U.S. patent application number 15/935094 was filed with the patent office on 2018-08-02 for methods and uses for ex vivo tissue culture systems.
This patent application is currently assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE. The applicant listed for this patent is Children's Medical Center Corporation, President And Fellows Of Harvard College. Invention is credited to Geraldine Hamilton, Donald E. Ingber, Akiko Mammoto, Tadanori Mammoto, Catherine Spina, Yusuke Torisawa.
Application Number | 20180216074 15/935094 |
Document ID | / |
Family ID | 47259847 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180216074 |
Kind Code |
A1 |
Ingber; Donald E. ; et
al. |
August 2, 2018 |
METHODS AND USES FOR EX VIVO TISSUE CULTURE SYSTEMS
Abstract
The technology described herein is directed to methods and
devices that can be used to induce functional organ structures to
form within an implantation device by implanting it in vivo within
the body of a living animal, and allowing cells and tissues to
impregnate the implantation device and establish normal
microenvironmental architecture and tissue-tissue interfaces. Then
the contained cells and tissues can be surgically removed intact
and either transplanted into another animal or maintained ex vivo
by perfusing it through one or more of the fluid channels with
medium and/or gases necessary for cell survival.
Inventors: |
Ingber; Donald E.; (Boston,
MA) ; Torisawa; Yusuke; (Kyoto, JP) ;
Hamilton; Geraldine; (Cambridge, MA) ; Mammoto;
Akiko; (Brookline, MA) ; Mammoto; Tadanori;
(Brookline, MA) ; Spina; Catherine; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President And Fellows Of Harvard College
Children's Medical Center Corporation |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Assignee: |
PRESIDENT AND FELLOWS OF HARVARD
COLLEGE
Cambridge
MA
CHILDREN'S MEDICAL CENTER CORPORATION
Boston
MA
|
Family ID: |
47259847 |
Appl. No.: |
15/935094 |
Filed: |
March 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14122273 |
Mar 7, 2014 |
9951313 |
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PCT/US2012/040188 |
May 31, 2012 |
|
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15935094 |
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61492609 |
Jun 2, 2011 |
|
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61601745 |
Feb 22, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/04 20180101; A61P
35/00 20180101; C12N 5/0669 20130101; A61K 35/28 20130101; G01N
27/44791 20130101; A61P 13/12 20180101; A61P 37/06 20180101; A01N
1/0247 20130101; A61P 7/00 20180101; A61P 37/04 20180101; A61P 7/06
20180101; A61P 35/02 20180101 |
International
Class: |
C12N 5/077 20100101
C12N005/077; A61K 35/28 20150101 A61K035/28; A01N 1/02 20060101
A01N001/02 |
Claims
1-29. (canceled)
30. A method comprising: a) providing one or more organoids and a
microfluidic device, said device comprising a growth chamber; and
b) inducing said one or more organoids to colonize said growth
chamber.
31. The method of claim 30, wherein said growth chamber comprises a
channel.
32. The method of claim 30, wherein said organoids comprise bone
marrow organoids.
33. The method of claim 30, further comprising implanting said
microfluidic device in vivo in a subject.
34. The method of claim 32, further comprising contacting said bone
marrow organoids with at least one of demineralized bone powder and
bone morphogenic protein (BMP).
35. The method of claim 30, further comprising providing perfusion
fluid.
36. The method of claim 35, wherein providing a perfusion fluid
comprises connecting the microfluidic device to a microfluidic
system.
37. The method of claim 30, wherein the microfluidic device
comprises a porous separation component.
38. The method of claim 36, wherein the microfluidic device
comprises at least one inlet port and at least one outlet port
connecting to the microfluidic system.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation under 35 U.S.C. .sctn.
120 of co-pending U.S. application Ser. No. 14/122,273 filed Mar.
7, 2014, which is a 35 U.S.C. .sctn. 371 National Phase Entry
Application of International Application No. PCT/US2012/040188
filed May 31, 2012, which designates the U.S., and which claims
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application No. 61/492,609 filed Jun. 2, 2011 and U.S. Provisional
Application No. 61/601,745 filed Feb. 22, 2012, the contents of
each of which are incorporated herein by reference in their
entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 13, 2013, is named 002806-069763-US_SL.txt and is 7,311
bytes in size.
TECHNICAL FIELD
[0003] The technology described herein relates to methods and uses
for ex vivo systems of living organs, tissues, and cells.
BACKGROUND
[0004] One limitation of tissue engineering and in vitro tissue
growth strategies has been the challenge of recapitulating the
natural structures of tissues and organs. In order to grow even the
simplest of tissues, an exquisite balance of a complex mixture of
growth factors, signaling molecules, nutrients, extracellular
matrix scaffolds and mechanical forces that vary over time must be
achieved, or cells will fail to organize correctly into tissue and
organ structures. Alternatively, removing existing tissue or organ
structures from a subject in order to study or propagate the tissue
ex vivo can cause damage to the tissues and reestablishing the
proper flow of nutrients into the tissue in culture or during
transport for transplantation has proven difficult.
[0005] For example, hematopoietic stem cells, useful as a source of
therapeutic bone marrow material for transplantation or for
manufacturing differentiated blood cell replacements (e.g.
erthyrocytes, platelets, or leukocytes) have been intractable to in
vitro culture. Some investigators have attempted to culture and
expand hematopoietic stem cells (HSCs) in vitro, but long-term
engraftment and host hematopoietic reconstitution from cultured
HSCs has been extremely inefficient (Csaszar, E. et al. Cell Stem
Cell 10, 218-229 (2012); Boitano, A. E. et al. Science 329,
1345-348 (2010); Cook, M. M. et al. Tissue Eng. 18, 319-328
(2012)). Multi-potent HSCs are difficult to maintain in vitro
because they commonly differentiate when removed from the complex
bone marrow niche that contains numerous chemical, structural,
mechanical and spatial signals that are required for maintenance of
their stem cell features (Takagi, M. J. Biosci. Bioeng. 99, 189-196
(2005); Nichols, J. E. et al. Biomaterials 30, 1071-1079 (2009);
Maggio, N. D. et al. Biomaterials 32, 321-329 (2011)).
[0006] Thus, there is a need for experimental tools and methods
that promote assembly of multi- cellular and multi-tissue
organ-like structures that exhibit the key structural organization
and physiological function of the tissues and organs being modeled
and that can survive and remain functional ex vivo.
SUMMARY
[0007] The technology described herein is directed to methods and
devices that can be used to induce functional cell, tissue and
organ structures to form and/or develop within an implantation
device by implanting it in vivo within the body of a living animal,
and allowing cells and tissues to impregnate the implantation
device and establish normal microenvironmental architecture,
tissue-tissue interfaces, and/or organ-like structures and
functions. Then the whole device, including the contained cells and
tissues, can be surgically removed. In some embodiments, the cell,
tissues, and/or organoids that formed and/or developed in the
implantation device while it was implanted or afterward, and
optionally, the implantation device, can be transplanted into
another animal. In some embodiments, the cell, tissues, and/or
organoids formed and/or developed in the implantation device while
it was implanted or afterward can be maintained as viable in vitro
by perfusing the cell, tissues, and/or organoids with media and/or
gases necessary for cell survival within the implantation device or
after being removed from the implantation device and placed in a
microfluidic device. The complex organ mimic can be maintained
viable in vitro through, for example, continuous perfusion via
microfluidic channels and be used to study highly complex cell and
tissue functions in their normal 3D context with a level of
complexity not possible using any existing in vitro model system.
It can also be used as a manufacturing strategy to produce
transplantable therapeutic micro-organs or as a manufacturing
device to manufacture cells or cell products.
[0008] In accordance with one aspect of the technology described
herein, an implantation device having at least one cell growth
chamber can be implanted in vivo into an animal. In some
embodiments, the implantation device contains compounds which
induce the growth of the desired tissue type or types in a chamber
that has one or more corresponding cell growth chamber openings
(e.g. ports) to the surrounding tissue space. By way of example, in
order to induce the growth of bone, the implantation device can
contain bone-inducing materials, i.e. demineralized bone powder
and/or bone morphogenic proteins (BMPs). As a result of wound
healing, connective tissues containing microcapillaries and
mesenchymal stem cells can grow into the cell growth chamber of the
implantation device and, due to the presence of the bone-inducing
material, can form bone with spaces that recruit circulating
hematopoietic precursor cells to form fully functional bone marrow.
In some embodiments, the implantation device can be implanted
subcutaneously. In some embodiments, the implantation device can be
implanted under the kidney capsule. In some embodiments, the
implantation device can be implanted intraperitoneally. In some
embodiments, the implantation device can be implanted
intramuscularly. In some embodiments, the implantation device can
be implanted subcutaneously with at least one cell growth chamber
opening (e.g. port allowing the entry and/or exist of cells) facing
the surface of a muscle.
[0009] In some embodiments, once the tissue ingrowth process is
complete, the surgical site can be reopened, the implantation
device with newly formed and/or developed organ-like tissue
composites can be dissected from the surface of the surrounding
tissues. In some embodiments, the cell, tissue and/or organoid, and
optionally, the implantation device can be transplanted into a
second animal. In some embodiments, the cell, tissue, and/or formed
and/or developed organoid can be removed from the implantation
device and placed in a microfluidic device to perfuse the newly
formed and/or developed tissues and structures with medium, oxygen,
nutrients and supportive factors necessary to maintain its
viability and function in vitro. In some embodiments, the cell,
tissue, and/or organoid and the implantation device can be removed
and coupled to a microfluidic device and/or system to perfuse the
newly formed and/or developed tissues and structures with medium,
oxygen, nutrients and supportive factors necessary to maintain its
viability and function in vitro.
[0010] In accordance with one aspect of the technology described
herein, the implantation device is a microfluidic device. In some
embodiments, a microfluidic device having at least one cell growth
chamber and at least one abutting, fluid flow channel, can be
implanted in vivo into an animal in which the implantation device
contains compounds which induce the growth of the desired tissue
type or types in a channel that has one or more corresponding cell
growth chamber openings (e.g. ports allowing cells to enter and/or
exit the device) to the surrounding tissue space. The cell growth
chamber is the portion of the microfluidic device where cells,
tissues, and/or organoids will form and/or develop. The fluid flow
channel is the portion of the microfluidic device through which
fluid will enter, pass through, and/or exit the microfluidic chip.
In some embodiments, the cell growth chamber is a portion of one or
more fluid flow channels (i.e. the fluid flow channel can comprise
the cell growth chamber). In some embodiments, the cell growth
chamber is connected to the one or more fluid flow channels, but
the fluid flow channels do not comprise the cell growth chamber.
The fluid flow channel could be closed during implantation by
closing its end ports or filling it with a solid removable
material, such as a solid rod or a plug. By way of example, in
order to induce the growth of bone, the implantation device can
contain bone-inducing materials, i.e. demineralized bone powder
and/or bone morphogenic proteins (BMPs). As a result of wound
healing, connective tissues containing microcapillaries and
mesenchymal stem cells can grow into the cell growth chambers of
the implantation device and, due to the presence of the
bone-inducing material, can form and/or develop bone with spaces
that recruit circulating hematopoietic precursor cells to form
fully functional bone marrow. In some embodiments, the microfluidic
device can be implanted subcutaneously. In some embodiments, the
microfluidic device can be implanted under the kidney capsule. In
some embodiments, the micrfluidic device can be implanted
intraperitoneally. In some embodiments, the microfluidic device can
be implanted intramuscularly. In some embodiments, the microfluidic
device can be implanted subcutaneously with at least one cell
growth chamber opening facing the surface of a muscle.
[0011] In some embodiments, once the tissue ingrowth process is
complete, the surgical site can be reopened, and the fluid flow
channel can be reopened by removing the rod or plugs and can then
be connected to a fluid reservoir so that culture medium containing
nutrients and gases necessary for cell survival can be pumped
through the fluid flow channel and passed through the pores of the
separator component (e.g., membrane or micropillars) into the cell
growth chamber containing the formed tissue. The entire
microfluidic device can then be cut free from the surrounding
tissue, and with the medium flowing into the microfluidic device,
could be removed from the animal and passed to a tissue culture
incubator and maintained in culture with, for example, continuous
fluid flow through the second channel, and additional flow can be
added to the first channel as well if desired by connecting to
their inlet and outlet ports.
[0012] The cells, tissue and/or organ structures contained within
the implantation device can form and/or develope the correct tissue
structure and organization and thus create a complex three-
dimensional microenvironment that more closely mimics in vivo
conditions than other technologies for culturing cells in
vitro.
[0013] In some embodiments, the tissue contained in the
implantation device could then be used to study intact tissue
function in vitro as in a controlled environment. In some
embodiments, the tissue contained in the implantation device could
then be used to screen compounds for interactions with that tissue,
including toxicity, or modulation of development, function,
structure, growth, survival and/or differentiation.
[0014] In some embodiments, the tissue and/or cells contained in
the implantation device could then be used to produce cells or
cell-derived factors. In some embodiments, the cells or
cell-derived factors thus produced can be administered to a
subject. In some embodiments, the tissue contained in the
implantation device comprises bone marrow. In some embodiments, the
hematopoietic cells or bone marrow-derived factors can be
administered to a subject. In some embodiments, the subject can
have a cancer, a hematopoietic disease, or a compromised immune
system. In some embodiments, the subject can have undergone
chemotherapy or radiation therapy.
[0015] In some embodiments, the implantation device can be
implanted into an animal having human or humanized cells, thereby
providing human or humanized tissue and/or cells in the
implantation device. In some embodiments, the human cells in the
animal can be obtained from a cancer or a human having a disease
which affect the cell type growing in the implantation device.
[0016] In some embodiments, the implantation device can be
implanted into an animal having human or humanized bone marrow
and/or hematopoietic cells, thereby providing human or humanized
bone marros tissue and/or hematopoietic cells in the implantation
device. In some embodiments, the human cells in the animal can be
obtained from the bone marrow of a healthy human subject or of a
human with a cancer or other disease which affects hematopoietic
cells.
[0017] In some embodiments, the tissue and/or cells contained in
the implantation device or the tissue and/or cells removed from the
implantation device could be transplanted to a second subject. In
some embodiments, the bone marrow tissue and/or hematopoietic cells
contained in the implantation device or the bone marrow tissue
and/or hematopoietic cells removed from the implantation device
could be transplanted to a second subject. In some embodiments, the
subject can have a cancer, a hematopoietic disease, radiation
toxicity, or a compromised immune system. In some embodiments, the
subject can have undergone chemotherapy or radiation therapy.
[0018] In some embodiments, more than one implantation device
containing tissue and/or cells or more than one tissue can be
implanted into the second subject. By way of non-limiting example,
human bone marrow in an implantation device could be implanted into
a mouse and human cancer cells could be implanted, either as a
biopsy or contained in a second implantation device, at a second
site in the same mouse. This model can be used to study human
immune response to cancer. A further non-limiting example is the
implantation of human bone marrow in a chip and a second human
tissue implant, such as skin, at a second site in the subject in
order to study autoimmune disease.
[0019] In some embodiments, the first subject in which the
implantation device is implanted can be a non-human subject having
human or humanized cells. In some embodiments, the first subject
and the second subject can be the same subject and the method
comprises an additional step of maintaining the implantation device
containing tissue and/or cells in vitro between removal from the
site of implantation and transplantation back into the subject. In
some embodiments maintaining can comprise freezing, refrigeration
and/or connecting the implantation device to a microfluidic system
and providing a perfusion fluid to the implantation device after
removing the chip from the site of implantation and before
transplanting it back into the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
[0021] FIGS. 1A-1C show diagrams and a photograph of one embodiment
(the Open Channel Format) of the microfluidic device described
herein.
[0022] FIGS. 2A-2B show two embodiments of the microfluidic device
described herein. FIG. 2A shows an optical top view and a diagram
of a vertical cross-section of a Single Channel Format device. FIG.
2B shows an optical top view and a diagram of a vertical
cross-section of a Closed Channel Format device.
[0023] FIGS. 3A-3D show two embodiments of the implantation device
described herein. FIG. 3A shows a Well Format implantation device
next to a dime for perspective. FIG. 3B shows a Well Format
implantation device with two openings and a Well Format
implantation device with one opening after in vivo culture. FIG. 3C
shows a diagram of a Well Format implantation device with two
openings. FIG. 3D shows a diagram of a Well Format implantation
device with one opening.
[0024] FIGS. 4A-4C show photographs depicting the subcutaneous
implantation of an implantation device in a mouse. FIGS. 4A-4B
depict PDMS wells sutured to muscle in subcutaneous sites in mice.
FIG. 4C depicts an optical image of the implant in the PDMS well
after 8 weeks of subcutaneous implantation.
[0025] FIG. 5 shows diagrams and images of Well Format implantation
devices having one opening or two openings. The photographs show
the implantation devices after 4 weeks of subcutaneous in vivo
growth in a mouse.
[0026] FIGS. 6A-6C show H&E stains of the contents of a Well
Format implantation device with one opening 8 weeks after
implantation. FIG. 6A is oriented such that the opening of the
implantation device would be below each image. Scale bars as
shown.
[0027] FIGS. 7A-7D show H&E stains of the contents of a Well
Format implantation device with two openings 4 weeks (7A-7B) and 8
weeks (7C-7D) after implantation. Scale bars as shown.
[0028] FIG. 8 shows a diagram of the hematopoietic stem cell
lineage and the markers that can be used to identify each cell
type.
[0029] FIGS. 9A-9E show the results of FACS analysis to detect
hematopoietic stem cells. 9A shows the profile obtained from mouse
bone marrow (mBM), 9B shows the profile obtained from mouse
peripheral blood (mPB) that the red blood cells have been lysed, 9C
shows the profile obtained for tissue recovered from the
implantation device after 4 weeks of in vivo growth (sBM 4 w) and
9D shows the profile obtained for tissue recovered from the
implantation device after 8 weeks of in vivo growth (sBM 8w).
Antibodies were used which recognized the markers indicated on x-
and y-axes in FIGS. 9A-9D. FIG. 9E compares the populations on one
graph.
[0030] FIGS. 10A-10E show the results of FACS analysis to detect
hematopoietic progenitor cells. 10A shows the profile obtained from
mouse bone marrow (mBM), 10B shows the profile obtained from mouse
peripheral blood (mPB) that the red blood cells have been lysed,
10C shows the profile obtained from tissue recovered from the
implantation device after 4 weeks of in vivo growth (sBM 4 w) and
10D shows the profile obtained from tissue recovered from the
implantation device after 8 weeks of in vivo growth (sBM 8 w).
Antibodies were used which recognized the markers indicated on x-
and y-axes in FIGS. 10A-10D. FIG. 10E compares the populations on
one graph.
[0031] FIGS. 11A-11E show the results of FACS analysis to determine
the proportion of red blood cells and leukocytes. 11A shows the
profile obtained from mouse bone marrow (mBM), 11B shows the
profile obtained from mouse peripheral blood (mPB), 11C shows the
profile obtained from tissue recovered from the implantation device
after 4 weeks of in vivo growth (sBM 4 w) and 11D shows the profile
obtained from tissue recovered from the implantation device after 8
weeks of in vivo growth (sBM 8 w). Antibodies were used which
recognized the markers indicated on x- and y-axes in FIGS. 11A-11D.
FIG. 11E compares the populations on one graph.
[0032] FIGS. 12A-12E show the results of FACS analysis to detect
leukocytes. 12A shows the profile obtained from mouse bone marrow
(mBM), 12B shows the profile obtained from mouse peripheral blood
(mPB), 12C shows the profile obtained from tissue recovered from
the implantation device after 4 weeks of in vivo growth (sBM 4w),
and 12D shows the profile obtained from tissue recovered from the
implantation device after 8 weeks of in vivo growth (sBM 8w).
Antibodies were used which recognized the markers indicated on x-
and y-axes in FIGS. 12A-12D. FIG. 12E compares the populations on
one graph.
[0033] FIG. 13 shows diagrams of a further embodiment of the
implantation device having multiple layers which can be separated
by a porous membrane.
[0034] FIGS. 14A-14B show a Biopsy Format device. FIG. 14A is a
diagram of the microfluidic device and FIG. 14B is an optical image
of the microfluidic device with a biopsy sample present in the cell
growth chamber.
[0035] FIG. 15 depicts an implantation device after the in vivo
implantation stage is complete and the implantation device has been
removed from the first subject, with bone marrow tissue visible in
the center of the implantation device, and coupled to a
microfluidic system.
[0036] FIGS. 16A-16D depict the engineering of bone marrow by
inducing bone formation in vivo. The top image in FIG. 16A depicts
an implantation device filled with bone-inducing material and the
growth of bone marrow after 4 and 8 weeks of implantation. FIG. 16B
depicts high magnification micrographs of natural mouse bone marrow
from a femur and the synthetic bone marrow from the implantation
device of FIG. 16A. FIGS. 16C-16D depict graphical representations
of hematopoietic stem and progenitor populations (FIG. 16C) and
differentiated blood cell populations (FIG. 16D) present in a mouse
femur (mBM), the engineered bone marrow 4 weeks (eBM 4wk), and 8
weeks (eBM 8wk) after being implanted into a mouse, compared to
mouse peripheral blood (mPB).
[0037] FIG. 17 depicts the microfluidic culture of synthetic bone
marrow and transplantation to a lethally irradiated mouse. The
bottom right graph depicts the cell type distribution of the
cultured synthetic bone marrow at day 0 and day 4 of ex vivo
culture. The bottom left chart shows the contribution of the
transplanted bone marrow to peripheral blood populations 6 weeks
after transplantation.
[0038] FIG. 18 depicts a further embodiment of the implantation
device.
[0039] FIGS. 19A-19D depict micro computed tomography (micro-CT)
images of engineered bone marrow 4 (FIG. 19A) and 8 weeks (FIG.
19B) following implantation and a mouse vertebra (FIG. 19C). (FIG.
19D) Comparison of a cross-section of the engineered bone marrow 8
weeks following implantation with a cross-section of the mouse
vertebra. The micro-CT images demonstrate the structure and extent
of bone mineralization.
[0040] FIGS. 20A-20E depict the outcomes of a transplant
experiment. FIG. 20A depicts the extent of engraftment of lethally
irradiated mice transplanted with mouse femur bone marrow cells or
engineered bone marrow cells following 4 days of in vitro culture.
Engraftment was assessed 6 and 16 weeks post transplantation. FIG.
20B depicts the distribution of differentiated blood cells within
the engrafted CD45+ population. (FIG. 20C) Viability of blood cells
following 4 days of culture, compared to freshly isolated mouse
femur bone marrow (mBM) and engineered bone marrow (eBM). The
engineered bone marrow was cultured in the device for 4 days (eBM,
D4). Bone marrow cells isolated from a mouse femur were cultured on
a stromal cell layer in a dish for 4 days (mBM, D4). (FIGS.
20D-20E) Graphical representation of hematopoietic stem and
progenitor populations (FIG. 20D) and a long-term hematopoietic
stem cell population (Lin-CD150+CD48-) (FIG. 20E).
[0041] FIGS. 21A-21B depict graphs of the results of human cord
blood cell culture. The engineered bone marrow containing hCB cells
was cultured in the microfluidic device for 4 days (eBM, D4) or 7
days (eBM, D7) while perfusing culture media to maintain HSCs. hCB
cells were also cultured in a dish for 4 days (mBM, D4) or 7 days
(mBM, D7). (FIG. 21A) Viability of hCBs following 4 days of
culture, compared to freshly isolated hCB. (FIG. 21B) Graphical
representation of hematopoietic stem cell population
(Lin-CD34+CD38-CD90+).
DETAILED DESCRIPTION
Definitions
[0042] For convenience, the meaning of some terms and phrases used
in the specification, examples, and appended claims, are provided
below. If there is an apparent discrepancy between the usage of a
term in the art and its definition provided herein, the definition
provided within the specification shall prevail.
[0043] Definitions of common terms in cell biology and molecular
biology can be found in "The Merck Manual of Diagnosis and
Therapy", 18th Edition, published by Merck Research Laboratories,
2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8); The ELISA guidebook (Methods in molecular biology
149) by Crowther J. R. (2000); Fundamentals of RIA and Other Ligand
Assays by Jeffrey Travis, 1979, Scientific Newsletters; Immunology
by Werner Luttmann, published by Elsevier, 2006. Definitions of
common terms in molecular biology are also be found in Benjamin
Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007
(ISBN-13: 9780763740634); Kendrew et al. (eds.), Molecular Biology
and Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols
in Protein Sciences 2009, Wiley Intersciences, Coligan et al.,
eds..
[0044] Unless otherwise stated, the experiments, assays, and
methods described herein were performed using standard procedures,
as described, for example in Methods in Enzymology, Volume 289:
Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B.
Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13:
978-0121821906); U. S. Pat. Nos: 4,965,343, and 5,849,954; Maniatis
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et
al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);
Davis et al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1986); or Methods in Enzymology:
Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A.
R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987);
Current Protocols in Protein Science (CPPS) (John E. Coligan, et.
al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell
Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and
Sons, Inc.), and Culture of Animal Cells: A Manual of Basic
Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition
(2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol.
57, Jennie P. Mather and David Barnes editors, Academic Press, 1st
edition, 1998) which are all incorporated by reference herein in
their entireties.
[0045] The terms "decrease" , "reduced", "reduction" , "decrease"
or "inhibit" are all used herein generally to mean a decrease by a
statistically significant amount. However, for avoidance of doubt,
"reduced", "reduction" or "decrease" or "inhibit" means a decrease
by at least 10% as compared to a reference level, for example a
decrease by at least about 20%, or at least about 30%, or at least
about 40%, or at least about 50%, or at least about 60%, or at
least about 70%, or at least about 80%, or at least about 90% or up
to and including a 100% decrease (e.g. absent level or
non-detectable level as compared to a reference sample), or any
decrease between 10-100% as compared to a reference level. In the
context of a disease marker or symptom is meant a statistically
significant decrease in such level. The decrease can be, for
example, at least 10%, at least 20%, at least 30%, at least 40% or
more, and is preferably down to a level accepted as within the
range of normal for an individual without such disorder.
[0046] The terms "increased" ,"increase" or "enhance" or "activate"
are all used herein to generally mean an increase by a statically
significant amount; for the avoidance of any doubt, the terms
"increased", "increase" or "enhance" or "activate" means an
increase of at least 10% as compared to a reference level, for
example an increase of at least about 20%, or at least about 30%,
or at least about 40%, or at least about 50%, or at least about
60%, or at least about 70%, or at least about 80%, or at least
about 90% or up to and including a 100% increase or any increase
between 10-100% as compared to a reference level, or at least about
a 2-fold, or at least about a 3-fold, or at least about a 4-fold,
or at least about a 5-fold or at least about a 10-fold increase, or
any increase between 2-fold and 10-fold or greater as compared to a
reference level.
[0047] As used herein, a "subject" means a human or animal. Usually
the animal is a vertebrate such as a primate, rodent, domestic
animal or game animal. Primates include chimpanzees, cynomologous
monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents
include mice, rats, woodchucks, ferrets, rabbits and hamsters.
Domestic and game animals include cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, canine species, e.g.,
dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and
fish, e.g., trout, catfish and salmon. Subject includes any subset
of the foregoing, e.g., all of the above, but excluding one or more
groups or species such as humans, primates or rodents. In some
embodiments, the subject is a mammal, e.g., a primate, e.g., a
human. The terms, "individual," "patient" and "subject" are used
interchangeably herein.
[0048] Preferably, the subject is a mammal. The mammal can be a
human, non-human primate, mouse, rat, dog, cat, horse, or cow, but
are not limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
bone and/or bone marrow formation and growth. A subject can be male
or female.
[0049] As used herein, "hematopoietic disease," "hematopoietic
condition" or "hematopoietic disorder"are used interchangeably and
refer to a condition in which any cell type of the hematopoietic
lineage is overproduced, underproduced, or displays aberrant
function or behavior.
[0050] As used herein, a "hematopoietic stem cell" refers to a cell
capable of self-renewal and differentiating to become one of the
mature cell types of the hematopoietic cell lineage.
[0051] As used herein, a "hematopoietic progenitor cell" refers to
a cell capable of differentiating to become at least one of the
mature cell types of the hematopoietic cell lineage. Hematopoietic
progenitor cells include, but are not limited to, pre-T cells,
pre-B cells, blast colony-forming cells, megakaryocyte erythrocyte
progenitor (MEP), common multipotent progenitor (CMP),
granulocyte/macrophage progenitor (GMP), and granulocyte-macrophage
colony-stimulating factor cells.
[0052] As used herein, a "hematopoietic cell" includes any mature
cell of the hematopoietic lineage, any hematopoietic progenitor
cell, and/or any hematopoietic stem cell.
[0053] As used herein, "maintain" refers to continuing the
viability of a tissue or population of cells. A maintained tissue
will have a population of metabolically active cells. The number of
these cells can be roughly stable over a period of at least two
weeks or can grow.
[0054] As used herein, the term "implantation device" or "chip"
refers to a structure or substrate which contains therein or
thereon at least a cell growth chamber and one port. The
implantation device can be implanted in a subject and be colonized
by cells, tissues, and/or organoids as described herein. In some
embodiments, an implantation device can refer to a microfluidic
device, microfluidic chip, or a portion thereof. In some
embodiments, the implantation device can be coupled to one or more
additional structures to create a microfluidic device or chip.
[0055] As used herein, the terms "microfluidic device" and
"microfluidic chip" are used interchangeably and refer to a
structure or substrate having microfluidic structures contained
therein or thereon. In some embodiments, the chip can be detachably
connected to a microfluidic system.
[0056] As used herein, the term "channel" refers to any capillary,
channel, tube, or groove that is deposed within or upon a
substrate. A channel can be a microchanel; a channel that is sized
for passing through microvolumes of liquid.
[0057] As used herein, the term "cell growth chamber" refers to a
void within an implantation device that can be shaped to control
the growth of ingrowth cells, tissues, and/or organoids so that the
cells, tissues and/or organoids take on a specified 3D form. In
some embodiments, cells, tissues, and/or organoids are placed in a
cell growth chamber. In some embodiments, cells, tissues, and/or
organoids are induced to colonize a cell growth chamber. In some
embodiments a cell growth chamber can be a channel
[0058] As used herein, the term "port" refers to a portion of the
implantation device or microfluidic chip which provides a means for
fluid and/or cells to enter and/or exit the device and/or chip. A
port can allow passage of fluid and/or cells into and/or out of the
device and/or chip during in vivo growth. The port can be of a size
and shape to accept and/or secure a connection with tubes,
connections, or adaptors of a microfluidic system and allow passage
of fluid and/or cells when attached to a microfluidic system. A
port can be configured to allow entry of only fluid or only cells
into and/or out of the device or can be configured to allow entry
of either fluid and/or cells into and/or out of the device. A port
that provides a means for cells to enter and/or exit the cell
growth chamber from the exterior of the device and/or chip is also
referred to herein as a "cell growth chamber opening."
[0059] As used herein, the term "microfluidic system" refers to a
machine capable of the manipulation of microliter and/or nanoliter
volumes of fluids.
[0060] The term "statistically significant" or "significantly"
refers to statistical significance and generally means a two
standard deviation (2SD) below normal, or lower, concentration of
the marker. The term refers to statistical evidence that there is a
difference. It is defined as the probability of making a decision
to reject the null hypothesis when the null hypothesis is actually
true. The decision is often made using the p-value.
[0061] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages can mean .+-.1%.
[0062] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. Although methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of this disclosure, suitable methods and materials are
described below. The abbreviation, "e.g." is derived from the Latin
exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
[0063] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the technology
described herein. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
Implantable Devices
[0064] FIGS. 1A-1C depict one embodiment of the implantation
device, or chip, described herein. The embodiments shown in FIGS.
1A-1C shall be referred to herein as the Open Channel Format. FIG.
1A shows a PDMS chip 10 next to a dime for perspective, although
the implantation device can be larger or smaller according to the
application of the implantation device. As shown, the chip can be
formed in an octagonal shape, although other shapes can be used. In
some embodiments of the technology described herein, the size and
shape of the chip 10 can be selected to enable the chip to be used
in a particular microfluidic system. In some embodiments, the size,
shape and configuration of the chip 10 can be selected so that the
chip can be used as a replacement for to other chips provided by
manufacturers or suppliers for a particular microfluidic system. In
some embodiments, the chip 10 can include one or more inlet ports
12 connected to one or more outlet ports 14 by one or more
microfluidic channels 20. The ports 12, 14 can be provided in the
appropriate size and shape necessary to accept the tubes and/or
connectors of a particular microfluidic system. In some
embodiments, the inlet port(s) 12 and the outlet port(s) 14 can be
connected to enable fluid entering the inlet port(s) 12 to pass
through some or all of the fluid channel(s) 20 before reaching the
outlet port(s). In some embodiments, multiple ports can be
connected to a fluid channel
[0065] FIG. 1B shows a top view of one embodiment of the technology
described herein. In accordance with some embodiments of the
technology described herein, the chip 10 can include one or more
fluid channels 24, 26 and one or more cell growth chamber(s) 22. In
accordance with one embodiment of the technology described herein,
each channel 22, 24, 26 can be at least 100 .mu.m in width, at
least 200 .mu.m in width, at least 300 .mu.m in width, at least 500
.mu.m in width, at least 750 .mu.m in width, at least 1000 .mu.m in
width, or at least 2000 .mu.m in width. In accordance with one
embodiment of the technology described herein, two fluid channels
24, 26 can be separated by a cell growth chamber 22. The boundary
between the cell growth chamber and a fluid channel can be created
by micropillars 16. In this embodiment, the gaps between the
micropillars 16 can be adjusted to provide a predefined amount of
fluid exchange between the fluid channel(s) 24, 26 and the cell
growth chamber 22 to sustain and promote cell growth. A micropillar
can be, for example, at least 25 .mu.m in width, at least 50 .mu.m
in width, at least 100 .mu.m in width, at least 250 .mu.m in width,
at least 500 .mu.m in width, or at least 1000 .mu.m in width. The
gaps between the micropillars can be roughly the same size as the
micropillars. The gaps between the micropillars can be, for
example, at least 25 .mu.m in width, at least 50 .mu.m in width, at
least 100 .mu.n in width, at least 250 .mu.m in width, at least 500
.mu.m in width, or at least 1000 .mu.m in width.
[0066] FIG. 1C shows a cross-section of the fluid channels 24, 26
and cell growth chamber 22 in a microfluidic device according to an
embodiment of the technology described herein. In accordance with
some embodiments of the technology described herein, there can be
one fluid channel abutting the cell growth chamber. In alternative
embodiments, there can be two, three, four, five, six, or more
fluid channels abutting the cell growth chamber. The fluid channels
can extend parallel to the cell growth chamber 22 and positioned
along side as well as above and/or below the cell growth chamber
22. In some embodiments, different fluid channels can abut the cell
growth chamber at different locations along the the cell growth
chamber. In some embodiments, the cell growth chamber is abutted by
fluid channels on approximately 180 degrees total. In some
embodiments, the cell growth chamber is abutted by fluid channels
on approximately 45 degrees, approximately 90 degrees,
approximately 135 degrees, approximately 180 degrees, approximately
225 degrees, approximately 270 degrees, or approximately 315
degrees. In some embodiments the fluid channels and/or cell growth
chamber have a square cross-section. In other embodiments, the
fluid channels and/or cell growth chamber can each have an
approximately circular cross-section, a partly circular
cross-section, an ovoid cross-section, a rectangular, a pentagonal,
a hexagonal, a septagonal, an octagonal, a nonagonal, or a
decagonal cross-section. In some embodiments, as shown in FIG. 1C,
at least one side of the cell growth chamber (6) is not bounded by
any other component of the PDMS chip (1) and is able to be directly
exposed to the environment surrounding the chip.
[0067] In accordance with some embodiments, each channel 22, 24, 26
can be at least 500 .mu.m in height, at least 200 .mu.m in height,
at least 300 .mu.m in height, at least 500 .mu.m in height, at
least 750 .mu.m in height, at least 1000 .mu.m in height, or at
least 2000 .mu.m in height. The depth of the cell growth chamber
can be at least 50 .mu.m in height, at least 200 .mu.m in height,
at least 300 .mu.m in height, at least 500 .mu.m in height, at
least 750 .mu.m in height, at least 1000 .mu.m in height, or at
least 2000 .mu.m in height. The thickness, i.e. the height
dimension of the chip or carrying microfluidic device can be
sufficient to provide structural integrity to the implantation
device for its intended use and purpose. In some embodiments, the
microfluidic device can range in thickness from at least 250 .mu.m
in height, at least 500 .mu.m in height, at least 1 mm in height,
at least 2 mm in height, at least 5mm in height, or at least 10 mm
in height.
[0068] In the embodiment shown in FIGS. 1A-1C, the chip is 500
.mu.m depth. The fluid channels 24, 26 are 750 .mu.m-1 mm in width.
The cell growth chamber 22 is 3 mm long, 500 .mu.m in width and 250
.mu.m depth. The posts 16 are 100 .mu.m.times.200 .mu.m with 100
.mu.m gaps between the posts.
[0069] FIGS. 2A-2B and FIGS. 3A-3D show other embodiments of an
implantation device according to the technology described herein.
FIG. 2A depicts a top view and a horizontal cross-section of the
Single Closed Channel Format microfluidic device. In this
embodiment, the chip 10 can have 4 ports 12, 14; two being located
through the top surface of the chip and two being located through
the bottom surface of the chip. The ports can connect to one or
more channels (3/4), which serves as both a cell growth chamber and
a fluid channel
[0070] FIG. 2B depicts a top view and a horizontal cross-section of
the Closed Channel Format microfluidic device in accordance with an
alternative embodiment. In this embodiment, the chip 10 can include
4 ports 12, 14; two being located on the top surface of the chip
and two being located on the bottom surface of the chip. The ports
connect to a cell growth chamber 22 flanked by two fluid channels
24, 26. In this embodiment, micropillars 16 can be used to separate
the cell growth chamber 22 from the fluid channels 24, 26 but
enable transfer of fluids, agents and factors between the fluid
channels and the growth channel In other embodiments, different
separation elements can be used including walls with windows, holes
or slots, membranes, and other permeable materials.
[0071] In the embodiment shown in FIG. 2A, the chip 10 is 8-10 mm
in width, 10-15 mm in length and 2 mm in height. The ports 12, 14
are 2 mm in diameter. The cell growth chamber 22 is 3-5 mm long, 1
mm in width, and 400 um in depth. In the embodiment shown in FIG.
2B, the chip 10 is 8mm wide and 12 mm in length. The ports 12, 14
are 2 mm in diameter. The cell growth chamber 22 is 3 mm long. The
fluid channels 24, 26 are 750 .mu.m-1 mm in width. The posts 16,
are 150 .mu.m.times.200 .mu.m, with 100 .mu.m gaps between the
posts.
[0072] FIGS. 3A-3D depict two Well Format or cartridge embodiments.
FIG. 3A shows a photograph of a Well Format chip next to a dime for
perspective, although the implantation device can be larger or
smaller according to the application of the implantation device.
FIG. 3B shows two Well Format chips after the in vivo implantation
phase. The implantation device can include holes 18 for suture
attachment. FIG. 3C shows a diagram of a Well Format chip having a
cell growth chamber 22 connected to two openings (or ports), one
through the top of the implantation device and another through the
bottom of the implantation device. In this embodiment, the ports 12
(which can, in this embodiment, function as cell growth chamber
openings) and cell growth chamber 22 can be coextensive and the
fluid channel (not shown) and cell growth chamber 22 can be
combined, for example, where the fluid channel extends into or
through the cell growth chamber. FIG. 3D shows an embodiment of a
Well Format chip 10 having one opening. In this embodiment, the
port 12 and cell growth chamber opening can be, at least in part,
coextensive. In the embodiments depicted the cell growth chamber 22
is 4 mm in diameter with a depth ranging from 1 mm to 2 mm, the
chip 10 is 2 mm in depth and 8 mm in diameter. The holes 18, can
be, for example 1 mm in diameter.
[0073] The embodiments shown in FIGS. 3A-3D can be part of a
cartridge based system. In these embodiments, the chip or device 10
can be, for example, round or circular, as shown in FIGS. 3B-3D or
any other polygonal shape, such as a square or rectangle as shown
in FIG. 3A. In addition, the cell growth chamber or channel 22 can
be, for example, round as shown in FIGS. 3A-3D or any other shape,
including oval, square, rectangular, or other polygonal shape. In
this embodiment, the chip or device can service as cartridge that
is adapted to be inserted into a microfluidic device having one or
more fluid channels that enable fluid to flow adjacent the cell
growth chamber. In some embodiments, several cartridges 10 can be
inserted into a single device.
[0074] A further embodiment is shown in FIG. 13. In this
embodiment, the chip 10 consists of two PDMS layers 30, 32
separated by a PDMS porous membrane 34. The upper layer 32 has a
microfluidic channel 24 to allow medium perfusion, whereas the
lower layer has a cylindrical cell growth chamber 22. The
implantation device filled with bone-inducing material can be
implanted subcutaneously in a subject, and then surgically removed
after in vivo growth. Prior to connecting to a microfluidic system,
a bottom fluid channel 26 can be bound to the bottom of the
implantation device to perfuse culture medium. In the embodiment
depicted, the chip 10 ranges from 10-15 mm in length and 8-10 mm in
width. The ports 12, 14 are 1 mm in diameter where they connect to
the microfluidic system. The bottom layer 30 is 500 .mu.m in depth
with a 3 mm diameter cell growth chamber 22. The top layer 32 is
10-15 mm in both width and length with a depth of 1 mm. The fluid
channel 24 is 200 .mu.m in depth and 3 mm wide. The ports 14, 16
are 1 mm in width and can range in length from 1 mm to 7 mm. The
aspect containing the bottom fluid channel 26 can be identical to
the upper layer 32. The PDMS membrane 34 is 10 .mu.m in depth.
[0075] A further embodiment is shown in FIG. 18. In this
embodiment, the chip 10 comprises a middle PDMS layer 30 comprising
a Well Format chip with a cell growth chamber 22 as described above
herein. The cell growth chamber 22 in the middle layer 30, filled
with bone-inducing material, can be implanted subcutaneously in a
subject and then surgically removed after in vivo growth. After
removal, each opening of the cell growth chamber 22 of the middle
layer 30 can be covered with a PDMS porous membrane 34. The PDMS
membranes 34 can then be covered with an upper PDMS layer 32 and a
bottom PDMS layer 36. The upper layer 32 has can include a
microfluidic channel 24 to allow medium perfusion and ports 12, 14
to connect to a microfluidic system. At least two of the ports
connect to the top microfluidic channel 24 and at least two of the
ports connect to a bottom microfluidic channel 26 in the bottom
layer 36. With the bottom layer 36 and upper layer 32 are in place,
the entire chip 10 can be connected to a microfluidic system. In
the embodiment depicted, the chip 10 can range from 10-15 mm in
length and 8-12 mm in width. The ports 12, 14 can be 0.25 to 3 mm,
preferably 1 mm, in diameter where they connect to the microfluidic
system, e.g., the ports 12, 14 can be 0.5 to 2 mm in diameter, 0.75
to 2 mm in diameter, or 0.8 to 1.5 mm in diameter where they
connect to the microfluidic system. The middle layer 30 can be 400
to 600 .mu.m in depth, e.g. 450 to 550 .mu.m in depth or 475 to 525
.mu.m in depth and can have a 2 to 6 mm diameter cell growth
chamber 22, e.g. a 2.5 to 5.5 mm diameter cell growth chamber, a 3
to 5 mm diameter cell growth chamber, or an about 4 mm diameter
cell growth chamber. The top layer 32 can be 8-16 mm in both width
and length, e.g. 9 to 15 mm, or 10 to 14 mm, or 11 to 13 mm in both
width and length and can range from 3 to 7 mm in depth, e.g. from 4
to 6 mm or about 5 mm in depth. The fluid channel 24 can range from
50 to 400 .mu.m in depth, e.g. from 100 to 300 .mu.m, or from 150
to 250 .mu.m or about 200 .mu.m in depth and range from 2 to 6 mm
in width, e.g. from 3 to 5 mm, or from 3.5 to 4.5 mm or about 4 mm
in width. The ports 12, 14 can be be 0.25 to 3 mm, preferably 1 mm,
in diameter , e.g., the ports 12, 14 can be 0.5 to 2 mm in
diameter, 0.75 to 2 mm in diameter, or 0.8 to 1.5 mm in diameter.
The bottom layer 36 containing the bottom fluid channel 26 can have
identical dimensions as the upper layer 32. The PDMS membrane 34
can be from 2 to 20 .mu.m in depth, e.g. 5 to 15 .mu.m, 8 to 13
.mu.m, 9 to 11 .mu.m or about 10 .mu.m in depth.
[0076] A further embodiment is shown in FIGS. 14A-14B. In this
embodiment, the chip 10 (3 mm depth, 50 mm in length) can include 5
ports 12, 14, 18. Four ports 12, 14 connect to the two fluid
channels 24, 26. The fifth port 18, a biopsy entry port, connects
to the cell growth chamber 22 which is 500 .mu.m in width, 10 mm in
length, and range from 200-350 .mu.m in depth. In this embodiment,
micropillars 16 (ranging in size from 50-150 .mu.m.times.100-200
.mu.m, with a 50-100 .mu.m gap between micropillars) can be used to
separate the cell growth chamber 22 from the fluid channels 24, 26
(1 mm width) but enable transfer of fluids, agents and factors
between the fluid channels and the growth channel. A 500 .mu.m
diameter biopsy can be introduced into the middle channel while
maintaining negative pressure on the outflow openings.
[0077] As a complete system, the chip 10 described herein allows,
for example, bone marrow to be cultured in vitro while maintaining
bone marrow structure and viability either for extended culture,
during transport for transplantation into a subject or transport to
a microfluidic system.
[0078] The cell growth chamber of the implantation device described
herein provides a space which can be colonized by, for example,
bone marrow cells or bone marrow precursor cells during in vivo
implantation of the implantation device. The open side of the cell
growth chamber can allow for migration of cells into the cell
growth chamber. When the implantation device is removed from the
subject, the chip can be placed into or connected to a compatible
microfluidic supply system and a flow of desired fluid can be
established by connecting the microfluidic supply system to the
inlet and outlet ports (see FIG. 13). The provided fluid flows
through the fluid channels and the micropillars or other separation
component enables fluid exchange to occur between the cell growth
chamber and the fluid channels. In this way, nutrients, growth
factors, hormones, test compounds, small molecules, etc can be
provided to the cells in the cell growth chamber in order to
maintain their viability and/or support their in vitro growth and
enable testing and evaluation. Additionally, products and/or waste
materials of the cells can be removed from the cell growth chamber
and exit into the microfluidic system where they can be disposed of
and/or analyzed. The fluid channels also provide a means of
delivering test compounds to the bone marrow cells in the cell
growth chamber. The bone marrow cells in the cell growth chamber
may also give rise to cells which will enter the fluid channels and
can be collected by the microfluidic system for subsequent
analysis. In accordance with the various embodiments, a porous
separation component can be used to separate the cell growth
chamber or channel from the fluid channel(s) be chosen to
selectively allow fluids, molecules and cells to flow between the
cell growth chamber or area and the fluid channel(s). For example,
in some embodiments, the micropillars and the gaps between them can
be selected to allow at least some of the cells in the cell growth
chamber to remain in place and not be removed from the chip by the
fluid flow. The size of the pillars and gaps selected can be used
to determine the extent of the fluid and material exchange which
the cells in the cell growth chamber are subjected to and can be
varied to achieve the degree of fluid exchange which is desired
and/or necessary for a particular application. In some embodiments
the gaps can be made adjustable, for example, using a set of
sliding windows (gap) or membrane having adjustable
permeability.
[0079] The body of the chip or device 10 can be made of a flexible
material or a non-flexible material according to the design and
application requirements. It should be noted that the microchannel
design is exemplary and not limited to the configuration shown in
the figures. The chip 10 can be made of a flexible biocompatible
material, including but not limited to, a biocompatible material
such as polydimethyl siloxane (PDMS), polyurethane or polyimide.
The chip 10 can also be made of non-flexible materials like glass,
silicon, polysulfone, hard plastic, and the like, as well as
combinations of these materials. The chip 10 can also be fabricated
using any suitable biocompatible and/or biodegradable materials,
such as poly-lactide-co-glycolide acid (PLGA) and in some
embodiments, the organ mimic device can be used for transplantation
or implantation in vivo.
[0080] In accordance with the some embodiments of the technology
described herein, the cell growth chamber can be separated from the
fluid channel by a separation component (for example, a plurality
of micropillars) that can be formed, such as by etching, 3-D
printing or micro-machining. Alternatively, the separation
component can include a selectively permeable or semi-permeable
membrane, for example, also formed from PDMS or porous
polycarbonate filter. In other embodiments, the membrane can be
made of from other materials or a combination of materials
including PDMS.
[0081] A biocompatible polymer refers to materials which do not
have toxic or injurious effects on biological functions.
Biocompatible polymers include natural or synthetic polymers.
Examples of biocompatible polymers include, but are not limited to,
collagen, poly(alpha esters) such as poly(lactate acid),
poly(glycolic acid), polyorthoesters and polyanhydrides and their
copolymers, polyglycolic acid and polyglactin, cellulose ether,
cellulose, cellulosic ester, fluorinated polyethylene, phenolic,
poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,
polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,
polyester, polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene,
polyfluoroolefin, polyimide, polyolefin, polyoxadiazole,
polyphenylene oxide, polyphenylene sulfide, polypropylene,
polystyrene, polysulfide, polysulfone, polytetrafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinyl,
polyvinylidene fluoride, regenerated cellulose, silicone, urea-
formaldehyde, polyglactin, or copolymers or physical blends of
these materials.
[0082] A biocompatible material can also be, for example, ceramic
coatings on a metallic substrate. But any type of coating material
and the coating can be made of different types of materials:
metals, ceramics, polymers, hydrogels or a combination of any of
these materials. Biocompatible materials include, but are not
limited to an oxide, a phosphate, a carbonate, a nitride or a
carbonitride. Among the oxide the following ones are preferred:
tantalum oxide, aluminum oxide, iridium oxide, zirconium oxide or
titanium oxide. Substrates are made of materials such as metals,
ceramics, polymers or a combination of any of these. Metals such as
stainless steel, Nitinol, titanium, titanium alloys, or aluminum
and ceramics such as zirconia, alumina, or calcium phosphate are of
particular interest.
[0083] The biocompatible material can also be biodegradable in that
it will dissolve over time and may be replaced by the living
tissue. Such biodegradable materials include, but are not limited
to, poly(lactic acid-co-glycolic acid), polylactic acid,
polyglycolic acid (PGA), collagen or other ECM molecules, other
connective tissue proteins, magnesium alloys, polycaprolactone,
hyaluric acid, adhesive proteins, biodegradable polymers,
synthetic, biocompatible and biodegradable material, such as
biopolymers, bioglasses, bioceramics, calcium sulfate, calcium
phosphate such as, for example, monocalcium phosphate monohydrate,
monocalcium phosphate anhydrous, dicalcium phosphate dihydrate,
dicalcium phosphate anhydrous, tetracalcium phosphate, calcium
orthophosphate phosphate, calcium pyrophosphate, alpha-tricalcium
phosphate, beta-tricalcium phosphate, apatite such as
hydroxyapatite, or polymers such as, for example,
poly(alpha-hydroxyesters), poly(ortho esters), poly(ether esters),
polyanhydrides, poly(phosphazenes), poly(propylene fumarates),
poly(ester amides), poly(ethylene fumarates), poly(amino acids),
polysaccharides, polypeptides, poly(hydroxy butyrates),
poly(hydroxy valerates), polyurethanes, poly(malic acid),
polylactides, polyglycolides, polycaprolactones,
poly(glycolide-co-trimethylene carbonates), polydioxanones, or
copolymers, terpolymers thereof or blends of those polymers, or a
combination of biocompatible and biodegradable materials. One can
also use biodegradable glass and bioactive glass self-reinforced
and ultrahigh strength bioabsorbable composites assembled from
partially crystalline bioabsorbable polymers, like polyglycolides,
polylactides and/or glycolide/lactide copolymers.
[0084] The biocompatible polymer may be shaped using methods such
as, for example, solvent casting, compression molding, filament
drawing, meshing, leaching, weaving and coating. In solvent
casting, a solution of one or more polymers in an appropriate
solvent, such as methylene chloride, is cast as a branching pattern
relief structure. After solvent evaporation, a thin film is
obtained. In compression molding, a polymer is pressed at pressures
up to 30,000 pounds per square inch into an appropriate pattern.
Filament drawing involves drawing from the molten polymer and
meshing involves forming a mesh by compressing fibers into a
felt-like material. In leaching, a solution containing two
materials is spread into a shape close to the final form of the
RUG. Next a solvent is used to dissolve away one of the components,
resulting in pore formation. (See Mikos, U.S. Pat. No. 5,514,378,
hereby incorporated by reference). In nucleation, thin films in the
shape of a RUG are exposed to radioactive fission products that
create tracks of radiation damaged material. Next the polycarbonate
sheets are etched with acid or base, turning the tracks of
radiation-damaged material into pores. Finally, a laser may be used
to shape and bum individual holes through many materials to form a
RUG structure with uniform pore sizes. Coating refers to coating or
permeating a polymeric structure with a material such as, for
example liquefied copolymers (poly-DL-lactide co-glycolide 50:50 80
mg/ml methylene chloride) to alter its mechanical properties.
Coating may be performed in one layer, or multiple layers until the
desired mechanical properties are achieved. These shaping
techniques may be employed in combination, for example, a polymeric
matrix may be weaved, compression molded and glued together.
Furthermore different polymeric materials shaped by different
processes may be joined together to form a composite shape. The
composite shape may be a laminar structure. For example, a
polymeric matrix may be attached to one or more polymeric matrixes
to form a multilayer polymeric matrix structure. The attachment may
be performed by gluing with a liquid polymer or by suturing. In
addition, the polymeric matrix may be formed as a solid block and
shaped by laser or other standard machining techniques to its
desired final form. Laser shaping refers to the process of removing
materials using a laser.
[0085] In some embodiments, the chip 10, comprising one or more
layers, can be mounted onto or between one or more acrylic plates
40. The acrylic plate(s) can have one or more holes to permit
microfluidic tubes access to the ports 12, 14 of the chip 10.
Alternatively, the acrylic plate can have a central opening that
does not cover the entire surface of the chip 10. The acrylic
plates can be connected using screws, bolts, pins, adhesives or any
other fastener known in the art. By way of non-limiting example, in
the embodiment depicted in FIG. 15, the chip 10 can be placed
between two acrylic plates 40. Each acrylic plate 40 depicted in
FIG. 15 can be 25 mm in diameter and 2 mm in thickness. The acrylic
plates 40 can protect the chip 10 from damage and/or contamination.
The acrylic plates 40 can be of a size and shape to mount in a
microfluidic system.
[0086] Methods
[0087] In accordance with various embodiments of the technology
described herein, the chip described herein can be filled with a
hydrogel, such as a collagen gel, mixed with other agents or
formulations to induce, attract and/or support cell migration
and/or colonization in the cell growth chamber. In some
embodiments, the cells colonizing the cell growth chamber can be
stem cells. In some embodiments, the cells colonizing the cell
growth chamber can be progenitor cells. In some embodiments, the
stem and/or progenitor cells colonizing the cell growth chamber can
differentiate and give rise to one or more differentiated cell
types. In some embodiments, the cells colonizing the cell growth
chamber can form tissue structures or miniature organ structures.
The cell and/or tissue types which can colonize or arise from stem
cells within the cell growth chamber include, but are not limited
to, mesenchymal stem cells, adipose-derived stem cells, endothelial
stem cells, hematopoietic stem cells, bone cells, bone marrow
cells, liver cells, blood cells, adipose cells, muscle cells, stem
cells, vascular cells, immune cells, differentiated cells, diseased
cells, connective tissue, muscle tissue, nervous tissue, and/or
epithelial tissue.
[0088] In some embodiments, the hydrogel can include compositions
which enhance or stimulate bone growth. In some embodiments these
compositions can include demineralized bone powder and/or the
proteins bone-morphogenic protein 2 (BMP-2) (SEQ ID NO: 01, NCBI ID
NO: NP001191.1) and/or BMP-4 (SEQ ID NO: 02, NCBI ID NO: NP_001193,
NP_570911, and NP_570912). In some embodiments these compositions
can include demineralized bone powder and/or the proteins BMP-2
and/or BMP-4 and optionally, other bone-inducing material, i.e. a
material such as a small molecule, peptide, or protein that
contributes to providing a matrix for cell growth, attracting cell
migration into the implantation device, promoting cell growth,
promoting bone cell growth, promoting bone marrow cell growth,
promoting bone cell differentiation, promoting bone marrow cell
differentiation, promoting bone tissue growth, and/or promoting
bone marrow tissue growth. In some embodiments, the gel mixture can
be placed in the ports, in the fluid channels and/or in the cell
growth chamber. In some embodiments, the bone-inducing material can
be placed in the cell growth chamber and the collagen can be placed
in the ports. In some embodiments, the bone-inducing material can
be placed in the cell growth chamber and the fluid channels and the
collagen can be placed in the ports.
[0089] Demineralized bone powder can be obtained commercially.
Alternatively, demineralized bone powder can be prepared by
excising, fragmenting, and sieving mouse femurs to obtain particles
less than 250 .mu.m in diameter. The powder can then be
demineralized using 0.5 N HCl.
[0090] An exemplary hydrogel gel mixture can be prepared using Type
I collagel gel (3 mg/mL), demineralized bone powder (0.1 mg/uL),
BMP-2 (5 ng/uL), and BMP-4 (5 ng/uL). BMP-2 and BMP-4 are available
commercially from Sigma Aldrich (St. Louis Mo.; Catalog Numbers
B3555 and B2680 respectively).
[0091] In other embodiments, the tissue grown in the chip can be a
hematopoietic or immune system tissue, including for example, bone
marrow, lymph nodes, or spleen and the collagen gel can include
compositions which enhance or stimulate the growth of, for example,
lymph node or spleen tissue. In some embodiments these compositions
can include interleukins, chemokines, interferons, tumor necrosis
factor, hematopoietins, granulocyte colony-stimulating factor
(G-CSF) and granulocyte-macrophage colony-stimulating factor
(GM-CSF).
[0092] In other embodiments, the tissue grown in the chip can be,
for example, connective tissue, vascular tissue, or fascial type
structures and the collagen gel can include compositions which
enhance or stimulate the growth of, for example, connective tissue,
vascular tissue, or fascial type structures. In some embodiments
these compositions can include VEGF (Cat #V7259, Sigma Aldrich, St.
Louis, Mo.) and PDGF (Cat #P8147, Sigma Aldrich, St. Louis,
Mo.).
[0093] In accordance with some embodiments of the technology
described herein, the chip can then be implanted in a subject. In
some embodiments, the chip can be implanted subcutaneously. In some
embodiments, the chip can be implanted intraperitoneally. In some
embodiments, the chip can be implanted intramuscularly. In some
embodiments, the chip can be sutured to muscle at the subcutaneous
site. The chip can be provided with one or more holes to allow
passage of a suture needle and/or thread through the implantation
device. In a preferred embodiment, the chip can be placed such that
the open side(s) and/or ports of the cell growth chamber face the
underlying muscle tissue of the subject.
[0094] In some embodiments, the in vivo implantation phase can last
at least 1 week, at least 2 weeks, at least 4 weeks, at least 6
weeks, at least 8 weeks, at least 10 weeks, or longer. The time
duration of the implantation phase can be determined as function of
the desired amount of migration and colonization of cells in the
cell growth chamber or area. This can be dependent on the subject
and the composition of collagen gel or other mixture provided in
the cell growth chamber or area.
[0095] In some embodiments, it is believed that when the chip is
implanted subcutaneously, wound healing processes result in
connective tissues containing microcapillaries, mesenchymal stem
cells, stem cells, vascular cells, immune cells, differentiated
cells, and/or diseased cells growing into the cell growth chambers
of the implantation device, and due to the presence of the
bone-inducing material, can form bone with spaces that recruit
circulating hematopoietic precursor cells to form fully functional
bone marrow.
[0096] In some embodiments, a chip 10 can be implanted in a
subject. In some embodiments, an implantation device comprising
part of a complete microfluidic chip, for example, the middle layer
30 of a chip 10 can be implanted in a subject. In some embodiments,
an implantation device which does not comprise part of a complete
microfluidic chip, for example, device having the structure of a
middle layer 30 (e.g. a cartridge), but which is not later coupled
to further structures to constitute a complete microfluidic chip
can be implanted in a subject. In some embodiments, the
implantation device can be implanted in the subject and after the
in vivo growth period is concluded, the implantation device can be
coupled to a microfluidic system without removing the cells,
tissues and/or organoid from the implantation device and/or chip.
In some embodiments, the implantation device can be implanted in
the subject and after the in vivo growth period is concluded, the
cells, tissues, and/or organoid can be removed from the
implantation device and placed in a microfluidic device which can
be coupled to a microfluidic system. In some embodiments, the
implantation device can be implanted in the subject and after the
in vivo growth period is concluded, the implantation device can be
transplanted to a subject without removing the cells, tissues
and/or organoid from the implantation device. In some embodiments,
the implantation device can be implanted in the subject and after
the in vivo growth period is concluded, the cells, tissues, and/or
organoid can be removed from the implantation device and
transplanted in a subject.
In Vitro /Ex Vivo Methods
[0097] Some embodiments of the technology described herein relate
to forming a functional organ composed of two or more
physiologically and structurally integrated tissues during the in
vivo implantation phase and maintaining these structural and
functional relationships during at least part of the in vitro
and/or ex vivo phase as described below herein.
[0098] In accordance with some embodiments of the technology
described herein, when the in vivo implantation phase is to be
ended, the chip can be removed from the subject and placed in or
connected to a microfluidic system. In some embodiments, the
microfluidic system can be used to provide continuous medium
perfusion, for example, using the one or more fluid channels, to
develop and/or maintain cell and tissue viability. In some
embodiments the chip can be removed from the site of implantation
and then connected to a microfluidic system where perfusion begins.
In other embodiments, the chip can be connected to a microfluidic
system prior to removal from the site of implantation and perfusion
begun at any time prior to complete detatchment of the chip and the
tissue contained therein from the tissue of the implantation
site.
[0099] In some embodiments, the chip can be placed into a
nutrient-rich medium after removal from the site of implantation in
order to transport the chip to a microfluidic system.
[0100] In accordance with some embodiments, the cells present in
the chip can be cultured under continuous perfusion conditions and
can differentiate and/or produce more tissue. In alternative
embodiments, the cells can be cultured using a wide range of
perfusion profiles and conditions as desired to control the
development and maintenance of the cultured cells. In accordance
with some embodiments, the cells present in the chip can be
cultured under continuous perfusion conditions and can
differentiate into bone marrow and/or produce more bone marrow
tissue. In some embodiments, the viability of the cells present in
the chip can be maintained through perfusion until the implantation
device is implanted into a second subject.
[0101] Appropriate media for perfusion, which may also be used as
the nutrient-rich medium for transport of the chip, are
commercially available and a medium appropriate for the particular
tissue being grown in the chip would be well known to one skilled
in the art. The medium can be supplemented with, for example,
additional nutrients, antibiotics, growth factors or other
compounds which enhance the growth of the desired cells in vitro.
By way of example only, an appropriate medium for growth of liver
tissue can be Dulbecco's Modified Eagle's Medium (DMEM) containing
10%(v/v) fetal bovine serum (FBS),100 U/mL penicillin, and 100 U/mL
streptomycin while an appropriate medium for the growth of bone
marrow can be MyeloCul M5300 Medium (Stem Cell Technologies)
containing non-Essenetial Amino Acid (Gibco) Sodium Pyrvate
(Gibco), 100 U/mL penicillin, and 100 U/mL streptomycin.
[0102] In some embodiments the cells and/or tissue grown in the
chip can be utilized in vitro to produce cells or stem cells for
therapeutic or research purposes. In some embodiments the cells
and/or tissue grown in the chip can be utilized in vivo to produce
hematopoietic cells or hematopoietic stem cells for therapeutic or
research purposes. In some embodiments, the hematopoietic cells can
be selected from the group consisting of red blood cells, white
blood cells, platelets, hematopoietic stem cells, lymphocytes,
eosinophils, neutrophils, monocytes, hematopoietic progenitor
cells, stromal cells, and a mixture of two or more of these cell
types. In some embodiments, the cells can be collected from the
effluent of the chip. In some embodiments, the cells can be
collected by removing the cells and/or tissue from the cell growth
chamber 22. In some embodiments, the cells can be utilized for
therapeutic or research purposes while present in the chip.
[0103] In some embodiments, growth factors or compounds that
enhance the production of the desired cell type(s) can be added to
the perfusion fluid. By way of non-limiting example, erythropoietin
stimulates the production of red blood cells, VEGF stimulates
angiogenesis, and thrombopoietin stimulates the production of
megakaryocytes and platelets.
[0104] Cells produced according to the methods described herein can
be used for therapeutic or research purposes without further
manipulation or can be manipulated prior to their final use. By way
of non-limiting example, manipulation of the cells can include
sorting according to cell type, expanding in culture, mixing with
other components of a pharmaceutical composition, contacting the
cells with a compound that alters the state, function, or
functionality of the cell (i.e. nucleic acid, a protein, a peptide,
and/or a small molecule), freezing, and/or refrigeration.
[0105] In some embodiments, the methods and devices directed to
production of a chip containing tissue and/or cells can also be
used to investigate the response of cells and/or tissue, such as
for example, bone marrow tissue and/or hematopoietic cells, to some
compounds (e.g. chemotherapies or radiation therapies). In some
embodiments, the chip containing tissue and/or cells can also be
used to investigate the mechanisms involved in control of
differentiation, formation, survival, growth, function, and/or
remodeling of cells, tissues and/or organ structures. In these
embodiments, it can be desirable to deliver some compounds to the
cells in the cell growth chamber at predefined time intervals or
delivery profiles (according to predefined concentrations,
quantities and time intervals) via the fluid channels (e.g.
hormones such as thrombopoietin which regulates production of
platelets or erythropoietin which controls red blood cell
production). Compounds which can be delivered to the cells in the
cell growth chamber via the fluid channel(s) include, but are not
limited to, hormones, enzymes, cells, gene silencing molecules,
inhibitors of some enzymes, small molecules, peptides, proteins,
nucleotides, antibodies, growth factors, viruses, bacteria,
parasites, markers and/or dyes.
[0106] In some embodiments, the tissue and/or cells present in the
chip can be used to screen compounds for toxicity. In some
embodiments, the bone marrow tissue and/or hematopoietic cells
present in the chip can be used to screen compounds for toxicity to
bone marrow. In some embodiments, the tissue and/or cells present
in the chip can be used to screen compounds for an ability to
increase the growth, development and/or function of the tissue or a
component thereof. In some embodiments, the bone marrow tissue
and/or hematopoietic cells present in the chip can be used to
screen compounds for an ability to increase the growth,
development, survival, growth and/or function of bone marrow or a
component thereof. In some embodiments, the tissue and/or cells
present in the chip can be used to screen compounds for an ability
to inhibit the growth, survival, development and/or function of the
tissue or a component thereof. In some embodiments, the bone marrow
tissue and/or hematopoietic cells present in the chip can be used
to screen compounds for an ability to inhibit the growth, survival,
development and/or function of bone marrow or a component thereof.
In some embodiments, the bone marrow tissue and/or hematopoietic
cells present in the chip can be used to screen compounds for an
ability to mobilize some cells and/or non-cellular products from
the bone marrow. In some embodiments, the bone marrow tissue and/or
hematopoietic cells present in the chip can be used to screen
compounds for an ability to inhibit the mobilization of some cells
and/or non-cellular products from the bone marrow. In some
embodiments, the tissue and/or cells present in the chip can be
used to screen compounds for suitability as imaging agents for the
tissue or a component thereof. In some embodiments, the bone marrow
tissue and/or hematopoietic cells present in the chip can be used
to screen compounds for suitability as imaging agents for bone
marrow or a component thereof In some embodiments, the tissue
and/or cells present in the chip can be used to characterize the
pharmacokinetics of a drug or lead compound. In some embodiments,
the bone marrow tissue and/or hematopoietic cells present in the
chip can be used to characterize the pharmacokinetics of a drug or
lead compound. In some embodiments, the tissue and/or cells present
in the chip can be used to study the microenvironment,
architecture, function and/or development of that tissue type. In
some embodiments, the bone marrow tissue and/or hematopoietic cells
present in the chip can be used to study the microenvironment,
architecture, function and/or development of bone marrow.
[0107] In some embodiments, the tissue and/or cells present in the
chip can be used to produce cells, and/or non-cellular factors or
compounds produced by the tissue for use in additional in vitro
systems and/or models. In some embodiments, the bone marrow tissue
and/or hematopoietic cells present in the chip can be used to
produce blood cells, immune cells, other bone marrow-derived cells,
and/or non-cellular factors or compounds produced by bone marrow
for use in additional in vitro systems and/or models. By way of
non-limiting example, a bone marrow-derived factor can be a
peptide, protein, small molecule, nucleotide, lipid, carbohydrate,
cytokine and/or growth factor. By way of non-limiting example, the
response of liver tissue to compounds can include an immune
component which is absent in in vitro models of liver tissue.
Immune cells produced by the bone marrow cells and/or tissue
present in the chip can be delivered to an in vitro model of liver
tissue in order to constitute a more complete model of liver
function and response. In some embodiments the products of bone
marrow can be collected from the chip as described elsewhere herein
and added to an additional in vitro system and/or model. In some
embodiments, the chip containing the bone marrow can be located in
the same microfluidic system as the additional in vitro model
and/or system. The perfusion fluid can be passed through the chip
containing the bone marrow and the effluent, containing the
products of bone marrow, can be delivered to the additional in
vitro model and/or system. The chip containing the bone marrow can
be upstream, downstream, or both with respect to the additional in
vitro model and/or system. All or part of the effluent of the chip
containing bone marrow can be delivered to the additional in vitro
model and/or system.
[0108] In some embodiments, the methods and devices described
herein can be used to study or produce cells and/or tissue at a
particular stage of development. By varying the time of
implantation, the identities and concentrations of tissue-inducing
material, the design of the chip, and the composition of the
perfusion fluid, the stage of development of the cells and/or
tissue present on the chip can be varied. In some embodiments this
can be used to study, for example, the process of bone marrow
development, the function of bone marrow constituents at some
points of development, the differential effects of compounds on
bone marrow over the course of development and/or the effects of
compounds on development of bone marrow. By way of non-limiting
example, the compound can be a chemotherapeutic, radiation therapy,
modifier of differentiation, formation, function or remodeling,
hormone, nucleic acid, peptide, protein, antibody, small molecule,
drug lead, enzyme, cell, virus, bacterium, parasite, nucleotide,
marker, dye, imaging agent, enzyme, nanoparticle, and/or gene
silencing molecule.
[0109] In some embodiments, the tissue and/or cells present in the
chip can be human cells and/or tissue without having been implanted
in a human. In these embodiments, the chip can be implanted into an
animal having human and/or humanized cells. By way of non-limiting
example, a mouse can be irradiated to eliminate its endogenous bone
marrow and then be given a graft of human bone marrow and/or human
hematopoietic stem cells. When a chip is implanted, whether before,
during or after the above-mentioned procedures, it will ultimately
be populated by human bone marrow cells. When removed from the site
of implantation and placed in a microfluidic system, the bone
marrow contained in the chip will be genetically human. The bone
marrow produced in this manner can be used in any of the
embodiments described herein.
[0110] In some embodiments, the source of the xenografted human
cells is a cancer tumor or cancerous tissue and/or cells. In some
embodiments the suitable types of cancers can include, but are not
limited to, leukemia, lymphoma, Hodgkin's lymphoma,
myeloproliferative disorders, Langerhans cell histiocytosis,
myeloma, and myelodysplastic syndromes. In some embodiments, the
xenografted human cells can be obtained from diseased tissue. In
some embodiments, the xenografted human cells can be obtained from
a human having a hematopoietic disease. Hematopoietic diseases
include, but are not limited to thalassemia, factor IX deficiency,
hemophilia, sickle cell disease, amyloidosis, agranulocytosis,
anemia, leucopenia, neutropenia, thrombocytopenia, panctyopenia,
Glanzmann's thrombasthenia, uremia, platelet storage pool disease,
Von Willebrand disease, afibrinogenemia and auto-immune
disease.
[0111] In some embodiments, when the chip is removed from the first
subject, some or all of the cells present in the chip can be
removed from the chip. By way of non-limiting example, bone marrow
cells that have colonized the bone marrow niche in the chip can be
removed by chemical and/or physical means. Cells in the cell growth
chamber of a chip can be removed from the chip and/or made
nonviable by any method known in the art for partially or
completely decellularizing a tissue. Non-limiting examples include
1) washing the chip and/or tissue with detergents or 2) washing the
chip with a buffer, then fixing the tissue with paraformaldehyde.
In some embodiments, the chip and/or tissue can be decellularized
by fixing with 4% paraformaldehyde (PFA) for 48 hours at 4.degree.
C. and then immersing the chip and/or tissue in 70% ethanol for 24
hours at 4.degree. C. and three times in PBS at 4.degree. C. for 2
hours each time to wash out the PFA. In some embodiments, the
internal marrow cavity of the chip can be removed by cutting a
small opening at the edge of the cavity and flusing with
medium.
[0112] The remaining decellularized bone marrow scaffold can be
used ex vivo and/or in vitro and be repopulated by bone marrow
and/or blood cells provided from another source. The tissue grown
in vivo in the chip and the second population of cells can be from
different species, e.g. the chip can be implanted in a mouse and
decellularized ex vivo, then repopulated with human bone marrow
cells.
In Vivo Methods
[0113] In some embodiments, the chip containing the tissue and/or
cells, or the tissue and/or cells removed from the chip, after
being removed from the first subject, can be implanted into a
second subject (e.g. human or animal) and used in vivo. In some
embodiments, the chip containing the tissue and/or cells, or the
tissue and/or cells removed from the chip can be implanted into a
second subject subcutaneously. Any amount of tissue and/or cells
from the chip can be used in vivo, e.g. from 0.1% to 100% of the
tissue and/or cells in the chip can be used in vivo. For example,
at least 0.1% of the tissue or cells in the chip, at least 1% of
the tissue or cells in the chip, at least 10% of the tissue or
cells in the chip, at least 50% of the tissue or cells in the chip,
at least 90% of the tissue or cells in the chip, or at least 95% or
more of the tissue or cells in the chip can be used in vivo. The
tissue and/or cells can be selected and/or separated prior to in
vivo use by, for example, cell sorting (e.g. FACS) or physical
separation of a portion of the tissue and/or cells.
[0114] In some embodiments, both the first and second subjects can
be animals. In some embodiments, both the first and second subjects
can be non-human animals of the same species, e.g. both subjects
can be mice. In some embodiments, both the first and second
subjects can be human. In some embodiments, the first and second
subjects can be the same subject. In some embodiments, the first
and second subjects can be of different species, e.g. the first
subject can be a mouse and the second subject can be a human.
[0115] In some embodiments, a chip can be implanted into a mouse
having human and/or humanized cells. After a period of in vivo
growth, the the chip containing the tissue and/or cells, or the
tissue and/or cells removed from the chip can be transplanted to a
human in need of a transplant of the type of cells grown in the
chip. In some embodiments, a chip can be implanted into a mouse
having human and/or humanized bone marrow or hematopoietic cells.
After a period of in vivo growth, the chip is transplanted to a
human in need of a transplant of bone marrow and/or hematopoietic
cells.
[0116] In some embodiments, the recipient (e.g. second) subject can
have a cancer, a hematopoietic disease, radiation toxicity, or a
compromised immune system. In some embodiments, the recipient
subject can have undergone chemotherapy or radiation therapy. In
some embodiments, the recipient is a subject diagnosed as having a
hematologic disease in which one or more of the blood cell types
found in the bone marrow are rendered dysfunctional. In some
embodiments, the recipient is a human who received a treatment that
damaged, compromised, or eliminated their endogenous bone marrow.
Such treatments can include, for example chemotherapy, radiation
therapy. In some embodiments, the human may be one who was exposed
to a source of radiation or a chemical, pollutant or infection that
damaged, compromised, or eliminated their endogenous bone marrow.
Examples of such chemicals, pollutants, or infections include, but
are not limited to, alcohol, benzene, hepatitis, Epstein-Barr
virus, cytomegalovirus, parvovirus B19 and HIV.
[0117] In further embodiments, tissue and/or stem cells can be
removed from a human prior to them receiving a treatment that will
damage, compromise, or eliminate that tissue and/or cell type. By
way of non-limiting example, bone marrow tissue and/or
hematopoietic stem cells can be removed from a human prior to them
receiving a treatment that will damage, compromise, or eliminate
their bone marrow. The bone marrow tissue and/or hematopoietic stem
cells removed from the human can be implanted into an
immunocompromised animal. A chip implanted in such a mouse will be
colonized by bone marrow tissue and/or hematopoietic cells that are
genetically identical to the human and the chip, after a period of
in vivo growth in the animal, can be implanted into the human, or
the cells and/or tissue contained in the chip can be implanted into
the human, thereby supplying a source of bone marrow tissue and/or
hematopoietic cells that is genetically identical to the human. In
some embodiments, the chip or the cells and/or tissue contained in
the chip can be stored, preserved, or maintained after removing
them from the site of implantation in the mouse and before
transplanting them to the human. In some embodiments, the chip or
the cells and/or tissue can be preserved by freezing. In some
embodiments, the chip or the cells and/or tissue can be preserved
by refrigeration. In some embodiments, the cells contained in the
chip can be maintained by removing the chip from the site of
implantation and connecting it to a microfluidic system as
described elsewhere herein. Perfusion fluid can be provided via the
microfluidic system and the cells contained in the chip maintain
their viability until it is desired to transplant the chip or the
bone marrow tissue and/or hematopoietic cells into the human.
[0118] In some embodiments, a chip can be implanted into a subject.
After a period of in vivo growth, the chip can be connected to a
microfluidic system and the tissue and/or cells contained on the
chip can be maintained by perfusion of a fluid as described herein.
After removal of the chip from the site of implantation, the
subject receives a treatment that damages, compromises, or
eliminates the cell and/or tissue type that was grown on the chip.
After such a treatment is concluded, the chip containing the tissue
and/or cells, or the tissue and/or cells removed from the chip can
be implanted into the subject, thereby supplying a source of tissue
and/or cells that is genetically identical to the subject. By way
of non-limiting example, after a period of in vivo growth where
bone growth was induced in the chip, the chip can be connected to a
microfluidic system and the bone marrow tissue and/or hematopoietic
cells contained on the chip can be maintained by perfusion of a
fluid as described herein. After removal of the chip from the site
of implantation, the subject receives a treatment that damages,
compromises, or eliminates their endogenous bone marrow. After such
a treatment is concluded, the chip containing the bone marrow
tissue and/or hematopoietic cells, or the bone marrow tissue and/or
hematopoietic cells removed from the chip can be implanted into the
subject, thereby supplying a source of bone marrow tissue and/or
hematopoietic cells that is genetically identical to the
subject.
[0119] In some embodiments, the chip or the tissue and/or cells
contained in the chip can be stored, preserved, or maintained after
removing them from the site of implantation in the first subject
and before returning them to the second subject and/or after
removing them from the site of implantation in the first subject
and before returning them to the first subject. In some
embodiments, the chip or the cells and/or tissue can be preserved
by freezing. In some embodiments, the chip or the cells and/or
tissue can be preserved by refrigeration. In some embodiments, the
cells contained in the chip can be maintained by removing the chip
from the site of implantation and connecting it to a microfluidic
system as described elsewhere herein. Perfusion fluid can be
provided via the microfluidic system and the cells contained in the
chip maintain their viability until it is desired to transplant the
chip or the bone marrow tissue and/or hematopoietic cells into the
human. In some embodiments, a chip and/or the tissue and/or cells
contained in the chip can be subject to multiple methods of
maintaining, storing, or preserving the cells and/or tissue ex
vivo, e.g. the tissue in a chip can be cultured in a microfluidics
device and then frozen and subsequently thawed before being
transplanted into a recipient subject.
[0120] In some embodiments, the chip and/or the tissue and/or cells
contained in the chip can be transplanted directly from the first
subject to the second subject without maintaining, storing, and/or
preserving the cells and/or tissue ex vivo.
[0121] In some embodiments, the tissue and/or cells contained
within the implantation device are genetically modified before
implantation into the second subject. In some embodiments, the
device is colonized by cells that are genetically modified, e.g.
the first subject comprises genetically modified cells. In some
embodiments, the first subject is a transgenic subject. In some
embodiments, the tissue and/or cells are genetically modified after
the implantation device and the tissue and/or cells contained
therein are removed from the first subject. In some embodiments,
the tissue and/or cells are genetically modified after the tissue
and/or cells are removed from the implantation device. In some
embodiments, the tissue and/or cells are genetically modified while
the tissue and/or cells are being maintained or cultured in a
microfluidic device or system. By way of non-limiting example,
tissue and/or cells can be genetically modified to increase or
decrease gene expression or to express an exogenous gene (e.g. a
marker gene). Methods of genetically modifying tissue and/or cells
are well known in the art and can include, but are not limited to,
viral vectors, plasmid vectors, homologous recombination, stable
integration, and transient expression.
[0122] Pharmaceutical Compositions
[0123] For administration to a subject, the chip containing cells
and/or tissue or the cells and/or tissue contained within the chip
or products of the cells and/or tissue contained within the chip
can be provided in pharmaceutically acceptable compositions. These
pharmaceutically acceptable compositions comprise the chip, cells,
tissues, and/or products of the cells or tissues, as described
above, formulated together with one or more pharmaceutically
acceptable carriers (additives) and/or diluents. As described in
detail below, the pharmaceutical compositions of the technology
described herein can be specially formulated for administration in
solid or liquid form, including those adapted for the following:
(1) oral administration, for example, drenches (aqueous or
non-aqueous solutions or suspensions), lozenges, dragees, capsules,
pills, tablets (e.g., those targeted for buccal, sublingual, and
systemic absorption), boluses, powders, granules, pastes for
application to the tongue; (2) parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; (3) topical application, for
example, as a cream, lotion, gel, ointment, or a controlled-release
patch or spray applied to the skin; (4) intravaginally or
intrarectally, for example, as a pessary, cream, suppository or
foam; (5) sublingually; (6) ocularly; (7) transdermally; (8)
transmucosally; or (9) nasally. Additionally, compositions can be
implanted into a patient or injected using a drug delivery system.
Coated delivery devices can also be useful. See, for example,
Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984);
Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals"
(Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; U.S. Pat.
No. 6,747,014; and U.S. Pat. No. 35 3,270,960.
[0124] As used here, the term "pharmaceutically acceptable" refers
to those compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0125] As used here, the term "pharmaceutically-acceptable carrier"
means a pharmaceutically- acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting the subject compound from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include, but are not
limited to: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose,
methylcellulose, ethyl cellulose, microcrystalline cellulose and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;
(7) lubricating agents, such as magnesium stearate, sodium lauryl
sulfate and talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)
esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)
buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)
isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)
pH buffered solutions; (21) polyesters, polycarbonates and/or
polyanhydrides; (22) bulking agents, such as polypeptides and amino
acids (23) serum component, such as serum albumin, HDL and LDL;
(22) C.sub.2-C.sub.12 alchols, such as ethanol; and (23) other
non-toxic compatible substances employed in pharmaceutical
formulations. Wetting agents, binding agents, fillers, lubricants,
coloring agents, disintegrants, release agents, coating agents,
sweetening agents, flavoring agents, perfuming agents,
preservative, water, salt solutions, alcohols, antioxidants,
polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose, polyvinylpyrrolidone and the like can also
be present in the formulation. The terms such as "excipient",
"carrier", "pharmaceutically acceptable carrier" or the like are
used interchangeably herein.
[0126] Many organized surfactant structures have been studied and
used for the formulation of drugs. These include monolayers,
micelles, bilayers and vesicles. Vesicles, such as liposomes, have
attracted great interest because of their specificity and the
duration of action they offer from the standpoint of drug delivery.
Liposomes are unilamellar or multilamellar vesicles which have a
membrane formed from a lipophilic material and an aqueous interior.
The aqueous portion contains the composition to be delivered.
Liposomes can be cationic (Wang et al., Biochem. Biophys. Res.
Commun., 1987, 147, 980-985), anionic (Zhou et al., Journal of
Controlled Release, 1992, 19, 269-274), or nonionic (Hu et al.
S.T.P.Pharma. Sci., 1994, 4, 6, 466). Liposomes can comprise a
numer of different phospholipids, lipids, glycolipids, and/or
polymers which can impart specific properties useful in some
applications and which have been described in the art (Allen et
al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,
53, 3765; Papahadjopoulos et al. Ann. N.Y. Acad. Sci., 1987, 507,
64; Gabizon et al. PNAS, 1988, 85, 6949; Klibanov et al. FEBS
Lett., 1990, 268, 235; Sunamoto et al. Bull. Chem. Soc. Jpn., 1980,
53, 2778; Illum et al. FEBS Lett., 1984, 167, 79; Blume et al.
Biochimica et Biophysica Acta, 1990, 1029, 91; U.S. Pat. Nos.
4,837,028; 5,543,152; 4,426,330; 4,534,899; 5,013,556; 5,356,633;
5,213,804; 5,225,212; 5,540,935; 5,556,948; 5,264,221; 5,665,710;
European Patents EP 0 445 131 B1; EP 0 496 813 B1; and European
Patent Publications WO 88/04924; WO 97/13499; WO 90/04384; WO
91/05545; WO 94/20073; WO 96/10391; WO 96/40062; WO 97/0478).
[0127] The compositions of the technology described herein can be
prepared and formulated as emulsions or microemulsions. Emulsions
are typically heterogeneous systems of one liquid dispersed in
another in the form of droplets usually exceeding 0.1 .mu.m in
diameter and have been described in the art. Microemulsion can be
defined as a system of water, oil and amphiphile which is a single
optically isotropic and thermodynamically stable liquid solution
and can comprise surfactants and cosurfactants. Both of these drug
delivery means have been described in the art (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,
Popovich N G., and Ansel H C., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 199, 245,
& 335; Higuchi et al., in Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa., 1985, p. 301; Leung and Shah, in:
Controlled Release of Drugs: Polymers and Aggregate Systems,
Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215;
Schott, in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa., 1985, p. 271; Constantinides et al.,
Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find.
Exp. Clin. Pharmacol., 1993, 13, 205; Ho et al., J. Pharm. Sci.,
1996, 85, 138-143; Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, p. 92; U.S. Pat. Nos. 6,191,105; 7,063,860;
7,070,802; 7,157,099).
[0128] In one embodiment, the liposome or emulsion formulation
comprises a surfactant. Surfactants find wide application in
formulations such as emulsions (including microemulsions) and
liposomes. The nature of the hydrophilic group (also known as the
"head") provides the most useful means for categorizing the
different surfactants used in formulations (Rieger, in
Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y.,
1988, p. 285). Suitable surfactants include fatty acids and/or
esters or salts thereof, bile acids and/or salts thereof. In some
embodiments the surfactant can be anionic, cationic, or nonionic.
The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0129] In some embodiments, the technology described herein employs
various penetration enhancers to effect the efficient delivery of
compounds across cell membranes. Penetration enhancers can be
classified as belonging to one of five broad categories, i.e.,
surfactants, fatty acids, bile salts, chelating agents, and
non-chelating non-surfactants all of which have been described
elsewhere (see e.g., Malmsten, M. Surfactants and polymers in drug
delivery, Informa Health Care, New York, N.Y., 2002; Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;
Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252; Touitou, E.,
et al Enhancement in Drug Delivery, CRC Press, Danvers, Mass.,
2006; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,
1992, 44, 651-654; Brunton, Chapter 38 in: Goodman & Gilman's
The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935; Swinyard, Chapter
39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Yamamoto et
al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J.
Pharm. Sci., 1990, 79, 579-583; Jarrett, J. Chromatogr., 1993, 618,
315-339; Katdare, A. et al., Excipient development for
pharmaceutical, biotechnology, and drug delivery, CRC Press,
Danvers, MA, 2006; Buur et al., J. Control Rel., 1990, 14,
43-51)
[0130] Oral formulations and their preparation are described in
detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and
U.S. Pat. No. 6,747,014, each of which is incorporated herein by
reference. Compositions and formulations for parenteral,
intraparenchymal (into the brain), intrathecal, intraventricular or
intrahepatic administration can include sterile aqueous solutions
which can also contain buffers, diluents and other suitable
additives such as, but not limited to, penetration enhancers,
carrier compounds and other pharmaceutically acceptable carriers or
excipients. Aqueous suspensions can further contain substances
which increase the viscosity of the suspension including, for
example, sodium carboxymethylcellulose, sorbitol and/or dextran.
The suspension can also contain stabilizers.
[0131] Aerosols for the delivery to the respiratory tract are known
in the art. See for example, Adjei, A. and Garren, J. Pharm. Res.,
1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm.,
114: 111-115 (1995); Gonda, I. "Aerosols for delivery of
therapeutic an diagnostic agents to the respiratory tract," in
Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313
(1990); Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324
(1989)) and have potential for the systemic delivery of peptides
and proteins as well (Patton and Platz, Advanced Drug Delivery
Reviews, 8:179-196 (1992)); Timsina et. al., Int. J. Pharm., 101:
1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29
(1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol
Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10
(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22:
263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22:
837-858 (1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995);
Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992);
Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S.,
et al., Controlled Release, 28: 15 79-85 (1994); Damms, B. and
Bains, W., Nature Biotechnology (1996); Niven, R. W., et al.,
Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al.,
Pharm. Res., 13(1): 80-83 (1996), contents of all of which are
herein incorporated by reference in their entirety.
[0132] The compositions of the technology described herein can
additionally contain other adjunct components conventionally found
in pharmaceutical compositions, at their art-established usage
levels. Thus, for example, the compositions can contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or can contain additional materials useful in physically
formulating various dosage forms of the compositions of the
technology described herein, such as dyes, flavoring agents,
preservatives, antioxidants, opacifiers, thickening agents and
stabilizers. However, such materials, when added, should not unduly
interfere with the biological activities of the components of the
compositions of the technology described herein. The formulations
can be sterilized and, if desired, mixed with auxiliary agents,
e.g., lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the compound(s) of the
formulation.
[0133] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While specific embodiments of, and examples for,
the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize. For example, while method steps or functions are
presented in a given order, alternative embodiments may perform
functions in a different order, or functions may be performed
substantially concurrently. The teachings of the disclosure
provided herein can be applied to other procedures or methods as
appropriate. The various embodiments described herein can be
combined to provide further embodiments. Aspects of the disclosure
can be modified, if necessary, to employ the compositions,
functions and concepts of the above references and application to
provide yet further embodiments of the disclosure. These and other
changes can be made to the disclosure in light of the detailed
description.
[0134] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
[0135] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the technology
described herein. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0136] The technology described herein is further illustrated by
the following examples which should not be construed as
limiting.
[0137] Some embodiments of the technology described herein can be
defined as any of the following numbered paragraphs: [0138] 1. A
method of maintaining tissue ex vivo, the method comprising:
implanting an implantation device in a subject whereby the
implantation device is colonized by at least one of stem cells,
vascular cells, immune cells, differentiated cells and diseased
cells; [0139] removing the implantation device and the tissue
contained in the implantation device from the subject; [0140]
providing a perfusion fluid to the tissue. [0141] 2. The method of
paragraph 1, further comprising the step of removing the tissue
from the implantation device prior to providing perfusion fluid.
[0142] 3. The method of paragraph 2, further comprising the step of
placing the tissue in a microfluidic system prior to providing
perfusion fluid. [0143] 4. The method of any of paragraphs 1-3,
wherein the implantation device includes at least one cell growth
chamber and at least one open port providing a passage to the cell
growth chamber to enable cells to enter the cell growth chamber for
cell colonization. [0144] 5. The method of paragraph 1, wherein the
implantation device is a microfluidic device. [0145] 6. The method
of paragraph 5, further comprising connecting the microfluidic
device to a microfluidic system. [0146] 7. The method of any of
paragraphs 5-6, wherein the microfluidic device includes at least
one cell growth chamber and at least one open port providing a
passage to the cell growth chamber to enable cells to enter the
cell growth chamber for cell colonization. [0147] 8. The method of
any of paragraphs 5-7, wherein the microfluidic device includes at
least one fluid channel separated from the at least one cell growth
chamber by a porous separation component. [0148] 9. The method of
any of paragraphs 5-8, wherein the microfluidic device includes at
least one inlet port and at least one outlet connecting the at
least one fluid channel to the microfluidic system and providing
perfusion fluid to the at least one cell growth chamber through the
porous separation component. [0149] 10. The method of any of
paragraphs 1-9, wherein the implantation device is implanted such
that at least one port faces the muscle tissue of the subject.
[0150] 11. The method of any of paragraphs 1-10, wherein the tissue
is bone marrow tissue. [0151] 12. The method of any of paragraphs
1-12, wherein the bone marrow tissue maintained ex vivo is used to
produce hematopoietic cells or bone marrow-derived factors. [0152]
13. The method of paragraph 12, wherein the hematopoietic cells are
selected from the group consisting of red blood cells, white blood
cells, platelets, hematopoietic stem cells, lymphocytes,
eosinophils, neutrophils, monocytes, hematopoietic progenitor
cells, stromal cells, and a mixture of two or more of these cell
types. [0153] 14. The method of paragraph 12, wherein the bone
marrow-derived factors are selected from the group consisting of a
peptide, protein, small molecule, nucleotides, lipids,
carbohydrates, cytokines and growth factors. [0154] 15. The method
of any of paragraphs 12-14, wherein the hematopoietic cells and/or
bone marrow-derived factors are administered to a subject. [0155]
16. The method of paragraph 15, wherein the subject has a condition
selected from the group consisting of a compromised immune system,
a cancer, an auto-immune disease, radiation toxicity, and a
hematopoietic disease. [0156] 17. The method of paragraph 16,
wherein the cancer is selected from the group consisting of
leukemia, lymphoma, Hodgkin's lymphoma, myeloproliferative
disorders, Langerhans cell histiocytosis, myeloma, and
myelodysplastic syndromes. [0157] 18. The method of paragraph 16,
wherein the hematopoietic disease is selected from the group
consisting of thalassemia, factor IX deficiency, hemophilia, sickle
cell disease, amyloidosis, agranulocytosis, anemia, leucopenia,
neutropenia, thrombocytopenia, panctyopenia, Glanzmann's
thrombasthenia, uremia, platelet storage pool disease, Von
Willebrand disease, and afibrinogenemia. [0158] 19. The method of
paragraph 15, wherein the subject has undergone chemotherapy and/or
radiation therapy. [0159] 20. The method of paragraph 12, wherein
the hematopoietic cells and/or bone marrow-derived factors are
provided to another tissue type being maintained in vitro. [0160]
21. The method of any of paragraphs 1-11, wherein the tissue
maintained ex vivo is used to test the effect of compounds on the
tissue or interaction of compounds with the tissue, where the
compound is selected from the group consisting of
chemotherapeutics, radiation therapies, modifiers of
differentiation, formation, function or remodeling, hormones,
nucleic acids, peptides, proteins, antibodies, small molecules,
drug leads, enzymes, cells, viruses, bacteria, parasites,
nucleotides, markers, dyes, imaging agents, enzymes, nanoparticles,
and gene silencing molecules. [0161] 22. The method of any of
paragraphs 1-11, wherein the tissue maintained ex vivo is at any
stage of development. [0162] 23. The method of any of paragraphs
1-22, wherein the implantation device is implanted into a non-human
subject. [0163] 24. The method of any of paragraphs 1-22, wherein
the implantation device is implanted into a non-human subject
having human or humanized hematopoietic cells. [0164] 25. The
method of paragraph 24, wherein the non-human subject has human
hematopoietic cells obtained from a cancer or a human having a
hematopoietic disease. [0165] 26. The method of paragraph 25,
wherein the cancer is selected from the group consisting of
leukemia, lymphoma, Hodgkin's lymphoma, myeloproliferative
disorders, Langerhans cell histiocytosis, myeloma, and
myelodysplastic syndromes. [0166] 27. The method of paragraph 25,
wherein the hematopoietic disease is selected from the group
consisting of thalassemia, factor IX deficiency, hemophilia, sickle
cell disease, amyloidosis, agranulocytosis, anemia, leucopenia,
neutropenia, thrombocytopenia, panctyopenia, Glanzmann's
thrombasthenia, uremia, platelet storage pool disease, Von
Willebrand disease, and afibrinogenemia. [0167] 28. A method of
producing tissue or cells for implantation into a subject, the
method comprising: implanting an implantation device in a first
subject whereby the implantation device is colonized by tissue or
cells; [0168] removing the implantation device and the tissue
contained in the implantation device from the first subject; [0169]
transplanting the implantation device or at least the tissue or
cells contained in the implantation device into a second subject
thereby providing tissue or cells to the second subject; [0170]
whereby the implanted tissue or cells exhibit cell growth and
function in the second subject. [0171] 29. The method of paragraph
28, wherein the implantation device includes at least one cell
growth chamber and at least one open port providing a passage to
the cell growth chamber to enable cells to entire the cell growth
chamber for cell colonization. [0172] 30. The method of paragraph
28, wherein the implantation device is a microfluidic device.
[0173] 31. The method of paragraph 30, wherein the microfluidic
device includes at least one cell growth chamber and at least one
open port providing a passage to the cell growth chamber to enable
cells to enter the cell growth chamber for cell colonization.
[0174] 32. The method of any of paragraphs 30-31, wherein the
microfluidic device includes at least one fluid channel separated
from the at least one cell growth chamber by a porous separation
component. [0175] 33. The method of any of paragraphs 30-32,
wherein the microfluidic device includes at least one inlet port
and at least one outlet connecting the at least one fluid channel
to the microfluidic system and providing perfusion fluid to the at
least one growth channel through the porous separation component.
[0176] 34. The method of any of paragraphs 28-33, wherein the
implantation device is implanted such that at least one port faces
the muscle tissue of the subject. [0177] 35. The method of any of
paragraphs 28-34, wherein the tissue or cells comprise bone marrow
tissue or hematopoietic cells. [0178] 36. The method of any of
paragraphs 28-35, wherein the second subject receiving the
transplant has been diagnosed with a condition selected from the
group consisting of a compromised immune system, a cancer, an
auto-immune disease, and a hematopoietic disease. [0179] 37. The
method of paragraph 36, wherein the cancer is selected from a group
consisting of leukemia, lymphoma, Hodgkin's lymphoma,
myeloproliferative disorders, Langerhans cell histiocytosis,
myeloma, and myelodysplastic syndromes. [0180] 38. The method of
paragraph 36, wherein the hematopoietic disease is selected from
the group consisting of thalassemia, factor IX deficiency,
hemophilia, sickle cell disease, amyloidosis, agranulocytosis,
anemia, leucopenia, neutropenia, thrombocytopenia, panctyopenia,
Glanzmann's thrombasthenia, uremia, platelet storage pool disease,
Von Willebrand disease, and afibrinogenemia. [0181] 39. The method
of any of paragraphs 28-38, wherein the second subject has
undergone chemotherapy and/or radiation therapy. [0182] 40. The
method of any of paragraphs 28-39, wherein the implantation device
is implanted into a non-human first subject having human or
humanized hematopoietic cells. [0183] 41. The method of any of
paragraphs 28-40, wherein the first and second subjects are the
same individual and the method comprises an additional step of
maintaining either the implantation device containing bone marrow
tissue and/or hematopoietic cells or the bone marrow tissue or
hematopoietic cells after removal from the implantation device
between removal from the site of implantation and transplantation
into the subject. [0184] 42. The method of any of paragraphs 28-40,
wherein the method comprises an additional step of maintaining
either the implantation device containing bone marrow tissue and/or
hematopoietic cells or the bone marrow tissue or hematopoietic
cells after removal from the implantation device between removal
from the site of implantation and transplantation into the second
subject. [0185] 43. The method of any of paragraph 28-42, wherein
maintaining the implantation device containing bone marrow tissue
or hematopoietic cells ex vivo comprises freezing or refrigerating
either the implantation device containing bone marrow tissue and/or
hematopoietic cells or the bone marrow tissue and/or hematopoietic
cells after removal from the implantation device. [0186] 44. The
method of any of paragraphs 28-42, wherein maintaining the
implantation device containing bone marrow tissue or hematopoietic
cells ex vivo comprises: connecting the implantation device to a
microfluidic systems; providing a perfusion fluid to the
implantation device. [0187] 45. The method of any of paragraphs
28-45, wherein the tissue or cells contained within the
implantation device are genetically modified before implantation
into the second subject. [0188] 46. A pharmaceutical composition
comprising bone marrow tissue, hematopoietic cells, or
differentiated blood cells obtained from a bone marrow tissue
maintained ex vivo according to the method of paragraph 1. [0189]
47. A pharmaceutical composition comprising bone marrow tissue
and/or hematopoietic cells obtained from a bone marrow tissue
maintained ex vivo accoding to the method of paragraph 28. [0190]
48. The composition of any of paragraphs 46-47, wherein the bone
marrow tissue and/or hematopoietic cells are contained within an
implantation device. [0191] 49. A method for producing or
manufacturing hematopoietic cells, the method comprising implanting
an implantation device in a subject whereby the implantation device
is colonized by stem cells, vascular cells, immune cells or
differentiated cells; [0192] removing the implantation device and
the tissue contained in the implantation device from the subject;
[0193] providing a perfusion fluid to the tissue; [0194] wherein
the implantation device includes at least one cell growth chamber
and at least one open port providing a passage to the cell growth
chamber to enable cells to enter the cell growth chamber for cell
colonization; [0195] wherein the tissue is bone marrow tissue;
[0196] and wherein the bone marrow tissue maintained ex vivo is
used to produce hematopoietic cells. [0197] 50. The method of
paragraph 49, wherein the hematopoietic cells are selected from the
group consisting of red blood cells, white blood cells, platelets,
hematopoietic stem cells, lymphocytes, eosinophils, neutrophils,
monocytes, a hematopoietic progenitor cell, and a mixture of two or
more of these cell types.
EXAMPLES
Example 1
Well Format Implantation Devices
[0198] In vivo culturing of bone tissue in an implantation device
was first investigated using a chip as shown in FIG. 3C, i.e. a
Well Format implantation device having two openings. The well was
filled with a collagen gel mixture comprising Type I collagen,
demineralized bone powder, BMP-2, and BMP-4 as described herein.
The implantation device was then implanted subcutaneously in a
mouse (FIGS. 4A-4B). Implantation devices were removed from the
subject at 4 weeks or 8 weeks after implantation and the contents
of the chip were examined to determine if bone marrow tissue was
present in the implantation device.
[0199] Histological staining via H&E stain revealed newly
generated bone marrow surrounded by a combination of new bone and
the original demineralized bone powder (FIGS. 7A-7D). Staining for
alkaline phosphatase activity using NBT/BCIP ready-to-use tables
(cat. #11-697-471-001, Roche applied science) indicated that tissue
growing in the chips contained zones of active bone formation as
compared to a section of mouse femur (data not shown). In the
presence of alkaline phosphatase, the BCIP
(5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt) is
dephosphorylated and then oxidized by NBT (Nitro blue tetrazolium
chloride) to yield a dark-blue indigo precipitating dye.
[0200] The presence of erythroid cells was evaluated by staining
with fluorescent markers (data not shown). Erythroid cells, as
detected by Ter119 staining, were found to be present in both
4-week and 8-week old in vivo grown tissues. The presence of
leukocytes was evaluated by staining with fluorescent markers (data
not shown). Leukocytes, as detected by CD45 staining, were found to
be present in both 4-week and 8-week old in vivo grown tissues.
Example 2
Comparison of Well Format Implantation Devices with One or Two
Openings
[0201] The bone marrow in the chips with two openings was dominated
by adipocytes, a cell type known to have an inhibitory effect on
the hematopoietic microenvironment. To improve the quality of the
engineered marrow, the polymer mold was designed to prevent
infiltration of adipocytes by blocking the end of the central
cavity of the polymer that faced the adipose tissue of the skin,
while retaining the opening facing the muscle surface (FIG. 3D).
The performance of Well Format chips having one opening or two
openings was compared. The chips were filled with a collagen
mixture as described herein and implanted subcutaneously in mice.
FIG. 5 shows a diagram of each chip type and photographs of the
chips after 4 weeks of in vivo growth. The chips were implanted
subcutaneously in mice and the chips having one opening were
oriented such that the opening faced the muscle of the mouse.
Histological staining via H&E stain revealed newly generated
bone marrow surrounded by new bone in both chip types after 8 weeks
(FIGS. 6A-6C and FIGS. 7C-7D). The newly formed marrow in the chips
with two openings was dominated by adipocytes and exhibited a low
level of cellularity (FIGS. 7A-7D), likely because fat cells
inhibit hematopoiesis (Naverias O, et al. Nature 460, 259-263
(2009)). Histological analysis of the bone marrow in the chip
having one opening revealed that a thick shell of well formed bone
surrounding a highly cellular marrow with a morphology nearly
identical to the marrow within a femur (FIG. 16B). Micro-computed
tomography (microCT) analyses show that the engineered bone marrow
was surrounded by mineralized cortical bone and permeabled by
trabecular bone with architectural properties similar to natural
trabecular bone seen in adult mouse vertebrae (FIGS. 19A-19D).
Example 3
FACS Analysis of Implantation Device-Grown Cells
[0202] The identity of the cells growing in these chips was further
investigated by FACS analysis designed to detect hematopoietic stem
cells and their differentiated descendant cell types. To determine
the hematopoietic cellular constitution of the bone marrow
compartment, the engineered bone marrow was dissociated immediately
upon surgical removal and analyzed using flow cytometry with
antibodies directed against Lineage cocktail (Lin), Sca-1, c-Kit,
CD34, CD135, Ter119, CD45, Mac-1, Gr-1, CD19, and CD3 to identify
HSCs (Lin-c-Kit+Sca-1+), progenitor cells (Lin-c-Kit+, Lin-Sca-1+,
Lin-CD34+, and Lin-CD135+), erythrocytes (Ter119+), leukocytes
(CD45+, macrophages (CD45+Mac-1+), granulocytes (CD45+Gr-1+), B
cells (CD45+CD19+), T cells (CD45+CD3+) (FIGS. 16C-16D). FIG. 8
shows the hematopoietic stem cell lineage and the markers used to
detect particular cell types. The cells were grown in a Well Format
chip having one opening implanted subcutaneously in a mouse such
that the opening faced the muscle tissue. The chips were harvested
from mice 4 weeks or 8 weeks after implantation. The tissue in the
implantation devices were removed, cut into small pieces, and
digested using 1 mg/mLcollagenase for 30 min to harvest the cells
inside the tissue. The cells were stained with antibodies as
indicated and analyzed using flow cytometry. One million cells were
stained with 100 .mu.L of staining solution for 30min. The solution
was washed out and changed into sorting buffer and then the cells
were analyzed using flow cytometry.
[0203] For the HSC panel the staining solution was: 20 .mu.L mouse
hematopoietic lineage eFluor 450 Cocktail, 2 .mu.L Anti-mouse CD34
FITC, 0.3 .mu.L Anti-mouse Ly-6A/E (Sca-1) APC, 5 .mu.L Anti-mouse
CD135 (Flt3) PE, 0.65 .mu.L Anti-mouse CD117 (c-Kit) APC-eFluor
780, and 72 .mu.L staining buffer. For the cell line panel the
staining solution was: 2.5 .mu.L Anti-mouse CD19 eFluor 450, 0.5
.mu.L Anti-mouse CD45 FITC, 0.65 .mu.L Anti-mouse Ly-6G (Gr-1) APC,
0.65 .mu.L Anti-mouse CD11b (Mac-1) PE, 2.5 .mu.L Anti-mouse Ter119
APC-eFluor 780, 5 .mu.L Anti-mouse CD3 PE-Cy5 and 90 staining
buffer. Staining buffer was 3% FBS and 0.05% sodium zide in PBS(-).
Sorting buffer was 0.1% Bovine serum albumin (BSA) and 0.5% FBS in
PBS(-). The antibodies were obtained from eBioscience or BD
Biosciences.
[0204] For analysis of HSC in peripheral blood samples, those
samples were first subjected to red blood cell lysis. Three mL of
Red blood cell lysing buffer (R7757, Sigma) was added to a
peripheral blood cell pellet and pipetted up and down once. After 5
min incubation at room temperature, 20 mL of PBS(-) solution was
added to dilute the buffer. The buffer was removed by centrifuging
(450 g for 7 min).
[0205] The tissue recovered from the chips after 4 weeks of in vivo
growth (FIGS. 9C, 10C, 11C, 12C) or 8 weeks of in vivo growth
(FIGS. 9D, 10D, 11D, 12D) was compared to the cellular profile of
mouse bone marrow (FIGS. 9A, 10A, 11A, 12A) and peripheral blood
(FIGS. 9B, 10B, 11B, 12B). The results of FACS analysis are shown
in FIGS. 9A-9E, 10A-10E, 11A-11E, and 12A-12E.
[0206] Devices harvested 4 or 8 weeks after implantation contain
all hematologic cell types, including hematopoietic stem and
progenitor cells and both differentiated red and white blood cell
lineages (FIGS. 16C-16D). While the number of hematopoietic stem
and progenitor cells were lower in the engineered marrow at 4 weeks
than that found in marrow from the adult mouse femur, marrow
harvested from implanted devices at 8 weeks exhibited a
distribution of hematopoietic stem cells, progenitors, and
differentiated blood cells from all lineages nearly identical to
that isolated from marrow within intact femur. These data
demonstrate that the hematopoietic compartment of the engineered
bone marrow faithfully recapitulates the full cellular components
of natural bone marrow.
[0207] The collagen gel mixture containing demineralized bone
powder and bone morphogenic proteins was able to recruit both bone
and bone marrow formation from the host in the subcutaneous site.
After 4 weeks of in vivo growth, bone marrow formation was observed
in the implantation device and about 4 million cells could be
harvested from the chip. The blood cell population formed in the
implantation device was quite similar to mouse bone marrow. At 8
weeks, the chip is mostly filled with bone marrow and over 10
million cells could be harvested from the chip. The blood cell
population formed in the chip was identical to mouse bone marrow.
These results indicate that functional engineered bone marrow is
present in the microfluidic chip after in vivo growth.
Example 4
Single Channel Format Chip
[0208] The performance of a Single Channel Format Chip (shown in
FIG. 2A) was evaluated. The single closed channel was filled with
demineralized bone powder, BMP-2, and BMP-4 in a collagen gel as
described elsewhere herein. The ports were filled with a Type I
collagen gel. The chip was implanted subcutaneously in a transgenic
mouse expressing GFP. After 4 weeks of in vivo growth, the chip was
removed and cell growth in the channel was examined. The channel
was populated by GFP-expressing cells and after removing the
bone-inducing material GFP-expressing cells remained adhered to the
channel (data not shown). A large number of GFP-expressing cells
were found in the channel and Ter119 staining for erythroid cells
revealed their presence in the channel (data not shown).
Histological examination by H&E staining also revealed growth
of bone tissue (data not shown).
Example 5
Closed Channel Format Chip
[0209] The performance of a Closed Channel Format chip (shown in
FIG. 2B) was also tested. The cell growth chamber was filled with
demineralized bone powder, BMP-2, and BMP-4 in a collagen gel as
described elsewhere herein. The ports were filled with a Type I
collagen gel. The chips were implanted subcutaneously in mice and
in vivo growth proceeded for 4 weeks. Chips were then removed and
examined. When examined optically, dark matter was present
predominantly in the cell growth chamber. The chips were stained
with Hoechst DNA stain and the bright fluorescent staining revealed
that cells are present at the ports and in the cell growth chamber
(data not shown).
Example 6
Tumor Biopsy
[0210] Mouse lung cancer cells transduced with Cherry (red color)
were injected subcutaneously into a GFP-mouse whose cells show
green fluorescence. The resulting tumor formed in the mouse was
harvested and then the biopsy was introduced into the cell growth
chamber of the microfluidic chip as described herein. Culture
medium was perfused continuously at 1 .mu.L/min. The medium used
was Dulbecco's Modified Eagle's Medium (DMEM) containing 10%(v/v)
fetal bovine serum (FBS),100 U/mL penicillin, and 100 U/mL
streptomycin. When examined 5 days after biopsy, bright red
fluorescence (cancer cells) and green fluorescence (mouse-derived
cells) was be observed, indicating that the cancer cells and
mouse-derived cells are alive (data not shown). Furthermore, the
cancer cells migrated out of the middle channel, suggesting that
the cancer cells continue proliferating in the microfluidic
device.
Example 7
Liver Biopsy
[0211] Mouse liver was harvested and then liver biopsy was
introduced into the cell growth chamber of the microfluidic chip as
described herein. Culture medium was perfused continuously at 1
.mu.L/min. The medium used was Dulbecco's Modified Eagle's Medium
(DMEM) containing 10%(v/v) fetal bovine serum (FBS),100 U/mL
penicillin, and 100 U/mL streptomycin. The shape of the liver
biopsy was maintained for 4 days in the cell growth chamber.
Staining of the liver biopsy stained with calcein-AM (green; live
cell stain) and ethidiumhomodimer (red; dead cell stain) 3 days
after harvest indicated that most cells were alive after 3 days of
in vitro growth (data not shown).
Example 8
Bone Marrow Transplantation
[0212] Bone-inducing material was implanted subcutaneously on the
back of a mouse using a chip as shown in FIGS. 16A and FIG. 17.
After 8 weeks implantation, bone and bone marrow formed in the
material. Visual examination after the device was removed from the
mouse revealed a red color due to the presence of red blood cells
in the tissue present in the chip.
[0213] The materials were characterized histologically (FIG. 16B).
The bottom image of FIG. 16 shows the cross-section of a mouse
femur. The orange color indicates bone marrow is inside of the bone
tissue. The top and middle images show the cross section of the
synthetic bone marrow after 4 weeks and 8 weeks implantation,
respectively. Bone containing bone marrow is observed. The
synthetic bone marrow is nearly identical to the mouse bone marrow.
Under high magnification as shown, the cells look exactly the
same.
[0214] To determine how similar the synthetic bone marrow actually
is to the mouse bone marrow, the cellular distribution was analyzed
using antibodies and flow cytometry. FIGS. 16C-16D show the
distribution of differentiated blood cells, and hematopoietic stem
and progenitor cells. After 4 days in culture, the bone marrow
cells from the device were harvested and compared to cells isolated
from the natural marrow of the mouse femur that were placed in a
Dexter culture with a stromal cell support layer, which is the most
common method currently used to maintain hematopoietic stem and
progenitor cells in vitro. Flow cytometric analysis demonstrated
that the distribution of hematopoietic stem and progenitor cells in
the engineered bone marrow (eBM, D4) remained similar to fresh
mouse femur bone marrow (FIG. 20D) whereas the composition of blood
cells in the Dexter culture (mBM, D4) was strikingly different from
freshly harvested mouse femur bone marrow, as previously reported.
After 4 days in the Dexter culture, the proportion of long-term
HSCs (Lin-CD150+CD48- staining cells) decreased and that of the
hematopoietic progenitor cells (Lin-c-Kit+, Lin-Sca-1+, Lin-CD34+,
and Lin-CD135+ staining cells) increased, compared to the number
found in normal mouse bone marrow, suggesting the HSCs are
differentiating and losing their multipotency under these
conditions. In contrast, after being cultured for 4 days in the
microfluidic marrow-on-a-chip, the engineered bone marrow
maintained its normal distribution of HSCs as well as progenitors.
These results demonstrate successfully engineered new bone
marrow.
[0215] The synthetic bone marrow was cultured in a microfluidic
device to keep the cells alive in vitro. Synthetic bone marrow
harvested from a mouse was placed in the microfluidic device
consisting of two channel layers. Culture media was perfused
through the top and bottom channels. Images of the system are shown
in FIG. 17. The synthetic bone marrow is placed in between the two
channels and is cultured while perfusing media. After 4 days in
culture, the synthetic bone marrow cells were harvested and
analyzed. The bottom right graph in FIG. 17 shows the distribution
of hematopoietic stem and progenitor cells. The distribution of the
cells after 4 days in culture is similar to when analyzed directly
after harvesting.
[0216] These data suggest that the engineered bone marrow system
described herein can maintain a functional hematopoietic niche
capable of supporting hematopoietic stem and progenitor cells in
vitro; however, to confirm that the engineered marrow remains
functional, it is necessary to demonstrate its functionality in
vivo. To meet this challenge, cells isolated from engineered marrow
after 4 days in culture and from natural marrow freshly isolated
from the femur of mice expressing stable green fluorescent protein
(GFP) were transplanted into lethally irradiated syngeneic mice.
The presence of short-term and long-term HSCs in the engineered
bone marrow was assessed by determining the ability of the
GFP-labeled marrow cells to reconstitute the cellular components of
peripheral blood 6 weeks and 16 weeks after transplantation,
respectively. Bone marrow harvested from the device following 4
days in the microfluidic culture system successfully engrafted in
the mice at a similar rate as freshly harvested mouse femur bone
marrow (FIG. 20A). Following transplantation, the cultured marrow
cells successfully produced all lineages of differentiated blood
cells in vivo. By 6 and 16 weeks after transplantation, 65% and 85%
(respectively) of the blood cells in the recipient were descended
from the transplanted cells. These data confirm that the bone
marrow that was engineered in vivo as described herein is fully
functional bone marrow, and that the engineered marrow cultured
within a microfluidic chip is capable of maintaining functional
HSCs in vitro.
[0217] After 4 weeks implantation, the bone-inducing materials
induced the formation of bone inside the device. After 8 weeks
implantation, the synthetic bone consists of cortical bone on the
outside and trabecular bone on the inside with similar
architectural properties to a mouse vertebra (FIGS. 19A-19D).
[0218] The bone marrow cells isolated from the synthetic bone
marrow after 4 days in the microfluidic culture system were
transplanted into lethally irradiated mice. The synthetic bone
marrow cells successfully engrafted the mice and populated all
lineages of differentiated cells 16 weeks after transplantation
(FIGS. 20A-20B). The engraftment rate of the synthetic bone marrow
cells 16 week after transplantation is identical to that of the
mouse bone marrow cells harvested from a mouse femur, demonstrating
that the device described herein is capable of maintaining
functional long-term hematopoietic stem cells ex vivo.
Example 9
Human Bone Marrow
[0219] The ultimate value of the bone marrow-on-a-chip technology
for clinical applications, such as production of blood cells, will
depend on the ability to adapt it for use with human hematpoietic
cells. To explore this possibility, a mouse bone disk was
engineered subcutaneously as described herein and surgically
removed from the animal. The internal marrow cavity chip was
exposed by cutting a small opening at its edge, and the cavity was
flushed with medium to clear it of non-adherent mouse cells after
fixing it with paraformaldehyde to retain the niche architecture.
Human umbilical cord blood cells (huCBCs) were injected into the
engineered bone marrow through the same opening at the edge, which
was subsequently sealed using Matrigel. The fixed engineered bone
containing huCBCs within its marrow cavity was cultured under flow
(1 .mu.L/min, 0.005 dyn/cm.sup.2) in a microfluidic device in
medium designed to maintain human HSCs. When cells were plated in a
standard planar culture dish, flow cytometry analysis revealed that
the viability of huCBCs and the number of human HSCs
(Lin-CD34+CD38-CD90+ staining cells) in the culture decreased
dramatically over 7 days. In contrast, when cultured in the bone
marrow microfluidic chip, viability of both huCBCs and human HSCs
was maintained for 7 days in vitro (FIGS. 21A-21B). These data
indicate that the engineered mouse bone marrow niche retains all of
the microenviromental signals required to sustain viability human
HSCs in culture, and that the microfluidic chip culture technique
can provide a way to sustain and expand these cells in culture for
experimental, therapeutic and blood cell manufacturing
applications.
[0220] There is a need to develop reliable in vitro systems that
can reconstitute the complex human bone marrow niche for clinical
and scientific purposes. Development of a microdevice that can
maintain viability and self-renewing function of short- and
long-term HSCs, as well as their ability to differentiate into the
various blood cell types would have great value for many
applications. Hematopoietic toxicity identified in animal models is
a major source of early drug candidate failure during the drug
development process; however, animal models are not always
predictive of results in humans. Bone marrow chips containing human
HSCs and their various lineages can provide an alternative way to
test hematopoietic effects of drugs, as well as toxins or radiation
exposure, on human marrow before entering clinical trials. They can
also provide a way to enhance current bone marrow transplantation
techniques by expanding cells isolated from a single clinical bone
marrow aspirate to large enough numbers in vitro that they can be
frozen and stored to be used for multiple transplants for the same
patient in the future.
[0221] As blood donor supplies are limited and complicated by
infection risk (e.g. HIV), there is also a need for high quality
sources of human blood cells (leukocytes, erythrocytes, platelets)
for therapeutic applications. Bone marrow chips that can maintain
human HSC viability in vitro and enable sustained production of
differentiated blood cell types can provide an alternative
manufacturing strategy.
[0222] Finally, understanding of hematopoietic niche physiology
still remains incomplete due to the limited availability of
relevant in vitro models that successfully recreate and/or mimic
the native bone marrow environment outside of a living animal. The
bone marrow chip microtechnology described here provides for
creation of an organ-on-a-chip device that reconstitutes a whole
functional bone marrow and a permeating trabecular bone matrix, as
well as providing a way to culture HSCs and generate various blood
lineages in vitro.
TABLE-US-00001 SEQUENCE LISTING SEQ ID NO: 1 BMP-2 amino acid
sequence, NCBI ID NO: NP_001191.1 1 mvagtrclla lllpqvllgg
aaglvpelgr rkfaaassgr pssqpsdevl sefelrllsm 61 glkqrptps rdavvppyml
dlyrrhsgqp gspapdhrle raasrantvr sfhheeslee 121 etsgkttr rfffnlssip
teefitsael qvfreqmqda lgnnssfhhr iniyeiikpa 181 tanskfpvtr
lldtrlvnqn asrwesfdvt pavmrwtaqg hanhgfvvev ahleekqgvs 241
krhvrisrsl hqdehswsqi rpllvtfghd gkghplhkre krqakhkqrk rlkssckrhp
301 lyvdfsdvgw ndwivappgy hafychgecp fpladhlnst nhaivqtlvn
svnskipkac 361 cvptelsais mlyldenekv vlknyqdmvv egcgcr SEQ ID NO: 2
BMP-4 amino acid sequence, NCBI ID NO: NP_001193, NP_570911, and
NP_570912 1 mipgnrmlmv vllcqvllgg ashaslipet gkkkvaeiqg haggrrsgqs
hellrdfeat 61 llqmfglar pqpsksavip dymrdlyrlq sgeeeeeqih stgleyperp
asrantvrsf 121 hheehlenip gtsensafrf lfnlssipen evissaelrl
freqvdqgpd wergfhrini 181 yevmkppaev vpghlitrll dtrlvhhnvt
rwetfdvspa vlrwtrekqp nyglaievth 241 lhqtrthqgq hvrisrslpq
gsgnwaqlrp llvtfghdgr ghaltrrrra krspkhhsqr 301 arkknkncrr
hslyvdfsdv gwndwivapp gyqafychgd cpfpladhln stnhaivqtl 361
vnsvnssipk accvptelsa ismlyldeyd kvvlknyqem vvegcgcr
Sequence CWU 1
1
21396PRTHomo sapiens 1Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu
Leu Leu Pro Gln Val 1 5 10 15 Leu Leu Gly Gly Ala Ala Gly Leu Val
Pro Glu Leu Gly Arg Arg Lys 20 25 30 Phe Ala Ala Ala Ser Ser Gly
Arg Pro Ser Ser Gln Pro Ser Asp Glu 35 40 45 Val Leu Ser Glu Phe
Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys 50 55 60 Gln Arg Pro
Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu 65 70 75 80 Asp
Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp 85 90
95 His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe
100 105 110 His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly
Lys Thr 115 120 125 Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro
Thr Glu Glu Phe 130 135 140 Ile Thr Ser Ala Glu Leu Gln Val Phe Arg
Glu Gln Met Gln Asp Ala 145 150 155 160 Leu Gly Asn Asn Ser Ser Phe
His His Arg Ile Asn Ile Tyr Glu Ile 165 170 175 Ile Lys Pro Ala Thr
Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu 180 185 190 Asp Thr Arg
Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp 195 200 205 Val
Thr Pro Ala Val Met Arg Trp Thr Ala Gln Gly His Ala Asn His 210 215
220 Gly Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser
225 230 235 240 Lys Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp
Glu His Ser 245 250 255 Trp Ser Gln Ile Arg Pro Leu Leu Val Thr Phe
Gly His Asp Gly Lys 260 265 270 Gly His Pro Leu His Lys Arg Glu Lys
Arg Gln Ala Lys His Lys Gln 275 280 285 Arg Lys Arg Leu Lys Ser Ser
Cys Lys Arg His Pro Leu Tyr Val Asp 290 295 300 Phe Ser Asp Val Gly
Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr 305 310 315 320 His Ala
Phe Tyr Cys His Gly Glu Cys Pro Phe Pro Leu Ala Asp His 325 330 335
Leu Asn Ser Thr Asn His Ala Ile Val Gln Thr Leu Val Asn Ser Val 340
345 350 Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser
Ala 355 360 365 Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val
Leu Lys Asn 370 375 380 Tyr Gln Asp Met Val Val Glu Gly Cys Gly Cys
Arg 385 390 395 2408PRTHomo sapiens 2Met Ile Pro Gly Asn Arg Met
Leu Met Val Val Leu Leu Cys Gln Val 1 5 10 15 Leu Leu Gly Gly Ala
Ser His Ala Ser Leu Ile Pro Glu Thr Gly Lys 20 25 30 Lys Lys Val
Ala Glu Ile Gln Gly His Ala Gly Gly Arg Arg Ser Gly 35 40 45 Gln
Ser His Glu Leu Leu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met 50 55
60 Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro
65 70 75 80 Asp Tyr Met Arg Asp Leu Tyr Arg Leu Gln Ser Gly Glu Glu
Glu Glu 85 90 95 Glu Gln Ile His Ser Thr Gly Leu Glu Tyr Pro Glu
Arg Pro Ala Ser 100 105 110 Arg Ala Asn Thr Val Arg Ser Phe His His
Glu Glu His Leu Glu Asn 115 120 125 Ile Pro Gly Thr Ser Glu Asn Ser
Ala Phe Arg Phe Leu Phe Asn Leu 130 135 140 Ser Ser Ile Pro Glu Asn
Glu Val Ile Ser Ser Ala Glu Leu Arg Leu 145 150 155 160 Phe Arg Glu
Gln Val Asp Gln Gly Pro Asp Trp Glu Arg Gly Phe His 165 170 175 Arg
Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala Glu Val Val Pro 180 185
190 Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg Leu Val His His Asn
195 200 205 Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pro Ala Val Leu
Arg Trp 210 215 220 Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Ala Ile
Glu Val Thr His 225 230 235 240 Leu His Gln Thr Arg Thr His Gln Gly
Gln His Val Arg Ile Ser Arg 245 250 255 Ser Leu Pro Gln Gly Ser Gly
Asn Trp Ala Gln Leu Arg Pro Leu Leu 260 265 270 Val Thr Phe Gly His
Asp Gly Arg Gly His Ala Leu Thr Arg Arg Arg 275 280 285 Arg Ala Lys
Arg Ser Pro Lys His His Ser Gln Arg Ala Arg Lys Lys 290 295 300 Asn
Lys Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val 305 310
315 320 Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe
Tyr 325 330 335 Cys His Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu
Asn Ser Thr 340 345 350 Asn His Ala Ile Val Gln Thr Leu Val Asn Ser
Val Asn Ser Ser Ile 355 360 365 Pro Lys Ala Cys Cys Val Pro Thr Glu
Leu Ser Ala Ile Ser Met Leu 370 375 380 Tyr Leu Asp Glu Tyr Asp Lys
Val Val Leu Lys Asn Tyr Gln Glu Met 385 390 395 400 Val Val Glu Gly
Cys Gly Cys Arg 405
* * * * *