e.g., a rotating
centrifuge. In this embodiment, the re-usable component is designed
connect to and interface with a portion of the processing chamber
(comprising a centrifuge chamber) of the disposable set as shown in
FIG. 15A. It is understood that the means for separating and
concentrating regenerative cells in the re-usable component is not
limited to a rotating centrifuge but may also include any other
configuration described herein, including a spinning membrane
filter. The re-usable component may also house the processing
device described herein which contains pre-programmed software for
carrying out several different tissue processing procedures and
selectively activating the various pumps and valves of the system
accordingly. The processor may also include data storage capability
for storing donor/patient information, processing or collection
information and other data for later downloading or compilation.
The re-usable component may be used with a variety of disposable
sets. The disposable set is connected to the re-usable component
through, e.g., an interlocking device or configuration to connect
the disposable set such that the disposable set is securely
attached to and interfaces with the re-usable hardware component in
a manner that the processing device present on the re-usable
component can control, i.e., send and receive signals to and from
the various components of the disposable set as well as various
components of the re-usable component and other associated devices
and systems.
[0148] In one embodiment, a disposable set for use in the system is
comprised of a collection chamber 20 which can accommodate about
800 mL of tissue; a processing chamber 30 which can process the
regenerative cell composition generated by about 800 mL of tissue
washed and digested in the collection chamber 20; an output chamber
50 which can accommodate at least 0.5 mL of regenerative cells; and
a waster container 40 which can accommodate about 10 L of waste. In
this embodiment, the hardware device is no larger than
24''L.times.18''W.times.36''H. Alternative dimensions of the
various components of the disposable sets as well as the hardware
device may be constructed as needed and are intended to be
encompassed by the present invention without limitation.
[0149] The disposable components of the system are easy to place on
the device. An illustration of a disposable set utilized assembled
together with a corresponding re-usable component is illustrated in
FIG. 15A. The system is preferably designed such that it can detect
an improperly loaded disposable component. For example, the
components of each disposable set may have color-guided marks to
properly align and insert the tubing, chambers etc. into
appropriate places in the system. In additional embodiments, the
system disclosed herein is a portable unit. For example, the
portable unit may be able to be moved from one location where
adipose tissue harvesting has occurred, to another location for
adipose tissue harvesting. In certain implementations, the portable
unit is suitable for harvesting and processing of adipose tissue by
a patient's bedside. Thus, a portable unit may be part of a system
which can be moved from patient to patient. Accordingly, the
portable unit may be on wheels which lock in place and, thus, can
be easily placed and used in a convenient location in a stable and
secure position throughout the procedure. In other embodiments, the
portable unit is designed for set-up and operation on a flat
surface such as a table top. The portable unit may also be enclosed
in a housing unit. The portable unit may further be comprised of
hangers, hooks, labels, scales and other devices to assist in the
procedure. All of the herein described re-usable components of the
system such as the centrifuge, processing device, display screen
may be mounted on the portable unit of the system.
[0150] Alternate manual embodiments for obtaining regenerative
cells are also within the scope of this invention. For example, in
one embodiment, adipose tissue may be processed using any
combination of the components of the system, equipment and/or
supplies described herein.
[0151] A particular example of the system embodying the present
invention is shown in FIG. 4. FIG. 4 illustrates an automated
system and method for separating and concentrating regenerative
cells from tissue, e.g., adipose tissue, suitable for re-infusion
within a patient. In certain embodiments of the system shown in
FIG. 4, the system further includes an automated step for
aspirating a given amount of tissue from the patient. The system
shown in FIG. 4 is comprised of the disposable set shown in FIG. 13
which is connected to the re-usable component of the system shown
in FIG. 14 to arrive at an automated embodiment of the system shown
in FIG. 15A. The disposable set is connected to the re-usable
component through, e.g., an interlocking or docking device or
configuration, which connects the disposable set to the re-usable
component such that the disposable set is securely attached to and
associated with the re-usable hardware component in a manner that
the processing device present on the re-usable component can
control and interface with, i.e., send and receive signals to and
from the various components of the disposable set as well as
various components of the re-usable component and other associated
devices and systems.
[0152] The user may connect the disposable set to the re-usable
component, input certain parameters using the user interface, e.g.,
the volume of tissue being collected, attach the system to the
patient, and the system automatically performs all of the steps
shown in FIG. 4 in an uninterrupted sequence using pre-programmed
and/or user input parameters. One such sequence is illustrated in
FIG. 15B. Alternatively, the tissue may be manually aspirated from
the patient by the user and transported to system for processing,
i.e., separation and concentration of regenerative cells.
[0153] Specifically, as shown in FIG. 4, tissue, e.g., adipose
tissue, may be withdrawn from the patient using conduit 12 and
introduced into collection chamber 20. A detailed illustration of
the collection chamber of FIG. 4 is shown in FIG. 5. As illustrated
in FIG. 5, the collection chamber 20 may be comprised of a vacuum
line 11 which facilitates tissue removal using a standard cannula.
The user may enter the estimated volume of tissue directed to the
collection chamber 20 at this point. The tissue is introduced into
the collection chamber 20 through an inlet port 21 which is part of
a closed fluid pathway that allows the tissue, saline and other
agents to be added to the tissue in an aseptic manner. An optical
sensor of the system, e.g., sensor 29, can detect when the user
input volume of tissue is present in the collection chamber 20. In
certain embodiments, if less tissue is present in the collection
chamber than the user input, the user will have the option to begin
processing the volume of tissue which is present in the collection
chamber 20. In certain embodiments, a portion of the tissue removed
from the patient may be directed to the sample chamber 60 through
the use of a pump, e.g., a peristaltic pump, via a conduit, which
may be activated via user input utilizing the user interface.
[0154] A sensor 29 can signal the processing device present in the
re-usable component to activate the steps needed to wash and
disaggregate the tissue. For example, the processing device may
introduce a pre-set volume of washing agent based on the volume of
tissue collected using automated valves and pumps. This cycle may
be repeated in the collection chamber until the optical sensor
determines that the effluent liquid is sufficiently clear and
devoid of unwanted material. For example, an optical sensor 29
along the conduit leading out of the collection chamber 12b or 12d
can detect that the unwanted materials have been removed and can
signal the processing device to close the required valves and
initiate the next step.
[0155] Next, the processing device may introduce a pre-programmed
amount of disaggregation agent based on the volume of tissue
collected. The processing device may also activate agitation of the
tissue in the collection chamber for a preset period of time based
on the initial volume of tissue collected or based on user input.
In the embodiment shown in FIG. 4, once the disaggregation agent,
e.g., collagenase, is added to the collection chamber 20 through
the collagenase source 24, the motor in the collection chamber 20
is activated via the processing device. The motor activates the
rotatable shaft 25 which is comprised of a magnetic stirrer and a
paddle-like device wherein one or more paddles 25a are rigidly
attached to the filter cage 27 of a filter prefixed to the
collection chamber 28. The paddles agitate the in the presence of
the disaggregation agent such that the regenerative cells separate
from the tissue.
[0156] The solution in the collection chamber 20 is allowed to
settle for a preset period of time. The buoyant portion of the
solution is allowed to rise to the top of the solution. Once the
preset period of time elapses, the necessary valves and pumps are
activated by the processing device to remove the non-buoyant
portion to the waste chamber 40. The transfer into the waste
chamber 40 continues until a sensor 29 along the conduit leading
out of the collection chamber 12b or 12d can detect that the
buoyant fraction of the solution is about to be transferred to the
waste chamber 30. For example, a sensor 29 along the conduit
leading out of the collection chamber 12b or 12d can detect that
the unwanted materials have been removed and can signal the
processing device to close the required valves.
[0157] At this time the non-buoyant fraction of the solution, i.e.,
the regenerative cell composition, is moved to the processing
chamber 30. This is accomplished through the use of the necessary
valves and peristaltic pumps. In certain embodiments, before
transfer of the regenerative cell composition to the processing
chamber 30, an additional volume of saline may be added to the
buoyant fraction of solution remaining in the collection chamber
20. Another wash cycle may be repeated. After this cycle, the
solution is allowed to settle and the non-buoyant fraction (which
contains the regenerative cells) is transported to the processing
chamber 30 and the buoyant fraction is drained to the waste chamber
40. The additional wash cycle is used to optimize transfer of all
the separated regenerative cells to the processing chamber 30.
[0158] Once the regenerative cell composition is transported to the
processing chamber 30 by way of conduits 12, the composition may be
subject to one or more additional washing steps prior to the start
of the concentration phase. This ensures removal of waste and
residual contaminants from the collection chamber 20. Similarly,
subsequent to the concentration step, the regenerative cell
composition may be subjected to one or more additional washing
steps to remove residual contaminants. The unwanted materials may
be removed from the processing chamber 30 to the waste chamber 40
in the same manner, i.e., control of valves and pumps via signals
from the processing device, as described above.
[0159] The various embodiments of the processing chamber 30 shown
in FIG. 4 are described in detail below. The processing chamber 30
shown in FIG. 4 is in the form of a centrifuge chamber. A detailed
illustration of the processing chamber of FIG. 4 is shown in FIGS.
7 and 8. Such a processing chamber 30 is generally comprised of a
rotating seal network 30.1 comprising an outer housing 30.2, one or
more seals 30.3, one or more bearings 30.4 and an attachment point
30.6 for connecting the processing chamber to the centrifuge device
present in the re-usable component of the system; one or more fluid
paths 30.5 in the form of conduits extending out from the rotating
seal and ending in a centrifuge chamber on each end which is in the
form of an output chamber 50 housed in a frame 53 wherein the frame
is comprised of one or more ports 52 and one or more handles to
manually re-position the output chamber 50.
[0160] The rotating seal network 30.1 is included to ensure that
the fluid pathways of the processing chamber can be maintained in a
sterile condition. In addition, the fluid pathways of the
processing chamber can be accessed in a sterile manner (e.g., to
add agents or washing solution) at any time, even while the
centrifuge chamber of the processing chamber is spinning.
[0161] The rotating seal network 30.1 shown in FIGS. 7 and 8
includes a rotating shaft comprised of two or more bearings 30.4,
three or more lip seals 30.3, and an outer housing 30.2. In this
embodiment, the bearings 30.4 further comprise an outer and inner
shaft (not shown) referred to herein as races. These races may be
separated by precision ground spheres. The races and spheres
comprising the bearings are preferably fabricated with material
suitable for contact with bodily fluid, or are coated with material
suitable for contact with bodily fluid. In a preferred embodiment,
the races and spheres are fabricated using, for example, silicone
nitride or zirconia. Furthermore, in this embodiment, the three lip
seals are comprised of a circular "U" shaped channel (not shown) as
well as a circular spring (not shown). The circular "U" shaped
channel is preferably fabricated using flexible material such that
a leakage proof junction with the rotating shaft of the rotating
seal network 30.1 is formed. Additionally, the lip seals are
preferably oriented in a manner such that pressure from the
regenerative cell composition flowing through the processing
chamber causes the seal assembly to tighten its junction with the
rotating shaft by way of increased tension. The seals may be
secured in position by way of one or more circular clips (not
shown) which are capable of expanding and/or collapsing as needed
in order to engage a groove in the outer housing 30.2 of the
rotating seal network 30.1. The heat generated by or near the
rotating seal network 30.1 must be controlled to prevent lysis of
the cells in the solution which is being moved through the passage.
This may be accomplished by, for example, selecting a hard material
for constructing the rotating shaft, polishing the area of the
rotating shaft which comes in contact with the seals and minimizing
contact between the rotating shaft and the seal.
[0162] In another embodiment the rotating seal network 30.1 is
comprised of a single rubber seal 30.3 and an air gasket (not
shown). This seal and gasket provide a tortuous path for any
biologic matter which could compromise the sterility of the system.
In another embodiment the rotating seal network 30.1 is comprised
of multiple spring loaded seals 30.3 which isolate the individual
fluid paths. The seals 30.3 are fabricated of a material which can
be sterilized as well as seal the rotating shaft without lubricant.
In another embodiment the rotating seal network 30.1 is compromised
of a pair of ceramic disks (not shown) which create the different
fluid paths and can withstand the rotation of the system and not
cause cell lysis. In another embodiment the fluid pathway is
flexible and is allowed to wind and unwind with respect to the
processing chamber. This is accomplished by having the flexible
fluid pathway rotate one revolution for every two revolutions of
the processing chamber 30. This eliminates the need for a rotating
seal altogether.
[0163] The regenerative cell composition is pumped from the
collection chamber 20 along a fluid path through the axis of
rotation of the rotating seal network 30.1 and then divides into a
minimum of two fluid pathways 30.5 each of which radiate outward
from the central axis of the processing chamber 30 and terminate
near the outer ends of the processing chamber 30, i.e., within the
centrifuge chambers which house the output chambers 50 (FIGS. 7 and
8). Accordingly, in a preferred embodiment, the processing chamber
30 is comprised of two or more output chambers 50 as shown in FIGS.
7 and 8. These output chambers 50 are positioned such that they are
in one orientation during processing 30.7 and another orientation
for retrieval of concentrated regenerative cells 30.8. For example,
the output changes are tilted in one angle during processing and
another angle for cell retrieval. The cell retrieval angle is more
vertical than the processing angle. The two positions of the output
chamber 50 may be manually manipulated through a handle 53 which
protrudes out of the processing chamber 30. The regenerative cells
can be manually retrieved from the output chambers 50 when they are
in the retrieval orientation 30.8 using a syringe. In another
embodiment, fluid path 30.5 is constructed such that it splits
outside the processing chamber and then connects to the outer ends
of the processing chamber 30, i.e., within the centrifuge chambers
which house the output chambers 50 (not shown). In this embodiment,
large volumes of regenerative cell composition and/or additives,
solutions etc. may be transported to the centrifuge chamber and/or
the output chambers directly.
[0164] With reference to FIGS. 4 and 7-9, between the collection
chamber 20 and the processing chamber 30, a pump 34 and one or more
valves 14 may be provided. In a preferred embodiment, the valves 14
are electromechanical valves. In addition, sensors, such as
pressure sensor 29, may be provided in line with the processing
chamber 30 and the collection chamber 20. The valves, pumps and
sensors act in concert with the processing device present on the
re-usable component (FIG. 14) to automate the concentration steps
of the system.
[0165] The sensors detect the presence of the regenerative cell
composition in the centrifuge chambers and activate the centrifuge
device through communication with the processing device of the
system. The regenerative cell composition is then subjected to a
pre-programmed load for a pre-programmed time based on the amount
of tissue originally collected and/or user input. In certain
embodiments, this step may be repeated either automatically or
through user input. For example, the composition is subjected to a
load of approximately 400 times the force of gravity for a period
of approximately 5 minutes. The output chamber 50 is constructed
such that the outer extremes of the chamber form a small reservoir
for the dense particles and cells. The output chamber 50 retains
the dense particles in what is termed a `cell pellet`, while
allowing the lighter supernatant to be removed through a fluid
path, e.g., a fluid path which is along the axis of rotation of the
rotating seal network 30.1 and travels from the low point in the
center of the processing chamber 30 through the rotating seal
network 30.1 to the waste container 40. The valves 14 and pumps 34
signal the processing device to activate steps to remove the
supernatant to the waste container 40 without disturbing the cell
pellet present in the output chamber 50.
[0166] The cell pellet that is obtained using the system shown in
FIG. 4 comprises the concentrated regenerative cells of the
invention. In some embodiments, after the supernatant is removed
and directed to the waste chamber 40, a fluid path 30.5 may be used
to re-suspend the cell pellet that is formed after centrifugation
with additional solutions and/or other additives. Re-suspension of
the cell pellet in this manner allows for further washing of the
regenerative cells to remove unwanted proteins and chemical
compounds as well as increasing the flow of oxygen to the cells.
The resulting suspension may be subjected to another load of
approximately 400 times the force of gravity for another period of
approximately 5 minutes. After a second cell pellet is formed, and
the resulting supernatant is removed to the waste chamber 40, a
final wash in the manner described above may be performed with
saline or some other appropriate buffer solution. This repeated
washing can be performed multiple times to enhance the purity of
the regenerative cell solution. In certain embodiments, the saline
can be added at any step as deemed necessary to enhance processing.
The concentrations of regenerative cells obtained using the system
shown in FIG. 4 may vary depending on amount of tissue collected,
patient age, patient profile etc. Exemplary yields are provided in
Table 1.
[0167] The final pellet present in the output chamber 50 may then
be retrieved in an aseptic manner using an appropriate syringe
after the output chamber 50 is positioned in the orientation
appropriate for cell removal. In other embodiments, the final
pellet may be automatically moved to a container in the in the
output chamber 50 which may be removed and stored or used as
needed. This container may be in any appropriate form or size. For
example, the container may be a syringe. In certain embodiments,
the output container 50 itself may be heat sealed (either
automatically or manually) and isolated from the other components
of the processing chamber for subsequent retrieval and use of the
regenerative cells in therapeutic applications as described herein
including re-infusion into the patient. The cells may also be
subject to further processing as described herein either prior to
retrieval from the output chamber or after transfer to a second
system or device. The re-usable component shown in FIG. 14 is
constructed such that it can be connected to one or more additional
systems or devices for further processing as needed.
[0168] 2. Methods of Manufacturing a Cell Carrier Portion of the
Device
[0169] The cell carrier portion of the present invention is
critical for maintaining the bone forming functions of the
regenerative cells. Specifically, the cell carrier, on which the
regenerative cells are placed, as further described herein, serves
to organize the cells in three dimensions. Accordingly, in
considering substrate materials, it is imperative to choose one
that exhibits clinically acceptable biocompatibility. In addition,
the mechanical properties of the cell carrier must be sufficient so
that it does not collapse during the patient's normal
activities.
[0170] A variety of cell carriers are known and used in the art and
are intended to be encompassed by the present invention. Cell
carriers in the form of gels, such as hydrogels are encompassed by
the present invention. Fiber based cell carriers are also
encompassed by the present invention and include woven meshes,
knitted meshes, non-woven meshes (felts) and polymer coated meshes
(to enhance strength). Methods for manufacture fiber based cell
carriers are known in the art. For example, cell carriers based on
knitted meshes can be made according to the methods described by H.
J. Buchsbaum, W. Christopherson, S. Lifshitz, and S. Bernstein.
Vicryl mesh in pelvic floor reconstruction. Arch. Surg 120
(12):1389-1391, 1985. Non-woven mesh based cell carriers can be
made according to the methods described by L. E. Freed, G.
Vunjak-Novakovic, R. J. Biron, D. B. Eagles, D. C. Lesnoy, S. K.
Barlow, and R. Langer. Biodegradable polymer cell carriers for
tissue engineering. Biotechnology (N Y) 12 (7):689-693, 1994.
Polymer coated meshes (to enhance strength) can be made according
to the methods described by W. S. Kim, J. P. Vacanti, L. Cima, D.
Mooney, J. Upton, W. C. Puelacher, and C. A. Vacanti. Cartilage
engineered in predetermined shapes employing cell transplantation
on synthetic biodegradable polymers. Plast Reconstr Surg 94
(2):233-237, 1994.
[0171] In a preferred embodiment, the cell carrier portion of the
device will be preformed into particulates. The sizes and shapes of
these particulates can range from less than 1 mm to up to 10 mm in
diameter and from abstract shaped granules to defined shapes,
respectively. These particulates can either be highly porous
(greater than 95% void volume) or non-porous. The cell carrier
particulates can be used to contain the cells into the center of or
around the outside of the containment device and can be preloaded
into or onto the containment device before packaging and
sterilization or can be packaged and sterilized separately from the
containment device.
[0172] Sponge or foam based cell carriers are also encompassed by
the present invention and can be manufactured using methods such as
solvent casted-particulate leached (SC-PL), melt molded-particulate
leached (where melt molding can include e.g., compression molding,
injection molding or extrusion), extrusion-particulate leaching,
emulsion-freeze drying, freeze drying combined with
particulate-leaching, solution casting, gel casting, atomized
foams, phase separation, phase separation combined with particulate
leaching, high pressure CO2, gas foaming of effervescent salts, 3D
printing, fused deposition modeling and methods using electrospun
nanofibers or other nanofibers. In a preferred embodiment, the cell
carrier portion of the present invention is manufactured using
solvent casting/freeze drying methods.
[0173] Methods for manufacturing the sponge or foam based cell
carrier of the invention are known in the art. For example, solvent
casted-particulate leached (SC-PL) cell carriers can be made
according to the methods disclosed in A. G. Mikos, A. J. Thorsen,
L. A. Czerwonka, Bao Y., and R. Langer. Preparation and
characterization of poly(L-lactic acid) foams. Polymer 35
(5):1068-1077, 1994 and P. X. Ma and J. W. Choi. Biodegradable
polymer cell carriers with well-defined interconnected spherical
pore network. Tissue Eng 7 (1):23-33, 2001. SC-melt molded cell
carriers can be made according to R. C. Thomson, M. J. Yaszemski,
J. M. Powers, and A. G. Mikos. Hydroxyapatite fiber reinforced
poly(alpha-hydroxy ester) foams for bone regeneration. Biomaterials
19 (21):1935-43, 1998. Extrusion-particulate leaching based cell
carriers can be made according to M. S. Widmer, P. K. Gupta, L. Lu,
R. K. Meszlenyi, G. R. Evans, K. Brandt, T. Savel, A. Gurlek, C. W.
Patrick, Jr., and A. G. Mikos. Manufacture of porous biodegradable
polymer conduits by an extrusion process for guided tissue
regeneration. Biomaterials 19 (21):1945-1955, 1998.
[0174] Freeze-drying (Phase separation) based cell carriers can be
made according to H. Lo, Ponticiello M. S., and K. W. Leong.
Fabrication of controlled release biodegradable foams by phase
separation. Tissue Eng 1 (1):15-28, 1995 and C. Schugens, V.
Maquet, C. Grandfils, R. Jerome, and P. Teyssie. Polylactide
macroporous biodegradable implants for cell transplantation. II.
Preparation of polylactide foams by liquid-liquid phase separation.
J Biomed Mater. Res 30 (4):449-461, 1996. Scaffolds manufactured
using freeze drying (Phase separation) combined with
particulate-leaching can be made according to J. H. de Groot, A. J.
Nijenhuis, Bruin P., A. J. Pennings, R. P. H. Veth, J. Klompmaker,
and H. W. B. Jansen. Use of porous biodegradable polymer implants
in meniscus reconstruction. 1. Preparation of porous biodegradable
polyurethanes for the reconstruction of meniscus lesions. Colloid
Polym. Sci. 268:1073-1081, 1990 and Q. Hou, D. W. Grijpma, and J.
Feijen. Preparation of interconnected highly porous polymeric
structures by a replication and freeze-drying process. J Biomed
Mater. Res 67B (2):732-740, 2003. Freeze-extraction based cell
carriers can be made according to M. H. Ho, P. Y. Kuo, H. J. Hsieh,
T. Y. Hsien, L. T. Hou, J. Y. Lai, and D. M. Wang. Preparation of
porous cell carriers by using freeze-extraction and freeze-gelation
methods. Biomaterials 25 (1):129-138, 2004. Emulsion--Freeze drying
based cell carriers can be made using the methods disclosed in K.
Whang, C. H. Thomas, and K. E. Healy. A novel method to fabricate
bioabsorbable cell carriers. Polymer 36 (4):837-842, 1995.
[0175] Scaffolds manufactured using gel casting or solution casting
methods can be manufactured using J. P. Schmitz and J. O.
Hollinger. A preliminary study of the osteogenic potential of a
biodegradable alloplastic-osteoinductive alloimplant. Clin Orthop.
(237):245-255, 1988, A. G. Coombes and J. D. Heckman. Gel casting
of resorbable polymers. 1. Processing and applications.
Biomaterials 13 (4):217-224, 1992 and the methods disclosed in U.S.
Pat. No. 5,716,416 (1998). Methods of manufacturing cell carriers
using atomized foams can found in H. Lo, Ponticiello M. S., and K.
W. Leong. Fabrication of controlled release biodegradable foams by
phase separation. Tissue Eng 1 (1):15-28, 1995. Methods using gas
Foaming-particulate leaching can be found in Y. S. Nam, J. J. Yoon,
and T. G. Park. A novel fabrication method of macroporous
biodegradable polymer cell carriers using gas foaming salt as a
porogen additive. J Biomed Mater. Res 53 (1):1-7, 2000.
[0176] The 3D printing (Theriform.TM. process) is described in, for
example, R. A. Giordano, B. M. Wu, S. W. Borland, L. G. Cima, E. M.
Sachs, and M. J. Cima. Mechanical properties of dense polylactic
acid structures fabricated by three dimensional printing. J
Biomater. Sci Polym. Ed 8 (1):63-75, 1996 and S. S. Kim, H.
Utsunomiya, J. A. Koski, B. M. Wu, M. J. Cima, J. Sohn, K. Mukai,
L. G. Griffith, and J. P. Vacanti. Survival and function of
hepatocytes on a novel three-dimensional synthetic biodegradable
polymer cell carrier with an intrinsic network of channels. Ann
Surg 228 (1):8-13, 1998. Fused deposition modeling based methods
can be found in D. W. Hutmacher, T. Schantz, I. Zein, K. W. Ng, S.
H. Teoh, and K. C. Tan. Mechanical properties and cell cultural
response of polycaprolactone cell carriers designed and fabricated
via fused deposition modeling. J Biomed Mater. Res 55 (2):203-216,
2001. Methods using electrospun nanofibers can be found in W. J.
Li, C. T. Laurencin, E. J. Caterson, R. S. Tuan, and F. K. Ko.
Electrospun nanofibrous structure: a novel cell carrier for tissue
engineering. J Biomed Mater. Res 60 (4):613-621, 2002. Methods
using nanofibers with porogen leaching can be found in R. Zhang and
P. X. Ma. Synthetic nano-fibrillar extracellular matrices with
predesigned macroporous architectures. J Biomed Mater. Res 52
(2):430-438, 2000.
[0177] Both natural (e.g., collagen, elastin, poly(amino acids);
and polysaccharides such as hyaluronic acid, glycosamino glycan,
carboxymethylcellulose; and ceramic based-cell carriers such as
porous hydroxyapatite, tricalcium phosphate, and chitosan) and
synthetic materials may be used to manufacture the cell carriers of
the present invention. In a preferred embodiment, the cell carrier
is constructed of a resorbable material to allow room for tissue
growth in the cell carrier while eliminating the need for a second
surgery to remove the cell carrier. Exemplary synthetic resorbable
polymers that may be used to manufacture the cell carriers of the
present invention include, poly(glycolic acid) (PGA), poly(L-lactic
acid) (PLLA), poly (D-lactide) (PDLA), poly (D,L-lactide)(PDLLA)
and their copolymers, as well as PCL, PDO and PTMC and their
co-polymers. These polymers offer distinct advantages in that their
sterilizability and relative biocompatibility have been well
documented. Also, their resorption rates can be tailored to match
that of new tissue formation. In a preferred embodiment, the cell
carrier is constructed of 70:30 poly(L-lactide-co-D,L-lactide). In
another embodiment, the cell carrier is constructed of 85:15 poly
(D,L-lactide-co-glycolide).
[0178] In certain embodiments, any of the scaffolds or cell carrier
portions described above may be coated with apatite using a
simulated body fluid (SBF) solution. The SBF solutions may be
prepared with ion concentrations approximately 0-10 times that of
human blood plasma and can be sterile filtered through a 0.22 .mu.m
PES membrane or a similar membrane filter. Methods of making
art-recognized SBF solutions and variations thereof for use in the
present invention can be found in, e.g., Chou et al. (2004) The
Effect of Biomimetic Apatite Structure on Osteoblast Vitality,
Proliferation and Gene Expression Biomaterials (In press); Oyane et
al. (2003) Preparation and Assessment of Revised Simulated Body
Fluids J. Biomed mater Res 65A: 188-195; Murphy et al. (1999)
Growth of Continuous Bonelike Mineral Within Porous
Poly(lactic-co-glycolide) Scaffolds In Vitro J. Biomed. Mater. Res.
50: 50-58. The scaffolds or cell carriers may be treated with glow
discharge, argon-plasma etching prior to being soaked in the SBF
solution to improve wettability and affinity for the SBF ions.
Different apatite microenvironments can be created on the scaffold
or cell carrier surfaces by controlling the SBF concentration,
components, pH and the duration of the scaffold or cell carrier in
each SBF solution. Vacuum or fluid flow (directed or non-directed)
can be used to force the SBF into the pores of the scaffold or cell
carrier portion.
[0179] It is understood in the art that desired resorption rates of
the cell carrier portion will vary based on the particular
therapeutic application. It is believed that the cell carrier
portion of the present invention having a thickness of 0.1
millimeters to about 5 millimeters should maintain its structural
integrity for a period in excess of about two weeks to multiple
months, preferably three to six months, before substantially
degrading, so that the bone forming can be achieved and
optimized.
[0180] The rates of resorption of the cell carrier may also be
selectively controlled. For example, the cell carrier portion may
be manufactured to degrade at different rates depending on the rate
of recovery of the patient from a surgical procedure. Thus, a
patient who recovers more quickly from a surgical procedure
relative to an average patient, may be administered an agent that
for example is selective for the polymeric material of the cell
carrier portion and causes the cell carrier portion to degrade more
quickly. Or, if the polymeric material is, for example, temperature
sensitive or is influenced by electrical charge, the area in which
the device has been implanted may be locally heated or cooled, or
otherwise exposed to an electrical charge that causes the device to
dissolve at a desired rate for the individual patient.
[0181] In addition to resorption rates, certain physical
characteristics of the cell carriers must also be considered.
Generally, the cell carrier must have large a large surface area to
allow cell attachment. One method of achieving this result may be
to create a highly porous polymer foam. In these foams, the pore
size should be large enough so that cells penetrate the pores, and
the pores must be interconnected to facilitate nutrient exchange by
cells deep within the construct. These characteristics (porosity
and pore size) are often dependent on the method of cell carrier
fabrication (Mikos, A. G., Bao, Y., Cima, L. G., Ingber, D. E.,
Vacanti, J. P. and Langer, R. (1993a). Preparation of Poly(glycolic
acid) bonded fiber structures for cell attachment and
transplantation. Journal of Biomedical Materials Research
27:183-189; Mikos, A. G., Sarakinos, G., Leite, S. M., Vacanti, J.
P. and Langer, R. (1993b) Mikos, A. G., Thorsen, A. J., Czerwonka,
L. A., Bao, Y., Langer, R., Winslow, D. N. and Vacanti, J. P.
(1994). Preparation and characterization of poly(L-lactic acid)
foams. Polymer 35:1068-1077; Nam, Y. S. and Park, T. G. (1999a).
Biodegradable polymeric microcellular foams by modified thermally
induced phase separation method. Biomaterials 20:1783-1790; Nam, Y.
S., Yoon, J. J. and Park, T. G. (2000). A novel fabrication method
of macroporous biodegradable polymer scaffolds using gas foaming
salt as a porogen additive. Journal of Biomedical Materials
Research (Applied Biomaterials) 53:1-7).
[0182] Optimal pore sizes for bone formation generally range from
40 to 400 microns. Several methods have been developed to create
highly porous cell carriers, including fiber bonding (Mikos et al.
1993 above), solvent casting/particulate leaching (Mikos et al.
1993 above; Mikos et al. 1994 above), gas foaming (Nam et al. 2000
above) and phase separation (Lo, H., Ponticiello, M. S. and Leong,
K. W. (1995). Fabrication of controlled release biodegradable foams
by phase separation. Tissue Engineering 1:15-28; Whang, K., Thomas,
C. H., Healy, K. E. and Nuber, G. (1995). A novel method to
fabricate bioabsorbable scaffolds. Polymer 36:837-842; Lo, H.,
Kadiyala, S., Guggino, S. E. and Leong, K. W. (1996). Poly(L-lactic
acid) foams with cell seeding and controlled-release capacity.
Journal of Biomedical Materials Research 30:475-484; Schugens, C.,
Maquet, V., Grandfils, C., Jerome, R. and Teyssie, P. (1996).
Polylactide macroporous biodegradable implants for cell
transplantation II. Preparation of polylactide foams for
liquid-liquid phase separation. Journal of Biomedical Materials
Research 30:449-461; Nam and Park 1999 above). Of these methods,
fiber bonding, solvent casting/particulate leaching, gas
foaming/particulate leaching and liquid-liquid phase separation
produce large, interconnected pores to facilitate cell seeding and
migration. The fiber bonding, solvent casting/particulate leaching
and gas foaming/particulate leaching methods exhibit clinically
acceptable biocompatibility.
[0183] All of the methods described herein, as well as all
art-recognized methods for manufacturing cell carriers, must be
appropriately modified to remove organic solvents, which could
reduce the ability of cells to form new tissues in vivo. Some
methods of manufacturing the cell carriers of the present invention
are described herein by way of example and not limitation.
[0184] For example, as set forth above, one technique used for
constructing three-dimensional cell carriers is known as "melt
molding," wherein a mixture of fine PLGA powder and gelatin
microspheres is loaded in a Teflon.RTM. mold and heated above the
glass-transition temperature of the polymer. The PLGA-gelatin
composite is removed from the mold and gelatin microspheres are
leached out by selective dissolution in distilled de-ionized water.
Other cell carrier manufacturing techniques include polymer/ceramic
fiber composite foam processing, phase separation, and
high-pressure processing.
[0185] Another technique for manufacturing cell carriers is known
as "solvent-casting and particulate-leaching." In this technique,
sieved salt particles, such as sodium chloride crystals, are
disbursed in a PLLA/chloroform solution which is then used to cast
a membrane. After evaporating the solvent, the PLLA/salt composite
membranes are heated above the PLLA melting temperature and then
quenched or annealed by cooling at controlled rates to yield
amorphous or semi-crystalline forms with regulated crystallinity.
The salt particles are eventually leached out by selective
dissolution to produce a porous polymer matrix.
[0186] Yet another technique for manufacturing the cell carriers of
the present invention is known as "fiber bonding", and involves
preparing a mold in the shape of the desired cell carrier and
placing fibers, such as polyglycolic acid (PGA) into the mold and
embedding the PGA fibers in a poly (L-lactic acid) (PLLA)/methylene
chloride solution. The solvent is evaporated, and the PLLA-PGA
composite is heated above the melting temperatures of both
polymers. The PLLA is then removed by selective dissolution after
cooling, leaving the PGA fibers physically joined at their
cross-points without any surface or bulk of modifications and
maintaining their initial diameter. Fiber bonding is particularly
useful for fabrication of thin cell carriers.
[0187] Another technique may be solid freeform fabrication (SFF)
which refers to computer-aided-design and
computer-aided-manufacturing (CAD/CAM) methodologies which have
been used in industrial applications to quickly and automatically
fabricate arbitrarily complex shapes. SFF processes construct
shapes by incremental material buildup and fusion of
cross-sectional layers. In these approaches, a three-dimensional
(3D) CAD model is first decomposed, or "sliced", via an automatic
process planner into thin cross-sectional layer representations
which are typically 0.004 to 0.020 of an inch thick. To build the
physical shape, each layer is then selectively added or deposited
and fused to the previous layer in an automated fabrication
machine. These and other cell carrier manufacturing techniques are
discussed in U.S. Pat. No. 6,143,293, the entire contents of which
are incorporated herein by this reference.
[0188] In accordance with another aspect of the present invention,
the cell carrier portion may comprise a substance for cellular
control, such as a chemotactic substance for influencing
cell-migration, an inhibitory substance for influencing
cell-migration, a mitogenic growth factor for influencing cell
proliferation, a growth factor for influencing cell
differentiation, and factors which promote angiogenesis (formation
of new blood vessels). Cellular control substances may be located
at one or more predetermined locations on the cell carrier. For
example, substances that generally inhibit or otherwise reduce
cellular growth and/or differentiation may be located on one
surface of the cell carrier (e.g., the surface that will be in
proximity to the area within the area of intended bone formation
where bone growth is not desired). Similarly, substances that
generally promote or otherwise enhance cellular growth and/or
differentiation may be located on one surface of the cell carrier
(e.g., the surface that will be in proximity to the area of
intended bone formation). Additionally, the inhibiting and
promoting substances may be interspersed through the cell carrier
at predetermined locations to help influence rates of cellular
growth at different regions over the surface of the device.
[0189] Preferably, an appropriate cell carrier portion of the
device will be pre-formed into specific shapes, configurations and
sizes that conform to the shape of the cell carrier containment
portion by the manufacturer (e.g., by MacroPore Biosurgery, Inc.)
before packaging and sterilization. However, a pre-formed cell
carrier portion can also be shaped at the time of surgery by
bringing the material to its glass transition temperature, using
heating iron, hot air, heated sponge or hot water bath methods. In
a certain embodiment, the cell carrier could be cut and
mechanically press-fit into a containment portion and held in place
by the resulting interference fit. In another embodiment, the
containment portion could be heated to glass transition temperature
to revert to a pre-determined and pre-formed geometry, resulting in
a clamping of the cell carrier, resulting in stabilization. In yet
another embodiment, the cell carrier may be affixed to the
containment portion by one or more appropriately sized mating
resorbable or non-resorbable screw, tack or pin inserted through
one or more of the apertures in said containment portion.
Resorbable screws, tacks or pins can be obtained from MacroPore
Biosurgery, Inc. (San Diego, Calif.). Alternatively, once inserted
into an intended area of bone formation, the influx of water to the
area could expand the cell carrier and shrink the containment
portion or shrink the cell carrier and expand the containment
portion, resulting in fixation. The cell carrier and the
containment portion could also be affixed to each other using
polymer solutions, solvents or appropriate glues.
i. Illustrated Embodiment of a Porous Cell Carrier Portion of the
Device
[0190] As shown in FIG. 5, a porous cell carrier portion of the
device of the invention 210 is of parabolic shape, having a top
face 211, a bottom face 212, side faces 213, a front end 214 and a
back end 215, and a plurality of interconnected pores 216.
[0191] 3. Methods of Adding Regenerative Cells to a Cell Carrier
Portion of the Device
[0192] The cell carrier must be appropriately seeded with the
regenerative cells to optimize bone formation in the intended area
of bone formation. The function of the cell carrier is to deliver
cells in a manner compatible with optimal cell mediated bone
formation at the intended site of bone formation. The carrier may
be a solid, resorbable or non-resorbable polymer based scaffold
with a fiber or sponge macrostructure into which cells are seeded,
or a hydrogel, e.g., fibrin, polyethylene glycol, in which cells
are encapsulated. The carrier may be modified by a chemical,
peptide, protein, or gene to support cell attachment, e.g., RGD,
cell differentiation, e.g., CBFA-1gene, calcification, e.g.,
phosphor group. The cells can either be seeded in vivo by injection
or other appropriate method at the treatment site, or seeded ex
vivo.
[0193] Ex vivo seeding methods for solid based scaffolds
encompassed in this invention include art-recognized methods such
as static seeding (or capillary action), injection, dynamic
seeding, including uni- and bidirectional perfusion, spinner flask,
orbital or random shaker, rotating or end-over-end bioreactor
seeding, and vacuum assisted seeding, or a mixture of the methods,
to name a few. Seeding methods for natural or synthetic gel based
carrier encompassed in this invention include art-recognized
methods such as mixing the cells with the liquid components of the
gel based carrier and forming a gel by physical interaction
crosslinks (e.g. hydrogen, van der waals forces, polar forces,
ionic bonds) or covalent crosslinks. Seeding using gel based
carriers can be accomplished either in vivo or ex vivo. If the cell
carrier portion is of a particulate form, the seeding methods may
be as described above, or may be different. For example, the cells
may simply be mixed with the cell carrier portion and place, press
fit or inject the cell/particulate mixture into the intended area
of bone formation, such as in the center of the containment device
and/or around the containment device.
[0194] The cells to be incorporated into the carrier may be
previously incubated or cultured with any substance that may
promote the cells' ability to adhere to the carrier or to stimulate
the cells' bone forming capacity, the latter exemplified by bone
growth promoting proteins or genes (e.g.) BMPs, or other bone
growth regulatory molecules (e.g., CBFA-1). In an ex vivo seeding
approach, the cell laden cell carriers are then placed into the
intended area of bone formation with the cell carrier containment
portion of the present invention to induce bone formation. Once
implanted, host cell influx and ADC proliferation, differentiation,
osteoid deposition, mineralization, and, in the case of resorbable
scaffolds, concomitant reabsorption of the scaffold, proceeds.
[0195] These and other methods of seeding the cell carriers are
disclosed in, for example, S. L. Ishaug, G. M. Crane, M. J. Miller,
A. W. Yasko, M. J. Yaszemski, and A. G. Mikos. Bone formation by
three-dimensional stromal osteoblast culture in biodegradable
polymer scaffolds. J Biomed Mater Res 36 (1):17-28, 1997; H. L.
Wald, G. Sarakinos, M. D. Lyman, A. G. Mikos, J. P. Vacanti, and R.
Langer. Cell seeding in porous transplantation devices.
Biomaterials 14 (4):270-278, 1993; N. S. Dunkelman, M. P. Zimber,
R. G. LeBaron, R. Pavelec, M. Kwan, and A. F. Purchio. Cartilage
production by rabbit articular chondrocytes on polyglycolic acid
scaffolds in a closed bioreactor system. Biotechnol Bioeng
46:299-305, 1995; L. E. Freed, A. P. Hollander, I. Martin, J. R.
Barry, R. Langer, and G. Vunjak-Novakovic. Chondrogenesis in a
cell-polymer-bioreactor system. Exp Cell Res 240 (1):58-65, 1998;
R. E. Schreiber, N. S. Dunkelman, G. Naughton, and A. Ratcliffe. A
method for tissue engineering of cartilage by cell seeding on
bioresorbable scaffolds. Ann N Y Acad Sci 875:398-404, 1999; G.
Vunjak-Novakovic, et al (1999) van Wachem P B, tronck J W,
oers-Zuideveld R, ijk F, and ildevuur C R. Vacuum cell seeding: a
new method for the fast application of an evenly distributed cell
layer on porous vascular grafts. Biomaterials 11 (8):602-606, 1990;
L. E. Freed, J. C. Marquis, G. Vunjak-Novakovic, J. Emmanual, and
R. Langer. Composition of cell-polymer cartilage implants.
Biotechnol. Bioeng. 43:605-614, 1994; J. L. van Susante, P. Buma,
G. J. van Osch, D. Versleyen, P. M. van der Kraan, W. B. van der
Berg, and G. N. Homminga. Culture of chondrocytes in alginate and
collagen carrier gels. Acta Orthop Scand 66 (6):549-56, 1995; J.
Elisseeff, W. McIntosh, K. Anseth, S. Riley, P. Ragan, and R.
Langer. Photoencapsulation of chondrocytes in poly(ethylene
oxide)-based semi-interpenetrating networks. J. Biomed. Mater. Res.
51:164-171, 2000.
[0196] Once seeded on the cell carrier, the device can be inserted
into an intended area of bone formation in the recipient.
Alternatively, the cells can be seeded onto the cell carrier
subsequent to insertion of the device (the cell carrier portion and
the containment portion) into an intended area of bone formation in
the recipient. Once the device is inserted, in-vivo cellular
proliferation and, in the case of resorbable cell carriers,
concomitant reabsorption of the cell carrier, proceeds.
[0197] In accordance with another aspect of the invention, the cell
carrier portion may further comprise a substance (e.g., a chemical
or biological molecule) to facilitate cell seeding, for example, an
adhesion peptide or other biological molecule to enhance cell
attachment, a substance that alters the hydrophilicity of the cell
carrier, a surface charge, a polar molecule, or a surfactant or
wetting agent to increase cell attachment or increase fluid uptake
into the cell carrier. The substance used to facilitate cell
seeding may be located on the surface of the cell carrier pores,
the outer regions of the cell carrier, interspersed in the bulk of
the cell carrier material, at predetermined locations, or any
combinations thereof.
i. Illustrated Embodiment of a Cell Carrier Seeded with
Regenerative Cells
[0198] As shown in FIG. 17, a porous cell carrier portion of the
device of the invention 210 is in the shape of a parabolic sleeve,
having a top face 211, a bottom face 212, side faces 213, a front
end 214 and a back end 215, a plurality of interconnected pores 216
and seeded with regenerative cells such as stem cells and
progenitor cells 217.
[0199] 4. Methods for Manufacturing a Cell Carrier Containment
Portion of the Device
[0200] In a broad embodiment, the cell carrier containment portion
of the device ("the containment portion") may be constructed from
any material effective to govern bone formation when used in
accordance with the methods disclosed herein. Preferably, the
containment portion is resorbable. For example, the containment
portion may be constructed from any biodegradable material, such as
resorbable polymers. In preferred embodiments, the containment
portion is constructed with materials that do not induce a
significant antigenic or immunogenic biological response.
[0201] In accordance with one embodiment, non-limiting polymers
which may be used to form containment portion of the present
invention include polymers (e.g., copolymers) of lactide (L, D, DL,
or combinations thereof), glycolide, trimethylene carbonate,
caprolactone and/or physical and chemical combinations thereof. In
one embodiment, the containment portion is comprised of a
polylactide, which can be a copolymer of L-lactide and D,L-lactide.
For example, the copolymer can comprise about 60-80% of L-lactide
and about 20-40% of D,L-lactide, and in a preferred embodiment the
copolymer comprises 70:30 poly (L-lactide-co-D,L-lactide).
[0202] In one embodiment, the containment portion is formed by
polymers (e.g., homo and/or copolymers) derived from one or more
cyclic esters, such as lactide (i.e., L, D, DL, or combinations
thereof), epsilon-caprolactone and glycolide. For instance, in one
embodiment, the containment portion can comprise about 1 to 99%
epsilon-caprolactone, or in another embodiment can comprise 20 to
40% epsilon-caprolactone. In one example, a membrane comprises
65:35 poly (L-lactide-co-epsilon-caprolactone). In other
embodiments, butyrolactone, valerolactone, or dimethyl
propiolactone can be used with or as a substitute for
epsilon-caprolactone. In another embodiment, the containment
portion can comprise a copolymer including lactide and glycolide
which is resorbed into the body more rapidly than the
above-mentioned poly (L-lactide-co-D,L-lactide).
[0203] The polymers (e.g., co-polymers) of the present invention
require relatively simple chemical reactions and formulations. The
containment portion of the present invention is preferably smooth
and substantially non-porous. Preferably, the containment portion
may be formed into specific shapes, configurations and sizes by the
manufacturer (e.g., MacroPore Biosurgery, Inc.) before packaging
and sterilization. However, a pre-formed containment portion can
also be shaped at the time of surgery by bringing the material to
its glass transition temperature, using heating iron, hot air,
heated sponge or hot water bath methods.
[0204] The containment portion may have a thickness of about 0.1
millimeters to about 5 millimeters. Preferably, the containment
portion has a thickness of about 2 millimeters to about 4
millimeters. The material of the containment portion may be
flexible enough to conform to a curvature of a bone and sufficient
strength to reduce macro-motion of the bone defect and limit
transmission of surrounding motion into the interior space when the
device is attached to the targeted area. The containment portion is
adapted for protecting the target area from a prolapse of adjacent
soft tissues into the target area during bone formation and,
further, is adapted for preventing resorption of bone due to stress
shielding associated with insertion of the device after the desired
new bone formation has occurred.
[0205] The containment portion may be provided in any shape which
may effectively promote bone growth. In one embodiment, the
containment portion may be provided in a parabolic shape. In
another embodiment, the containment portion may be provided in a
rectangular shape. In yet another embodiment, the containment
portion may be provided in a trapezoidal shape. In a further
embodiment, the containment portion may be provided in a
cylindrical shape. The containment portion of the present invention
may be sufficiently flexible to conform around anatomical
structures.
[0206] In one embodiment, the containment portion comprises two
opposing surfaces. On one side of the containment portion, there is
a bone-growing substantially-smooth side or surface, and on the
other side there is a non-bone growing substantially-smooth side or
surface. Preferably, the bone-growing substantially-smooth side is
positioned to face the area of intended bone formation, and the
non-bone growing substantially-smooth side is positioned to face
the area where bone formation is not desired.
[0207] As used herein, the term "non-porous" refers to a material
which is generally water tight and, in accordance with a preferred
embodiment, not fluid permeable. However, in a modified embodiment
of the invention micro-pores (i.e., fluid permeable but not cell
permeable) may exist in the containment portion of the present
invention, to the extent, for example, that they do not
substantially disrupt the smoothness of the surfaces of the
containment portion to cause scarring of tissue. In substantially
modified embodiments for limited applications, pores which are cell
permeable but not vessel permeable may be manufactured and used. As
presently preferred, the containment portion is manufactured using
an injection molding procedure to yield a substantially non-porous
containment portion. The containment portion may also be
manufactured using extrusion or compression molding procedures. The
containment portion may have a semi-rigid construction, and are
fully contourable when heated to approximately 55 degrees
Celsius.
[0208] Accordingly, in particular embodiments, the substantially
smooth surface of the containment portion may be comprised of a
plurality of apertures with a diameter in a range between 20
microns to 3000 microns. The apertures are adapted for allowing a
proliferation of vasculature and connective tissue cells
therethrough, while preventing the prolapse of adjacent soft
tissues into the biological tissue defect. In addition, the
apertures allow vital contributions of blood vessels from
surrounding tissues, musculature, and periosteum into the target
area. Blood vessels invading the target area through the device can
greatly enhance the generation of new bone. The ability for
capillaries from surrounding tissues to proliferate through the
device can help prevent migrating cells from the osseous bed and
the periosteum from outstripping their proliferating blood supply.
This proliferation of blood vessels can increase the potential of
bone formation within a given target area.
[0209] The surface of the containment portion may further be
comprised of a plurality of larger apertures with a diameter in a
range of 1 to 5 millimeters. Such larger apertures in the
containment portion are strategically placed to facilitate
insertion of an insertion and/or fixation device for securing the
device into an intended area of bone formation. The apertures can
also be strategically placed for a subsequent surgical procedure in
which additional regenerative cells are injected through the
aperture in the event of a delayed union or non-union. It is
understood in the art that the size of the larger apertures on the
containment portion may vary in accordance with the type of
insertion tool and/or fixation device used to secure the device in
the target area. In addition to art-recognized tools for insertion
of the device, the device may be frictionally secured in the target
area using, fixation devices such as clamps, staples, screws,
sutures, tacks, pins and other conventional means. In a preferred
embodiment, the fixation devices are resorbable, such as those
manufactured by MacroPore Biosurgery, Inc. In alternative
embodiments, a combination of insertion devices or fixation devices
may be used.
[0210] It is understood in the art that desired resorption rates
will vary based on the particular therapeutic application. It is
believed that a containment portion of the present invention having
a thickness of 1 millimeters to about 5 millimeters, preferably 2-4
millimeters, should maintain its structural integrity for a period
in excess of about one month, preferably in excess of about three
months, more preferably for about three to six months, before
substantially degrading, so that the bone forming can be achieved
and optimized. Similar to the rates of resorption of the cell
carrier portion of the device as set forth above, the rates of
resorption of the cell carrier containment portion of the device
may also be selectively controlled.
[0211] Exemplary materials and methods related to making and using
all aspects of the present invention are disclosed in, for example,
U.S. Pat. Nos. 5,919,234, 6,280,473, 6,269,716, 6,343,531,
6,477,923, 6,391,059, 6,531,146 and 6,673,362, the contents of
which are incorporated herein by this reference. Resorbable
containment portions of the devices of the present invention are
available from MacroPore Biosurgery, Inc. (San Diego, Calif.).
i. Illustrated Embodiment of a Cell Carrier Containment Portion of
the Device
[0212] As shown in FIG. 18, in one embodiment, a cell carrier
containment portion of the device of the invention is of parabolic
shape 218, having a top face 219, a bottom face 220, side faces
221, a front end 222 and a back end 223. The surfaces of top and
bottom faces 219 and 220 each have ridges 224 to help anchor the
containment portion of the device to the surrounding bone in the
target area. The recesses between the ridges 224 can enhance
mechanical stability that would aid in anchoring the containment
portion of the device in place. The cell carrier containment
portion 218 may contain apertures 225 of appropriate diameter, that
can be used to screw the device into place. The apertures 225 may
also be used to insert an art-recognized tool to insert the device
into a target area. The cell carrier containment portion has a
large center hole 226 for insertion of a cell carrier portion of
the present invention. FIG. 19 depicts an embodiment of the
invention wherein a porous cell carrier of parabolic shape has been
inserted into a containment portion of parabolic shape. FIG. 20
depicts an embodiment of the invention wherein a porous cell
carrier of parabolic shape seeded with the regenerative cells of
the invention has been inserted into a containment portion of
parabolic shape. FIG. 20 depicts an embodiment of the device and
the cells which is suitable for insertion into a recipient using
art-recognized surgical techniques.
[0213] As shown in FIG. 21, in one embodiment, a cell carrier
containment portion of the device of the invention is of
trapezoidal shape 227, having a top face 228, a bottom face 229,
side faces 230, a front end 231 and a back end 232. The surfaces of
top and bottom faces 228 and 229 each have ridges 233 to help
anchor the containment portion of the device to the surrounding
bone in the target area. The recesses between the ridges 233 can
enhance mechanical stability that would aid in anchoring the
containment portion of the device in place. The cell carrier
containment portion 227 may contain apertures 234 of appropriate
diameter, that can be used to screw the device into place. The
apertures 234 may also be used to insert an art-recognized tool to
insert the device into a target area. The cell carrier containment
portion has a large center hole 235 for insertion of a cell carrier
portion of the present invention.
[0214] As shown in FIG. 22, in one embodiment, a cell carrier
containment portion of the device of the invention is of
cylindrical shape 236, having a top face 237, a bottom face 238,
side faces 239, a front end 240 and a back end 241. The surfaces of
top and bottom faces 237 and 238 each have ridges 242 to help
anchor the containment portion of the device to the surrounding
bone in the target area. The recesses between the ridges 242 can
enhance mechanical stability that would aid in anchoring the
containment portion of the device in place. The cell carrier
containment portion 236 may contain apertures 243 of appropriate
diameter, that can be used to screw the device into place. The
apertures 243 may also be used to insert an art-recognized tool to
insert the device into a target area. The cell carrier containment
portion has a large center hole 244 for insertion of a cell carrier
portion of the present invention.
[0215] As shown in FIG. 23, in one embodiment, a cell carrier
containment portion of the device of the invention is of
rectangular shape 245, having a top face 246, a bottom face 247,
side faces 248, a front end 249 and a back end 250. The surfaces of
top and bottom faces 246 and 247 each have ridges 251 to help
anchor the containment portion of the device to the surrounding
bone in the target area. The recesses between the ridges 251 can
enhance mechanical stability that would aid in anchoring the
containment portion of the device in place. The cell carrier
containment portion 245 may contain apertures 252 of appropriate
diameter, that can be used to screw the device into place. The
apertures 252 may also be used to insert an art-recognized tool to
insert the device into a target area. The cell carrier containment
portion has a large center hole 253 for insertion of a cell carrier
portion of the present invention.
[0216] 5. Methods for Using the Device for Bone and/or Cartilage
Formation
[0217] As exemplified herein, the adipose derived regenerative
cells of the present invention are a rich source of bone or
cartilage precursor cells and they have been shown to form bone and
cartilage in vivo. Accordingly, the device of the present invention
can be used to treat bone related disorders, e.g., by promoting
bone and/or cartilage formation.
[0218] Accordingly, in one aspect of the present invention,
regenerative cells are extracted from a donor's adipose tissue, are
added to a porous resorbable cell carrier surrounded by a
resorbable cell carrier containment portion, and the combination of
the device and the cells is inserted into an intended area of bone
formation in a recipient to thereby elicit a therapeutic benefit by
promoting bone and/or cartilage formation. In a preferred
embodiment the cells are extracted from the adipose tissue of the
person into whom they are to be implanted, thereby reducing
potential complications associated with antigenic and/or
immunogenic responses to the transplant. Patients are typically
evaluated to assess bone related disorders by one or more of the
following procedures performed by a physician or other clinical
provider: patient's health history, physical examination, and
objective data including but not limited to radiographs.
[0219] In one embodiment, the harvesting procedure is performed
prior to the patient receiving any products designed to reduce
blood clotting in connection with treatment for the bone related
disorder, e.g., spinal fusion surgery. However, in certain
embodiments, the patient may have received drugs known to affect
coagulation (e.g., aspirin) prior to the harvesting procedure. In
addition, one preferred method includes collection of adipose
tissue prior to any attempted spinal fusion procedure. However, as
understood by persons skilled in the art, the timing of collection
is expected to vary and will depend on several factors including,
among other things, patient stability, patient coagulation profile,
provider availability, and quality care standards. Ultimately, the
timing of collection will be determined by the practitioner
responsible for administering care to the affected patient.
[0220] The volume of adipose tissue collected from the patient can
vary from about 1 cc to about 2000 cc and in some embodiments up to
about 3000 cc. The volume of fat removed will vary from patient to
patient and will depend on a number of factors including but not
limited to: age, body habitus, coagulation profile, hemodynamic
stability, co-morbidities, and physician preference.
[0221] The cells may be extracted in advance and stored in a
cryopreserved fashion or they may be extracted at or around the
time of defined need. As disclosed herein, the cells may be
administered to the patient, or applied directly to the damaged
tissue, or in proximity of the damaged tissue, without further
processing or following additional procedures to further purify,
modify, stimulate, or otherwise change the cells. For example, the
cells obtained from a patient may be administered to a patient in
need thereof via the resorbable cell carrier and resorbable cell
carrier containment portion without culturing the cells before
administering them to the patient. In one embodiment, the
collection of adipose tissue will be performed at a patient's
bedside. Hemodynamic monitoring may be used to monitor the
patient's clinical status.
[0222] In accordance with the invention disclosed herein, the
regenerative cells can be delivered to the patient soon after
harvesting the adipose tissue from the patient. For example, the
cells may be administered immediately via the device after
processing of the adipose tissue and obtaining a composition of
regenerative cells. Ultimately, the timing of delivery will depend
upon patient availability and the processing time required to
process the adipose tissue. In another embodiment, the timing for
delivery may be relatively longer if the cells to be re-infused to
the patient are subject to additional modification, purification,
stimulation, or other manipulation, as discussed herein. The number
of cells administered to a patient may be related to, for example,
the cell yield after adipose tissue processing. A portion of the
total number of cells may be retained for later use or
cyropreserved. In one embodiment of the invention, a number of
cells, e.g., unpurified cells, to be delivered to the patient is
expected to be about 5.5.times.10.sup.4 cells. However, this number
can be adjusted by orders of magnitude to achieve the desired
therapeutic effect.
[0223] Cells obtained after disaggregation from adipose tissue may
be further enriched for bone or cartilage progenitor cells by
passage over a biologic based component within or separate from the
device that either captures chondroprogenitor cells, osteoprecursor
cells or all cells but one of these cells. If the former, then an
additional step included within or external to the device would be
used to release the captured cells from the biologic based
component. An example of this chondroprogenitor or osteoprecursor
enrichment would be a chromatographic resin to which an antibody
that recognizes chondroprogenitor cells or osteoprecursor cells
(e.g., SH3 cells) is attached. An example of a release agent is
papain or pepsin that could cleave the antibody/cell complex from
the resin.
[0224] The cells may also be applied with additives to enhance,
control, or otherwise direct the intended therapeutic effect. Cells
may be administered following genetic manipulation such that they
express gene products that are believed to or are intended to
promote the therapeutic response(s) provided by the cells. Examples
of manipulations include manipulations to control (increase or
decrease) expression of factors promoting bone or cartilage
formation, expression of developmental genes promoting
differentiation into specific cartilage or bony cell lineages or
that stimulate cartilage or bony cell growth and proliferation.
[0225] The active cell population may be applied to the resorbable
cell carrier of the present device alone or in combination with
other cells, tissue, tissue fragments, growth factors (e.g., BMPs),
or other additive intended to enhance the delivery, efficacy,
tolerability, or function of the population. The cell population
may also be modified by insertion of DNA in a plasmid or viral
vector or by placement in cell culture in such a way as to change,
enhance, or supplement the function of the cells for derivation of
a structural or therapeutic purpose. For example, gene transfer
techniques for stem cells are known by persons of ordinary skill in
the art, as disclosed in (Morizono et al., 2003; Mosca et al.,
2000), and may include viral transfection techniques, and more
specifically, adeno-associated virus gene transfer techniques, as
disclosed in (Walther and Stein, 2000) and (Athanasopoulos et al.,
2000). Non-viral based techniques may also be performed as
disclosed in (Muramatsu et al., 1998).
[0226] In another aspect, the cells could be combined with a gene
encoding a pro-chondroprogenitor or pro-osteogenic growth factor(s)
which would allow cells to act as their own source of growth factor
during cartilage or bone formation. Genes encoding anti-apoptotic
factors or agents could also be applied. Addition of the gene (or
combination of genes) could be by any technology known in the art
including but not limited to adenoviral transduction, "gene guns,"
liposome-mediated transduction, and retrovirus or
lentivirus-mediated transduction, plasmid, adeno-associated virus.
Cells could be implanted along with a carrier material bearing gene
delivery vehicle capable of releasing and/or presenting genes to
the cells over time such that transduction can continue or be
initiated in situ. Particularly when the cells and/or tissue
containing the cells are administered to a patient other than the
patient from whom the cells and/or tissue were obtained, one or
more immunosuppressive agents may be administered to the patient
receiving the cells and/or tissue to reduce, and preferably
prevent, rejection of the transplant. As used herein, the term
"immunosuppressive drug or agent" is intended to include
pharmaceutical agents which inhibit or interfere with normal immune
function. Examples of immunosuppressive agents suitable with the
methods disclosed herein include agents that inhibit T-cell/B-cell
costimulation pathways, such as agents that interfere with the
coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as
disclosed in U.S. Patent Pub. No. 20020182211. A preferred
immunosuppressive agent is cyclosporine A. Other examples include
myophenylate mofetil, rapamicin, and anti-thymocyte globulin. In
one embodiment, the immunosuppressive drug is administered with at
least one other therapeutic agent. The immunosuppressive drug is
administered in a formulation which is compatible with the route of
administration and is administered to a subject at a dosage
sufficient to achieve the desired therapeutic effect. In another
embodiment, the immunosuppressive drug is administered transiently
for a sufficient time to induce tolerance to the ADC of the
invention.
[0227] In certain embodiments of the invention, the cells are
administered to a patient with one or more cellular differentiation
agents, such as cytokines and growth factors. Examples of various
cell differentiation agents are disclosed in (Gimble et al., 1995;
Lennon et al., 1995; Majumdar et al., 1998; Caplan and Goldberg,
1999; Ohgushi and Caplan, 1999; Pittenger et al., 1999; Caplan and
Bruder, 2001; Fukuda, 2001; Worster et al., 2001; Zuk et al.,
2001).
[0228] i. Methods of Using the Device for Intervertebral Disc
Repair or Interbody Spinal Fusion Procedures
[0229] As set forth herein, back pain remains a major public health
problem, especially among aged people. Persistent and severe back
pain often causes debility and disability. This pain is closely
associated with intervertebral disc abnormalities of the spine. By
way of background, the human spine is a flexible structure
comprised of thirty-three vertebrae. Intervertebral discs separate
and cushion adjacent vertebrae, act to minimize loading applied to
the spine, and allow motion between the vertebrae (flexion,
extension, rotation). An intervertebral disc comprises two major
components: the nucleus pulposus and the annulus fibrosis. The
nucleus pulposus is centrally located in the disc and occupies
25-40% of the disc's total cross-sectional area. The matrix of the
nucleus pulposis is largely composed of a thick gelatinous
substance made up of water, polysaccharides and protein molecules.
The annulus fibrosis surrounds the nucleus pulposus and resists
torsional and bending force applied to the disc. Vertebral
end-plates separate the disc from the vertebrae on either side of
the disc. The outer ring layer structurally consists mainly of
overlapping orthogonal layers of fibrocartilage which are mainly
made up of type I collagen in an extracellular matrix. This
crisscrossing pattern imparts outstanding structural resilience to
the pressure applied by the adjacent vertebrae. The inner core,
located in the middle of the annulus fibrosis, consists of a few
cells embedded in a matrix rich in hyaluronic acid and type II
collagen and acts as a biomechanical shock absorber.
[0230] As a result of exertion, injury, illness, accident or abuse,
one or more of the vertebrae and/or one or more discs may become
damaged. Specifically, disorders of the vertebrae and discs include
but are not limited to 1) disruption of the disc annulus such as
annular fissures; 2) chronic inflammation of the disc; 3) localized
disc herniations with contained or escaped extrusions; 4) relative
instability of the vertebrae surrounding the disc; and 5)
trauma.
[0231] Though intervertebral disc cells in both the annulus
fibrosus and the nucleus pulposus only constitute a small percent
of the disc volume, they have been shown to have the capacity to a
synthesize a proteoglycan rich tissue (similar to cartilage) which
comprises the extracellular matrix of the disc. In diseased discs,
a decrease in intervertebral disc cell density in the nucleus
pulposus is accompanied by a reduced production of the hydrophilic
extracellular matrix. A reduction in intervertebral disc cells thus
results progressively in the structural decrease of the
intervertebral disc space, contributing to degenerative disc
disease (DDD). Traditionally, cartilage is classified as either
hyaline, elastic, or fibrocartilage. The gel-like anatomical
characteristics of the nucleus pulposus (as well as it unique
notochordal embryonic origin) does not fit into any of the
cartilagenous categories, and is not classically considered a
cartilage. It is, nonetheless, permeated with a proteogylcan rich
extracellular matrix similar to that of cartilage. Based on the
ability of the regenerative cells of the present invention to
produce cartilage and bone, these cells can also stimulate the
formation of the cartilage like proteoglycan rich extracellular
matrix to thereby restore the intervertebral disc.
[0232] Various approaches have been developed to treat back pain.
Minor back pain can be treated with medication and other
non-invasive therapy. Mild to moderate degenerated discs may be
treated by restoring the damages tissues within the disc, nucleous
pulposis and annulus fibrosis. Accordingly, the regenerative cells
of the invention may be used to stimulate cartilage development and
thereby restore the intervertabral discs at various stages of
degeneration. For example, the regenerative cells of the invention
may be delivered via injection in a physiologic solution directly
into the disc's nucleus pulposis in cases of mild degeneration,
wherein the disc is largely still grossly intact, but beginning to
break down. In cases of moderate disc degeneration, where the disc
is macroscopically degenerating, the regenerative cells could be
mixed with a hydrogel liquid that gels in situ when injected into
the nucleus pulposis. The gelation could be thermal or
thermochemically mediated.
[0233] However, it is often necessary to remove at least a portion
of the damaged and/or malfunctioning back component. For example,
when a disc becomes ruptured, a discectomy surgical procedure can
be performed to remove the ruptured disc and to fuse the two
vertebrae between the removed disc together. Details regarding
typical implementations of methods for fusing vertebrae are
disclosed in U.S. Pat. Nos. 6,033,438 and 5,015,247, the contents
of which are incorporated in their entireties herein by
reference.
[0234] Spinal fusion is indicated to provide stabilization of the
spinal column for disorders such as structural deformity, traumatic
instability, degenerative instability, and post resection
iatrogenic instability. Fusion, or arthrodesis, can thus be
achieved, for example, by the formation of an osseous bridge
between adjacent motion segments. The fusion can be accomplished
either anteriorly between contiguous vertebral bodies or
posteriorly between consecutive transverse processes, laminae or
other posterior aspects of the vertebrae. Surgical techniques for
spinal fusion are well-known in the art. The present invention
provides an inherent advantage over the art-recognized techniques
because it eliminates the need for a second surgery to harvest
autologous bone graft. In addition, typically, bone graft materials
and/or growth factors are often needed to promote spinal fusions.
The present invention provides autologous regenerative cells that
promote bone growth cost-effectively and without fear of graft
rejection.
[0235] Surgical methods for spinal fusion and other surgical
techniques for treating bone related disorders are well known to
the ordinarily skilled artisan. All such art recognized techniques
are intended to be encompassed by the present invention.
[0236] The present invention is further illustrated by the
following examples which in no way should be construed as being
further limiting, The contents of all cited references, including
literature references, issued patents, published patent
applications, and co-pending patent applications, cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
[0237] The ADC or regenerative cells used throughout the examples
set forth below can be obtained by the method(s) described in the
instant disclosure and/or the method(s) described in U.S.
application Ser. No. 10/316,127, entitled SYSTEMS AND METHODS FOR
TREATING PATIENTS WITH PROCESSED LIPOASPIRATE CELLS, filed Dec. 9,
2002, which claims priority to U.S. Provisional Application Ser.
No. 60/338,856, filed Dec. 7, 2001, as well as well as the methods
described in U.S. application Ser. No. 10/887,822 entitled, SYSTEMS
AND METHODS FOR SEPARATING AND CONCENTRATING REGENERATIVE CELLS
FROM TISSUE, filed Jun. 25, 2004, which claims priority to U.S.
application Ser. No. 10/316,127, entitled SYSTEMS AND METHODS FOR
TREATING PATIENTS WITH PROCESSED LIPOASPIRATE CELLS, filed Dec. 9,
2002, which are all commonly assigned and the contents of all of
which are expressly incorporated herein by this reference.
Example 1: Human Adipose Tissue is a Source of Osteoprogenitor
Cells
Methods and Materials:
[0238] Liposuction:
[0239] Human adipose tissue was obtained from individuals
undergoing elective lipoplasty (liposuction). In general the
liposuction procedure involved creation of a 0.5 cm incision in the
skin of the abdomen and/or thigh followed by infiltration of the
subcutaneous space with tumescent solution (saline supplemented
with lidocaine and epinephrine). After approximately 10 minutes the
subcutaneous adipose tissue was aspirated through a cannula (3-5 mm
diameter according to Physician preference) under machine-assisted
suction. Adipose tissue, blood, and tumescent solution
(lipoaspirate) was collected into a disposable container and stored
at 4.degree. C. during transport to the laboratory.
[0240] Tissue Digestion:
[0241] Adipose tissue was washed several times with warm
(37.degree. C.) saline to remove excess blood and free lipid. The
tissue was then digested at 37.degree. C. with Blendzyme
collagenase (Blendzyme 1 for 45 minutes or Blendzyme 3 for 20
minutes; Roche, Indianapolis, Ind.) in approximately two volumes of
saline. Constant agitation was applied to ensure adequate mixing of
tissue and medium throughout the digestion. After digestion cells
non-buoyant cells were removed and washed three times to remove
residual enzyme. Cells were counted using a vital dye (propidium
iodide:) on a hemocytometer using a fluorescence microscope
[0242] CFU-AP Assay:
[0243] Washed cells plated in duplicate at 1,000, 5,000, and 25,000
cells/well in six multiwell plates (Corning, New York). Osteogenic
differentiation was induced by culturing cells for 3 weeks in
medium consisting of .alpha.-MEM (Cellgro, Herndon, Va.), 10%
FBS/5% Horse serum/1% Antibiotic-antimycotic solution (Omega
Scientific, Tarzana, Calif.) supplemented with 0.1 .mu.M
Dexamethasone (Sigma, St. Louis, Mo.), 10 mM
.beta.-glycerophosphate (Sigma, St. Louis, Mo.) and 50 .mu.M
L-Ascorbic acid 2-phosphate (Sigma, St. Louis, Mo.). After the
first week of induction the culture media was changed weekly with
Osteogenic Medium without dexamethasone. To detect Alkaline
Phosphatase (AP) activity, culture plates were rinsed with PBS and
stained with 1% Napthol AS-BI phosphate (Sigma, St. Louis, Mo.) and
1 mg/ml Fast Red TR (Sigma, St. Louis, Mo.) for 20 min at
37.degree. C. After staining, the cells were washed with PBS and
fixed with 10% Buffered Formalin Phosphate (Fisher Scientific,
Pittsburgh, Pa.) for 15 min. Colonies consisting of more than 50
cells with positive AP staining were defined as CFU-AP. The number
of CFU-AP was counted in each well and the average number of CFU-AP
from the duplicate sample was calculated.
[0244] Alkaline Phosphatase Assay:
[0245] Total alkaline phosphatase activity in day 21 cultures of
adipose tissue-derived cells was measured using a colorimetric
assay (Sigma Diagnostics, St. Louis, Mo.). Briefly, day 21 cultures
were harvested by scraping, cells were resuspended in saline, and
lysed by sonication. Cell lysates were then incubated with
p-nitropheyl phosphate at pH 10.2 for 15 minutes at 30.degree. C.
Absorbance at 405 nm was determined using a Turner SP-830
spectrophotometer (Turner Designs, Sunnyvale, Calif.).
Results:
[0246] Donor Characteristics:
[0247] The characteristics of the donor population is shown in
Table A below. This population is representative of the population
undergoing cosmetic lipoplasty. Data from the American Society for
Aesthetic Plastic Surgery indicate that 384,626 liposuction
procedures were performed in the USA in 2003 making this the most
common cosmetic surgery procedure for both men and women. Women
accounted for 84% of all lipoplasty procedures (81% in the present
study). Approximately half of all patients undergoing lipoplasty in
2003 were between 35 and 50 years of age; median age in this study
46.
TABLE-US-00004 TABLE A Donor Characteristics Gender 44F 10M Median
Age 46 (range 19-72) Mean Volume of Lipoaspirate 510 .+-. 47 ml
(range 50-1,500) Time from Liposuction to Tissue Processing <6
hours 17 Overnight 37 Body Mass Index Normal 20 Overweight 12 Obese
6 Unknown 16
[0248] Development and Validation of the CFU-AP Assay:
[0249] Tissue processing yielded an average of
7.3.+-.0.1.times.10.sup.5 viable non-buoyant nucleated cells per
unit volume (ml) of adipose tissue. These cells were then plated in
CFU-AP assays at multiple densities in order to validate this assay
for this cell source. Further, we have demonstrated that CFU-AP
count is linear with cell number plated across the range of 1,000
to 50,000 cells/well. The range of frequency detected is such that
for standard assays we routinely plate cells in triplicate at two
different cell concentrations in order to ensure that we have
cultures in which colony number per well falls into the range of
10-60 per well.
Osteoprogenitor Frequency and Yield
[0250] Using this approach we tested adipose tissue obtained from
54 donors (Table A). The data shows that CFU-AP frequency in
samples processed from fresh tissue (tissue processed within six
hours of collection) was significantly higher than that of samples
processed on the day following tissue collection (2.0.+-.0.3
CFU-AP/100 cells compared with 0.6.+-.0.2 CFU-AP/100 cells;
p=0.001). CFU-AP frequency did not correlate with donor age or
gender.
[0251] The average yield of osteoprogenitor cells was
7,225.+-.1,760 CFU-AP/ml adipose tissue (range 120-67,500). Unlike
CFU-AP frequency there was a trend towards reduced CFU-AP yield per
unit volume of adipose tissue with increasing donor age. However,
when the upper quartile of age (>48 yr) was compared with the
lower age quartile (<34 yrs) the difference did not reach
statistical significance (4,300.+-.1,500 cells/ml tissue for older
donors compared to 9,200.+-.1,800 for younger donors; p=0.09).
Alkaline Phosphatase Activity
[0252] In this study a colony was only counted for the purposes of
the assay if it was alkaline phosphatase-positive and contained at
least 50 cells. Colony size varied considerably with some colonies
consisting of several thousand cells. Total alkaline phosphatase
activity might represent a superior way to measure osteogenic
activity as it takes into consideration both total osteoblastic
cell number (a function of progenitor cell proliferation) and the
degree of differentiation of these cells. Thus, alkaline
phosphatase assays were performed in replicate wells in 15
consecutive donors. Total alkaline phosphatase activity exhibited a
linear relationship with cell number plated. When alkaline
phosphatase activity (normalized to cell number plated) was then
plotted against age the data demonstrate an inverse relationship
between age and alkaline phosphatase activity (FIG. 2;
r.sup.2=0.57).
Osteoprogenitor Cell Yield and Body Mass Index
[0253] Data on donor height and weight were available for 38
donors. This allowed calculation of Body Mass Index (BMI) and
stratification into three groups; 20 donors of normal BMI (between
18.5 and 24.9), 12 in the Overweight category (25.0-25.9), and 6
Obese donors (>30). The yield of CFU-AP per unit volume of
tissue was significantly higher in persons of normal BMI than for
those who were overweight (5,279.+-.780 in normal BMI donors versus
1,373.+-.1,150 in overweight donors; p<0.02). Obese donors
showed considerable variation in CFU-AP yield per unit volume of
tissue (867-18,462 CFU-AP/ml; FIG. 3). The average was higher than
for overweight donors but this did not reach statistical
significance (p=0.08).
Summary:
[0254] The foregoing results indicate that human adipose tissue may
be a rich source of osteprogenitor cells. Specifically, the results
indicate that human adipose contains an average of approximately
7,200 osteoprogenitor cells per gram of tissue. The frequency of
CFU-AP within this population is depended upon the time from tissue
collection to tissue processing such that tissue processed within
six hours of collection yielded a frequency of 2.0.+-.0.3
CFU-AP/100 nucleated cells. Studies with freshly harvested human
bone marrow have estimated CFU-AP yield at 200-2,000 CFU-AP/ml
marrow at a frequency of approximately 1 in 50,000 (Banfi, 2001;
Galotto, 1999; D'Ippolito 1999) Thus, this data indicates that
human adipose tissue contains substantially more osteoprogenitor
cells than a similar volume of marrow.
[0255] Adipose tissue utilized for the foregoing study was obtained
using liposuction procedures which are optimized for tissue
extraction and not for recovery of viable cells. However, using the
systems and methods of the present invention, in combination with
altered parameters for liposuction (i.e., aimed at recovery of
viable cells by for example altering the amount of suction force
applied and the style and size of the cannula used) will
undoubtedly result in an even greater regenerative cell yield.
Higher osteoprogenitor cell yield is particularly important in the
context of the device of the present invention. For example, a
higher frequency of osteoprogenitor cells will allow application of
a higher dose and potentially greater clinical efficiency in
promoting bone formation.
Example 2: Adipose Derived Regenerative Cells Form Cartilage and
Bone In Vivo
Methods and Materials:
Cell Scaffolds:
PLA Scaffold Fabrication and Preparation:
[0256] Porous polymer scaffolds having an approximate porosity of
90% were manufactured by a solvent-casting/particulate-leaching
method (see e.g., A. G. Mikos, A. J. Thorsen, L. A. Czerwonka, Bao
Y., and R. Langer. Preparation and characterization of
poly(L-lactic acid) foams. Polymer 35 (5):1068-1077, 1994.).
Briefly, sodium chloride was used as the leachable porogen and was
sieved into particles ranging in diameter of 300-500 .mu.m. A 3%
polymer solution was made by combining 70:30
poly(L-lactide-co-DL-lactide) with chloroform. The sieved salt and
polymer solution were mixed in Teflon Petri dishes to make a 9:1
weight ratio of salt to polymer and the solvent was allowed to
evaporate in the hood for 2 days. The salt was leached from the
scaffold by immersion in water for 3 days, with water changes every
8-12 hours, Residual solvent was removed by placing the porous
polymeric sponges under low vacuum for 2 days. Scaffolds were sized
for implantation by punching 8 mm diameter implants from the
polymer sheets. Scaffolds were rendered aseptic by overnight
submersion in 70% ethanol and exposure to ultraviolet light, rinsed
3 times with 0.9% injectable saline to, then the residual saline
was removed from the scaffold prior to cell seeding.
Demineralized Bone Matrix (DBM) Scaffold Preparation and Cell
Seeding:
[0257] Grafton.RTM. Demineralized Bone Matrix Putty (Osteotech, New
Jersey) implants were prepared for cell seeding by rinsing evenly
divided portions (approximately 70 mg per implant) with 0.9%
injectable saline, 3 times, prior to removal of residual saline
from the scaffold.
Adipose Derived Regenerative Cell Isolation, Culture, and Cell
Seeding:
[0258] ADCs were obtained by excising the white subcutaneous
adipose tissue from transgenic mice expressing Green Fluorescent
Protein [C57Bl/6-TgN(ACTbEGFP)1Osb]. Tissue was subjected to
enzymatic digestion. Briefly, cells were washed in 1.times.PBS and
digested in 0.075% Collagenase I (Sigma) for 45 min at 37.degree.
C. shaking, then neutralized with 10% FBS (Fisher) in Dulbecco's
Modified Essential Medium (Gibco), passed through a gradient of
cell straining filters (100 .mu.m, 70 .mu.m and 40 .mu.m), and
centrifuged at 400 g for 5 min. The cell pellet was resusupended in
osteogenic culture medium [.alpha.-MEM (Cellgro, Herndon, Va.), 10%
FBS, 1% Antibiotic-antimycotic solution (Omega Scientific, Tarzana,
Calif.) supplemented with 0.1 .mu.M Dexamethasone (Sigma, St.
Louis, Mo.), 10 mM .beta.-glycerophosphate (Sigma, St. Louis, Mo.)
and 50 .mu.M L-Ascorbic acid 2-phosphate (Sigma, St. Louis, Mo.)].
After the first 3 days of induction the culture media was changed
weekly with osteogenic medium without dexamethasone. Flasks were
passaged twice in a 3 week culture period. Cultured ADCs were then
incubated overnight with osteogenic medium in the presence or
absence of recombinant human BMP-2 (5 .mu.g/ml). Cells were then
resuspended in 0.9% injectable saline (5.times.10.sup.7 cells/ml)
and kept at 4.degree. C. until seeding onto PLA or Grafton DBM
scaffolds at 2.5.times.10.sup.6 cells per scaffold.
Surgical Procedure:
[0259] Athymic rats (approximately 200 g) were anesthetized with
ketamine/xylazine [100/10 mg/kg (TW Medical Veterinary Supply),
respectively]. The rats' abdomens were shaven, swabbed with
betadine and 70% isopropanol. Under sterile conditions, a
full-thickness incision, approximately 1.5 cm in length through the
skin of the leg close to the hip area was made. The tensor muscle
was exposed via blunt dissection and a 1.0-cm incision along the
tensor muscle was made, dissecting the fascia lata to expose the
rectus muscle. Using a microspatula, a channel was then created,
into which a single implant was placed. A single pass of 5-0 silk
sutures were then used to close off the channel, followed by
suturing of the tensor muscle incision with 5-0 coated Vicryl.RTM.
braided suture, and closure of the skin incision with 9-mm wound
clips. Following surgeries, allow animals recovered from anesthesia
on heating pads. Buprenorphine (0.02-0.05 mg/kg, subcutaneously)
was administered upon awakening, in order to relieve post-surgical
pain.
Analysis:
[0260] Two weeks after surgery, rats were euthanized via CO.sub.2
exposure, and implants were recovered. Tissues were fixed overnight
in 0.4% paraformaldehyde, embedded in paraffin, cut into 5 micron
sections, then stained with Toluidine Blue or Von Kossa to
visualize cartilage and mineralized tissue, respectively. Unstained
sections were evaluated for the presence of green fluorescent donor
cells.
Results:
[0261] Tissues implanted with osteoconductive (DBM) or
non-osteoconductive (PLA) scaffold seeded with ADCs that had been
primed ex situ with BMP-2 contained a mixture of cartilage,
osteoid, and mineralized bone. Cartilage was identified in
Toluidine Blue stained sections by the presence of
metachromatically stained cells and tissue. Chondrocytes were
present within lacunae. These were surrounded by a
proteoglycan-rich matrix. Osteoid was evident as a smooth matrix in
which osteocytes were embedded. Mineralized bone was evident in Von
Kossa stained sections. Early hematopoietic elements were
identified in bone by the presence of cell laden medullary
cavities. Cartilage was also evident in tissues with implants
composed of unprimed ADCs and DBM. In contrast, no cartilage or
bone was present on either scaffold type implanted without
ADCs.
Summary:
[0262] In summary, the foregoing results demonstrate that adipose
derived regenerative cells have the capacity to form cartilage and
bone when delivered into skeletal muscle on an osteoconductive
(DBM) or non-osteoconductive (PLA) scaffold. Thus, adipose derived
regenerative cells are in stimulating cartilage formation to
restore the intervertebral disc. Moreover, the foregoing study
supports the use of adipose derived regenerative cells in
stimulating bone formation in both intervertebral body and
intertransverse process spine fusion, both environments of which
are surrounded by skeletal muscle. It is likely that the skeletal
muscle, which contains satellite cells that are known
osteoprogenitors, act in concert with adipose derived regenerative
cells to stimulate robust bone formation. At a minimum, skeletal
muscle provides a supportive environment in which adipose derived
regenerative cells can stimulate bone formation.
Example 3: Regenerative Cells Form Cartilage, Bone and Skeletal
Muscle In Vitro
Materials and Methods:
Regenerative Cell Preparation
[0263] Human regenerative cells were harvested by enzymatic
digestion as described in herein. Isolated regenerative cells were
then separated into CD34 (+) and CD34 (-) populations using immuno
magnetic beads. Each sub-population of cells were cultured in
complete medium (DMEM, 10% fetal bovine serum, 5% horse serum, and
1% antibiotic, antimycotic solution) at multiple densities in
multi-well culture plates. At 3 weeks, CD34 (-) clones were
selected from these cultures and subcultured for approximately 2
months in one of the following culture mediums: complete medium,
osteogenic medium, or chondrogenic medium.
Analysis:
[0264] Differentiation of clonal cells towards cartilage or bone
was determined by staining with alcian blue or alkaline
phosphatase, respectively. Differentiation of clonal cells towards
muscle was assessed by immunohistochemical staining of CD34 (+) and
CD34 (-) cells using antibodies directed towards human myosin heavy
chain, as well as the skeletal muscle specific markers myf5 and
myoD1. RT-PCR was performed to assess gene expression of myosin
heavy chain, myf5, and myoD1.
Results:
[0265] Regenerative cells from CD34 (-) clonal colonies
differentiated to chondrocytes, osteoblasts, and muscle cells,
depending on the culture medium used. regenerative cells cultured
in osteogenic medium differentiated to osteoblasts, while
regenerative cells cultured in chondrogenic medium differentiated
to chondrocytes, as evidenced by histochemical staining with alcian
blue and alkaline phosphatase, respectively. Moreover, regenerative
cells from CD34 (-) clonal colonies that were cultured in complete
medium in the absence of any differentiation medium were
immunohistochemically positive for myosin heavy chain, Myf5 and
MyoD, demonstrating their differentiation to a myoblast phenotype.
Consistent with the immunohistochemical data, these muscle clones
expressed the genes for myosin heavy chain, Myf5, and MyoD.
Summary:
[0266] In summary, these data show that human cultured regenerative
cell colonies give rise to chondrocytes, osteoblasts, and skeletal
myoblasts. Thus, the regenerative cells of the present invention
have the ability to stimulate cartilage, bone, and skeletal muscle
formation to restore lost tissue, e.g., soft tissue tears in the
cartilage, tendon, or ligament, as well as in repairing hard tissue
breaks, such as in non-union long bone fractures. Additionally, the
regenerative cells of the present invention have the ability to
generate tissue de novo in order to impart mechanical support,
e.g., in spinal fusion applications by bone formation between the
transverse processes or vertebrae.
[0267] Any feature or combination of features described herein are
included within the scope of the present invention provided that
the features included in any such combination are not mutually
inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art. For purposes of summarizing the present invention, certain
aspects, advantages and novel features of the present invention
have been described herein. Of course, it is to be understood that
not necessarily all such aspects, advantages or features will be
embodied in any particular embodiment of the present invention.
Additional advantages and aspects of the present invention are
apparent in the following detailed description and claims.
[0268] The above-described embodiments have been provided by way of
example, and the present invention is not limited to these
examples. Multiple variations and modification to the disclosed
embodiments will occur, to the extent not mutually exclusive, to
those skilled in the art upon consideration of the foregoing
description. Additionally, other combinations, omissions,
substitutions and modifications will be apparent to the skilled
artisan in view of the disclosure herein. Accordingly, the present
invention is not intended to be limited by the disclosed
embodiments, but is to be defined by reference to the appended
claims.
[0269] A number of publications and patents have been cited
hereinabove. Each of the cited publications and patents are hereby
incorporated by reference in their entireties.
EQUIVALENTS
[0270] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *