U.S. patent application number 13/800175 was filed with the patent office on 2013-08-08 for tissue engineered blood vessel.
This patent application is currently assigned to CORDIS CORPORATION. The applicant listed for this patent is CORDIS CORPORATION. Invention is credited to Iksoo CHUN, David C. COLTER, Kevin COOPER, Sridevi DHANARAJ, Carrie H. FANG, Anna GOSIEWSKA, Agnieszka SEYDA, Chunlin YANG.
Application Number | 20130203168 13/800175 |
Document ID | / |
Family ID | 41110839 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130203168 |
Kind Code |
A1 |
COOPER; Kevin ; et
al. |
August 8, 2013 |
TISSUE ENGINEERED BLOOD VESSEL
Abstract
Compositions and methods of using tissue engineered blood
vessels to repair and regenerate blood vessels of patients with
vascular disease are disclosed.
Inventors: |
COOPER; Kevin; (Flemington,
NJ) ; CHUN; Iksoo; (Princeton, NJ) ; COLTER;
David C.; (Hamilton, NJ) ; DHANARAJ; Sridevi;
(Raritan, NJ) ; GOSIEWSKA; Anna; (Skillman,
NJ) ; SEYDA; Agnieszka; (Belle Mead, NJ) ;
FANG; Carrie H.; (Pittstown, NJ) ; YANG; Chunlin;
(Belle Mead, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CORDIS CORPORATION; |
Bridgewater |
NJ |
US |
|
|
Assignee: |
CORDIS CORPORATION
Bridgewater
NJ
|
Family ID: |
41110839 |
Appl. No.: |
13/800175 |
Filed: |
March 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12432994 |
Apr 30, 2009 |
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13800175 |
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61049067 |
Apr 30, 2008 |
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Current U.S.
Class: |
435/402 |
Current CPC
Class: |
C12M 21/08 20130101;
A61L 27/18 20130101; C08L 67/04 20130101; A61L 27/38 20130101; A61L
27/3604 20130101; C12N 5/0691 20130101; A61L 27/507 20130101; A61L
27/18 20130101; C12M 25/14 20130101 |
Class at
Publication: |
435/402 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. A method of making a tissue engineered blood vessel comprising
the steps of: a. Providing a tubular shaped scaffold comprising
poly(p-dioxanone); b. Applying said scaffold with cell lysate; and
then c. Lyophilizing the scaffold.
8. The method of claim 7 further comprising the steps of: a.
applying one or more of cells and minced tissue to the cell lysate
scaffold; and b. culturing the cell lysate scaffold having one or
more of cells and minced tissue in a bioreactor.
9. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a non-provisional filing of a
provisional application U.S. Pat. App. No. 61/049,067.
FIELD OF THE INVENTION
[0002] The invention relates to tissue engineered blood vessels for
treatment of vascular disease. In particular, the invention
provides tissue engineered blood vessels prepared from scaffolds,
and one or more of cells, cell sheets, cell lysate, minced tissue,
and bioreactor processes to repair or replace a native blood vessel
that has been damaged or diseased.
BACKGROUND OF THE INVENTION
[0003] Cardiovascular-related disorders are a leading cause of
death in developed countries. In the US alone, one cardiovascular
death occurs every 34 seconds and cardiovascular disease-related
costs are approximately $250 billion. Current methods for treatment
of vascular disease include chemotherapeutic regimens, angioplasty,
insertion of stents, reconstructive surgery, bypass grafts,
resection of affected tissues, or amputation. Unfortunately, for
many patients, such interventions show only limited success, and
many patients experience a worsening of the conditions or
symptoms.
[0004] These diseases often require reconstruction and replacement
of blood vessels. Currently, the most popular source of replacement
vessels is autologous arteries and veins. However, such autologous
vessels are in short supply or are not suitable especially in
patients who have had vessel disease or previous surgeries.
[0005] Synthetic grafts made of materials such, as PTFE and Dacron
are popular vascular substitutes. Despite their popularity,
synthetic materials are not suitable for small diameter grafts or
in areas of low blood flow. Material-related problems such as
stenosis, thromboembolization, calcium deposition, and infection
have also been demonstrated. Therefore, there is a clinical need
for biocompatible and biodegradable structural matrices that
facilitate tissue infiltration to repair/regenerate diseased or
damaged tissue. In general, the clinical approaches to repair
damaged or diseased blood vessel tissue do not substantially
restore their original function. Thus, there remains a strong need
for alternative approaches for tissue repair/regeneration that
avoid the common problems associated with current clinical
approaches.
[0006] The emergence of tissue engineering may offer alternative
approaches to repair and regenerate damaged/diseased tissue. Tissue
engineering strategies have explored the use of biomaterials in
combination with cells, growth factors, bioactives and bioreactor
processes to develop biological substitutes that ultimately can
restore or improve tissue function. The use of colonizable and
remodelable scaffolding materials has been studied extensively as
tissue templates, conduits, barriers, and reservoirs. In
particular, synthetic and natural materials in the form of foams,
and textiles have been used in vitro and in vivo to
reconstruct/regenerate biological tissue, as well as deliver agents
for inducing tissue growth.
[0007] Such tissue-engineered blood vessels (TEBVs) have been
successfully fabricated in vitro and have been used in animal
models. However, there has been very limited clinical success.
[0008] Regardless of the composition of the scaffold and the
targeted tissue, the template must possess some fundamental
characteristics. The scaffold must be biocompatible, possess
sufficient mechanical properties to resist the physical forces
applied at the time of surgery, porous enough to allow cell
invasion, or growth, easily sterilized, able to be remodeled by
invading tissue, and degradable as the new tissue is being formed.
Furthermore, the scaffold may be fixed to the surrounding tissue
via mechanical means, fixation devices, or adhesives. So far,
conventional materials, alone or in combination, lack one or more
of the above criteria. Accordingly, there is a need for scaffolds
that can resolve the potential pitfalls of conventional
materials.
SUMMARY OF THE INVENTION
[0009] The invention is a tissue engineered blood vessel (TEBV)
comprised of a biocompatible, bioabsorbable scaffold and one or
more of cells, cell sheets, cell lysate, minced tissue, and
cultured with or without a bioreactor process. Such tissue
engineered blood vessels may be used to repair or replace a native
blood vessel that has been damaged or diseased. In one embodiment,
the tissue engineered blood vessel is comprised of a biocompatible,
bioabsorbable scaffold and cells. In another embodiment the tissue
engineered blood vessel is comprised of a biocompatible,
bioabsorbable scaffold and cell sheets. In another embodiment the
tissue engineered blood vessel is comprised of a biocompatible,
bioabsorbable scaffold and cell lysate. In yet another embodiment
the tissue engineered blood vessel is comprised of a biocompatible,
bioabsorbable scaffold and minced tissue. In addition, various
combinations of cells, cell sheets, cell lysate and minced tissue
are combined with a biocompatible, bioabsorbable scaffold to form
the tissue engineered bloods vessel. These tissue engineered blood
vessels may be cultured with or without a bioreactor process. In
one embodiment, the tissue engineered blood vessel is enhanced by
combining with bioactive agents.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1. Human umbilical cord-derived cell (hUTC) attachment
and growth on a vascular graft biomaterial after 3 and 7 days in
culture. A: PDO-ESS scaffold after 3 days, B: PDO-ESS scaffold
after 7 days, C: PDO/collagen-ESS scaffold after 3 days, D:
PDO/collagen-ESS scaffold after 7 days. All images taken at
40.times. magnification
[0011] FIG. 2. Representative example of smooth muscle cell (UASMC)
cell attachment and growth on a vascular graft biomaterial after 3
and 7 days in culture. A: 100 mg/ml PDO-ESS scaffold after 3 days,
B: 100 mg/ml PDO-ESS scaffold after 7 days, C: 140 mg/ml PDO-ESS
scaffold after 3 days, D: 140 mg/ml PDO-ESS scaffold after 7 days.
All images taken at 40.times. magnification.
[0012] FIG. 3. Representative example of endothelial cell (HUVEC)
cell attachment and growth on a vascular graft biomaterial after 3
and 7 days in culture. A: 100 mg/ml PDO-ESS scaffold after 3 days,
B: 100 mg/ml PDO-ESS scaffold after 7 days, C: 140 mg/ml PDO-ESS
scaffold after 3 days, D: 140 mg/ml PDO-ESS scaffold after 7 days.
All images taken at 40.times. magnification.
[0013] FIG. 4. Cell attachment to lysate-augmented PDO-ESS
scaffolds. PDO-ESS scaffolds were loaded with hUTC cell lysate at
two different concentrations and lyophilized. Cells
(50,000/scaffold) were then seeded onto the scaffolds and cultured
for 3 and 7 days before Live/Dead staining (A) control scaffold
after 3 days (B) control scaffold after 7 days (C) 18 mg/ml
lysate-augmented scaffold after 3 days (D) 18 mg/ml
lysate-augmented scaffold after 7 days (E) 5.2 mg/ml lysate
augmented scaffold after 3 days (F) 5.2 mg/ml lysate-augmented
scaffold after 7 days. All images taken at 40.times.
magnification.
[0014] FIG. 5. DNA content of cells cultured on lysate-augmented
PDO-ESS scaffolds. PDO-ESS scaffolds were loaded with hUTC cell
lysate at two different concentrations (low=5.2 mg/ml, high=18
mg/ml) and lyophilized. Cells (50,000/scaffold) were then seeded
onto the scaffolds and cultured for 3 days before analysis of
cellular DNA content. Samples were washed, digested with papain and
DNA quantitated using the CyQuant NF assay kit.
[0015] FIG. 6. Tubular scaffolds coated with PBS or hUTC lysate
which were cultured with rat SMC for 24 hours showed Lysate coated
scaffolds showed more cell attachment from LIVE/DEAD and H&E
image
[0016] FIG. 7. H&E staining for the hUTC seeded PDO sheet
pre-coated with hUTC lysate after cultured 11 days after seeding.
While cells were only seeded on one side, cells migrated and
penetrated all over the scaffold. Box?
[0017] FIG. 8. The rolled tube cartoon shows the sampling areas for
Live-Dead staining experiment. In a static culture, position 1c2 is
the bottom of the tube, a and b are the side of the tube and 1d2
the top of the tube.
[0018] FIG. 9. H&E staining for the rolled PDO tube from an
rSMC seeded sheet (50 micron in thickness).
[0019] FIG. 10. Seeding of hUTC on 2 mm tubular scaffolds. PDO-ESS
(A) and PDO-ESS coated with rat tail type I collagen (B) tubular
scaffolds 2 mm in diameter were secured to LumeGen bioreactor
chambers and seeded by filling the lumen with a cell suspension at
5.5.times.10.sup.5 cells/ml. The chambers were then rotated at 0.4
rpm overnight and analyzed by Live/Dead staining All images taken
at 100.times. magnification.
[0020] FIG. 11. Seeding of rat aortic smooth muscle cells on
tubular scaffolds. PDO-ESS (A) and PDO/collagen-ESS (B) tubular
scaffolds were secured to LumeGen bioreactor chambers and seeded by
filling the lumen with a cell suspension at 2.times.10.sup.6
cells/ml. The chambers were then rotated at 0.4 rpm overnight and
analyzed by Live/Dead staining All images taken at 100.times.
magnification.
[0021] FIG. 12. Exposure of rat aortic smooth muscle cells on a
tubular scaffold to fluid flow or 7 days of static culture. A
PDO-ESS tubular scaffolds .about.5 cm long were secured to LumeGen
bioreactor chambers and seeded by filling the lumen with a cell
suspension at 2.times.10.sup.6 cells/ml. The chambers were then
rotated at 0.4 rpm overnight. The next day, the tubular scaffolds
were connected to the LumeGen bioreactor and exposed to flow at a
rate of 10 ml/min for 2 hours. One tubular scaffold was then
analyzed by Live/Dead staining (A) Left side of the tube. (B) Right
side of the tube. The other tubular scaffold was incubated
statically for an additional 7 days then analyzed by Live/Dead
staining (C) Left side of the tube. (D) Right side of the tube. All
images taken at 100.times. magnification.
[0022] FIG. 13. Exposure of rat aortic smooth muscle cells on a
tubular scaffold to dynamic or static culture conditions. PDO-ESS
tubular scaffolds .about.5 cm long were secured to LumeGen
bioreactor chambers and seeded by filling the lumen with a cell
suspension at 2.times.10.sup.6 cells/ml. The chambers were then
rotated at 0.4 rpm overnight. The next day, the tubular scaffolds
were connected to the LumeGen bioreactor and exposed to flow at a
rate of 20 ml/min and pulsatile pressure ranging from 120-80 mm Hg
at a frequency of 1 Hz for 3 days. Another tubular scaffold was
cultured under static conditions for the same time period. Both
tubes were then analyzed by Live/Dead staining (A) Left side of the
tube cultured statically. (B) Right side of the tube cultured
statically. (C) Left side of the tube cultured under dynamic
conditions. (D) Right side of the tube cultured under dynamic
conditions. All images taken at 100.times. magnification.
[0023] FIG. 14. Image showed minced rat muscle tissue were
distributed uniformly over tubular constructs with different amount
of tissue
[0024] FIG. 15. After 72 hours culturing, image showed minced
tissue still attached to the tubular scaffolds
[0025] FIG. 16. Rat Smooth Muscle Cells seeded (static) on PDO
tubes for 4 days followed by 10 days in bioreactor (H&E
staining)
DETAILED DESCRIPTION OF INVENTION
[0026] The invention is a tissue engineered blood vessel (TEBV)
comprised of a biocompatible, bioabsorbable scaffold and one or
more of cells, cell sheets, cell lysate, minced tissue, and
cultured with or without a bioreactor process. Such tissue
engineered blood vessels may be used to repair or replace a native
blood vessel that has been damaged or diseased. In tissue
engineering, the rate of resorption of the scaffold by the body
preferably approximates the rate of replacement of the scaffold by
tissue. That is to say, the rate of resorption of the scaffold
relative to the rate of replacement of the scaffold by tissue must
be such that the structural integrity, e.g. strength, required of
the scaffold is maintained for the required period of time. If the
scaffold degrades and is absorbed unacceptably faster than the
scaffold is replaced by tissue growing therein, the scaffold may
exhibit a loss of strength and failure of the device may occur.
Additional surgery then may be required to remove the failed
scaffold and to repair damaged tissue. Thus, devices of the present
invention advantageously balance the properties of
biodegradability, resorption, structural integrity over time and
the ability to facilitate tissue in-growth, each of which is
desirable, useful or necessary in tissue regeneration or repair.
Such devices provide synergistic improvements over devices of the
prior art.
[0027] In general, a suitable biodegradable polymer for preparing
the scaffold is desirably configured so that it has mechanical
properties that are suitable for the intended application, remains
sufficiently intact until tissue has in-grown and healed, does not
invoke a minimal inflammatory response or toxic response, is
capable of withstanding long-term hemodynamic stress without
material failure, resistant to both thrombosis and infection and is
metabolized in the body after fulfilling its purpose, is easily
processed into the desired final product to be formed, demonstrates
acceptable shelf-life, and is easily sterilized.
[0028] The biocompatible, biodegradable scaffold may be comprised
of natural, modified natural or synthetic biodegradable polymers,
including homopolymers, copolymers and block polymers, linear or
branched, segmented or random; as well as combinations thereof.
Particularly well suited synthetic biodegradable polymers are
aliphatic polyesters which include but are not limited to
homopolymers and copolymers of lactide (which includes lactic acid
D-,L- and meso lactide), glycolide (including glycolic acid),
epsilon-caprolactone, p-dioxanone (1,4-dioxan-2-one), and
trimethylene carbonate (1,3-dioxan-2-one). In one embodiment the
polymers are poly(p-dioxanone), poly(lactide-co-glycolide)
(PLA/PGA) copolymers (95/5, 85/15, 10/90 mole-mole %), and
poly(glycolide-co-caprolatone) (PGA/PCL) 65/35 copolymers, and
poly(lactide-co-caprolatone) (PLA/PCL) (60/40 mole-mole %)
copolymers.
[0029] Suitable natural polymers include, but are not limited to
collagen, atelocollagen, elastic, and fibrin and combinations
thereof. In one embodiment, the natural polymer is collagen. In yet
another embodiment, the combination of natural polymer is a
acellular omental matrix.
[0030] The scaffold has dimensions that reflect desired ranges
that, in combination with the one or more of cells, cell sheets,
cell lysate, minced tissue, and a bioreactor process will replace a
small diameter, damaged or diseased vein or artery blood vessel.
Desirable dimensions include but are not limited to: internal
diameter (3-7 mm preferable, 4-6 mm most preferable); wall
thickness (0.1-1 mm preferable, 0.2-0.7 mm most preferable); and
length (1-20 cm preferable, 2-10 cm most preferable). The table
below shows how the properties of our PDO construct align with
those of a natural vessel.
TABLE-US-00001 Dimensions Physical Properties Wall Burst Suture
Tensile ID Thickness Length Compliance Pressure retention (peak
(mm) (mm) (cm) (%) (mmHg) (gmf) stress) PDO 2 & 5 0.5 1-20
0.5-1 1500-2500 310 5 MPa Vessel 2 & 5 0.5-0.7 1-20 0.2-10
1500-4500 100-500 2-20 MPa
[0031] The scaffold has physical properties that reflect desired
ranges that, in conjunction with one or more of cells, cell sheets,
cell lysate, minced tissue tissue, and a bioreactor process will
replace a small diameter, damaged or diseased vein or artery blood
vessel. Desirable physical properties include but are not limited
to: compliance (0.2-10% preferable, 0.7-7% most preferable); suture
retention strength (100 gm-4 Kg preferable, 100-300 gm most
preferable); burst strength/pressure (1000-4500 mm Hg preferable,
1500-4500 mm Hg most preferable with greater than 100 mmHg during
the bioreactor process); kink resistance (resist kinking during
handling during all stages of process-cell seeding, bioreactor,
implantation, life of patient); and in-vitro strength retention (1
day-1 yr maintain enough strength until cell and ECM growth
overcomes physical property losses of scaffold; 1 day-3 mos under
bioreactor "flow" conditions preferable). The scaffold should also
have desirable tensile properties (radial and axial) that include
but are not limited to: elastic modulus (MPa) of longitudinal/axial
(1-200 preferable; 5-100 most preferable) and orthogonal/radial
(0.1-100 preferable, 0.5-50 most preferable) and random (0.1-100
preferable, 0.5-50 most preferable) and wet/longitudinal (5-100
preferable, 25-75 preferable); a peak stress (MPa) of
longitudinal/axial (1-30 preferable; 2-20 most preferable) and
orthogonal/radial (0.5-15n preferable, 1-10 most preferable) and
random (0.5-15 preferable, 1-10 most preferable) and wet/long (1-30
preferable; 2-20 most preferable); failure strain (%) of
longitudinal/axial (1-200 preferable; 5-75 most preferable) and
orthogonal/radial (5-400 preferable, 10-300 most preferable) and
random (5-400 preferable, 10-300 most preferable) and wet/long
(1-200 preferable; 20-100 most preferable).
[0032] The scaffold has morphology that reflects desired ranges
that, in conjunction with one or more of cells, cell sheets, cell
lysate, minced tissue, and a bioreactor process will replace a
small diameter, damaged or diseased vein or artery blood vessel.
Desirable morphology includes but is not limited to: pore size
(1-200 um preferable, most preferable less than 100 um); porosity
(40-98% preferable, most preferable 60-95%); surface area/vol
(0.1-7 m.sup.2/cm.sup.3 preferable, most preferable 0.3-5.5
m.sup.2/cm.sup.3); water permeability (1-10 ml cm.sup.2/min @80-120
mmHg preferable, most preferable <5 ml cm.sup.2/min @120 mmHg);
and orientation of polymer/fibers (allows proper cell seeding,
adherence, growth, and ECM formation). Polymer/fiber orientation
will also allow proper cell migration; also important for the
minced tissue fragments such that cells will migrate out of the
fragments and populate the scaffold.
[0033] The scaffold has biocompatibility that reflects desired
properties for a scaffold that, in conjunction with one or more of
cells, cell sheets, cell lysate, minced tissue and a bioreactor
process will replace a small diameter, damaged or diseased vein or
artery blood vessel. Desirable biocompatibility includes but is not
limited to: absorption (1 wk-4-yrs preferable, most preferable 4
wks-30 wks to allow greatest vol of scaffold to be occupied by
cells and ECM); tissue reaction (minimal); cell compatibility
(adherence, viability, growth, migration and differentiation not
negatively impacted by scaffold); residual solvent (minimal);
residual EtO (minimal); and hemocompatible (non-thrombogenic).
[0034] The scaffold has other factors that reflect desired
properties for a scaffold that, in conjunction with one or more of
cells, cell sheets, cell lysate, minced tissue, tissue and a
bioreactor process will replace a small diameter, damaged or
diseased vein or artery blood vessel. Desirable factors includes
but are not limited to: surface energy (allows proper cell seeding,
adherence, growth, migration and ECM formation); surface chemistry
(addition of factors such as oxygen, surface roughness or
topography (can be utilized to affect cell attachment and other
cell functions), nanoscale features on the surface preferably in a
size range of 10-1200 nanometers; more preferably 25-900 nanometers
(allow preferential endothelial cell attachment), NO, free-radical
scavengers allows proper cell seeding, adherence, growth, and ECM
formation); cell mediators (addition of factors such as matrix
proteins allows proper cell seeding, adherence, growth, migration
and ECM formation); hydrophobicity/hydrophilicity (proper balance
of hydrophobicity/hydrophilicity allows proper cell seeding,
adherence, growth, and ECM formation). Other surface modifications
include providing electrical microcurrent in a form of coating a
surface with galvanic materials. In particular, zinc and copper
(0.01 microns-0.1 microns) can act as an electrical current source
enhancing endothelial and smooth muscle cell attachment and
proliferation.
[0035] Non-limiting examples of a scaffold that may be used in the
present invention include textile structures such as felts, weaves,
knits, braids, meshes, non-wovens, warped knits; foams, including
porous foams and semi-porous foams; perforated films or sheets;
patterned films or sheets or fibers; and combinations thereof. As
used herein, the term "nonwoven fabric" includes, but is not
limited to, bonded fabrics, formed fabrics, or engineered fabrics,
that are manufactured by processes other than spinning, weaving or
knitting. More specifically, the term "nonwoven fabric" refers to a
porous, textile-like material, usually in flat sheet form, composed
primarily or entirely of staple fibers assembled in a web, sheet or
batt. The fiber diameter is preferably 10 nm to 100 um and more
preferably 25 nm to 10 um. In one embodiment, the scaffold is a
textile, a foam and combinations thereof.
[0036] In another embodiment, the scaffold is a textile comprised
of fibers prepared by electrostatic spinning, extrusion, injection
molding, as well as any pre- or post-processes (ex. laser cutting
to form pours in extruded tube). In one embodiment, the scaffold is
a textile prepared by electrostatic spinning. In the
electrostatically spun scaffold process, an electrical force is
applied to the polymeric solution that overcomes the surface
tension of the solution, forming a charged jet. This jet of
solution is ejected, dried and solidified onto a substrate to form
a sheet, tube or other construct comprised of the electrostatically
spun fibers. Spinnability of the polymeric solution is controlled
by several parameters that include but are not limited to:
concentration (a concentration that allows polymer/solvent solution
to be spun and yield fibers that form a proper scaffold (1-50 w/v %
preferable); solvent (a solvent that dissolves the polymer in the
given concentration range, HFIP preferable); solution viscosity
(10-300 mg/ml preferable, 25-250 mg/ml most preferable (50-3000
centipoise)). By controlling the spinning conditions, the resulting
fibers can range from about 0.1 .mu.m to about 10 m and preferably
will range from about 0.3 .mu.m to about 5.0 .mu.m.
[0037] Other processing parameters for electrostatic spinning that
are important include but are not limited to: voltage potential
(10-100 kV preferable, most preferable 15-30 kV); flow rate (0.1-20
ml/hr preferable, 1-15 ml/hr most preferable); gap/tip distance
(1-35 cm preferable, 2.5-25 cm most preferable); rotation/mandrel
rate/speed (10-5,000 rpm preferable, 50-3000 rpm most preferable).
The fibers can also be spun from the melt.
[0038] Optionally, the strength of the electrostatically spun
scaffold (ESSC) may be improved by bonding the fibers of the
aforementioned construct. Bonding of the fibers may be accomplished
by coating the ESSC with a low melting materials such as PCL and
low molecular PLGA copolymers. After coating the scaffold, a post
process using a heat press may be performed. The process melts the
coated layer on the reinforcement fibers. The molten coating
between the electrospun fibers is compressed and fuses the fibers
together upon cooling to room temperature. Alternatively, during
the electrospinning process, electrospun fibers are exposed with
the vapor of the solvent. Upon curing, the fibers fuse together
thereby strengthening the scaffold.
[0039] In one embodiment the textiles and ESSC scaffolds are
prepared from polymers including, but not limited to are
poly(p-dioxanone), poly(lactide-co-glycolide) (PLA/PGA) copolymers
(95/5, 85/15, 10/90 mole-mole %), and collagen.
[0040] In another embodiment the scaffold is a foam. In one
embodiment, the foam scaffolds are prepared from elastomeric
copolymers. Suitable bioabsorbable, biocompatible elastomeric
copolymers include but are not limited to copolymers of
epsilon-caprolactone and glycolide (preferably having a mole ratio
of epsilon-caprolactone to glycolide of from about 30:70 to about
70:30, preferably 35:65 to about 65:35, and more preferably 45:55
to 35:65); elastomeric copolymers of epsilon-caprolactone and
lactide, including L-lactide, D-lactide blends thereof or lactic
acid copolymers (preferably having a mole ratio of
epsilon-caprolactone to lactide of from about 35:65 to about 65:35
and more preferably 45:55 to 30:70;) elastomeric copolymers of
p-dioxanone (1,4-dioxan-2-one) and lactide including L-lactide,
D-lactide and lactic acid (preferably having a mole ratio of
p-dioxanone to lactide of from about 40:60 to about 60:40);
elastomeric copolymers of epsilon-caprolactone and p-dioxanone
(preferably having a mole ratio of epsilon-caprolactone to
p-dioxanone of from about 30:70 to about 70:30); elastomeric
copolymers of p-dioxanone and trimethylene carbonate (preferably
having a mole ratio of p-dioxanone to trimethylene carbonate of
from about 30:70 to about 70:30); elastomeric copolymers of
trimethylene carbonate and glycolide (preferably having a mole
ratio of trimethylene carbonate to glycolide of from about 30:70 to
about 70:30); elastomeric copolymer of trimethylene carbonate and
lactide including L-lactide, D-lactide, blends thereof or lactic
acid copolymers (preferably having a mole ratio of trimethylene
carbonate to lactide of from about 30:70 to about 70:30) and blends
thereof. In one embodiment, the elastomeric copolymer is
poly(glycolide-co-caprolatone) (PGA/PCL) 65/35 copolymer or a
poly(lactide-co-caprolatone) (PLA/PCL) (60/-40 mole-mole %)
copolymer.
[0041] Foam scaffolds may be prepared by conventional processes
such as, lyophilization, supercritical solvent foaming (i.e., as
described in EP 464,163 B1), gas injection extrusion, gas injection
molding or casting with an extractable material In one embodiment,
the foams are prepared by lyophilization. Suitable methods for
lyophilizing elastomeric polymers such as 65/35 PGA/PCL to form
foams is described in the examples of U.S. Pat. No. 6,355,699,
"Process for Manufacturing Biomedical Foams", assigned to Ethicon,
Inc incorporated herein by reference in its entirety.
[0042] In another embodiment, leachables can be introduced into the
scaffold as an additional method to form pores. Suitable leachable
solids include nontoxic leachable materials such as salts (e.g.,
sodium chloride, potassium chloride, calcium chloride, sodium
tartrate, sodium citrate, and the like), biocompatible mono and
disaccharides (e.g., glucose, fructose, dextrose, maltose, lactose
and sucrose), polysaccharides (e.g., starch, alginate, chitosan),
water soluble proteins (e.g., gelatin and agarose).
[0043] The foams have microstructures suitable for tissue
engineering. The features of such foams can be controlled to suit a
desired application by choosing the appropriate conditions to form
the foam during lyophilization. These features in absorbable
polymers have distinct advantages over the prior art where the
scaffolds are typically isotropic or random structures. However, it
is preferred that foams used in tissue engineering (i.e. repair or
regeneration) have a structure that provides organization at the
microstructural level that provides a template that facilitates
cellular organization and regeneration of tissue that has the
anatomical, biomechanical, and biochemical features of normal
tissues. These foams can be used to repair or regenerate tissue
(including organs) in animals such as domestic animals, primates
and humans.
[0044] The features of such foams can be controlled to suit desired
application by selecting the appropriate conditions for
lyophilization to obtain one or more of the following properties:
(1) interconnecting pores of sizes ranging from about 10 to about
200 .mu.m (or greater) that provide pathways for cellular ingrowth
and nutrient diffusion; (2) a variety of porosities ranging from
about 20% to about 98% and preferably ranging from about 50% to
about 95%; (3) gradient in the pore size across one direction for
preferential cell culturing; (4) channels that run through the foam
for improved cell invasion, vascularization and nutrient diffusion
(5) micro-patterning of pores on the surface for cellular
organization; (6) tailorability of pore shape and/or orientation
(e.g. substantially spherical, ellipsoidal, columnar); (7)
anisotropic mechanical properties; (8) composite foams with a
polymer composition gradient to elicit or take advantage of
different cell response to different materials; (9) blends of
different polymer compositions to create structures that have
portions that will break down at different rates; (10) foams
co-lyophilized or coated with pharmaceutically active compounds;
(11) and the ability to make 3 dimensional shapes and devices with
preferred microstructures. The inner, or luminal, layer of the
scaffold may be optimized for endothelialization through control of
the porosity of the surface and the possible addition of a surface
treatment. The outermost, or adventitial, layer of the scaffold may
be tailored to support smooth muscle cell growth, again by
optimizing the porosity (percent porosity, pore size, pore shape
and pore size distribution) and by incorporating bioactive factors,
pharmaceutical agents, or cells. There may or may not be a barrier
layer with low porosity disposed between these two porous layers to
increase strength and decrease leakage. Such structural features of
the scaffold described herein can also be found in textiles such
as, electrostatically spun scaffolds and other scaffolds described
herein.
[0045] In one embodiment, the scaffold is a combination of foams
and textiles. Textiles include woven, knitted, warped knitted
(i.e., lace-like), non-woven, and braided structures that act as a
reinforcement for the scaffold. The reinforcement should have a
sufficient density to permit suturing, but the density should not
be so great as to impede proper bonding between the foam and the
textile. The reinforcing material may also be formed from a thin,
perforation-containing elastomeric sheet with perforations to allow
tissue ingrowth.
[0046] For example, the present invention also provides a composite
scaffold comprising a first layer that is a textile layer and a
second layer of biocompatible foam or ESSC. This composite
structure allows for the creation of structures with unique
mechanical properties. In one embodiment the textile layer could
allow the use of sutures, staples or various fixation devices to
hold the composite in place. Generally, the textile has a thickness
in the range of about 1 micron to 500 microns. The textile layer
allows the composite to have variable mechanical strength depending
on the design, a different bioabsorption profile, and a different
microenvironment for cell invasion and seeding, which are
advantageous in a variety of medical applications. The textile
layer may be made from a variety of biocompatible polymers and
blends of biocompatible polymers, which are preferably
bioabsorbable. The biocompatible foam or ESSC may be either contain
gradients or channels. The gradient structure has a substantially
continuous transition in at least one characteristic selected from
the group consisting of composition, stiffness, flexibility,
bioabsorption rate, pore architecture and/or microstructure. The
gradient structure can be made from a blend of absorbable polymers
that form compositional gradient transitions from one polymeric
material to a second polymeric material. In situations where a
single chemical composition is sufficient for the application, the
invention provides a composite that may have microstructural
variations in the structure across one or more dimensions that may
mimic the anatomical features of the tissue. The channeled
structure provides channels that extend through the foam to
facilitate cell migration and nutrient flow throughout the
channeled structure.
[0047] In another embodiment the foam or ESSC may have a textile
fused to the top or bottom surface. This way, surface properties of
the structure can be controlled such as porosity, permeability,
degradation rate and mechanical properties. The textile can be
produced via conventional techniques, described herein, and in
which a textile can be built up on a lyophilized foam surface. The
textile can be produced via an electrostatic spinning process in
which a ESSC can be built up on a lyophilized foam surface.
[0048] The scaffold may include one or more layers of each of the
foam or ESSC and reinforcement components. Preferably, adjacent
layers of foam or ESSC are also integrated by at least a partial
interlocking of the pore-forming webs or walls in the adjacent
layers.
[0049] In one embodiment the scaffold may be coated with natural
polymers to enhance cellular compatibility. Suitable natural
polymers include, but are not limited to collagen, atelocollagen,
elastin, hyaluronic acid and fibrin and combinations thereof. In
one embodiment, the natural polymer is collagen. In yet another
embodiment, the combination of natural polymer is acellular omental
matrix.
[0050] In one embodiment, the tissue engineered blood vessel is
comprised of a biocompatible, bioabsorbable scaffold and cells. The
scaffold is as described herein above.
[0051] Suitable cells that may be combined with the scaffold
include, but are not limited to, stem cells such as multipotent or
pluripotent stem cells; progenitor cells, such as smooth muscle
progenitor cells and vascular endothelium progenitor cells;
embryonic stem cells; postpartum tissue derived cells such as,
placental tissue derived cells and umbilical tissue derived cells,
endothelial cells, such as vascular endothelial cells; smooth
muscle cells, such as vascular smooth muscle cells; precursor cells
derived from adipose tissue; and arterial cells such as, cells
derived from the radial artery and the left and right internal
mammary artery (IMA), also known as the internal thoracic
artery.
[0052] In one embodiment, the TEBV comprises a scaffold as
described herein above and human umbilical tissue derived cells
(hUTCs). The methods for isolating and collecting human umbilical
tissue-derived cells (hUTCs) (also referred to as umbilical-derived
cells (UDCs)) are described in copending U.S. application Ser. No.
10/877,012 incorporated herein by reference in its entirety. In
another embodiment, the TEBV comprises a scaffold as described
herein above, human umbilical tissue derived cells (hUTCs) and one
or more other cells. The one or more other cells includes, but is
not limited to vascular smooth muscle cells (SMCs), vascular smooth
muscle progenitor cells, vascular endothelial cells (ECs), or
vascular endothelium progenitor cells, and/or other multipotent or
pluripotent stem cells. hUTCs in combination with one or more other
cells on the scaffold may enhance the seeding, attachment, and
proliferation of for example ECs and SMCs on the scaffold. hUTCS
may also promote the differentiation of the EC or SMC or progenitor
cells in the scaffold construct. This may promote the maturation of
TEBVs during the in vitro culture as well as the engraftment during
the in vivo implantation. hUTCs may provide trophic support, or
provide and enhance the expression of ECM proteins. The trophic
effects of the cells, including hUTCs, can lead to proliferation of
the vascular smooth muscle or vascular endothelium of the patient.
The trophic effects of the cells, including hUTCs, may induce
migration of vascular smooth muscle cells, vascular endothelial
cells, skeletal muscle progenitor cells, vascular smooth muscle
progenitor cells, or vascular endothelium progenitor cells to the
site or sites of the regenerated blood vessel.
[0053] Cells can be harvested from a patient (before or during
surgery to repair the tissue) and the cells can be processed under
sterile conditions to provide a specific cell type. One of skill in
the art is aware of conventional methods for harvesting and
providing the cells as describe above such as described in
Osteoarthritis Cartilage 2007 February; 15(2):226-31 and
incorporated herein by reference in their entirety.
[0054] The cells can be seeded on the scaffolds of the present
invention for a short period of time, e.g. less than one day, just
prior to implantation, or cultured for longer a period, e.g.
greater than one day, to allow for cell proliferation and
extracellular matrix synthesis within the seeded scaffold prior to
implantation. In one embodiment, a single cell type is seeded on
the scaffold. In another embodiment, one or more cell types are
seeded on the scaffold. Various cellular strategies could be used
with these scaffolds (i.e., autologous, allogenic, xenogeneic cells
etc.).
[0055] In another embodiment the cells are genetically modified to
express genes of interest responsible for pro-angiogenic activity,
anti-inflammatory activity, cell survival, cell proliferation or
differentiation or immunemodulation.
[0056] In another embodiment the tissue engineered blood vessel is
comprised of a biocompatible, bioabsorbable scaffold and cell
sheets. Cell sheets may be made of hUTCs or other cell types.
Methods of making cell sheets are as described in copending U.S.
application Ser. No. 11/304,091 incorporated herein by reference in
its entirety. The cell sheet is generated using thermoresponsive
polymer coated dishes that allow harvesting intact cell sheets with
the decrease of the temperature. Alternatively, other methods of
making cell sheets include, but are not limited to growing cells in
a form of cell sheets on a polymer film. Selected cells may be
cultured on a surface of glass, ceramic or a surface-treated
synthetic polymer. For example, polystyrene that has been subjected
to a surface treatment, like gamma-ray irradiation or silicon
coating, may be used as a surface for cell culture. Cells grown to
over 85% confluence form cell sheet layer on cell growth support
device. Cell sheet layer may be separated from cell growth support
device using proteolysis enzymes, such as trypsin or dispase.
Non-enzymatic cell dissociation could also be used. A non-limiting
example includes a mixture of chelators sold under the trade name
CELLSTRIPPER (Mediatech, Inc., Herndon, Va.), a non-enzymatic cell
dissociation solution designed to gently dislodge adherent cells in
culture while reducing the risk of damage associated with enzymatic
treatments.
[0057] Alternatively, the surface of the cell growth support
device, from which cultured cells are collected, may be a bed made
of a material from which cells detach without a proteolysis enzyme
or chemical material. The bed material may comprise a support and a
coating thereon, wherein the coating is formed from a polymer or
copolymer which has a critical solution temperature to water within
the range of 0.degree. C. to 80.degree. C.
[0058] In one embodiment, one or more cells sheets are combined
with the scaffold as described herein above by wrapping the cell
sheet or sheets around the scaffold. The one or more cell sheets
may be of the same cell type or of different cell types as
described herein above. In one embodiment, multiple cell sheets
could be combined to form a robust vascular construct. For example,
cell sheets made of endothelial cells and smooth muscle cells could
be combined with the scaffold to form TEBVs. Alternatively, other
cell types such as hUTC cell sheets could be combined with
endothelial cell sheets and the scaffold to form TEBV. Furthermore,
cell sheets made of hUTCs can be wrapped around a pre-formed TEBV
composed of a scaffold, ECs, and SMCs to provide trophic factors
supporting maturation of the construct.
[0059] Cell sheets may be grown on the scaffold to provide
reinforcement and mechanical properties to the cell sheets.
Reinforced cell sheets can be formed by placing biodegradable or
non-biodegradable reinforcing members at the bottom of support
device prior to seeding support device with cells. Reinforcing
members are as described herein above. Cell sheet layer that
results will have incorporated the reinforcing scaffold providing
additional strength to cell sheet layer, which can be manipulated
without the requirement for a backing layer. A preferred
reinforcing scaffold is a mesh comprised of poly(dioxanone). The
mesh can be placed at the bottom of a Corning.RTM. Ultra low
attachment dish. Cells can then be seeded on to the dishes such
that they will form cell-cell interactions but also bind to the
mesh when they interact with the mesh. This will give rise to
reinforced cell sheets with better strength and handling
characteristics. Such reinforced cell sheets may be rolled into
TEBV or the reinforced cell sheet layer may be disposed on a
scaffold (as described above).
[0060] In another embodiment, the cell sheet is genetically
engineered. The genetically engineered cell sheet comprises a
population of cells wherein at least one cell of the population of
cells is transfected with an exogenous polynucleotide such that the
exogenous polynucleotide expresses express diagnostic and/or
therapeutic product (e.g., a polypeptide or polynucleotide) to
assist in tissue healing, replacement, maintenance and diagnosis.
Examples of "proteins of interest" (and the genes encoding same)
that may be employed herein include, without limitation, cytokines,
growth factors, chemokines, chemotactic peptides, tissue inhibitors
of metalloproteinases, hormones, angiogenesis modulators either
stimulatory or inhibitory, immune modulatory proteins,
neuroprotective and neuroregenerative proteins and apoptosis
inhibitors. More specifically, preferred proteins include, without
limitation, erythropoietin (EPO), EGF, VEGF, FGF, PDGF, IGF, KGF,
IFN-.alpha., IFN-.delta., MSH, TGF-a, TGF-.beta., TNF-.alpha.,
IL-1, BDNF, GDF-5, BMP-7 and IL-6.
[0061] In another embodiment the tissue engineered blood vessel is
comprised of a biocompatible, bioabsorbable scaffold and cell
lysate. Cell lysates may be obtained from cells including, but not
limited to stem cells such as multipotent or pluripotent stem
cells; progenitor cells, such as smooth muscle progenitor cells and
vascular endothelium progenitor cells; embryonic stem cells;
postpartum tissue derived cells such as, placental tissue derived
cells and umbilical tissue derived cells, endothelial cells, such
as vascular endothelial cells; smooth muscle cells, such as
vascular smooth muscle cells; precursor cells derived from adipose
tissue; and arterial cells such as, cells derived from the radial
artery and the left and right internal mammary artery (IMA), also
known as the internal thoracic artery. The cell lysates and cell
soluble fractions may be stimulated to differentiate along a
vascular smooth muscle or vascular endothelium pathway. Such
lysates and fractions thereof have many utilities. Use of lysate
soluble fractions (i.e., substantially free of membranes) in vivo,
for example, allows the beneficial intracellular milieu to be used
allogenically in a patient without introducing an appreciable
amount of the cell surface proteins most likely to trigger
rejection, or other adverse immunological responses. Methods of
lysing cells are well-known in the art and include various means of
mechanical disruption, enzymatic disruption, or chemical
disruption, or combinations thereof. Such cell lysates may be
prepared from cells directly in their growth medium and thus
containing secreted growth factors and the like, or may be prepared
from cells washed free of medium in, for example, PBS or other
solution.
[0062] In yet another embodiment the tissue engineered blood vessel
is comprised of a biocompatible, bioabsorbable scaffold and minced
tissue. Minced tissue has at least one viable cell that can migrate
from the tissue fragments onto the scaffold. More preferably, the
minced tissue contains an effective amount of cells that can
migrate from the tissue fragments and begin populating the
scaffold. Minced tissue may be obtained from one or more tissue
sources or may be obtained from one source. Minced tissue sources
include, but are not limited to muscle tissue, such as skeletal
muscle tissue and smooth muscle tissue; vascular tissue, such as
venous tissue and arterial tissue; skin tissue, such as endothelial
tissue; and fat tissue.
[0063] The minced tissue is prepared by first obtaining a tissue
sample from a donor (autologous, allogeneic, or xenogeneic) using
appropriate harvesting tools. The tissue sample is then finely
minced and divided into small fragments either as the tissue is
collected, or alternatively, the tissue sample can be minced after
it is harvested and collected outside the body. In embodiments
where the tissue sample is minced after it is harvested, the tissue
samples can be washed three times in phosphate buffered saline. The
tissue can then be minced into small fragments in the presence of a
small quantity, for example, about 1 ml, of a physiological
buffering solution, such as, phosphate buffered saline, or a matrix
digesting enzyme, such as 0.2% collagenase in Ham's F12 medium. The
tissue is minced into fragments of approximately 0.1 to 1 mm.sup.3
in size. Mincing the tissue can be accomplished by a variety of
methods. In one embodiment, the mincing is accomplished with two
sterile scalpels cutting in parallel and opposing directions, and
in another embodiment, the tissue can be minced by a processing
tool that automatically divides the tissue into particles of a
desired size. In one embodiment, the minced tissue can be separated
from the physiological fluid and concentrated using any of a
variety of methods known to those having ordinary skill in the art,
such as for example, sieving, sedimenting or centrifuging. In
embodiments where the minced tissue is filtered and concentrated,
the suspension of minced tissue preferably retains a small quantity
of fluid in the suspension to prevent the tissue from drying
out.
[0064] The suspension of minced living tissue can be used to create
a TEBV according to the present invention by depositing the
suspension of living tissue upon a biocompatible scaffold, such
that the tissue and the scaffold become associated. Preferably, the
tissue is associated with at least a portion of the scaffold. The
TEBV can be implanted in a subject immediately, or alternatively,
the construct can be incubated under sterile conditions that are
effective to maintain the viability of the tissue sample.
[0065] In another aspect of the invention, the minced tissue could
consist of the application of two distinct minced tissue sources
(e.g., one surface could be loaded with minced endothelial tissue
and the other surface could be loaded with mince smooth muscle
tissue).
[0066] In one embodiment, the tissue engineered blood vessels
comprising a scaffold and one or more of cells, cell sheets, cell
lysate, or minced tissue is enhanced by combining with bioactive
agents. Suitable bioactive agents include, but are not limited to
an antithrombogenic agent, an anti-inflammatory agent, an
immunosuppressive agent, an immunomodulatory agent, pro-angiogenic,
an antiapoptotic agent, antioxidants, growth factors, angiogenic
factors, myoregenerative or myoprotective drugs, conditioned
medium, extracellular matrix proteins, such as, collagen,
atelocollagen, laminin, fibronectin, vitronectin, tenascin,
integrins, glycosaminoglycans (hyaluronic acid, chondroitin
sulfate, dermatan sulfate, heparan sulfate, heparin, keratan
sulfate and the like), elastin and fibrin; growth factors and/or
cytokines, such as vascular endothelial cell growth factors,
platelet derived growth factors, epidermal growth factors,
fibroblast growth factors, hepatocyte growth factors, insulin-like
growth factors, and transforming growth factors.
[0067] Conditioned medium from cells as described previously herein
allows the beneficial trophic factors secreted by the cells to be
used allogeneically in a patient without introducing intact cells
that could trigger rejection, or other adverse immunological
responses. Conditioned medium is prepared by culturing cells in a
culture medium, then removing the cells from the medium.
Conditioned medium prepared from populations of cells, including
hUTCs, may be used as is, further concentrated, for example, by
ultrafiltration or lyophilization, or even dried, partially
purified, combined with pharmaceutically-acceptable carriers or
diluents as are known in the art, or combined with other bioactive
agents Conditioned medium may be used in vitro or in vivo, alone or
combined with autologous or allogenic live cells, for example. The
conditioned medium, if introduced in vivo, may be introduced
locally at a site of treatment, or remotely to provide needed
cellular growth or trophic factors to a patient. This same medium
may also be used for the maturation of the TEBVs. Alternatively,
hUTC or other cells conditioned medium may also be lyophilized onto
the scaffolds prior to seeding with both ECs and SMCs.
[0068] From a manufacturing perspective, hUTCs or other cells, or
conditioned medium may shorten the time for the in vitro culture or
fabrication of TEBVs. This will also result in the use of less
starting cells making autologous sources of ECs and SMCs a more
viable option.
[0069] In one embodiment, the tissue engineered blood vessels
comprising a scaffold and one or more of cells, cell sheets, cell
lysate, or minced tissue is enhanced by combining with bioactive
agents. These tissue engineered blood vessels may be cultured with
or without a bioreactor process. The TEBV scaffolds may be cultured
using various cell culture bioreactors, including but not limited
to a spinner flask, a rotating wall vessel (RWV) bioreactor, a
perfusion-based bioreactor or combination thereof. In one
embodiment the cell culture bioreactor is a rotating wall vessel
(RWV) bioreactor or a perfusion-based bioreactor. The
perfusion-based bioreactor will consist of a device for securing
the TEBV scaffolds and allow culture medium to flow through the
lumen of the scaffolds, and may also allow for seeding and
culturing of cells on both the inner (lumen) and outer surfaces of
the scaffolds. The perfusion bioreactors may also have the
capability of generating pulsatile flow and various pressures for
conditioning of the cell-seeded scaffolds prior to implantation.
pulsatile flow stress during bioreactor process (1-25
dynes/cm.sup.2 over lday-lyr preferable; more preferably a gradual
increase from 1-25 dynes/cm.sup.2 over 2-4-wks).
[0070] The scaffolds having cells, cell sheets, cell lysate, or
minced tissue and optionally bioactive agents may be cultured for
longer a period, e.g. greater than one day, to allow for cell
proliferation and matrix synthesis within the scaffold prior to
implantation. Cells cell sheets, cell lysate, or minced tissue are
applied to the scaffold as described herein above and transferred
to the bioreactor for longer term culture, or more preferably,
seeded and cultured within the bioreactor. Multiple bioreactors may
be also used sequentially, e.g. one for initial seeding of cells,
and another for long-term culture.
[0071] The process of seeding and culturing cells with a scaffold
using a bioreactor may be repeated with multiple cell types
sequentially, e.g. smooth muscle cells are seeded and cultured for
a period of time, followed by seeding and culture of endothelial
cells, or simultaneously (e.g. smooth muscle cells on the outer
surface, and endothelial cells with on the inner surface (lumen) of
the scaffolds). The construct may or may not be cultured for a
period of time to promote maturation. The bioreactor conditions can
be controlled as to promote proper maturation of the construct.
Following the culture period the construct can be removed and
implanted into a vascular site in an animal or human.
[0072] General cell culture conditions include temperatures of
37.degree. C. and 5% CO.sub.2. The cell seeded constructs will be
cultured in a physiological buffered salt solution maintained at or
near physiological pH. Culture media can be supplemented with
oxygen to support metabolic respiration. The culture media may be
standard formulations or modified to optimally support cell growth
and maturation in the construct. The culture media may contain a
buffer, salts, amino acids, glucose, vitamins and other cellular
nutrients. The media may also contain growth factors selected to
establish endothelial and smooth muscle cells within the construct.
Examples of these may include VEGF, FGF2, angiostatin, endostatin,
thrombin and angiotensin II. The culture media may also be perfused
within the construct to promote maturation of the construct. This
may include flow within the lumen of the vessel at pressures and
flow rates that may be at or near values that the construct may be
exposed to upon implant.
[0073] The media is specific for the cell type being cultured
(i.e., endothelial medium for endothelial cells, and smooth muscle
cell medium for SMCs). For the perfusion bioreactor especially,
there are other considerations taken into account such as but not
limited to shear stress (related to flow rate), oxygen tension, and
pressure.
[0074] The TEBVs can be also be electrically stimulated to enhance
the attachment or proliferation of the different cell types. The
electrical stimulation can be performed during the culture and
expansion of the cells prior to the fabrication of the TEBV, during
the maturation phase of the TEBV, or during implantation. Cells,
including hUTCs may also be electrically stimulated during the
production of conditioned medium.
[0075] The present invention also provides a method for the repair
or regeneration of tissue inserting the TEBV described above at a
location on the blood vessel in need of repair. These TEBV
structures are particularly useful for the regeneration of tissue
between two or more different types of tissues. For a
multi-cellular system in the simplest case, one cell type could be
present on one side of the scaffold and a second cell type on the
other side of the scaffold. Examples of such regeneration can be
(a) vascular tissue with smooth muscle on the outside and
endothelial cells on the inside to regenerate vascular
structures.
[0076] The invention also relates to methods of treating tissue
using the TEBV prepared by the methods described herein. The TEBV
can be used in arteriovenous grafting, coronary artery grafting or
peripheral artery grafting. For example, in a typical AV
(arteriovenous) surgical procedure used for the treatment of
end-stage renal failure patients, the surgeon makes an incision
through the skin and muscle of the forearm. An artery and a vein
are selected (usually the radial artery and the cephalic vein) and
an incision is made into each. The TEBV is then used to anastomos
the ends of the artery and the vein. The muscle and skin are then
closed. After the graft has properly healed (4-6 weeks), the
successful by-pass can be used to treat the patients blood.
[0077] In a coronary by-pass (CABG) procedure, a TEBV would be used
for patients suffering from arteriosclerosis, a common arterial
disorder characterized by arterial walls that have thickened, have
lost elasticity, and have calcified. This leads to a decrease in
blood supply which can lead to damage to the heart, stroke and
heart attackes. In a typical CABG procedure, the surgeon opens the
chest via a sternotomy. The heart's functions are taken over by a
Heart and Lung machine. The diseased artery is located and one end
of the TEBV is sewn onto the coronary arteries beyond the blockages
and the other end is attached to the aorta. The heart is restarted,
the sternum is wired together and the incisions are sutured closed.
Within a few weeks, the successful by-pass procedure is fully
healed and the patient is functioning normally.
[0078] The following examples are illustrative of the principles
and practice of this invention, although not limited thereto.
Numerous additional embodiments within the scope and spirit of the
invention will become apparent to those skilled in the art once
having the benefit of this disclosure.
EXAMPLES
Example 1
Tubular Scaffolds of Bioabsorbable Polymers Fabricated by
Electrospinning Processes
PDO Electrospun Tubes
[0079] 1) From High Concentration (140 mg/ml)
[0080] Solutions of 140 mg/mL of poly(p-dioxanone) (PDO) (Ethicon,
Inc., Somerville, N.J.) were made with
1,1,1,3,3,3-hexafluoro-2-propanol (HFP, TCI America Inc., Portland,
Oreg.) solvent. Solutions were left in a box (dark environment)
overnight on a shaker plate to ensure that all PDO had dissolved
and formed a homogenous solution. 4 mL of polymer solution were
then drawn into a plastic syringe (5 ml) (Beckton Dickinson,
Franklin Lakes, N.J.) and placed in a syringe pump (KD Scientific
Model 100, Holliston, Mass.) to be dispensed at a rate of 8 ml/h. A
high voltage power supply (Spellman CZE1000R; Spellman High Voltage
Electronics Corporation, Hauppauge, N.Y.)) was used to apply a
voltage of +25 kV to a blunt tip 18 gauge needle fixed to the
solution containing syringe. Solutions were electrospun onto a 5 mm
diameter cylindrical grounded mandrel placed 8 inches from the
needle tip and rotating at a rate of .about.400 rpm to produce a
scaffold of randomly oriented fibers. Mandrel translation distance
was 18 cm, with a translational speed of 18 cm/s to ensure even
coverage along the length of the mandrel. Immediately after
electrospinning, the mandrel and scaffold were quickly immersed in
an ethanol bath, and the scaffold was carefully slid off the
mandrel. The tube (inner diameter: 5 mm, thickness: .about.500
microns, length: 10 cm) was then placed in a fume hood for 30
minutes to allow for the evaporation of any residual ethanol.
2) From Medium Concentration (100 mg/ml)
[0081] Solutions of 100 mg/ml of PDO were made by placing the
polymer in HFP solvent and leaving the solution overnight in the
dark on a shaker plate to ensure that all PDO was dissolved and
forms a homogenous solution. The desired volume of polymer solution
is then drawn into a plastic Beckton Dickinson syringe and placed
in the syringe pump to be dispensed at a rate of 10 ml/hr. Two high
voltage power supplies were used. One was used to apply a voltage
of +20 kV to a blunt tip 18 gauge needle fixed to the solution
containing syringe, while the other provides -8 kV to a flat metal
target 5'' in diameter placed 6'' behind the grounded mandrel (2 or
5 mm in diameter). The grounded mandrel was placed 8'' from the
needle tip and rotating at a rate of .about.400 rpm to produce a
scaffold of randomly oriented fibers. Mandrel translation distance
was 18 cm, with a translational speed of 18 cm/s. For tubular
constructs, immediately after electrospinning the mandrel and
scaffold were quickly immersed in an ethanol bath to assist in
sliding the tube off the mandrel. Tubes were then placed in a fume
hood for 30 minutes to allow for the evaporation of any residual
ethanol.
3) From Low Concentration (60 mg/ml)
[0082] Solutions of 60 mg/mL of PDO were made with HFP solvent.
Solutions were left in a box (dark environment) overnight on a
shaker plate to ensure that all PDO had dissolved and formed a
homogenous solution. 15 mL of polymer solution were then drawn into
a plastic Beckton Dickinson syringe (30 ml) and placed in the
syringe pump to be dispensed at a rate of 12 ml/h. Two high voltage
power supplies were used. One was used to apply a voltage of +22 kV
to a blunt tip 18 gauge needle fixed to the solution containing
syringe, while the other provided -10 kV to a flat metal target
placed 6 inches behind the grounded mandrel. Solutions were
electrospun onto a 5 mm diameter cylindrical grounded mandrel
placed 12 inches from the needle tip and rotating at a rate of
.about.400 rpm to produce a scaffold of randomly oriented fibers.
Mandrel translation distance was 18 cm, with a translational speed
of 18 cm/s. Immediately after electrospinning, the mandrel and
scaffold were quickly immersed in an ethanol bath, and the scaffold
was carefully slid off the mandrel. The tube (inner diameter: 5 mm,
thickness: .about.500 microns, length: 10 cm) was then placed in a
fume hood for 30 minutes to allow for the evaporation of any
residual ethanol.
85/15 PLGA Electrospun Tubes
[0083] 1) From High Concentration (120 mg/ml)
[0084] Solutions of 120 mg/mL of poly(lactide-co-glycolide) (Purac,
Linolnshire, Ill.) having a mole percent ratio of lactide to
glycolide of 85/15 (85/15 PLGA) were made with HFP solvent.
Solutions were left in a box (dark environment) overnight on a
shaker plate to ensure that all 85/15 PLGA had dissolved and formed
a homogenous solution. 5 mL of polymer solution were then drawn
into a plastic Beckton Dickinson syringe (5 ml) and placed in the
syringe pump to be dispensed at a rate of 8 ml/h. Two high voltage
power supplies were used. One was used to apply a voltage of +22 kV
to a blunt tip 18 gauge needle fixed to the solution containing
syringe, while the other provided -10 kV to a flat metal target
placed 6 inches behind the grounded mandrel. Solutions were
electrospun onto a 5 mm diameter cylindrical grounded mandrel
placed 8 inches from the needle tip and rotating at a rate of
.about.400 rpm to produce a scaffold of randomly oriented fibers.
Mandrel translation distance was 18 cm, with a translational speed
of 18 cm/s. Prior to electrospinning, the mandrel was wrapped with
a small section of aluminum foil to aid in tube removal. Upon
completion of electrospinning, the foil liner was slid off the
mandrel, and carefully removed from the inside of the tube (inner
diameter: 5 mm, thickness: .about.500 microns, length: 10 cm).
2) From Low Concentration (50 mg/ml)
[0085] Solutions of 50 mg/mL of 85/15 PLGA were made with HFP
solvent. Solutions were left in a box (dark environment) overnight
on a shaker plate to ensure that all 85/15 PLGA had dissolved and
formed a homogenous solution. 15 mL of polymer solution were then
drawn into a plastic Beckton Dickinson syringe (30 ml) and placed
in the syringe pump to be dispensed at a rate of 12 ml/h. Two high
voltage power supplies were used. One was used to apply a voltage
of +22 kV to a blunt tip 18 gauge needle fixed to the solution
containing syringe, while the other provided -5 kV to a flat metal
target placed 6 inches behind the grounded mandrel. Solutions were
electrospun onto a 5 mm diameter cylindrical grounded mandrel
placed 8 inches from the needle tip and rotating at a rate of
.about.400 rpm to produce a scaffold of randomly oriented fibers.
Mandrel translation speed was set to 18 cm/s. Prior to
electrospinning, the mandrel was wrapped with a small section of
aluminum foil to aid in tube removal. Upon completion of
electrospinning, the foil liner was slid off the mandrel, and
carefully removed from the inside of the tube (inner diameter: 5
mm, thickness: .about.500 microns, length: 10 cm).
Example 2
Tubular Scaffolds of Bioabsorbable Polymers and Collagen Fabricated
by Electrospinning Processes
1) Collagen Electrospun Tubes
[0086] Collagen (Bovine Collagen Type I, Kensey Nash, Exton, Pa.)
was electrospun at a concentration of 120 mg/ml in HFP. Collagen
solutions were mixed and allowed to sit overnight inside a dark box
placed on a shaker plate to ensure that all collagen was dissolved.
For collagen tubes, a small volume (0.2-0.5 ml depending on mandrel
diameter) of collagen solution was drawn into a 1 ml Beckton
Dickinson syringe and electrospun onto the rotating mandrel to aid
in tube removal. This preliminary coating of collagen was dispensed
through a blunted 18 gauge needle at a rate of 3 ml/hr. The two
high voltage power supplies were connected to the needle tip and
the 5'' diameter back target placed 6'' behind the mandrel (2 or 5
mm in diameter), and are set to +25 kv and -10 kv, respectively.
The grounded mandrel was placed 8'' from the charged needle tip,
and rotates at a rate of .about.400 rpm to produce a scaffold of
randomly oriented fibers. Mandrel translation distance was 18 cm,
with a translational speed of 18 cm/s. Upon completion of the
preliminary sacrificial layer of collagen, the initial syringe was
disposed of and a new syringe containing the desired volume of
collagen solution was placed on the syringe pump. This solution was
electrospun using the same parameters as the sacrificial layer.
Upon completion of the electrospinning process, the mandrel was
removed from the electrospinning chamber, and the graft was
carefully slid off the mandrel. During this process the initial
layer was torn away from the graft, leaving a thin layer of
collagen still on the mandrel.
2) PDO and Collagen Electrospun Tubes
[0087] 50:50 PDO:collagen scaffolds are scaffolds composed of a
50:50 ratio by volume of 100 mg/ml PDO and 120 mg/ml collagen
(Bovine Collagen Type I, Kensey Nash, Exton, Pa.) solutions. The
two polymer solutions were made in separate scintillation vials
under conditions identical to those of electrospinning the polymers
individually by placing the polymers in HFP solution overnight in a
dark box on a shaker plate. Once the polymers had completely
dissolved equal volumes of the two solutions were combined together
in a new scintillation vial, vortexed for 30 seconds, and placed on
a shaker. While the two solutions were mixing, a small volume of
pure collagen solution is electrospun onto the grounded mandrel to
serve as a sacrificial layer using a process identical to that in
the above protocol for electrospinning pure collagen tubes. Once
the preliminary collagen layer had been electrospun, the desired
volume of blended PDO:collagen solution was drawn into a Beckton
Dickinson syringe and electrospun onto the rotating mandrel (2 or 5
mm in diameter) to aid in tube removal. Upon completion of the
preliminary sacrificial layer of collagen, the initial syringe was
disposed of and a new syringe containing the desired volume of
collagen solution was placed on the syringe pump. This solution was
electrospun using the same parameters as the sacrificial layer and
as described in example 2, part 1.
3) Cross-Linking Collagen and PDO:Collagen ESS Tubes with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC)
[0088] Pure collagen scaffolds, as well as blends of PDO and
collagen prepared in example 2, parts 2 and 3 were cross-linked
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) in pure ethanol. Samples were soaked in a 40 mM (50.times.
the molar concentration of collagen in HFP) solution of EDC in
ethanol for 18 hrs, followed by a 2 hr rinse in 0.1 M disodium
phosphate solution to hydrolyze any unreacted O-isoacylurea
intermediates. After cross-linking, samples were rinsed in
de-ionized water, frozen, and lyophilized overnight to remove any
residual moisture.
Example 3
PDO Sheet Scaffolds of Bioabsorbable Polymers Fabricated by
Electrospinning Processes
[0089] Solutions of 100 mg/ml of PDO were made by placing the
polymer in HFP solvent and leaving the solution overnight in dark
box on a shaker plate to ensure that all PDO was dissolved and
forms a homogenous solution. The desired volume of polymer solution
was then drawn into a plastic Beckton Dickinson syringe and placed
in the syringe pump to be dispensed at a rate of 10 ml/hr. Two high
voltage power supplies were used. One was used to apply a voltage
of +20 kV to a blunt tip 18 gauge needle fixed to the solution
containing syringe, while the other provides -8 kV to a metal
target placed 6'' behind the grounded mandrel (2.5 cm in diameter).
The grounded mandrel was placed 8'' from the needle tip and
rotating at a rate of .about.400 rpm to produce a scaffold of
randomly oriented fibers. Mandrel translation distance was 18 cm,
with a translational speed of 18 cm/s. Immediately after
electrospinning the mandrel and scaffold are quickly immersed in an
ethanol bath to assist in sliding the tube off the mandrel. The
tube is cut to form a sheet and then placed in a fume hood for 30
minutes to allow for the evaporation of any residual ethanol.
Example 4
50:50 PDO:Collagen Sheet Scaffolds of Bioabsorbable Polymers and
Collagen Fabricated by Electrospinning Processes
[0090] 50:50 PDO:collagen scaffolds are scaffolds composed of a
50:50 ratio by volume of 100 mg/ml PDO and 120 mg/ml collagen
solutions and were made by a process as described in Example 3.
[0091] The 50:50 PDS:Collagen sheets were then cross-linked using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
in pure ethanol. Samples were soaked in a 40 mM (50.times. the
molar concentration of collagen in HFP) solution of EDC in ethanol
for 18 hrs, followed by a 2 hr rinse in 0.1 M disodium phosphate
solution to hydrolyze any unreacted O-isoacylurea intermediates.
After cross-linking, samples were rinsed in de-ionized water,
frozen, and lyophilized overnight to remove any residual
moisture.
Example 5
Tubular Scaffolds of Bioabsorbable Polymers Fabricated by a
Lyophilization Processes
[0092] 1) 35/65 poly(caprolactone-co-glycolide) (35/65 PCL/PGA)
lyophilized tubes (10 wt % solution in 1,4-dioxane)
[0093] This example describes the making of a tube containing
porous structures that would provide pathways for nutrient
transport and guided tissue regeneration. Hence, a 10% wt./wt.
polymer solution was prepared by dissolving 1 part 35/65 PCL/PGA
(Ethicon, Inc., Somerville, N.J.) with 9 parts of solvent
1,4-dioxane. The solution was prepared in a flask with a magnetic
stir bar. To dissolve the copolymer completely, the mixture was
gently heated to 60.degree. C. and continuously stirred overnight.
A clear homogeneous solution was then obtained by filtering the
solution through an extra coarse filter (Pyrex brand extraction
thimble with fritted disc).
[0094] A lyophilizer (DURA-STOP, FTS Systems, Stone Ridge, N.Y.)
was used to then form the tubes from the polymer solution. The
freeze dryer was powered up and the shelf chamber was maintained at
-17.degree. C. for approximately 30 minutes. Thermocouples to
monitor the shelf temperature were attached for monitoring. The
homogeneous polymer solution prepared in Step A was poured into a
gap between the barrel of a 1 ml Beckton Dickinson (BD) syringe and
the plunger of 1 ml BD syringe, thereby using the syringe as a mold
to form the tube. The mold was placed into the lyophilizer
maintained at -17.degree. C. (pre-cooling). The lyophilization
cycle was started and the shelf temperature was held at -17.degree.
C. for 15 minutes and then, held at -15.degree. C. for 120 minutes.
A vacuum was applied to initiate drying of the dioxane by
sublimation. The shelf temperature was raised to -5.degree. C. and
held at this temperature for 60 minutes. The shelf temperature was
raised to 5.degree. C. and held for 60 minutes. The shelf
temperature was raised again to 20.degree. C. and held at that
temperature for 60 minutes. A second stage of drying was started
and the shelf temperature was held at 20.degree. C. for an
additional 120 minutes. At the end of the second stage, the
lyophilizer was brought to room temperature and atmospheric
pressure. The thin scaffold was removed from the plunger of the
syringe.
2) 35/65 poly(caprolactone-co-glycolide) (35/65 PCL/PGA)
lyophilized tubes (5 wt % solution in 1,4-dioxane)
[0095] This example describes the making of a tube containing
porous structures that would provide pathways for nutrient
transport and guided tissue regeneration. Hence, a 5% wt./wt.
polymer solution was prepared by dissolving 1 part 35/65 PCL/PGA
with 9 parts of solvent 1,4-dioxane. The solution was prepared in a
flask with a magnetic stir bar. To dissolve the copolymer
completely, the mixture was gently heated to 60.degree. C. and
continuously stirred overnight. A clear homogeneous solution was
then obtained by filtering the solution through an extra coarse
filter (Pyrex brand extraction thimble with fitted disc).
[0096] A lyophilizer (DURA-STOP, FTS systems, Stone Ridge, N.Y.)
was used to then form the tubes from the polymer solution. The
freeze dryer was powered up and the shelf chamber was maintained at
-17.degree. C. for approximately 30 minutes. Thermocouples to
monitor the shelf temperature were attached for monitoring. The
homogeneous polymer solution prepared in Step A was poured into a
gap between the barrel of a 1 ml BD syringe and the plunger of 1 ml
BD syringe, thereby using the syringe as a mold to form the tube.
The mold was placed into a lyophilizer maintained at -17.degree. C.
(pre-cooling). The lyophilization cycle was started and the shelf
temperature was held at -17.degree. C. for 15 minutes and then,
held at -15.degree. C. for 120 minutes. A vacuum was applied to
initiate drying of the dioxane by sublimation. The shelf
temperature was raised to -5.degree. C. and held at this
temperature for 60 minutes. The shelf temperature was raised to
5.degree. C. and held for 60 minutes. The shelf temperature was
raised again to 20.degree. C. and held at that temperature for 60
minutes. A second stage of drying was started and the shelf
temperature was held at 20.degree. C. for an additional 120
minutes. At the end of the second stage, the lyophilizer was
brought to room temperature and atmospheric pressure. The thin
scaffold was removed from the plunger of the syringe.
Example 6
Preparation of Poly(Lactide) and Poly(Glycolide) Non-Woven
Tubes
[0097] Nonwoven tubes possessing a length of approximately 50 mm
and an internal diameter of approximately 4 mm and a wall thickness
of approximately 0.5-1.0 mm were fabricated from various
bioresorbable filaments. Specifically, filaments were comprised of
poly(lactide) (PLA) and poly(glycolide) (PGA) and a copolymer of
PGA and PLA in a 90:10 molar ratio (90/10 PGA/PLA). These samples
were fabricated using a dry lay nonwoven technique to first produce
a non-woven batt from filaments approximately 20 microns in
diameter and approximately 50 mm in length. This batt was then
consolidated via needle punching using a mandrel.
Example 7
Preparation of a Collagen Coated Absorbable, Synthetic Tissue
Engineered Tubular Scaffold
[0098] Highly purified atelocollagen (Colbar, a Johnson &
Johnson Co., Israel) is used for coating a tissue engineered
tubular scaffold, prepared as described in Examples 1-6 and 8-11.
The tissue engineered tubular scaffold (Example 1, part 2) was
placed on a mandrel and immersed in 10 mM HCl solution containing 1
mg/ml collagen. The tissue engineered tubular scaffold was soaked
in the collagen solution for 30 minutes at room temperature. The
tissue engineered tubular scaffold was removed from the solution
and dried at room temperature for 8 hours.
Example 8
Preparation of an Acellular Omental Matrix Coated Tissue Engineered
Tubular Scaffold
[0099] Pig omentum is placed in 0.9% saline after harvest. After
rinsing in the saline solution 3 times to rinse off blood and other
extraneous debris, the omentum is placed in 70% ethanol for 30
minutes. Following the treatment with 70% ethanol, the tissue is
dehydrated in 100% ethanol for 30 minutes with two changes into
fresh ethanol. The tissue is then transferred to acetone for 180
minutes, using fresh solution every 60 minutes. Subsequently, the
tissue is placed in a 50:50 acetone-hexane mixture for 60 minutes,
followed by a 20:80 mixture of the same for 24-48 hours (with 3
changes of fresh solution) for lipid removal. The tissue is then
transferred to 100% ethanol for 30 minutes and subsequently to 70%
ethanol where, if necessary, it could be stored at 4.degree. C.
until the decellularization process is initiated. The tissue is
then immersed in a decellularization buffer comprising TRITON.RTM.
X-100 (1% w/V; a nonionic detergent) (Sigma-Aldrich, St. Louis,
N.J.) and MgCl.sub.2 (1%) dissolved in 50 mM Tris-HCl (pH 7.2), for
30 minutes. This is followed by treatment in an enzyme solution
comprising endonuclease (BENZONASE; 41.8 U/ml, Sigma-Aldrich, St.
Louis, N.J.) mixed with the decellularization buffer. The tissue is
spun in this solution for 20 hours. The tissue is then washed twice
(2 hours each) in a solution comprising 50 mM Tris-HCl (pH 7.2), 5
mM MgCl.sub.2 and 1% (W/V) TRITON.RTM. X-100. The tissue is then
placed in a cell extracting solution comprising 1M NaCl, 20 mm
EDTA, 0.2% (W/V) TRITON.RTM. X-100 pH 7.0 for 1 hr, following which
the tissue is washed with ultra pure water (4 times, 5 minutes
each). The tissue is transferred to disinfection solution
comprising 80:20 water:ethanol (200 proof) with 0.15% peracetic
acid (or acetic acid) for 1 hour after washing in water (4 times,
20 minutes each), the tissue is stored in 70% alcohol at 4.degree.
C.
[0100] The acellular omentum is air dried and then cryogenically
milled into a powder. The powder is then dispersed into 10 mM HCl
at a concentration of 5 mg/ml. A tissue engineered tubular
scaffold, as described in Examples 1-6 and 9-11 is placed onto a
mandrel and immersed in the acellular omentum suspension and dried
by lyophilization at -20.degree. C. for 24 hours. The omental
matrix on the impregnated tissue engineered tubular scaffold is
then cross linked by thermal dehydration at 120.degree. C.
overnight under vacuum.
Example 9
Generation of Cell Sheets on PCL/PGA Films
[0101] This example describes the generation of cells sheets
comprised of Human umbilical vein endothelial cells (HUVECs) on
films. These sheets can then be fabricated into tubular structures
leading to TEBV comprised solely of human umbilical vein
endothelial cells (HUVEC). Human umbilical vein endothelial cells
(HUVEC) were seeded onto PCL/PGA films to obtain cell sheets. To do
this, films were cast by adding 2.5 ml of the polymer solution
(45/55 PCL/PGA 10% (w/w) in dioxane or 35/65 PCL/PGA 10% (w/w) in
dioxane) onto a 60 mm culture dishes. After casting, films were
sterilized by washing in ethanol and air-dried. HUVEC were
harvested by trypsinization and counted using a Guava instrument
(Guava Technologies, Hayward, Calif.). Cells were seeded onto the
films at a density of 5000 cells/cm.sup.2 (141,350 cells/60 mm
dish) and then placed in a 37.degree. C. incubator for 9 days.
Cells were visualized by microscopy or by calcein staining. The
HUVEC cells grew to a confluent layer on a
poly(caprolactone-co-glycolide 35/65 mole-mole %) (35/65 PCL/PGA)
film or a poly(caprolactone-co-glycolide 45/55 mole-mole %) (45/55
PCL/PGA) film providing a cell sheet. Microscopic images confirmed
there was a confluent monolayer of cells. Calcein staining showed
cells attached and proliferated at days 9 with little to no
evidence of dead cells.
[0102] The sheets can be rolled into a tube to form a construct
that can be used as a tissue engineered blood vessel alone or in
combination with a mechanical strut such as the scaffolds described
in Examples 1-8 or 11. By similar methods (see examples 18 and 19),
cell sheets can be formed into a tube directly.
Example 10
Generation of Cell Sheets on PCL/PGA Films
[0103] This example describes the generation of cells sheets
comprised of human umbilical tissue derived cells (hUTCs) on films.
Human umbilical tissue-derived cells are obtained by methods
described in U.S. Pat. No. 7,510,873 incorporated by reference in
its entirety. These sheets are fabricated into tubular structures
leading to TEBV comprised solely of hUTC. hUTCs are seeded onto
PCL/PGA films to obtain cell sheets. To do this, films are cast by
adding 2.5 ml of the polymer solution onto a 60 mm culture dishes.
After casting, films are sterilized by washing in ethanol and
air-dried. hUTCs are harvested by trypsinization and counted using
a Guava instrument. Cells are seeded onto the films at a density of
5000 cells/cm.sup.2 (141,350 cells/60 mm dish) and are then placed
in a 37.degree. C. incubator. Cells are visualized by microscopy or
by calcein staining Cell sheets comprised of hUTCs and a
poly(caprolactone-co-glycolide 35/65 mole-mole %) (35/65 PCL/PGA)
film or a poly(caprolactone-co-glycolide 45/55 mole-mole %) (45/55
PCL/PGA) film are prepared.
[0104] The sheets can be rolled into a tube to form a construct
that can be used as a tissue engineered blood vessel alone or in
combination with a mechanical strut such as the scaffolds described
in Examples 1-8 or 11. By similar methods (See examples 18 and 19),
cell sheets can be formed into a tube directly.
Example 11
Lyophilized, Decellularized Cell Sheets
[0105] This example relates to the use of lyophilized or
decellularized cell sheets to fabricate TEBV. The cell sheets will
be generated in vitro and then lyophilized or decellularized. When
needed, the required type of cell sheets can be thawed and then
formed into tubular structures to produce TEBV. Decellularized cell
sheets, on the other hand, can be wrapped around other cell sheets
to enhance the construction of TEBV by providing trophic factor
support or extracellular matrix proteins.
[0106] Cell sheets will be generated as in Examples 9, 10 and 17.
Alternative methods of obtaining cell sheets will include culturing
cells on decellularized omentum (Example 8), on tissue-culture
plastic, or on thermoresponsive dishes (CellSeed, Inc, Tokyo,
Japan). Cell types used for obtaining cell sheets will include
endothelial cells, smooth muscle cells, skeletal muscle cells, or
hUTC. Cells will be maintained in culture until a monolayer is
achieved. The resulting cell sheets will then be processed for
vitrification by cryopreservation and subsequent lyophilization
(Core Dynamics, Orangeburg, N.Y.).
Example 12
Attachment and Growth of Human Umbilical Tissue-Derived Cells on
PDO ESS Scaffolds
[0107] This example relates to the use of human umbilical
tissue-derived cells (hUTC) to produce tissue-engineered blood
vessels (TEBVs). TEBVs can be generated by seeding vascular grafts
or scaffold materials with human umbilical tissue-derived cells. It
is envisioned that seeding hUTC onto the TEBVs will enhance the
seeding, attachment, and proliferation of endothelian cells (ECs)
and smooth muscle cells (SMCs) when seeded in vitro or onto the
vascular grafts after implantation. hUTC may also promote the
infiltration and subsequent differentiation of the EC or SMC
progenitor cells into the graft construct. This may promote the
maturation and the engraftment of TEBVs during the in vivo
implantation by providing trophic support, or providing the
expression of ECM proteins.
[0108] Attachment and growth of hUTC on PDO and PDO/collagen
(50/50, crosslinked) (Examples 1 and 2) ESS scaffolds were
assessed. Biopsy punches 5 mm in diameter were made from the
scaffold materials and pre-wet in complete growth medium. hUTC were
then trypsinized, counted and resuspended at a concentration of
200,000 cells/ml in complete growth medium. The scaffold punches
were placed in 96-well low cluster plates and seeded with 100
microliters of the cell suspension (20,000 cells/punch). Cells were
allowed to attach for 3 hours at 37.degree. C., and then the
scaffolds were transferred to 24-well low cluster plates containing
1 ml of complete growth medium. The scaffolds were cultured for 7
days with a medium change after 3 days.
[0109] On day 3 and day 7 post-seeding, the scaffolds were
transferred to fresh low cluster 24-well dishes containing 1 ml
serum-free DMEM. The scaffolds were then washed with an additional
1 ml serum-free DMEM. A stock solution of Live/Dead stain
(Invitrogen, Carlsbad, Calif.) containing 2 micromolar calcein AM
and 4 micromolar ethidum homodimer was prepared and 0.5 ml was
added to each well. After incubation at room temperature for 5
minutes, cell attachment and viability of cells was assessed by
fluorescence microscopy.
[0110] Results:
[0111] The hUTC attached and grew on the TEBV scaffolds.
PDO/collagen-ESS scaffolds exhibited more significant increase in
the number of cells from day 3 to day 7 as compared to the PDO-ESS
scaffolds (FIG. 1).
Example 13
Attachment and Growth of Human Umbilical Artery Smooth Muscle Cells
(UASMCs) and Human Umbilical Vein Endothelial Cells (HUVECs) on PDO
ESS Scaffolds
[0112] Attachment and growth of human umbilical artery smooth
muscle cells (UASMCs) and human umbilical vein endothelial cells
(HUVECs) on PDO ESS scaffolds (100 mg/ml and 140 mg/ml, Example 1)
was assessed. UASMCs (Lonza Rockland Inc., Rockland, Me.) and
HUVECs (Lonza Rockland Inc., Rockland, Me.) were seeded onto PDO
ESS scaffolds, and at specified time points (day 3 and day 7) cells
grown on the different surfaces were assessed for viability by
Live/Dead staining Sterile PDO scaffolds (5 mm biopsy punches) were
placed into empty low cluster 96-well dishes, washed with PBS, and
then soaked in appropriate medium (EGM-2 for HUVECs, and SmGM for
UASMCs) while trypsinizing cells. UASMCs and HUVECs were harvested
by trypsinization, counted and resuspended to a final density of
5.times.10.sup.5 cells/ml in SmGM (UASMC) or EGM-2 (HUVEC) medium.
One hundred microliters (50,000 cells) of this stock cell
suspension was aliquotted onto the scaffolds, and the cells were
allowed to attach for 3 hours in 37.degree. C. incubator. The
scaffolds were then transferred to 24-well dishes containing 1 ml
of the appropriate medium and cultured for 3 and 7 days.
[0113] The scaffolds were transferred to fresh low cluster 24-well
dishes containing 1 ml serum-free DMEM. The graft materials were
then washed with an additional 1 ml serum-free DMEM. A stock
solution of Live/Dead stain containing 2 micromolar calcein AM and
4 micromolar ethidum homodimer in was prepared serum-free DMEM and
0.5 ml was added to each well. After incubation at room temperature
for 5 minutes, cell attachment and viability of cells was assessed
by fluorescence microscopy.
[0114] Results:
[0115] All samples showed cell attachment and growth over the 7 day
culture period (FIGS. 2 and 3). The endothelial cells formed an
intact monolayer on the surface of the scaffolds. The 100 mg/ml PDO
scaffold exhibited the best results, with a high number of attached
cells at day 3 and increases in cell number on day 7. The 140 mg/ml
PDO scaffold showed a high number of attached cells on day 3, but
the increase in cells by day 7 was not as dramatic as the 100 mg/ml
scaffold.
Example 14
Effect of hUTCs on Proliferation and Migration of HUVECs
[0116] This example relates to the use of human umbilical
tissue-derived cells (hUTCs) to produce tissue-engineered vascular
grafts (TEBVs). TEBVs can be generated by seeding vascular grafts
or scaffold materials with human endothelial cells (ECs) and human
smooth muscle cells (SMCs). It is envisioned that hUTCs will
enhance the seeding, attachment, and proliferation of ECs and SMCs
on the vascular grafts. hUTCS may also promote the differentiation
of the EC or SMC or progenitor cells in the graft construct. This
may promote the maturation of TEBVs during the in vitro culture as
well as the engraftment during the in vivo implantation. hUTCs may
provide trophic support, or provide and enhance the expression of
ECM proteins.
[0117] As proof of principle, the effects of hUTC on the
proliferation and migration of HUVEC are investigated in vitro. For
studies of proliferation, the effects of hUTC lot #120304 were
tested and three endothelial cell types from different vascular
beds were used as responder cells (human umbilical vein endothelial
cells[HUVEC], human coronary artery endothelial cells [HCAEC], and
human iliac artery endothelial cells [HIAEC]). Co-culture with hUTC
resulted in enhanced proliferation of endothelial cells. Co-culture
with mesenchymal stem cells (MSC) or fibroblasts resulted in cell
numbers comparable to media controls (Table 1).
[0118] Migration was quantitated by counting the number of cells
that were on the underside of a transwell and both HUVEC and HCAEC
were used as responder cells. Unlike the studies with
proliferation, the migratory responses of these cells are slightly
different. HUTC lot #120304 induced the migration of both HUVEC and
HCAEC. MSC did not induce the migration of HUVEC suggesting
specificity of this response to hUTC (Table 2).
TABLE-US-00002 TABLE 1 Effect of hUTC lot#120304, MSC, and
fibroblasts on the proliferation of endothelial cells. Endothelial
cells (human umbilical vein endothelial cells, human iliac artery
endothelial cells, human coronary artery endothelial cells) were
seeded onto the bottom of a 24-well tissue culture dish at a
density of 5000 cells/cm.sup.2 (10,000 cells/well) and hUTC
lot#120304, MSC, or fibroblasts inside transwell inserts at a
density of 5000 cells/cm.sup.2 (1,650 cells/insert) in co-culture
media (Hayflick 80% + EGM-2MV 20% or Hayflick 50% + EGM-2MV 50%).
After 7 days of co-culture, cells were harvested and counted using
a Guava instrument. Endothelial cells were also maintained in
EGM-2MV media as positive control. HUVEC HIAEC HCAEC average std
dev average std dev average std dev EGM-2MV 100% 36511.33 1307.32
18100 1609.9413 27328 3802 Hay80/EGM20 (H80) 6532.33 625.94
8770.6667 187.37752 7391 978 hUTC 120304 (H80) 13394.67 2011.56
10961.667 1678.5 12957 445 MSC (H80) 5674.33 716.29 9555.6667
933.66286 8136 620 fibroblasts (H80) ND ND 8630 1049.4805 ND ND
Hay50/EGM50 (H50) 6778.5 1175.92 21847.5 2947.9282 7818 837 hUTC
120304 (H50) 26595.667 4398.96 24577.333 3421.4854 16056 4225 MSC
(H50) 5554.67 2801.54 16065 2181.5799 8035 2198 fibroblasts (H50)
ND ND 12158 2113.0894 ND ND
TABLE-US-00003 TABLE 2 Effect of hUTCs and MSCs on the migration of
endothelial cells. HUVEC or HCAEC were seeded inside transwell
inserts at a density of 5000 cells/cm.sup.2 (23,000 cells/insert)
and hUTC lot#120304 or MSC onto the bottom of a 6-well tissue
culture dish at a density of 5000 cells/cm.sup.2 (48,000
cells/well) in co-culture media (Hayflick 50% + EGM-2MV 50%). After
7 days of co-culture, cells that were on the underside of the
transwell insert were harvested and counted using a Guava
instrument. Endothelial cells were also maintained in EGM-2MV media
as control. HUVEC HCAEC average std dev average std dev EGM-2MV
3125.67 1849.46 848.33 539.13 Hayflick 50% 805.33 323.96 1926.67
280.42 hUTC 120304 2402.33 880.1 9071.67 3792.28 MSC 383 124.65 ND
ND
Example 15
Preparation of a Cell Lysate-Augmented Biopsy Punches
[0119] In this example, hUTC were culture expanded, harvested,
lysed by repeated freeze-thaw cycles, and applied to the
bioabsorbable scaffolds of Examples 1-11 and lyophilized. The cell
lysate augmented scaffolds can be implanted as such or seeded with
cells (as in Examples 16, 17) or minced tissue (Examples 25) and
cultured to create tissue engineered blood vessels.
[0120] Umbilical Postpartum cells hUTC (Passage 11, lot 120304)
were cultured at 5,000 cells per cm squared in T225 cm.sup.2 cell
culture flasks (CORNING, Cat No 431082, Corning, N.Y.) with
complete growth media: DMEM-low glucose (GIBCO, Cat No 11054
Invitrogen, Carlsbad, Calif.), 15% Fetal Bovine Serum (HyClone Cat
No SH30070-03 Logan, Utah) and Pen/Strep solution (GIBCO, Cat No
15070). After cells expanded to approximately 25,000 cells per
cm.sup.2, cells were harvested by TrypLE Select (GIBCO cat No
12563) and collected in 50 ml conical tubes, centrifuged at 300 rcf
for 5 minutes and removed the supernatant. The cell pellets were
washed 3 times with PBS and then re-suspended in PBS at
1.times.10.sup.7 cells/ml for a total volume of 14 ml. This
solution was snap frozen in liquid nitrogen, thawed in a 37.degree.
C. water bath, centrifuged to remove cell debris, and the resulting
supernatant (10 ml) removed and stored. The remaining material was
again snap frozen, thawed, and centrifuged as above. The
supernatant (2 ml) was removed and stored. The protein
concentration of the two supernatants was determined by measuring
samples diluted in PBS with the Bradford protein assay kit (BIO-RAD
Laboratories Hercules, Calif.). The concentration of the first
lysate supernatant (Lot #082908) was 5.2 mg/ml (Lot #082908 low)
and the second lysate supernatant was 18 mg/ml (Lot #082908
high).
[0121] Biopsy punches 5 mm in diameter were taken from PDO-ESS
scaffold sheets (Example 3). These scaffolds were placed into 96
well ultra low cluster plate (COSTAR, cat No 3474 Fisher
Scientific, Pittsburgh, Pa.), and 25 microliters cell lysate was
loaded onto each disc at protein concentration 5.2 mg/ml (low) or
18 mg/ml (high). The scaffold punches were then lyophilized for 48
hours to remove water.
[0122] Cell Attachment: The lysate-augmented scaffolds were placed
into 96-well low cluster plates and rehydrated with 25 microliters
of EGM-2 medium (Lonza Walkersville, Md.). Human umbilical vein
endothelial cells (HUVECs) were tyrpisinized, counted and
resuspended to a concentration of 500,000 cells/ml. Each scaffold
was seeded with 100 microliters of this cell suspension (50,000
cells) and the cells were allowed to attach for 3 hours at
37.degree. C. After this attachment period, the scaffolds were
transferred to 24-well low cluster plates containing 1 ml of EGM-2
medium. The scaffolds were cultured for 3 and 7 days.
[0123] At day 3 and day 7 post-seeding scaffolds were analyzed for
cell attachment using Live/Dead stain and for cell number using the
CyQuant assay (Invitrogen) to measure cellular DNA. For the
Live/Dead stain, the scaffolds were transferred to fresh low
cluster 24-well dishes containing 1 ml serum-free DMEM. The
scaffolds were then washed with an additional 1 ml serum-free DMEM.
A stock solution of Live/Dead stain containing 2 micromolar calcein
AM and 4 micromolar ethidum homodimer was prepared and 0.5 ml was
added to each well. After incubation at room temperature for 5
minutes, cell attachment and viability of cells was assessed by
fluorescence microscopy.
[0124] For the measurement of cellular DNA, the scaffolds were
washed in PBS, then frozen in 150 microliters of PBS in
microcentrifuge tubes. The scaffolds were then lyophilized to
dryness and resuspended in 150 microliters of papain digestion
solution. The samples were then digested overnight at 60.degree. C.
The next day, 10 microliters was used to assay for DNA content
using the CyQuant NF assay kit (Invitrogen).
[0125] Results:
[0126] An increase in cell number was observed for both
concentrations of lysate tested and at both day 3 and day 7
post-seeding. Live/Dead staining shows a greater number of cells
and more of the scaffold surface covered at both timepoints
examined (FIG. 4). After 3 days there was an approximate 4-fold
increase in cellular DNA compared to control scaffolds (FIG.
5).
Example 16
Preparation of Cell Lysate on Tube Scaffolds
[0127] PDO tubular scaffolds (Example 1, part 2) were dip-coated
into hUTC lysate solution (Example 15) at a protein concentration
of 5.2 mg/ml (Lot #082908 Low) for 5 minutes then lyophilized for
24 hours using a lyophilizer DURA-STOP, FTS system. The freeze
dryer was powered up and the shelf chamber was maintained at
-40.degree. C. for approximately 15 minutes. Thermocouples to
monitor the shelf temperature were attached for monitoring. The
scaffold tubes were placed into a lyophilizer maintained at
-40.degree. C. (pre-cooling). The lyophilization cycle was started
and the shelf temperature was held at -40.degree. C. for 15 minutes
and then, held at -37.degree. C. for 60 minutes. A vacuum was
applied. The shelf temperature was maintained -40.degree. C. and
held at this temperature for 180 minutes.
[0128] The shelf temperature was raised to -25.degree. C. and held
for 500 minutes. The shelf temperature was raised to -15.degree. C.
and held for 180 minutes. The shelf temperature was raised to
-5.degree. C. and held for 180 minutes. The shelf temperature was
raised to 5.degree. C. and held for 120 minutes. The shelf
temperature was held at 20.degree. C. for 120 minutes. The shelf
temperature was held to -20.degree. C. for 120 minutes. After
lyophilization, the tubular scaffolds were evaluated for cell
attachment.
[0129] Individual tubular scaffolds were placed into 100 mm
untreated plates (from Corning, Cat No 430591). Rat smooth muscle
cells were seeded statically onto the tubular scaffolds coated with
hUTC lysate or phosphate buffered saline (PBS) (as a control) at a
seeding density of 5.times.10.sup.6/scaffold. Cell seeded scaffolds
were incubated in 37.degree. C. humidified air for one hour prior
to refeeding the dish with 15 milliters smooth muscle growth media.
The scaffolds were cultured for 24 hours. After 24 hours, the
scaffolds were evaluated by Live/Dead kit (from Invitrogen, Cat No
L3224). Live/Dead staining on tubular scaffolds coated with PBS or
hUTC lysate and cultured with rat SMC for 24 hours showed more cell
attached to the lysate-coated scaffold (FIG. 6).
Example 17
Generation of Cell Sheets on the Lysate Treated PDO Sheet
[0130] This example is to demonstrate hUTCs seeded on the lysate
treated PDO sheet could attach, migrate and penetrate into the
scaffold. These sheets could be fabricated into tubular structures
leading to TEBV comprised solely of hUTC (see next example) for
implantation right away at the site of injured vessel in vivo or
further maturation by culturing in a bioreactor. A PDO sheet (2.0
cm.times.2.0 cm.times.0.01 .mu.m) (Lot #3904-78) (Example 3) was
soaked with 40 .mu.l lysate (containing 5.2 mg/ml total protein,
Lot #082908) followed by air drying at 4.degree. C. for overnight.
The treated scaffold was then seeded with hUTCs, cultured and
obtained as described in Example 15, at a density of
1.75.times.10.sup.5 cells/cm.sup.2. The cell seeded scaffold was
then cultured in the same condition as for cells described in
Example 15. At 11 and 14 days after seeding, the cell sheets were
fixed for H&E staining. As shown, while cells were seeded on
only one side, cells spread all over the surface as well as inside
the scaffold, indicating hUTCs attach, migrate and proliferate
within the lysate treated PDO scaffold (FIG. 7).
Example 18
Tissue Engineered Graft of Rolled ESS Sheet with hUTC Cells and
hUTC Cell Lysate
[0131] Two PDO sheets (2.times.5.times.0.05 cm, Lot #5-6-08-2 sheet
3904-50-3) prepared as described in Example 3 were loaded with 700
.mu.l PBS or hUTC lysate (containing 5.2 mg/ml total protein, Lot
#082908 Low) and dried out by storing in -20.degree. C. for 3 days.
Each sheet was hydrated with 350 .mu.l growth medium (15% FBS in
DMEM). 350 .mu.l hUTCs (5.times.10.sup.6 cells/ml) were obtained as
described in Example 15 and loaded at a density
1.75.times.10.sup.5/cm.sup.2 (Cell lot #120304, P.9). The
cell-loaded sheets were cultured in growth medium. At day 4, each
sheet was cut into two (2.times.2.5.times.0.05 cm). One set was
rolled to a single layer tube with 5 mm in diameter and 2.5 cm in
length while the other set stayed in sheet format.
[0132] Fibrin sealant (0.5 ml) (Omrix, Biopharmaceutices LTD, Tel
Aviv Israel) was applied to the edge of rolled PDO ESS sheet
followed by Thrombin (0.5 ml) (Omrix, Biopharmaceutices LTD, Tel
Aviv Israel) to glue the end of the scaffold onto itself and keep
the scaffold in a tube shape. The glued rolled tube was removed
from the mandrel. A second sheet was kept as a sheet. All tubes and
sheets were continued to culture at 37.degree. C. The viability,
attachment and proliferation of hUTCs in tubes and sheets were
evaluated at 1 and 4 days after culture. The results showed that
hUTC attached and grew to confluence on PDO sheet with or without
lysate. Cell attachment is stronger with the lysate treated PDO
sheet. While the cell layer formed on PDO sheet is disturbed during
a rolling process since the cell density was low when evaluated on
day 1 after the tube was formed, hUTC uniformly and densely
distributed throughout the luminal surface of the tube, with more
proliferation found for the PDO material treated with hUTC lysate
(FIG. 8).
Example 19
Tissue Engineered Graft of Rolled ESS Sheet with rSMC Cells and
hUTC Cell Lysate
[0133] A PDO sheet (2.times.2.5.times.0.005 cm, lot #3904-72-23) as
described in Example 3 was loaded with 60 .mu.l hUTC lysate
(containing 5.2 mg/ml total protein, lot #082908 Low).
Approximately 250 .mu.l rSMCs (Cell Applications, San Diego,
Calif.) at 3.75.times.10.sup.6 cells/ml were seeded on the PDO
sheet at a density of 1.75.times.10.sup.5 cells/cm.sup.2. After 2
hrs at 37.degree. C., the seeded material was immersed with SMC
growth medium (GM). After 5 days in culture, the sheet was rolled
to a tube (-4 layers) around a mandrel (.phi.=2 mm).
[0134] A laboratory scale machine was fabricated to roll an ESS
sheet into a graft that can be used to develop a tissue engineered
blood vessel. The machine has a chuck to which a mandrel was
connected that was rotated to allow the sheet to be rolled into a
tube. A cell cultured PDO ESS sheet was placed on the mandrel (5 mm
in diameter or 2 mm in diameter). The mandrel was slowly rotated to
form 5 layers of a cell containing scaffold. The tube was sealed
with fibrin sealant as described in example 17. The construct was
cultured in GM for 5 days and switched to SMC Differentiation
Medium (DM) for 4 more days. The construct was cut into 2 segments,
one for Live-dead Staining and the other for H&E by fixing in
10% buffered formalin.
[0135] The results show rSMC cells penetrate into the scaffold from
both sides of the scaffold and attach and proliferate well within
the scaffold throughout the thickness of the material. Some areas
(right panel) show the integration of two layers (FIG. 9).
Example 20
Seeding of hUTC on 2 Mm Diameter Tissue-Engineered Blood Vessel
Scaffolds
[0136] This example relates to the uniform seeding of human
umbilical cord cells (hUTC) into PDO-ESS tubes of varying inner
diameters, followed by culture to allow cell growth and matrix
production. This growth can include static culture or culture under
physiological conditions in a bioreactor, such as luminal flow with
or without pressure and/or pulsatile flow. The PDO-ESS tubes can be
first coated with type I collagen or other extracellular matrix
component.
[0137] PDO-ESS tubes (100 mg/ml, Example 1, part 2) approximately
3.5 cm in length and 2 mm in inner diameter were coated with
collagen by soaking in a solution of 50 micrograms/ml rat tail type
I collagen (BD Biosciences, Bedford, Mass.). A collagen-coated tube
and a non-coated control were secured to barbs within a LumeGen
bioreactor chamber (Tissue Growth Technologies, Minnetonka Minn.)
using silk sutures and the chambers were sealed. The outer chamber,
which bathes the tubular scaffolds in medium, was filled with
complete growth medium. hUTC were trypsinized, counted and
resuspended to a concentration of 5.5.times.10.sup.5 cells/ml in
complete growth medium. Seeding rings were then attached to the
LumeGen chamber. These rings allow for rotation of the chamber when
placed on to a standard tissue culture bottle roller. The cell
suspension was injected into the lumen of the PDO-ESS tubes using a
syringe in a way that eliminated all air bubbles. The ends of the
luminal chambers were sealed and the chambers were placed on the
bottle roller and incubated overnight at 37.degree. C. with a
rotation of approximately 0.4 rpm. After this overnight incubation,
the tubes were cut open and 5 mm biopsy punches taken to examine
the distribution of cells within the TEBV scaffolds. The biopsies
of the scaffolds were transferred to fresh low cluster 24-well
dishes containing 1 ml PBS. A stock solution of Live/Dead stain
containing 2 micromolar calcein AM and 4 micromolar ethidum
homodimer was prepared and 0.5 ml was added separate wells. The
scaffold punches were then transferred to the wells containing the
Live/Dead solution. After incubation at room temperature for 5
minutes, cell attachment and viability of cells was assessed by
fluorescence microscopy.
[0138] Results:
[0139] Profuse cell attachment was observed for all PDO-ESS tubes
seeded with hUTC. There was a dramatic increase in the number of
cells attached to the collagen-coated PDO-ESS tube compared to the
uncoated PDO-ESS tube. Very few dead cells were observed in either
sample (FIG. 10).
Example 21
Bioreactor Processes for Tissue Engineered Blood Vessel
Development
(Static Culture)
[0140] This example related to the uniform seeding of smooth muscle
cells into PDO-ESS tubes of varying inner diameters, followed by
culture to allow cell growth and matrix production. This growth can
include static culture or culture under physiological conditions in
a bioreactor, such as luminal flow with or without pressure and/or
pulsatile flow.
[0141] PDO-ESS tubes (100 mg/ml) or PDO/collagen-ESS tubes (Example
1, part 2 and Example 2, part 2) approximately 5 cm in length were
secured to barbs within a LumeGen bioreactor chamber (Tissue Growth
Technologies, Minnetonka Minn.) using silk sutures. After the
chamber was sealed, the outer chamber which bathes the tubular
scaffolds was filled with smooth muscle growth medium (Cell
Applications, Inc., San Diego, Calif.). Rat aortic smooth muscle
cells (Cell Applications, Inc.) were trypsinized, counted and
resuspended to a concentration of 2.times.10.sup.6 cells/ml in
growth medium. Seeding rings were then attached to the LumeGen
chamber. These rings allow for rotation of the chamber when placed
on to a standard tissue culture bottle roller. The cell suspension
was injected into the lumen of the PDO-ESS tube using a syringe.
The ends of the luminal chamber were sealed and the chamber was
placed on the bottle roller and incubated overnight at 37.degree.
C. with a rotation of approximately 0.4 rpm. After this overnight
incubation, some tubes were cut open and 5 mm biopsy punches taken
to examine the distribution of cells within the TEBV scaffolds. The
biopsies of the scaffolds were transferred to fresh low cluster
24-well dishes containing 1 ml serum-free DMEM. The scaffolds were
then washed with an additional 1 ml serum-free DMEM. A stock
solution of Live/Dead stain containing 2 micromolar calcein AM and
4 micromolar ethidum homodimer was prepared and 0.5 ml was added to
each well. After incubation at room temperature for 5 minutes, cell
attachment and viability of cells was assessed by fluorescence
microscopy.
[0142] Cell-seeded PDO-ESS scaffolds in the LumeGen chambers were
then connected to the LumeGen bioreactor that is capable of
generating physiological flow rate, pulsatile flows and pressures.
The pulsatile flow comes partly from a peristaltic pump, while the
pressure and pulses can be adjusted by crimping the outlet media
flow tubing from either the lumen or chamber. In addition, pulses
can optionally be added through a mechanism that compresses the
graft in a pulsatile manner. This is controlled through a computer
interface. Flow was initiated and the cells were exposed to a flow
rate of 10 ml/min for 2 hours. After this flow period, one TEBV
scaffold was removed form the chamber, and analyzied using
Live/Dead stain as above. Another cell-seeded PDO-ESS scaffold was
then cultured statically within the LumeGen chamber for 7 days
followed by analysis with Live/Dead stain as above.
[0143] Results:
[0144] The Live/Dead stain results show that the rotating seeding
method within the LumeGen chamber enables cells to attach and
spread on the PDO-ESS scaffolds with a homogeneous distribution of
cells throughout the length of the scaffold (FIG. 11). Furthermore,
the exposure of cells to 10 ml/min flow did not shear the cells
form the luminal surface of the scaffold. Incubating the seeded
scaffold for 7 days following flow resulted in an increase in cell
number without affecting the viability of the cells (FIG. 12).
Example 22
Bioreactor Processes for Tissue Engineered Blood Vessel Development
(Pulsatile Flow Physiological Condtions-Short Term Culture)
[0145] This example relates to the uniform seeding of smooth muscle
cells into PDO-ESS tubes of varying inner diameters, followed by
culture under dynamic conditions to allow cell growth and matrix
production. These dynamic conditions can include culture under
physiological or non-physiological conditions in a bioreactor, such
as luminal flow with or without pressure and/or pulsatile flow. In
addition, flow can be introduced to the outer chamber, which bathes
the outside of the tissue engineered blood vessel construct in
media.
[0146] PDO-ESS tubes (100 mg/ml, Example 1, part 2) approximately 5
cm in length and 4 mm in diameter were secured to barbs within a
LumeGen bioreactor chamber (Tissue Growth Technologies, Minnetonka
Minn.) using silk sutures and the chambers were sealed. The outer
chambers, which bathe the tubular scaffolds in medium, were filled
with smooth muscle growth medium (Cell Applications, Inc). Rat
aortic smooth muscle cells (Cell Applications, Inc.) were
trypsinized, counted and resuspended to a concentration of
2.times.10.sup.6 cells/ml in growth medium. Seeding rings were then
attached to the LumeGen chamber. These rings allow for rotation of
the chamber when placed on to a standard tissue culture bottle
roller. The cell suspension was injected into the lumen of the
PDO-ESS tube using a syringe. The ends of the luminal chamber were
sealed and the chamber was placed on the bottle roller and
incubated overnight at 37.degree. C. with a rotation of
approximately 0.4 rpm.
[0147] One cell-seeded PDO-ESS scaffold in the LumeGen chamber was
then connected to the LumeGen bioreactor that is capable of
generating physiological flow conditions, including pulsatile flows
and pressures. The pulsatile flow comes partly from a peristaltic
pump, while the pressure and pulses can be adjusted by crimping the
outlet media flow tubing from either the lumen or chamber. In
addition, pulses can be added through a mechanism that compresses
the graft in a pulsatile manner. This is controlled through a
computer interface. Flow was initiated and the cells seeded on the
tubular scaffold were exposed to a flow rate of 20 ml/min and a
pulsatile pressure of 120-80 mm Hg at a frequency of 1 Hz. As a
control, another cell-seeded tube in the bioreactor chamber was
cultured statically with a luminal media change after 24 hours.
[0148] After three days of culture, the tubular scaffolds were
removed from the chambers, cut open, and 5 mm biopsy punches taken
to examine the number, distribution, and morphology of cells within
the TEBV scaffolds. The biopsy punches of the scaffolds were
transferred to fresh low cluster 24-well dishes containing 1 ml
PBS. A stock solution of Live/Dead stain containing 2 micromolar
calcein AM and 4 micromolar ethidum homodimer was prepared and 0.5
ml was added to separate wells. The scaffold punches were then
transferred to the wells containing the Live/Dead solution. After
incubation at room temperature for 5 minutes, cell attachment and
viability of cells was assessed by fluorescence microscopy.
[0149] Results:
[0150] The Live/Dead stain results show that the rotating seeding
method within the LumeGen chamber enables cells to attach and
spread on the PDO-ESS scaffolds with a homogeneous distribution of
cells throughout the length of the scaffold. Furthermore, the
exposure of cells to 20 ml/min flow and physiological 120-80 mm Hg
pulsatile pressure resulted in a dramatic increase in the number of
cells on the surface of the scaffolds. In addition, the morphology
of the cells was altered to align in the direction of the flow
(FIG. 13).
Example 23
Bioreactor Processes for cell seeded Tissue Engineered Blood Vessel
Development (Pulsatile Flow Physiological Condtions-Long Term
Culture)
[0151] Cells (umbilical artery smooth muscle cells--UASMCs) are
seeded on a scaffold as described in Examples 1-11 in a perfused
rotating wall vessel bioreactor (Synthecon, Inc., Houston Tex.).
The bioreactor has a central rotating core with barbs that the
scaffolds will be connected to. The core allows for medium to be
perfused through the lumen of the scaffold, while the exterior is
bathed in medium. The entire assembly is rotated horizontally as
above, again minimizing shear stress. For seeding of cells onto the
luminal surface of the scaffolds, a cell suspension (10.sup.6
cells/ml) is pumped into the lumen of the scaffold. The flow is
stopped and rotation continues. As the bioreactor rotates, cells
are attached to the surface of the rotating lumen in a uniform
manner. After the 2 to 4 hour incubation period for attachment, the
remaining unattached cells are flushed from the lumen, and growth
medium perfused through the lumen. This culture period is continued
for .about.14 days, allowing the cells to grow and migrate into the
pores of the scaffold (the actual time of culture will be
determined empirically).
[0152] Growth of cells into the scaffolds will be examined by
confocal microscopy following calcein staining and or actin
staining using rhodamine-phalloidin. Similar techniques would be
used to determine the depth of cell growth and/or migration into
scaffold biomaterials. Cross-sectional images will be examined and
the depth of cell ingrowth will be measured using image analysis
tools.
[0153] Other cell-seeded scaffolds can be transferred to a second
bioreactor (Tissue Growth Technologies, Inc.) that is capable of
generating physiological flow rate, pulsatile flows and pressures.
The pulsatile flow comes partly from a peristaltic pump, while the
pressure and pulses can be adjusted by crimping the outlet media
flow tubing from either the lumen or chamber. In addition, pulses
can be added through a mechanism that compresses the graft in a
pulsatile manner. This is controlled through a computer interface.
The bioreactor has built-in pressure sensors, as well as a laser
micrometer that measures the graft outer diameter. Flow rate,
pressure and the graft outer diameter are graphed on the computer
in real time. This allows the user to apply physiological pressures
and pulsatile waves to a graft and to alter them in any way.
[0154] The cell-seeded grafts are cultured in this bioreactor for
an additional period of time dependent on the desired physical and
biological characteristics desired. During the first period of
time, the pressure and flow rate are slowly increased to eventually
reached the desired physiological levels. The final parameters
depend on the eventual location of the scaffold. These levels are
maintained during the final stages. For 1 hour each day, the
following measurements will be recorded: pressure fluctuation
within the bioreactor chamber; flow rate; and fluctuation in the
outer diameter of the scaffold. At the end of the culture period,
the pressure may be increased until graft failure to determine the
burst-strength of construct.
Example 24
Bioreactor Processes for Cell Sheet Tissue Engineered Blood Vessel
Development
[0155] Cell sheets rolled into a tube as described in Examples
17-18 can be further bioprocessed to form a tissue engineered blood
vessel using bioreactor processes. The cell sheets will be seeded
with cells such as hUTCs or IMAs in a perfused rotating wall vessel
bioreactor (Synthecon, Inc.). The bioreactor has a central rotating
core with barbs that the scaffolds will be connected to. The core
allows for medium to be perfused through the lumen of the scaffold,
while the exterior is bathed in medium. The entire assembly is
rotated horizontally as above, again minimizing shear stress. For
seeding of cells onto the luminal surface of the cell sheets, a
cell suspension (10.sup.6 cells/ml) is pumped into the lumen of the
tube. The flow is stopped and rotation continues. As the bioreactor
rotates, cells are attach to the surface of the rotating lumen in a
uniform manner. After the 2 to 4 hour incubation period for
attachment, the remaining unattached cells are flushed from the
lumen, and growth medium perfused through the lumen. This culture
period is continued for .about.14 days, allowing the cells to grow
and migrate into the pores of the scaffold (the actual time of
culture will be determined empirically).
[0156] Growth of cells into the scaffolds will be examined by
confocal microscopy following calcein staining and or actin
staining using rhodamine-phalloidin. Similar techniques would be
used to determine the depth of cell growth and/or migration into
scaffold biomaterials. Cross-sectional images will be examined and
the depth of cell ingrowth will be measured using image analysis
tools.
[0157] The cell-seeded scaffolds can be transferred to a second
bioreactor (Tissue Growth Technologies, Inc.) that is capable of
generating physiological flow rate, pulsatile flows and pressures.
The pulsatile flow comes partly from a peristaltic pump, while the
pressure and pulses can be adjusted by crimping the outlet media
flow tubing from either the lumen or chamber. In addition, pulses
can be added through a mechanism that compresses the graft in a
pulsatile manner. This is controlled through a computer interface.
The bioreactor has built-in pressure sensors, as well as a laser
micrometer that measures the graft outer diameter. Flow rate,
pressure and the graft outer diameter are graphed on the computer
in real time. This allows the user to apply physiological pressures
and pulsatile waves to a graft and to alter them in any way.
[0158] The cell-seeded grafts are cultured in this bioreactor for
an additional period of time dependent on the desired physical and
biological characteristics desired. During the first period of
time, the pressure and flow rate are slowly increased to eventually
reached the desired physiological levels. The final parameters
depend on the eventual location of the scaffold. These levels are
maintained during the final stages. For 1 hour each day, the
following measurements will be recorded: pressure fluctuation
within the bioreactor chamber; flow rate; and fluctuation in the
outer diameter of the scaffold. At the end of the culture period,
the pressure may be increased until graft failure to determine the
burst-strength of construct.
Example 25
Preparation of Minced Tissue on Tubular Construct
[0159] Two small biopsy tissues were harvested from rat muscle
(Lewis rat from Harlan, Indianapolis, Ind.) by 5 mm diameter biopsy
punch (Miltex, REF No 33-35). Each biopsy weighed around 50-60 mg
and was placed in PBS supplemented with penicillin at standard
concentrations (100 U/ml). The tissue was rinsed three times in PBS
and minced into small pieces. Minced tissue was weighed and divided
into 25 mg and 50 mg and then spread evenly on the outer surface of
the each tubular construct (FIG. 14). The tissue fragments were
held on the scaffold by using fibrin glue (EVICEL, Cat No 3905,
Ethicon, Somerville, N.J.). The tubular construct was loaded with
minced tissue and placed in an incubator at 37.degree. C. for 2
hours and 72 hours (FIG. 15).
Example 26
Preparation of a Minced Tissue-Seeded TEBV Construct with Two
Sources of Minced Tissue
[0160] This example describes the preparation of a
tissue-engineered blood vessel created from a bioabsorbable
scaffold seeded with minced autologous tissue as the cell source. A
tubular or flat bioresorbable scaffold with dimensions as outlined
in Examples 1-11 is prepared. A small biopsy of tissue containing
smooth muscle cells from tissue source e.g. muscle layers in the
walls of hollow organs (such as the digestive tract, lower part of
the esophagus, stomach and intestines, the walls of the bladder,
the uterus, various ducts of glands and the walls of blood vessels)
is obtained. The biopsied tissue is placed in PBS supplemented with
penicillin at standard concentrations (100 U/ml). The tissue is
rinsed three times in PBS and then minced with the help of scalpels
to obtain minced tissue. The tissue is then distributed evenly on
the outer surface of the tubular or flat construct. Another tissue
biopsy is obtained from an endothelial tissue source for. e.g. the
lining of the blood vessel. The tissue is placed in PBS
supplemented with penicillin at standard concentrations (100 U/ml).
The tissue is minced and distributed on the inner surface of the
tubular construct or the inner surface of the flat construct. The
tissue fragments in both cases can be held on the scaffold by using
cell friendly glues for e.g. fibrin glue. Scaffold constructs that
are flat can now be sutured into tubular constructs. The constructs
can then be cultured in medium containing DMEM with Pen/Strep and
15% FBS for 4 to 8 weeks in low cell attachment dishes, during
which smooth muscle cells and endothelial cells will migrate from
the minced tissue on to the scaffolds. The engineered vessel can
then be further cultured in a bioreactor for several weeks or
months in an atmosphere of 10% CO.sub.2 at a temperature of
37.degree. C. in DMEM supplemented with 20% FBS, penicillin G (100
U/ml), 5 mM HEPES, ascorbic acid (0.05 mg/ml), CuSO.sub.4 (3
ng/ml), proline (0.05 mg/ml), alanine (0.03 mg/ml), and glycine
(0.05 mg/ml) as described in Example 27.
Example 27
Preparation of a Minced Tissue-Seeded TEBV Construct with a Single
Source of Minced Tissue
[0161] In another example, constructs seeded with minced tissue can
be prepared as outlined in Example 25. The source of minced tissue
can, however, be a single source such that the single tissue source
contains both the smooth muscle cells and endothelial cells and the
same minced tissue fragments are applied to the inner and outer
surface of the scaffolds.
Example 28
Bioreactor Processes for Cell Lysate or Minced Tissue Engineered
Blood Vessel Development
[0162] Constructs containing minced tissue or cell lysate and
scaffolds as described in Examples 1-11, 24-26 can be further
bioprocessed to form a tissue engineered blood vessel using
bioreactor processes. The constructs containing minced tissue and
scaffolds will be seeded with cells such as hUTCs or IMAs in a
perfused rotating wall vessel bioreactor (Synthecon Inc., Houston
Tex.). The bioreactor has a central rotating core with barbs that
the scaffolds will be connected to. The core allows for medium to
be perfused through the lumen of the scaffold, while the exterior
is bathed in medium. The entire assembly is rotated horizontally as
above, again minimizing shear stress. For seeding of cells onto the
luminal surface of the constructs containing minced tissue and
scaffolds, a cell suspension (10.sup.6 cells/ml) is pumped into the
lumen of the construct. The flow is stopped and rotation continues.
As the bioreactor rotates, cells are attach to the surface of the
rotating lumen in a uniform manner. After the 2 to 4 hour
incubation period for attachment, the remaining unattached cells
are flushed from the lumen, and growth medium perfused through the
lumen. This culture period is continued for .about.14 days,
allowing the cells to grow and migrate into the pores of the
scaffold (the actual time of culture will be determined
empirically).
[0163] Growth of cells into the scaffolds will be examined by
confocal microscopy following calcein staining and or actin
staining using rhodamine-phalloidin. Similar techniques would be
used to determine the depth of cell growth and/or migration into
scaffold biomaterials. Cross-sectional images will be examined and
the depth of cell ingrowth will be measured using image analysis
tools.
[0164] The cell-seeded scaffolds can be transferred to a second
bioreactor (Tissue Growth Technologies, Inc.) that is capable of
generating physiological flow rate, pulsatile flows and pressures.
The pulsatile flow comes partly from a peristaltic pump, while the
pressure and pulses can be adjusted by crimping the outlet media
flow tubing from either the lumen or chamber. In addition, pulses
can be added through a mechanism that compresses the graft in a
pulsatile manner. This is controlled through a computer interface.
The bioreactor has built-in pressure sensors, as well as a laser
micrometer that measures the graft outer diameter. Flow rate,
pressure and the graft outer diameter are graphed on the computer
in real time. This allows the user to apply physiological pressures
and pulsatile waves to a graft and to alter them in any way.
[0165] The cell-seeded grafts are cultured in this bioreactor for
an additional 7 days. During the first 3 days, the pressure and
flow rate are slowly increased to eventually reached the desired
physiological levels. The final parameters depend on the eventual
location of the scaffold. These levels are maintained for the final
4 days. For 1 hour each day, the following measurements will be
recorded: pressure fluctuation within the bioreactor chamber; flow
rate; and fluctuation in the outer diameter of the scaffold. At the
end of the culture period, the pressure may be increased until
graft failure to determine the burst-strength of construct.
Example 29
In-Vivo Efficacy Study of TEBV
[0166] TEBVs are surgically implanted in the femoral arteries of 14
adult dogs. 5 to 10 mm sections of the native vessel are removed
and replaced with the experimental TEBV using standard surgical
techniques. Anastomoses are performed using standard suture
techniques. The vessel lumen are irrigated with a standard heparin
solution. The muscle and skin are closed by standard techniques.
Postoperatively, the patency is monitored by standard
Ultrasound.
[0167] The TEBVs are explanted after 4 weeks and the patency is
assessed by direct inspection. Patency is confirmed by excising the
TEBV and evaluating the lumen histologically.
Example 30
Use of TEBV in the Treatment of Coronary Heart Disease Patient
[0168] In a coronary by-pass (CABG) procedure, a TEBV would be used
for patients suffering from arteriosclerosis, a common arterial
disorder characterized by arterial walls that have thickened
(blockages), have lost elasticity, and have calcified. This leads
to a decrease in blood supply which can lead to damage to the
heart, stroke and heart attacks.
[0169] Thus, a PDO tubular scaffold fabricated by electrospinning
processes described in Example 1 and then cell seeded and
bioreactor processed as described in Example 10, forms a TEBV that
is then sterilized, packaged and delivered to an operating room. In
a typical CABG procedure, the surgeon opens the chest via a
sternotomy. The heart's functions are taken over by a Heart and
Lung machine. The diseased artery is located and one end of the
TEBV is sewn onto the coronary arteries beyond the blockages and
the other end is attached to the aorta. The heart is restarted, the
sternum is wired together and the incisions are sutured closed.
Within a few weeks, the successful by-pass procedure is fully
healed and the patient is functioning normally.
[0170] The above description is merely illustrative and should not
be construed to capture all consideration in decisions regarding
the optimization of the design and material orientation. Although
shown and described is what is believed to be the most practical
and preferred embodiments, it is apparent that departures from
specific designs and methods described and shown will suggest
themselves to those skilled in the art and may be used without
departing from the spirit and scope of the invention. The present
invention is not restricted to the particular embodiments described
and illustrated, but should be constructed to cohere with all
modifications that may fall within the scope for the appended
claims.
Example 31
Mammary Artery Derived Cells: Isolation and Characterization
Mammary Artery Derived Cell Isolation
[0171] Internal mammary artery (IMA) will be obtained from the
National Disease Research Interchange (NDR1, Philadelphia, Pa.). To
remove blood and debris, the artery will be trimmed and washed in
Dulbecco's modified Eagles medium (DMEM-low glucose; Invitrogen,
Carlsbad, Calif.) or phosphate buffered saline (PBS; Invitrogen).
The artery will then be mechanically dissociated in tissue culture
plates until the tissue is minced to a fine pulp. The tissue will
then be transferred to a 50-milliliter conical tube. The tissue
will then be digested in an enzyme mixture containing 0.25
Units/milliliter collagenase (Serva Electrophoresis, Heidelberg,
Germany), 2.5 Units/milliliter dispase (Roche Diagnostics
Corporation, Indianapolis Ind.) and 1 Units/milliliter
hyaluronidase (Vitrase, ISTA Pharmaceuticals, Irvine, Calif.). The
enzyme mixture will then be combined with growth medium (DMEM-low
glucose (Gibco), penicillin (50 Units/milliliter) and streptomycin
(50 ug/mL, Gibco)) containing 1% fetal bovine serum (FBS). The
conical tube containing the tissue, medium and digestion enzymes
will be incubated at 37.degree. C. in an orbital shaker at 225 rpm
for 2 hours.
[0172] The digest is centrifuged at 150.times.g for 5 minutes, the
supernatant will then be aspirated. The pellet will then be
resuspended in 20 milliliters of medium. The cell suspension will
then be filtered through a 40-micron nylon BD FALCON Cell strainer
(BD Biosciences, San Jose, Calif.). The filtrate will then be
resuspended in medium (total volume 50 milliliters) and centrifuged
at 150.times.g for 5 minutes. The supernatant will then be
aspirated and the cells will be resuspended in another 50
milliliters of fresh culture medium. This washing procedure will be
repeated twice more.
[0173] After the final centrifugation, cells will be plated in
growth medium containing either 1% FBS or 10% FBS and cultured at
37.degree. C. and 5% CO2). Fragments of IMA will also be cultured
as explants in coated or non-coated tissue culture flasks. Cells
that migrate out of the tissue fragments, under media selection,
will be harvested using trypsin or other non-enzymatic methods.
[0174] For karyotype analysis, passage 4 and passage 10 mammary
artery derived cells will be plated into T25 flasks and allowed to
attach overnight. Flasks will then be filled with REGM and sent to
the University of Medicine and Dentistry of New Jersey for
karyotype analysis.
Analysis of Growth Potential
[0175] IMA derived cells will be plated at 5000 cells/cm.sup.2 onto
T75 flasks in growth medium and cultured at 37.degree. C. in 5%
carbon dioxide. Cells will be passaged every 3-5 days. At each
passage, cells are counted and viability is measured using a Guava
instrument (Guava Technologies, Hayward, Calif.). Population
doublings [ ln(final cell yield/initial number of cells plated)/ln
2] are then calculated.
Flow Cytometry
[0176] Flow cytometry analysis will be performed on IMA derived
cells. Cells will be expanded to passage four and ten in growth
medium on T225 flasks at 37.degree. C. and 5% carbon dioxide.
Adherent cells will be washed in PBS and detached with Trypsin/EDTA
(Gibco). Cells will be harvested, centrifuged and resuspended in 3%
(v/v) FBS in PBS at a concentration of 1.times.10.sup.7 cells/mL.
The specific antibody will be added to 100 microliters of cell
suspension and the mixture is incubated in the dark for 30-45
minutes at 4.degree. C. After incubation, cells will be washed with
PBS and centrifuged to remove excess antibody. Cells will be
resuspended in 500 microliters PBS and analyzed by flow cytometry.
Flow cytometry analysis will be performed with a Guava instrument.
Antibodies to be used are shown in Table 3.
TABLE-US-00004 TABLE 3 Antibodies to be used in characterizing cell
surface markers of IMA derived cells. Antibody Manufacture Catalog
number CD34 BD Pharmingen 555821 CD44 BD Pharmingen 555478 CD45R BD
Pharmingen 555489 CD117 BD Pharmingen 340529 CD141 BD Pharmingen
559781 CD31 BD Pharmingen 555446 CD133 Miltenyi Biotech 120-001-243
SSEA4 R&D Systems FAB1435P CD105 SantaCruz Biotech SC-21787
CD104 BD Pharmingen 555720 CD166 BD Pharmingen 559263 CD29 BD
Pharmingen 555442 IgG-FITC BD Pharmingen 555748 IgG-PE BD
Pharmingen 555749
Total RNA Isolation
[0177] RNA will be extracted from IMA derived cells. (RNeasy Mini
Kit; Qiagen, Valencia, Calif.). RNA will be eluted with 50 .mu.L
DEPC-treated water and stored at -80.degree. C.
Reverse Transcription
[0178] RNA will be reversed transcribed using random hexamers with
the TaqMan reverse transcription reagents (Applied Biosystems,
Foster City, Calif.) At 25.degree. C. For 10 minutes, 37.degree. C.
for 60 minutes and 95.degree. C. for 10 minutes. Samples will be
stored at -20.degree. C. Selected genes (see table below) will be
investigated using conventional PCR.
PCR
[0179] PCR reactions (with the exception of GAPDH--see chart below)
will be performed on cDNA samples using RT.sup.2 PCR Primer sets
(SuperArray Biosciences Corp, Frederick Md.). All primers shown
below will be sequence verified.
TABLE-US-00005 GENE CATALOG NUMBER Oct 4 PPH02394A Rex 1 PPH02395A
Sox2 PPH02471A Human TERT (hTERT) PPH00995A FGF4 PPH00356A
[0180] Primers will be mixed with 1 .mu.L of cDNA and 2.times.
ReactionReady.TM. SYBR Green PCR Master Mix (SuperArray
Biosciences) according to manufacturer's instructions and PCR will
be performed using an ABI Prism 7000 system (Applied Biosystems,
Foster City, Calif.). Thermal cycle conditions will be initially
50.degree. C. for 2 min and 95.degree. C. for 10 min followed by 34
cycles of 95.degree. C. for 15 sec and 60.degree. C. for 1 min. For
GAPDH, PCR will be performed using GAPDH primers from Applied
Biosystems (cat #: 402869) 1 .mu.L of cDNA solution and
1.times.AmpliTaq Gold universal mix PCR reaction buffer (Applied
Biosystems, Foster City, Calif.) according to manufacturer's
protocol. Primer concentration in the final PCR reaction will be
0.5 .mu.M for both the forward and reverse primer and the TaqMan
probe is not added. Samples will be run on 2% (w/v) agarose gel and
stained with ethidium bromide (Sigma, St. Louis, Mo.). Images will
be captured using a 667 Universal Twinpack film (VWR International,
South Plainfield, N.J.) using a focal-length Polaroid.TM. camera
(VWR International, South Plainfield, N.J.).
ELISA
[0181] IMA derived cells will be thawed at passage four and passage
ten and seeded onto T75 flasks at 5000 cells/cm.sup.2 each
containing 15 milliliters of growth medium. Cells will be cultured
for 24 hours at 37.degree. C. in 5% carbon dioxide and atmospheric
oxygen. The medium will be changed to a serum-free medium (DMEM-low
glucose (Gibco), 0.1% (w/v) bovine serum albumin (Sigma),
penicillin (50 Units/milliliter) and streptomycin (50 ug/mL,
Gibco)) and further cultured for 8 hours. Conditioned, serum-free
medium is then collected at the end of incubation by centrifugation
at 14,000.times.g for 5 min and stored at -20.degree. C.
[0182] To estimate the number of cells in each flask, cells will be
washed with PBS, detached using 2 milliliters trypsin/EDTA (Gibco)
and counted with a Guava instrument (Guava Technologies Hayward,
Calif.). Samples are then assayed for the following factors: tissue
inhibitor of metalloproteinase-1 (TIMP1), tissue inhibitor of
metalloproteinase-2 (TIMP2), platelet-derived epithelial growth
factor bb (PDGFbb), keratinocyte growth factor (KGF), hepatocyte
growth factor (HGF), fibroblast growth factor (FGF), vascular
endothelial growth factor (VEGF), Heparin-binding epidermal growth
factor (HB-EGF), monocyte chemotactic protein-1 (MCP1),
interleukin-6 (IL6), interleukin-8 (IL8), transforming growth
factor alpha (TGFa), brain-derived neurotrophic factor (BDNF),
stromal-derived factor 1B (SDF 1B), cilliary neurotrophic factor
(CNTF), basic nerve growth factor (bNGF), neurotrophin-3 (NT3) with
the Searchlight Proteome Arrays (Pierce Biotechnology Inc.).
Example 32
Seeding of Rat Smooth Muscle Cells R354-05 into PDO Scaffolds
[0183] This example relates to the uniform static seeding of rat
smooth muscle cells R354-05 (Cell Applications) into PDO-ESS tube
(Example 1, part 2). Cells are cultured to allow cell growth and
matrix production. This growth can include static culture or
culture under physiological conditions in a bioreactor, such as
luminal flow with or without pressure and/or pulsatile flow.
[0184] PDO tubes were cut in 2 cm length and placed in a 60 mm
tissue culture dish. Rat aortic smooth muscle cells (Cell
Applications, Inc.) were trypsinized, counted and resuspended in
rat smooth muscle growth medium at a concentration of
2.times.10.sup.6 cells/ml. Using a 200 ul pipet tip cells were
gently dripped onto the PDO tube. The PDO tubes containing the
cells were left at room temperature for one hour before being
placed in a 37.degree. C. humidified environment for 4 days.
[0185] After four days of static culture PDO tubes were placed in a
rotary cell culture system (Synthecon) for ten days at 7.0 rpm to
allow cell growth and matrix product. At 14 days in culture, the
distribution and morphology of the cells is evaluated. PDO tubes
were transferred to a 60 mm tissue culture dish. 5 mm biopsy
punches were harvested and placed in a 24 well plate containing
PBS. To test the viability of the cells, a live/dead assay
(Molecular Probes) was performed. Ten mls of PBS containing 2
micromolar calcein AM and 4 micromolar ethidum homodimer were
prepared. One ml of the live/dead stain was added to the biopsies.
After incubation at room temperature for 5 minutes, cell attachment
and viability of cells was assessed by fluorescence microscopy.
Similar punch biopsies were fixed in 10% neutral buffered formalin,
embedded in paraffin, sectioned and stained with H&E to
evaluate cell morphology (FIG. 16) and Masson's Trichome stain
which is specific for the formation of extracellular matrix.
[0186] Results:
[0187] Rat smooth muscle cells are viable throughout the culture
period with the PDO tubes supporting cell viability. Static seeding
of the rat smooth muscle cells for four days leads to an even cell
distribution. Cells attach on both sides of the scaffolds. Cell
infiltration into the scaffolds is observed.
* * * * *