U.S. patent application number 15/667986 was filed with the patent office on 2017-11-23 for bioreactor system and method of enhancing functionality of muscle cultured in vitro.
The applicant listed for this patent is Wake Forest University Health Sciences. Invention is credited to Anthony Atala, George Christ, Joel D. Stitzel, JR., James Yoo.
Application Number | 20170333177 15/667986 |
Document ID | / |
Family ID | 37115704 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170333177 |
Kind Code |
A1 |
Yoo; James ; et al. |
November 23, 2017 |
BIOREACTOR SYSTEM AND METHOD OF ENHANCING FUNCTIONALITY OF MUSCLE
CULTURED IN VITRO
Abstract
A method of producing organized skeletal muscle tissue from
precursor muscle cells in vitro comprises: (a) providing precursor
muscle cells on a support in a tissue media; then (b) cyclically
stretching and relaxing the support at least twice along a first
axis during a first time period; and then (c) optionally but
preferably maintaining the support in a substantially static
position during a second time period; and then (d) repeating steps
(b) and (c) for a number of times sufficient to enhance the
functionality of the tissue formed on the support and/or produce
organized skeletal muscle tissue on the solid support from the
precursor muscle cells.
Inventors: |
Yoo; James; (Winston-Salem,
NC) ; Stitzel, JR.; Joel D.; (Winston-Salem, NC)
; Atala; Anthony; (Winston-Salem, NC) ; Christ;
George; (Crozet, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wake Forest University Health Sciences |
Winston-Salem |
NC |
US |
|
|
Family ID: |
37115704 |
Appl. No.: |
15/667986 |
Filed: |
August 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15298382 |
Oct 20, 2016 |
9757225 |
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15667986 |
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12429385 |
Apr 24, 2009 |
9506025 |
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15298382 |
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11279671 |
Apr 13, 2006 |
9493735 |
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12429385 |
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60671600 |
Apr 15, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61F 2002/0894 20130101;
A61L 27/3826 20130101; A61L 2430/30 20130101; C12N 2527/00
20130101; A61L 27/3683 20130101; C12M 35/04 20130101; A61L 2430/34
20130101; A61L 27/3691 20130101; A61L 27/3604 20130101; C12N 5/0658
20130101; A61L 27/3895 20130101; A61F 2/08 20130101; C12N 2539/00
20130101; C12M 21/08 20130101; A61L 27/367 20130101 |
International
Class: |
A61F 2/08 20060101
A61F002/08; C12M 3/00 20060101 C12M003/00; C12N 5/077 20100101
C12N005/077; C12M 1/42 20060101 C12M001/42; A61L 27/36 20060101
A61L027/36; A61L 27/38 20060101 A61L027/38 |
Claims
1. A method of culturing organized skeletal muscle tissue from
precursor muscle cells, comprising: (a) providing precursor muscle
cells on a support in a tissue media; then (b) cyclically
stretching and relaxing said support at least twice along a first
axis during a first time period; and then (c) maintaining said
support in a substantially static position during a second time
period; and then (d) repeating steps (b) and (c) for a number of
times sufficient to enhance the functionality of the muscle tissue
or produce organized skeletal muscle tissue on said solid support
from said precursor muscle cells.
2. The method of claim 1, wherein said cyclically stretching and
relaxing is carried out at least three times during said first time
period.
3. The method of claim 1, wherein said stretching comprises
extending said support to a dimension at least 5% greater in length
than said static position.
4. The method of claim 1, wherein said relaxing comprises
retracting said support to a dimension not greater in length than
said static position.
5. The method of claim 1, wherein said relaxing comprises
retracting said support to a dimension at least 5% lesser in length
than said static position.
6. The method of claim 1, wherein said first time period is from 2
to 30 minutes in duration.
7. The method of claim 1, wherein said second time period is from
10 to 100 minutes in duration.
8. The method of claim 1, wherein said repeating of steps (b) and
(c) is carried out for a time of five days to three weeks.
9. Cultured muscle tissue produced by the process of claim 1.
10. The cultured muscle tissue of claim 9, wherein said tissue is
characterized by a contractile response to KCl-induced
depolarization in vitro.
11. The cultured muscle tissue of claim 10, wherein said tissue is
further characterized by: cells that exhibit unidirectional
orientation on histological examination; the presence of
multinucleated myofibril cells; cells that express muscle markers
as confirmed by immunohistochemistry; and extracellular matrices as
confirmed by Mason's Trichrome.
12.-22. (canceled)
23. The cultured muscle tissue of claim 9, wherein said tissue is
suturable and is 1 to 50 cm in length.
24. The cultured muscle tissue of claim 9, wherein said tissue
contracts in response to electrical stimulation four weeks after of
in vivo implantation.
25. A method of reconstructing a muscle in a subject in need
thereof, comprising implanting muscle tissue of claim 9 in said
subject in an orientation effective to reconstruct said muscle.
26. A method of building soft tissue in a subject in need thereof,
comprising implanting muscle tissue of claim 9 in said subject in
an orientation effective to build soft tissue.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/298,382, filed Oct. 20, 2016, now allowed,
which is a divisional of U.S. patent application Ser. No.
12/429,385, filed Apr. 24, 2009, now U.S. Pat. No. 9,506,025, which
is a divisional of U.S. patent application Ser. No. 11/279,671,
filed Apr. 13, 2006, now U.S. Pat. No. 9,493,735, which claims the
benefit of U.S. Provisional Patent Application Ser. No. 60/671,600,
filed Apr. 15, 2005, the disclosure of each of which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns methods and apparatus for the
growth of skeletal muscle in vitro.
BACKGROUND OF THE INVENTION
[0003] Loss of functional skeletal muscle due to traumatic injury,
tumor excision, etc., produces a physiological deficit for which
there is still no effective clinical treatment. Tissue engineering
of skeletal muscle in vitro for functional tissue replacement in
vivo may provide a potential therapeutic solution to this unmet
medical need. In fact, significant progress has been made during
last 15 years in understanding some of the basic requirements for
creating tissue engineered skeletal muscle constructs in vitro.
Early studies necessarily focused mainly on the production of
highly differentiated muscle constructs and characterizing their
properties in terms of response to stretch and other mechanical
stimulation in a 2-D tissue culture system (Vandenburgh, Mechanical
forces and their second messengers in stimulating cell growth in
vitro. Am J Physiol. 262(3 Pt 2):R350-5 (March 1992); Mechanical
stimulation of skeletal muscle generates lipid-related second
messengers by phospholipase activation. J Cell Physiol.
155(1):63-71 (April 1993).
[0004] The majority of recent work on 3-D cultures of skeletal
muscle myoblasts has been performed using gel-based matrix and
mechanical strainers; as biodegradable scaffolds are thought to
possess too much of a development barrier (both structural and
nutritional) to clinical development. Recently, 3-D cultures of
myoblasts have been successfully established and isometric
contractile responses in these 3-D constructs, termed myoids, were
measured (Dennis R G, Kosnik P E. Excitability and isometric
contractile properties of mammalian skeletal muscle constructs
engineered in vitro. In Vitro Cell and Dev Biol Animal. 36:327-335
(2000)). Additionally, fibrin-based gels were suggested as another
novel method to engineer 3-D functional muscle tissue. The latter
achieved muscle structures of 100-500 .mu.m diameter with measured
maximal tetanic force of 805.8.+-.55 .mu.N (Huang Y et al., Rapid
formation of functional muscle in vitro using fibrin gels. J Appl
Physiol 98: 706-713 (2005)). In short, tissue engineered 3-D
skeletal muscle constructs composed of collagen or fibrin gels have
clearly improved the understanding of skeletal muscle organogenesis
and provide a reasonable model for studying the developmental
physiology of skeletal muscle micro-structures in vitro.
[0005] However, while muscle constructs developed with synthetic
scaffolds can support the contractile portion of the muscle tissue,
and furthermore, can be maintained in culture for several months,
this approach still has significant limitations for clinical
utility. For example, implantation of tissue engineered skeletal
muscle constructs will require that they be of relevant size and
mechanical strength to be amenable to the rigors of the requisite
surgical procedures. Clearly, gel-based constructs are currently
too small and too fragile for such surgical manipulation.
[0006] As such, one of the major barriers to engineering clinically
applicable functional muscle tissues for reconstructive procedures
is the lack of a bioreactor system and methodology that would
accelerate cellular organization, tissue formation and
function.
SUMMARY OF THE INVENTION
[0007] A first aspect of the invention is a method of culturing
organized skeletal muscle tissue from precursor muscle cells. In
general the method comprises: (a) providing precursor muscle cells
on a support in a tissue media; then (b) cyclically stretching and
relaxing the support at least twice along a first axis during a
first time period; and then (c) optionally but preferably
maintaining the support in a substantially static position during a
second time period; and then (d) repeating steps (b) and (c) for a
number of times sufficient to enhance the functionality of the
muscle tissue (e.g., its ability to contract), or produce organized
skeletal muscle tissue, on the solid support from the precursor
muscle cells.
[0008] An alternate embodiment of the foregoing includes the step
of cyclically stretching and relaxing said support at least twice
along a second axis during said first time period (with stretching
and relaxing along still additional axes being possible if
desired).
[0009] A second aspect of the invention is cultured skeletal muscle
tissue produced by a process as described herein.
[0010] A third aspect of the invention is cultured skeletal muscle
tissue. The tissue is characterized by cells that exhibit, or its
ability to exhibit, a reproducible contractile response to
KCl-induced depolarization in vitro. In some embodiments the tissue
is further characterized by a unidirectional orientation on
histological examination; the presence of multinucleated myofibril
cells; cells that express muscle markers as confirmed by
immunohistochemistry (e.g., alpha actin and myosin heavy chain);
contains and cells that produce extracellular matrices as confirmed
by Masson's Trichrome.
[0011] A further aspect of the invention is a device useful for
carrying out a method as described herein. The device preferably
comprises a container, a pair of engaging members in said container
for engaging tissue supports or other tissue constructs, an
actuator mounted on the container and operatively associated with
one of said engaging members to provide controlled cyclic strain to
attached tissue supports or other tissue constructs, a motor
connected to said actuator, and a controller operatively associated
with said motor. All are positioned so that supports or constructs
carried by the engaging members may be immersed in a suitable
growth or culture media in the container. The controller is
configured to implement a method as described herein. The engaging
members preferably include a plurality of points of attachment so
that a plurality of tissue supports or tissue constructs, each with
its own volume of space within the container, are provided.
[0012] The foregoing and other objects and aspects of the present
invention are explained in greater detail in the drawings herein
and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram of an apparatus of the present
invention.
[0014] FIG. 2 is a top view of an apparatus of the present
invention. The bioreactor engineered tissue is cyclically stretched
by a linear motor that is, in turn, connected to a computer control
system. There is great flexibility in the equipment and computer
software for satisfying the desired biological boundary
conditions.
[0015] FIGS. 3A-3C. H&E staining of bioengineered skeletal
muscle following 5 days in the bioreactor. Note the dramatic change
in orientation of the myocytes during static (FIG. 3A) growth
versus culturing in the bioreactor (FIG. 3B). (FIG. C)
Representative tracing of the contractile response of bioengineered
muscle to KCl-induced depolarization in organ bath studies. A
similar response was observed on two other strips. Myocytes seeded
on a static scaffold in the same incubator for the same time
period, exhibited no detectable contractile response to addition of
KCl.
[0016] FIGS. 4A-4B show staining of control skeletal muscle (FIG.
4A) and skeletal muscle cultured 7 days (FIG. 4B) in a bioreactor
in accordance with methods of the present invention.
[0017] FIGS. 5A-5C. FIGS. 5A & 5B provide representative
examples of the contractile responses to electrical field
stimulation (EFS) observed 2 and 4 weeks after implantation of
bioengineered skeletal muscle on the latissimus dorsi of nu/nu
mice. FIG. 5C is normal muscle as a control.
[0018] FIG. 6. Graphical summary of the results of physiological
experiments performed at the 4 week time point on isolate skeletal
muscle tissue strips in vitro.
[0019] FIG. 7. Graphical summary of the results of all
physiological experiments performed on isolate skeletal muscle
tissue strips in vitro.
[0020] The present invention is explained in greater detail in the
drawings herein and the specification below. The disclosures of all
United States patent references cited herein are to be incorporated
by reference herein in their entirety.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] Subjects to be treated by the methods of the present include
both human subjects and other animal subjects (particularly
mammalian subjects such as dogs and cats) for veterinary
purposes.
[0022] Muscle cells used to carry out the present invention are
preferably mammalian muscle cells, including primate muscle cells,
including but not limited to human, pig, goat, horse, mouse, rat,
monkey, baboon, etc. In general such cells are skeletal muscle
cells. Muscle cells of other species, including birds, fish,
reptiles, and amphibians, may also be used if so desired. The
muscle cells are, in general, precursor cells, or cells that are
capable of differentiating into muscle cells, specifically skeletal
muscle cells, under appropriate culture conditions and stimuli as
described herein. Muscle precursor cells are known. See, e.g., U.S.
Pat. No. 6,592,623.
[0023] "Supports" on which muscle cells may be seeded and grown to
produce cultured muscle tissue of the present invention include any
suitable support. See, e.g., U.S. Pat. Nos. 6,998,418; 6,485,723;
6,206,931; 6,051,750; and 5,573,784. Collagen supports or
decellularized tissue supports (e.g., obtained from smooth muscle
or skeletal muscle, such as a decellularized mammalian (e.g.,
porcine) bladder, are currently preferred.
[0024] Any suitable culture media can be used to grow cells in the
present invention, including medias comprising serum and other
undefined constituents, defined medias, or combinations thereof,
such as RPMI, DMEM, etc. If desired an angiogenic compound such as
VEGF can be included in the media to facilitate the formation of
vascular cells or vasculature in the muscle tissue.
[0025] A device of the present invention is schematically
illustrated in FIG. 1. The device comprises an actuator mounted on
a tissue culture-compatible container and configured to provide
controlled cyclic strain to attached scaffolds or other tissue
constructs 21. These constructs are normally stretched in one
direction. Constructs are supported between two holders or support
engaging members 10 and 11, which can be formed from any structure
for attaching a scaffold or developed tissue for tissue culture.
The device has a removable lid, 12, which is a necessary component
for setting up tissues for culturing and necessary to cover the box
fully during culturing. The device has a single pass-through 13
consisting of a water-resistant bearing and mounting hardware. The
device contains vertical adjustments both on the actuator
connecting piece 14 (facilitated by a vertical slot and bolt, not
shown) and on the pieces on the opposite side of the box 15 (also
facilitated by vertical slot and bolt, not shown). The linear
actuator stator (17) is controlled by a linear motor controller
system (18) and a control computer (19), used for programming and
subsequent monitoring of the actuator. The shaft (16) gives the
motion to the support connecting piece 14, moving the horizontal
piece 10 which provides the cyclic deformation to the constructs
21. The crosspieces 10, 11 can have any number of connecting points
22 for constructs. The connecting points are spaced so that each
construct has its own volume immersed (fully or partially) in the
culture medium within the apparatus.
[0026] While the device is shown with a single motor and actuator,
it will be appreciated that an additional motor and engaging member
may also be included to provide for elongation and relaxation along
a second axis, if desired.
[0027] The apparatus may be used in a method of culturing organized
skeletal muscle tissue from precursor muscle cells. As noted above,
the method comprises: (a) providing precursor muscle cells on a
support (e.g., a collagen support) in a tissue media; then (b)
cyclically stretching and relaxing the support at least two or
three times, up to 5, 10 or 20 times or more, along a first axis or
direction of travel during a first time period. A preferred
embodiment comprises (c) maintaining the support in a substantially
static position during a second time period; and then (d) repeating
steps (b) and (c) for a number of times sufficient to produce
organized skeletal muscle tissue on the solid support from the
precursor muscle cells.
[0028] If desired an angiogenic compound such as VEGF can be seeded
on or carried by the solid support to facilitate the formation of
vascular cells or vasculature in the muscle tissue.
[0029] The length of stretching of the solid support may be to a
dimension at least 5% greater in length than the static position,
and the relaxing may comprise retracting the support to a dimension
not greater in length than the static position. In some embodiments
the "static position" may be intermediate between the stretched and
relaxed position, and in such cases the relaxing may comprise
retracting the support to a dimension at least 5% lesser in length
than the static position.
[0030] The first time period, during which the stretching and
relaxing occurs, may be of any suitable length, for example from 2
or 3 minutes up to 10, 20 or 30 minutes in duration or more.
[0031] The second time period during which the support is
maintained in a static position, may be of any suitable duration.
In some embodiments the second time period is shorter than the
first time period, and may be from 1 or 2 minutes in duration up to
10 or 20 minutes in duration. In other embodiments the second time
period is longer than the first time period, and may be from 10 or
20 minutes in duration up to 40, 60 or 90 minutes in duration, or
more. In some embodiments, such as where the first time period
contains comparatively long intervals between stretching and
relaxing, the need for a second time period may be obviated
altogether.
[0032] In one preferred embodiment, the support is cyclically
stretched and relaxed during a first "active" time period to a
dimension of 10 percent greater and lesser in length than the
static dimension at a rate of 3 cycles per minute for a total of
five minutes, followed by a 55 minute "rest" second time period,
continuously for 1 to 3 weeks of in vitro culture.
[0033] A particular advantage and application of the present
invention is its ability to speed, accelerate or enhance the
functional maturation or performance of muscle such as skeletal
muscle grown in vitro (e.g., as exhibited by the ability of the
muscle tissue to contract in response to contact to a 60 milliMolar
KCl solution in vitro). Thus in some embodiments the total
culturing time of the tissue, such as the repeating of steps (b)
and (c) is carried out for a time of up to five days, or a time of
up to one, two or three weeks, after which time a contractile
response is preferably observed, with shorter culture times being
preferred.
[0034] Skeletal muscle tissue produced as described herein may be
used in vitro, in the apparatus described herein or in a separate
apparatus, to examine the pharmacological or toxicological
properties of compounds of interest (e.g., by adding the compound
of interest to a culture medium in which the tissue is immersed,
and examining the histological or mechanical properties of the
tissue as compared to a control tissue).
[0035] Skeletal muscle tissue (with or without support) produced by
the methods of the present invention is preferably "suturable" in
that it has sufficient structural integrity to be surgically
sutured or otherwise fastened at either end when implanted and
thereafter develop tension upon contraction.
[0036] Skeletal muscle tissue produced as described herein may be
used for the reconstruction of damaged tissue in a patient, e.g., a
patient with a traumatic injury of an arm or leg. Such tissue may
be utilized on the support (which is also implanted) or removed
from the support and implanted into the subject. The skeletal
muscle tissue may be implanted to "build" soft tissue (e.g., at the
interface between an amputated limb and a prosthetic device) or to
reconstruct (partially or totally) a damaged muscle (e.g., a muscle
of the face, hand, foot, arm, leg, back or trunk). The cultured
skeletal muscle tissue preferably has, in some embodiments, a size
or volume of at least 1, 2, or 3 or more cubic centimeters (not
counting the volume of the support if present), and/or a length of
1 cm to 50 cm, to provide sufficient tissue mass for implantation
in a patient (e.g., in association with an existing muscle of the
patient) and reconstruction of a skeletal muscle involved in, for
example, movement of fingers.
[0037] For allogenic transplant into a patient, skeletal muscle as
described herein may be matched or tissue-typed in accordance with
known techniques, and/or the subject may be administered immune
suppressive agents to combat tissue transplant rejection, also in
accordance with known techniques.
[0038] The present invention is explained in greater detail in the
following non-limiting Examples.
Example 1
[0039] A bioreactor system consisted of an actuator mounted on a
tissue culture-compatible container which was designed to provide
controlled cyclic strain to muscle tissue scaffolds, as shown in
FIG. 2. The linear actuator was controlled by a linear motor
controller system and a control computer, used for programming and
subsequent monitoring of the actuator. Primary human skeletal
muscle precursor cells were isolated, grown and expanded in
culture. The cells were seeded onto collagen-based muscle scaffold
strips derived from porcine bladder tissue (1.0.times.0.3.times.0.3
cm.sup.3). After two days of static culture, the muscle cell seeded
scaffolds were placed in the bioreactor system and programmed
linear stretching cycles were applied (LinMot.RTM.). The controlled
cycle strain was programmed to exert .+-.10% of the initial length
of the cell seeded scaffolds at a frequency of 3 times per minute
for the first 5 minutes of every hour. The bioreactor was
continuously operated for up to 3 weeks after the initial set up.
Muscle cell seeded scaffolds without cyclic stimulation served as
controls. The muscle cell constructs were assessed for structural
and functional parameters using scanning electron microscopy,
histo- and immunohistochemistry, and physiologic tissue bath
studies.
[0040] As shown in FIGS. 3A, 3B, and 3C, the bioreactor engineered
muscle produced viable tissue with appropriate cellular
organization. Scanning electron microscopy of the bioreactor
stimulated muscle tissue showed a uniform attachment of muscle
cells on the scaffold surface. Histologically, the bioreactor
stimulated engineered muscle demonstrated unidirectional
orientation by 5 days and continued to mature with time Presence of
multinucleated myofibrils was evident within the tissue construct.
The cells expressed muscle markers and produced extracellular
matrices over time, as confirmed by immunohistochemistry and by
Masson's Trichrome, respectively. The control scaffolds (myoblasts
incubated under identical incubator conditions, but not in the
bioreactor) showed disorganized muscle cells without any
directional orientation. Physiologic organ bath studies of the
bioreactor applied engineered muscle exhibited a reproducible
contractile response to KCl-induced depolarization (p<0.05). The
control scaffolds without the bioreactor stimulation failed to show
any detectable contractile response.
[0041] This study demonstrates that an organized functional muscle
tissue can be engineered using a unidirectional tissue bioreactor
system. Muscle cell seeded scaffolds that are exposed to a constant
cyclic biomechanical stimulation are able to achieve enhanced
cellular organization and demonstrate significant contractile
function. The use of this bioreactor system allowed for an enhanced
cellular orientation and may accelerate muscle tissue formation for
the bioengineering of clinically relevant sized muscle tissues.
Example 2
[0042] This example presents the results of more detailed studies
with the bioreactor system described above. The system, in
overview, consisted of a linear actuator mounted on a tissue
container to provide a controlled cyclic strain to muscle tissue
scaffolds. Primary human muscle cells were seeded onto scaffolds
and placed in the bioreactor system and subjected to cyclic strain
equivalent to .apprxeq..+-.10% stretch of the original scaffold
length; strain was applied 3 times/min for the first 5 min/hour for
periods ranging from 5 days to 3 weeks. Following this conditioning
protocol, the cell constructs were assessed for structural and
functional parameters in vitro; cell and scaffold constructs under
static culture conditions (i.e., no cyclic strain) were run in
parallel. In a separate in vivo experiments, both the structures
conditioned in the bioreactors for 5 days and control tissue
structures maintained under static culture conditions were
implanted onto the latissimus dorsi muscle of nude mice. At 3, 5,
and 7 days after implantation, structures were retrieved and
assessed for structural characteristics, while at 1, 2, 3 and 4
weeks, functional parameters were assessed.
Materials and Methods
[0043] Preparation of Acellular Tissue Matrices.
[0044] A cellular tissue matrices were prepared from porcine
bladder as previously described (Ref). Briefly, excised porcine
bladder tissues were placed in Triton X 1% (Sigma-Aldrich,
Taufkirchen, Germany) for 24-48 h in the presence of 0.1% sodium
azide, while agitated in a water bath at 37.degree. C. The
extraction of all cellular elements was confirmed histologically.
Prepared acellular matrix was cut into the 1.5 cm.times.1.5 cm.
Additional longitudinal incisions were created to increase the
surface area for cell seeding and sutures were placed at both ends
in order to secure the scaffold in the bioreactor. Scaffolds were
sterilized by soaking them in Betadine.RTM. solution for 1 day, and
subsequently washed with 1% antibiotic PBC solution for 5 days
before use in the these experiments.
[0045] Cell Isolation, Culture and Characterisation.
[0046] Primary human skeletal muscles cells were isolated by
surgical biopsy from (i.e., psoas muscle) healthy volunteers ages
25-35 under the guidelines of Institutional Clinical Review Board
of Wake Forest University Health Sciences School of Medicine.
Muscles were washed 3-4 times with sterile PBS to remove debris
before being cut into small pieces. Muscle tissues were plated onto
35 mm culture dishes with myogenic medium [340 mL low glucose DMEM
(GIBCO Life Science, catalog no. CC-3161), 100 mL FBS (Fetal Bovine
Serum), 50 mL HS (Horse Serum), 5 mL CEE (Chicken Embryo Extract)
and 5 mL Penicillin/Streptomycin. When cells had achieved
confluence they were further expanded on 150 mm culture dishes.
Cells were passaged at confluence and always used before P10. Using
this methodology we observed that .apprxeq.75-85% of the cells were
desmin positive, confirming their myogenic phenotyped. P5 to P10
cells were transferred and seeded on the surfaces of acellular
scaffold (with dimensions of .apprxeq.1.5 cm.sup.3). The cell
seeded scaffolds were then incubated in DMEM for 24 h.
[0047] Mechanical Strain.
[0048] A linear motor-driven stimulator device (Linmot, Virginia
Tech) was used for applying the mechanical stimulation, which
consisted of cyclic unidirectional stretch and relaxation. The
bioreactor system itself consisted of an actuator mounted on a
tissue culture container in which the cell seeded scaffolds were
secured. The linear actuator was, in turn, calibrated, controlled
and programmed by a computer. To permit application of the cyclic
stretch protocol, one end of the cell-seeded scaffold (i.e., tissue
construct) was tied via sutures on a stationary bar, while the
other end was secured to the movable bar that was attached to the
linear motor and computer controller. As currently designed, the
container can hold up to 10 tissue constructs at one time, with the
maximal distance between the two bars of .apprxeq.10 cm. The media
were changed every 3 days and tissue constructs were continuously
provided with 95% air-5% CO.sub.2, at 37.degree. C. in an
incubator.
[0049] Cyclic Strain Protocol.
[0050] In this study, primary human skeletal muscle cells-seeded
scaffolds were subjected to stretch and relaxation of .apprxeq.10%
of their initial basal length. The exact protocol was as follows:
tissue constructs were stretched 3 times/min (i.e., the entire
stretch and relaxation protocol took 20 s) for the first 5 minutes
of every hour for periods ranging from 5 days to 3 weeks. Muscle
cell seeded scaffolds without cyclic stimulation were placed in the
incubator on 150 mm culture dishes and served as controls.
[0051] Experimental Design for In Vitro Studies.
[0052] Primary cultured myoblasts (600.times.10.sup.6
cells/cm.sup.3) were seeded onto the collagen-based scaffold
(1.5.times.0.3.times.0.3 cm.sup.3) derived from porcine bladder
tissue. After 2 days of static culture, the cell-seeded scaffolds
were placed in the bioreactor system described above. After periods
ranging from 5 days to 3 weeks of mechanical stimulation, the
muscle cell constructs were removed from the bioreactor system and
assessed for structural and functional parameters. Again, muscle
cell seeded scaffolds without cyclic stimulation served as
controls.
[0053] Experimental Design for In Vivo Studies.
[0054] Primary cultured myoblasts (600.times.10.sup.6
cells/cm.sup.3) were seeded onto the collagen-based scaffold
(1.5.times.0.3.times.0.3 cm.sup.3) derived from porcine bladder
tissue (see Methods above). After 2 days of static culture, the
cell-seeded scaffolds were moved into the bioreactor system.
Following 1 week of mechanical stimulation, the muscle cell
constructs and control tissue constructs without cyclic stimulation
were implanted onto the latissimus dorsi muscle of the nude mice).
At 3, 5, and 7 days after implantation, the constructs were
harvested and were assessed for structural and histological
characteristics. At 1, 2, 3 and 4 weeks after implantation in vivo,
the constructs were harvested and the contractility of the tissue
constructs was assessed and evaluated to normal latissimus dorsi
muscle of the same nude mice.
[0055] Contractility Test.
[0056] The KCl-induced contractile response was examined following
3 weeks of bioreactor conditioning in vitro. Contractility testing
was also performed following 1 week of bioreactor preconditioning
and then after 7, 14, 21 and 28 post-implantation in the latissimus
dorsi of nude mice (5 tissue constructs/group). The contractile
responses of the bioreactor-preconditioned muscle tissue was
compared to that of statically seeded bioengineered tissue (i.e., 1
week of in vitro cell culture with no bioreactor preconditioning,
as well as to the contractile responses observed in normal
latissimus dorsi muscle of similar dimensions that was harvested
from the same nude mouse. The procedures for electrical field
stimulation (i.e., EFS) followed previously published methods (5,6)
with extensor digitorum longus (EDL) muscle (Radnoti Glass
Technology Inc, Monrovia, Calif.).
[0057] After harvesting of the individual tissue constructs, the
existing suture was used to attach the one end of the construct to
a force transducer (Radnoti Model TRN001, Monrovia, Calif.) that
was mounted on the spindle of a non-rotating micrometer head and
then connected to an amplifier. The other end was attached to the
glass hook at the bottom of the field-stimulating electrode
(Radnoti model 160151). This configuration resulted in a vertically
oriented muscle suspended between the two parallel platinum
electrodes. The entire preparation was then submerged in a 25-mL
organ chamber (Radnoti model 158326) filled with Krebs solution of
the following composition: (pH 7.4; concentration in mM: 122.0
NaCl, 4.7 KCl, 1.2 MgCl.sub.2, 2.5 CaCl.sub.2, 15.4 NaHCO.sub.3,
1.2 KH.sub.2PO.sub.4, and 5.5 glucose). The solution was aerated
with a 95% O.sub.2-5% CO.sub.2 gaseous mixture and maintained at
37.degree. C. with the help of a polystat circulator (Cole-Parmer
Instruments, Chicago, Ill.), and changed at 15-min intervals. After
a 10-minute period of temperature equilibration, the optimal muscle
length (i.e., Lmax) was determined by adjusting the stretch of the
muscle through movement of the micrometer head. After determining
the Lmax, isometric maximal twitch force was determined by
gradually increasing voltage at 10 mV increments up to 100 mV,
until maximal twitch force was achieved and recorded. Tetanic
contractile force was then measured at frequencies of 40, 70, 100,
and 120 Hz, with 1.5 s for each stimulation. The voltage used for
tetanic testing was the same used to create maximal twitch force,
with a duration of 2 ms and a delay of 2 ms. A 3-min rest period
followed each stimulation. Electrical stimulation was provided by a
neurostimulator (model S44B, Grass Instruments, Quincy, Mass.) and
delivered to the constructs through platinum electrodes. Data were
recorded and stored using a computerized data acquisition software
(Mac Lab hardware and software; ADI Instruments, Natick, Mass.). At
the conclusion of the contractile measurements, all muscles were
weighed. All force measurements were observed on a digital display
and recorded on a chart recorder.
[0058] Results:
[0059] Compared to control structures under static culture
conditions, structures derived from the bioreactor conditioning
protocol (i.e., engineered tissue) produced viable muscle tissue
with appropriate cellular organization. The engineered muscle
showed unidirectional orientation within 5 days of bioreactor
conditioning, and continued to mature with time. The presence of
organized myofibrils was evident with the expression of muscle
markers in the bioreactor stimulated structures. Extending the
bioreactor conditioning period to 3 weeks produced a bioengineered
tissue capable of generating a contractile response to
depolarization with KCl. Finally, implantation of the engineered
muscle tissue into the latissimus dorsi of nude mice following 3
days to 4 weeks of bioreactor conditioning yielded tissues with
numerous structural and histological similarities to skeletal
muscle, and 4 weeks after implantation the bioengineered muscle
tissue showed a reproducible contractile response to EFS
(p<0.05) that was approximately 30-50% of the response observed
on comparably sized control segments from the same animal. No
detectable contractile responses were observed on statically seeded
constructs at any time point studied following implantation. In
addition, in all cases, scaffolds maintained under static culture
conditions showed disorganized tissue formation and
multidirectional orientation of muscle cells both in vitro and in
vivo.
[0060] FIG. 4 shows staining of control skeletal muscle (A) and
skeletal muscle cultured 7 days (B) in a bioreactor in accordance
with methods of the present invention. Note the unidirectional
orientation of cultured muscle (B) as compared to control muscle
(A).
[0061] FIG. 5, panels A and B provide representative examples of
the contractile responses to electrical field stimulation (EFS)
observed 2 and 4 weeks after implantation of bioengineered skeletal
muscle on the latissimus dorsi of nu/nu mice. Compare these
responses with that observed in native latissimus dorsi muscle from
an nu/nu mouse. Of major importance, only one month after
implantation of bioengineered muscle, we observed 30-50% of the
contractile response produced in native skeletal muscle.
[0062] FIG. 6 provides a graphical summary of the results of
physiological experiments performed at the 4 week time point on
isolate skeletal muscle tissue strips in vitro. Where: Normal
denotes experimental results obtained with native skeletal muscle;
Active denotes retrieved bioreactor preconditioned bioengineered
skeletal muscle (i.e., active) 4 weeks after implantation on the
latissimus dorsi; Control denotes experimental results obtained on
bioengineered skeletal muscle that was NOT preconditioned in the
bioreactor (cells+scaffold only and kept in an incubator under
static conditions prior to implantation). See Methods for details.
As illustrated, although the normal tissue produced greater
contractile responses under all experimental conditions studied,
the bioreactor preconditioned bioengineered skeletal muscle was
able to generate contractile responses that ranged from
.apprxeq.30-50% of normal muscle. In stark contrast, in the absence
of bioreactor preconditioning (i.e., Control), no detectable
contractile responses were observed at 4 weeks (or any other time
point). Actual mean.+-.SEM values can be found in the corresponding
Table 1.
TABLE-US-00001 TABLE 1 Changes of Contractility at 50 V Normal
Active (4 weeks) Control No. Animal 5 5 5 40 Hz 4.09 .+-. 1.59 2.18
.+-. 0.98 0 70 Hz 9.55 .+-. 2.04 2.78 .+-. 1.12 0 100 Hz 15.49 .+-.
4.25 4.66 .+-. 1.37 0 120 Hz 16.8 .+-. 4.09 5.40 .+-. 1.52 0 Max
twitch 9.16 .+-. 2.12 (100 V) 3.46 .+-. 1.05 (90 V) 0 Data are
expressed as Mean and SD.
[0063] FIG. 7 provides a graphical summary of the results of all
physiological experiments performed on isolate skeletal muscle
tissue strips in vitro. As illustrated, within 2 weeks after
implantation, bioreactor preconditioned bioengineered skeletal
muscle strips are capable of generating measurable contractile
responses. Again, while the bioengineered skeletal muscle generated
significantly less tension than native latissimus dorsi at all time
points thus far studied, these are the first data that we are aware
of which document force generation of this magnitude in
bioengineered human skeletal muscle. Actual mean.+-.SEM values can
be found in the corresponding Table 2.
TABLE-US-00002 TABLE 2 Time-dependent changes in contractility of
bioreactor-conditioned engineered muscle tissue Normal 1 wk. 2 wks.
3 wks. 4 wks. No. Animal 5 5 5 5 5 40 Hz 4.09 .+-. 1.59 0 0.32 .+-.
0.13 0.19 .+-. 0.1 2.18 .+-. 1.31 70 Hz 9.55 .+-. 2.04 0 0.67 .+-.
0.63 1.73 .+-. 0.72 2.78 .+-. 1.12 100 Hz 15.49 .+-. 4.25 0 1.75
.+-. 1.31 4.21 .+-. 1.70 4.66 .+-. 1.47 120 Hz 16.8 .+-. 4.09 0
2.83 .+-. 1.42 3.64 .+-. 1.89 5.40 .+-. 1.53 Data are expressed as
Mean and SD. There was no contractile response in all control
(cells, but not bioreactor conditioning) constructs of 1 wk, 2 wks,
3 wks and 4 wks.
[0064] Conclusion:
[0065] This study demonstrates that organized functional muscle can
be engineered using a computerized bioreactor system on a
biodegradable scaffold (matrix). That is, following isolation and
expansion, muscle cell seeded scaffolds that are exposed to a
cyclic stimulation protocol are able to achieve enhanced cellular
organization and accelerated tissue formation/maturation both in
vitro and in vivo, with significant contractile function in all
cases. The use of this bioreactor system may accelerate muscle
formation for reconstructive or replacement surgery in patients
with localized functional skeletal muscle deficits.
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[0083] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
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