WO1996008213A1 - Cultures tridimensionnelles de cellules humaines sur des structures de valvules cardiaques, et leurs utilisations - Google Patents

Cultures tridimensionnelles de cellules humaines sur des structures de valvules cardiaques, et leurs utilisations Download PDF

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Publication number
WO1996008213A1
WO1996008213A1 PCT/US1995/011395 US9511395W WO9608213A1 WO 1996008213 A1 WO1996008213 A1 WO 1996008213A1 US 9511395 W US9511395 W US 9511395W WO 9608213 A1 WO9608213 A1 WO 9608213A1
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WIPO (PCT)
Prior art keywords
cells
heart valve
stromal
stromal cells
fibroblasts
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PCT/US1995/011395
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English (en)
Inventor
Gail K. Naughton
Brian A. Naughton
Anthony F. Purchio
Lee K. Landeen
Joan Zeltinger
Todd D. Campbell
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Advanced Tissue Sciences, Inc.
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Application filed by Advanced Tissue Sciences, Inc. filed Critical Advanced Tissue Sciences, Inc.
Priority to AU35855/95A priority Critical patent/AU700911B2/en
Priority to JP8510252A priority patent/JPH10511563A/ja
Priority to EP95933062A priority patent/EP0781116A4/fr
Priority to NZ293419A priority patent/NZ293419A/en
Priority to KR1019970701618A priority patent/KR970705951A/ko
Publication of WO1996008213A1 publication Critical patent/WO1996008213A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
    • A61F2/2412—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body with soft flexible valve members, e.g. tissue valves shaped like natural valves
    • A61F2/2415—Manufacturing methods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels

Definitions

  • the invention relates to growing in vitro, human cells such as fibroblasts on a three-dimensional
  • scaffold comprising porcine aortic leaflets and walls, intact heart valves, other biological scaffolding
  • a valve or valve components for example, including but not limited to the pericardium or the small intestinal submucosa, and biodegradable frameworks, such that the scaffold is populated with viable human cells having normal function, and
  • fibroblasts are stimulated to produce human matrix proteins to supplement and replace the existing matrix on the scaffold.
  • the resulting three-dimensional tissue constructs have a variety of applications ranging from
  • Valve replacement surgical therapy is required for the treatment of various types of valvular heart
  • aortic stenosis aortic regurgitation
  • mitral stenosis mitral stenosis
  • valve replacement Two general types of valve replacement are available: the artificial, mechanical prosthesis or valve, and tissue biological prosthesis or valve.
  • mechanical prosthesis such as the ball valve, the tilting disk and the central flow disk.
  • tissue prostheses including preserved homografts and stent-mounted, porcine valve heterografts.
  • the primary advantage of the mechanical prosthesis is durability, whereas the disadvantage is a requirement that patients be on an anticoagulant therapy to reduce the risk of thromboembolic complications. This is because artificial mechanical heart valves are prone to occlusions by thrombus, and are subject to mechanical failures. Thromboembolism and anticoagulated hemorrhage are still the frequent causes for reoperation and patient death. Moreover, mechanical failure can occur suddenly and without warning resulting in emergency surgical interventions for replacement of the device.
  • Xenogeneic biological valves usually porcine or bovine in origin, have the advantage of being identical in design and structure to those valves being replaced, but are fixed with glutaraldehyde and, therefore, are non-living.
  • the glutaraldehyde-treated tissues calcify over time and do not allow infiltration and colonization by host cells, which is necessary for remodeling. Consequently, these xenogeneic valves degrade with time and eventually malfunction.
  • Autologous human tissue i.e., derived from the recipient
  • autologous human tissue is used for coronary and peripheral bypass procedures, third degree burns and reconstructive
  • tissue-based heart valves for treating diabetes
  • transplantation is necessary due to unmet patient demands to improve upon existing heart valve technologies, which are mechanical valves requiring the constant use of anticoagulants and glutaraldehyde fixed tissue valves which eventually experience calcification.
  • cytotoxic agents According to Orton, the addition of bFGF to the tissue in vitro is critical in this system, and is essential for causing the graft-populating cells to migrate into the tissue and proliferate in response to the growth factor, and populate the tissue.
  • Orton only demonstrates the system on small pieces of the valve fixed in a petri dish and does not show production of a functional heart valve or any of the alleged
  • Livesey et al., U.S. Patent No. 5,336,616 relates to a method of producing a transplantable tissue graft for processing and preserving acellular, collagen-based tissue matrix for transplantation.
  • the method described involved processing biological tissues with a stabilizing solution to prevent osmotic, hypoxic, autolytic and proteolytic degradation and to control contamination.
  • the tissues were decellularized with EDTA, CHAPS or a zwitterionic detergent, SDS or anionic/nonionic
  • a cryoprotectant such as DMSO, propylene glycol, butanediol, raffinose, polyvinyl pyrrolidone, dextran or sucrose and vitrified in liquid nitrogen. Thereafter, the tirssues were
  • the present invention relates to transplantable cardiac tissue or bioprosthetic grafts composed of human cells grown on three-dimensional frameworks, scaffolds or matrices, a method of culturing human cells on such frameworks and uses of such three-dimensional cell cultures.
  • stromal cells including but not limited to human fibroblasts, are inoculated and grown on a three-dimensional
  • the preferred three-dimensional framework may be prepared from intact porcine heart valves, aortic wall tissue, or leaflets which are decellularized (at -20°C to -70°C or with detergents and enzymes) and sterilized by: chemical methods including, but not limited to, ethylene oxide and peracetic acid; irradiation including, but not limited to, gamma and electron beam; and steam
  • sterilization including, but not limited to autoclaving. No viable cells remain in the decellularized/sterilized tissue samples which are used as a scaffold or framework for culturing the stromal cells.
  • the stromal cells which are inoculated onto the scaffold may include dermal or cardiac fibroblasts, and/or cells capable of producing collagen types I and III, and in some instances, elastin, which are typically produced in heart valves. (See Table I).
  • the stromal cells and connective tissue proteins naturally secreted by the stromal cells attach to and substantially envelope the three-dimensional framework or construct, having interstitial spaces bridged by the stromal cells.
  • the living stromal tissue so formed provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of stromal cells in culture and/or cultures implanted in vivo. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts in vivo.
  • the stromal cells can be genetically engineered to express a gene product beneficial for successful and/or improved
  • the stromal cells can be genetically engineered to express anticoagulation gene products to reduce the risk of thromboembolism, or anti- inflammatory gene products to reduce the risk of failure due to inflammatory reactions.
  • the stromal cells can be genetically engineered to express tissue plasminogen activator (TPA), streptokinase or urokinase to reduce the risk of clotting.
  • TPA tissue plasminogen activator
  • streptokinase or urokinase to reduce the risk of clotting.
  • the stromal cells can be engineered to express anti- inflammatory gene products, e.g., peptides or
  • the cells are engineered to express such gene products transiently and/or under inducible control during the post-operative recovery period, or as a chimeric fusion protein anchored to the stromal cell, e.g., a chimeric molecule composed of an intracellular and/or transmembrane domain of a receptor or receptorlike molecule, fused to the gene product as the
  • the stromal cells can be genetically engineered to "knock out" expression of factors or surface antigens that promote clotting or rejection.
  • factors or surface antigens that promote clotting or rejection.
  • expression of fibrinogen, von Willebrands factor or any cell surface molecule that binds to the platelet ⁇ 2B ⁇ -3 receptor can be knocked out in the stromal cells to reduce the risk of clot
  • MHC class II molecules can be knocked out in order to reduce the risk of rejection of the graft.
  • the three-dimensional culture system of the invention may afford a vehicle for introducing genes and gene products in vivo to assist or improve the results of the
  • genes that prevent or ameliorate symptoms of valvular disease such as thrombus formation, inflammatory reactions, fibrosis and calcification, may be used.
  • the level of gene activity in the patient may be increased or decreased, respectively, by gene replacement therapy by adjusting the level of the active gene product in genetically engineered stromal cells.
  • human dermal fibroblasts were grown in the three-dimensional culture systems of the
  • Porcine aortic walls and leaflets were chosen because they are currently used in replacement therapy of heart valves. Particular benefits were achieved in porcine aortic wall and leaflet cultures where
  • tissue similar to human matrix proteins in the aortic walls and leaflets was detected. These characteristics were monitored by analyzing the
  • the present invention thus, relates to a method of repopulating porcine aortic walls and leaflets with human fibroblasts to produce human matrix proteins in which the porcine aortic leaflets and walls are first sterilized with peracetic acid (or by other chemical means such as ethylene oxide) or by radiation with an electron beam (or by gamma irradiation) or by steam (autoclaving).
  • peracetic acid or by other chemical means such as ethylene oxide
  • an electron beam or by gamma irradiation
  • steam autoclaving
  • human fibroblasts were grown in culture on frameworks or constructs, composed of porcine aortic valves, walls and leaflets which had been decellularized and sterilized.
  • frameworks or constructs When implanted in vivo, such frameworks or constructs allow adequate nutrient and gas exchange to the cells until engraftment and vascularization at the site of
  • the advantage of adding human fibroblasts to the three-dimensional, decellularized porcine scaffolds or biodegradable constructs is that colonization of the porcine scaffolding results in a valve implant with living cells which produce biological factors that may stimulate host cells to endothelize the implant and stimulate host cardiac fibroblasts to integrate into the implant. The net result is
  • Another advantage of adding human fibroblasts is that cultures can be
  • heart valves colonized with functional human cells are less likely to be subject to immunological rejection and thus are superior to those heart valves which are covered with xenogeneic cells prepared for use in replacement therapy.
  • a heart valve from human foreskin or cardiac fibroblasts and porcine heart valve and/or aortic walls and leaflets, which no longer contains porcine cells but becomes a humanized porcine heart valve or a
  • recellularized heart valve suitable for transplantation in humans.
  • Such an approach provides an improved method and means of designing, constructing and utilizing aortic walls and leaflets, intact heart valves other biological scaffolding suitable for reconstructing a valve or valve component (e.g., pericardium, small intestinal submucosa, etc.) and biodegradable frameworks, as scaffolding for growth and implantation of human fibroblasts in vitro.
  • a valve or valve component e.g., pericardium, small intestinal submucosa, etc.
  • biodegradable frameworks e.g., as scaffolding for growth and implantation of human fibroblasts in vitro.
  • a heart valve consisting of human cells and human tissue matrix proteins made by human dermal or cardiac fibroblasts and a completely or nearly complete bioresorbable/biocompatible polymer scaffolding in the shape of different types of valves or their components, for example, but not limited to aortic, pulmonary, mitral, and tricuspid valves.
  • Such an approach provides bioprosthetic or transplantable tissues, which can be utilized for cell growth, both in vitro and in vivo, to replace or reconstruct degenerated and dysfunctional heart valves in human patients.
  • a valve or valve components e.g., pericardium, small intestinal submucosa, etc.
  • bioactive molecules normally produced in the body by the cells of the aortic walls and leaflets or the intact heart valve or the pulmonary, mitral, and tricuspid valves.
  • the present invention relates to methods and
  • valves for the treatment of valvular heart disease including, but not limited to, aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, pulmonary valve disease, tricuspid valve disease, multivalvular disease, tricuspid valve disease, Marfan syndrome and artificial valve disease.
  • Figure 1 is a photograph of autoradiographed proteins synthesized by human dermal fibroblasts post seeding onto porcine aortic leaflets and walls.
  • Figure 2 is a photograph of hematoxylin and eosin stained tissue sections: a) a fresh porcine leaflet (the cardiac fibroblast nuclei native to the tissue appear purple in coloration); b) a detergent and/or enzyme extracted porcine leaflet (no porcine cell nuclei are detected after chemical treatment); c) a detergent and/or enzyme extracted porcine leaflet cultured with human fibroblasts for 18 weeks (the dermal human fibroblasts are present in the porcine matrix). (Stained with Hematoxylin/Eosin.) (10x).
  • Figure 3 is a photograph of a porcine leaflet seeded with human dermal fibroblasts and cultured for 4 weeks.
  • Figure 4 is a bar graph showing that in three sample sets (#1-3) of detergent and/or enzyme extracted leaflets with or without fibroblasts, only the leaflets which are grown with fibroblasts incorporated 3 H-thymidine, indicating that the fibroblasts were proliferating.
  • Figure 5 is a SDS gel autoradiograph analysis showing protein bands: non-viable porcine leaflet (lane 1) and wall biopsy (lane 2) seeded with human fibroblasts show protein synthesis, whereas unseeded, non-viable porcine leaflet (lane 3) and porcine wall biopsy (lane 4) show no activity.
  • Fresh, viable porcine leaflet (lane 5) and wall biopsy (lane 6) seeded with human fibroblasts have similar patterns to fresh, viable, unseeded porcine leaflet (lane 7) and wall biopsy (lane 8).
  • FIG. Porcine leaflet and wall (negative controls) a,b, respectively) stained with serum only and showed no background staining. Porcine leaflet stained with human tenascin (c) and porcine wall stained with human
  • fibroblast antibody (d). Both c and d show no species cross reactivity.
  • Whole humanized porcine valve constructs cultured for 4 weeks under dynamic flow showed positive staining for human tenascin in the leaflet, wall, and muscle bar (e,g,i) and positive staining for human fibroblasts in the leaflet, wall, and muscle bar (f,h,j).
  • Figure 7 is a photograph of autoradiographed protein incorporation of human fibroblasts after dynamic culture on porcine aortic leaflets.
  • Figure 8 is a photograph depicting human fibroblast proliferation on a porcine matrix which was previously decellularized by detergent and/or enzyme treatment.
  • the proliferating cells were labeled with Brdu and detected using an antibody to Brdu and a visualization kit.
  • the labeled cells proliferating on the tissues were grown under dynamic flow conditions. Brdu labeling occurred during the last 72 hr of a 4 week culture period.
  • Figure 9 is a photograph depicting decellularized
  • the present invention relates to transplantable cardiac tissue constructs or bioprosthetic grafts grown in three-dimensional frameworks, a method of culturing human cells on such frameworks and uses of such three- dimensional, recellularized tissue constructs grown in cultures.
  • stromal cells including but not limited to human fibroblasts, are inoculated and grown on a three-dimensional framework or construct of intact heart valves, aortic walls and leaflets or other biological scaffolding suitable for reconstructing a valve or valve components, for example, including but not limited to the pericardium or the small intestinal submucosa or biodegradable frameworks.
  • Cells grown on a three-dimensional framework grow to form a cellular tissue-matrix which resembles tissue found in vivo to a greater degree than previously described.
  • the three- dimensional cell culture system treated with human stromal cells is applicable to the proliferation of different types of cells and formation of a number of different tissues, including but not limited to aortic walls and leaflets, or intact heart valves, pulmonary, mitral, and tricuspid valves.
  • the stromal cells grown in the system may be genetically engineered to produce gene products beneficial to transplantation, e.g., anti-coagulation factors, e.g., TPA, streptokinase, etc., or anti-inflammatory factors, e.g., anti-TNF, anti- IL-2, etc.
  • the stromal cells may be genetically engineered to "knock out” expression of native gene products that promote platelet binding and clot formation, e.g., fibrinogen, von Willebrands factor, or "knock out” expression of MHC in order to lower the risk of rejection.
  • the stromal cells may be genetically engineered for use in gene therapy to adjust the level of gene activity in a patient to assist or improve the results of the transplantation.
  • human foreskin fibroblasts in the three- dimensional tissue constructs has a variety of advantages and applications.
  • the three-dimensional tissue constructs can be produced at a rapid rate and may itself be transplanted or implanted into a living organism without undue delay.
  • the three- dimensional tissue constructs may also be used in vitro for testing the effectiveness or cytotoxicity of pharmaceutical agents, screening compounds for use in treatment of clotting or thromboembolism, as
  • anticoagulants as anti-inflammatory agents, as anti- calcification agents or as endothelialization agents.
  • the three-dimensional tissue construct system may be cellularized within a "bioreactor" to produce a valve or valve component with leaflet mobility and full valve function.
  • a bioreactor for example, an intact valve comprising of leaflets attached to the wall, may be assembled as a three-dimensional framework, inoculated with human stromal cells and maintained in recirculating culture medium regulated by a peristaltic or pneumatic pump which also keeps the leaflets or tissue sheets/patches in a dynamic state.
  • the bioreactor provides a closed system free from problems of
  • the methods for culturing cells including human dermal fibroblasts on aortic walls and leaflet cells or intact heart valves or other biological scaffolding suitable for reconstructing a valve or valve components for example, but not limited to the pericardium or the small intestinal submucosa or biodegradable frameworks, as a three-dimensional biological or synthetic framework or construct which can be used in accordance with the invention are described in applicants' co-pending
  • Methods for the treatment of valvular heart disease including, but not limited to, aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, pulmonary valve disease, tricuspid valve disease,
  • the three-dimensional framework for use in the present invention may be of any material and/or shape that: (a) allows cells to attach to it (or can be
  • allogeneic and . xenogeneic aortic walls and leaflets or intact heart valves or other biological scaffolding suitable for reconstructing a valve or valve components for example, but not limited to the pericardium or the small intestinal submucosa or biodegradable frameworks, obtained from a variety of mammals, including but not limited to, man, pig, cow, sheep or dog, may be used.
  • porcine leaflets and aortic biopsies may be used in the following forms: irradiated or chemically treated or steam treated (sterilized); decellularized (for example, detergent and/or enzyme treated), extracted and
  • valve tissue with nonviable cells and other biological tissues for example, but not limited to, pericardium or small intestinal submucosa
  • the methods for decellularizing the aortic walls and leaflets or intact valves or other biological scaffolding suitable for reconstructing a valve or valve components include, but are not limited to the methods described in U.S. Patent No. 5,336,616 and U.S. Patent No. 4,776,853, which are incorporated herein by reference in their entirety.
  • the tissues can be decellularized with EDTA, CHAPS or a zwitterionic detergent, followed by treatment with a cryoprotectant such as DMSO, propylene glycol, butanediol, raffinose, polyvinyl pyrrolidone, dextran or sucrose and vitrified in liquid nitrogen.
  • a cryoprotectant such as DMSO, propylene glycol, butanediol, raffinose, polyvinyl pyrrolidone, dextran or sucrose and vitrified in liquid nitrogen.
  • the tissue sample can be subjected to enzymatic digestion and/or extracting with reagents that break down the cellular membranes and allow removal of cell contents.
  • reagents include non-ionic detergents (for example, TRITON X-100, o ⁇ tylphenoxy polyethoxyethanol, (Rohm and Haas); BRIJ-35, a
  • polyethoxyethanol lauryl ether (Atlas Chemical Co.), TWEEN 20, a polyethoxyethanol sorbitan monolaureate (Rohm and Haas), LUBROL-PX, or polyethylene lauryl ether (Rohm and Haas)); and ionic detergents (for example, sodium dodecyl sulphate, sulfated higher aliphatic alcohol, sulfonated alkane and sulfonated alkylarene containing 7 to 22 carbon atoms in a branched or unbranched chain).
  • the enzymes used may include nucleases (for example, deoxyribonuclease and ribonuclease), proteases,
  • tissue in the invention can also be decellularized using physical procedures such as ultrasonic treatment or osmotic shock, or by chemical treatment using peracetic acid.
  • the three-dimensional framework may also be composed of completely or nearly complete bioreeorbable/
  • bioc ⁇ mpatible polymer scaffolding in the shape of various different types of valves, including but not limited to, aortic, pulmonary, mitral, and tricuspid valves and valve components of each type.
  • the biodegradable scaffolds, constructs, frameworks or matrices may be composed of materials such as polyglycolic acid, catgut suture material, hyaluronic acid, cellulose, collagen (in the form of sponges, braids, or woven threads, etc.),
  • Such frameworks or constructs may be molded into the shape of heart valves or repair sheets/patches prior to inoculation of human cells. Where possible, however, it is most preferable to use a three-dimensional construct of the tissue of origin, for example, the aortic walls and leaflets or intact heart valves.
  • the invention is based in part, on the discovery that the three-dimensional system supports the
  • hormones even though not absolutely necessary in the present invention, may be used to further enhance the reconstitution of the porcine or other biological
  • the three-dimensional framework provides a greater surface area for protein attachment
  • stromal cells Because of the three-dimensionality of the framework, stromal cells continue to actively grow, in contrast to many cells in monolayer cultures, which grow to confluence, exhibit contact inhibition, and cease to grow and divide.
  • the elaboration of extracellular matrix proteins and secretion of growth and regulatory factors by replicating stromal cells may be partially responsible for stimulating proliferation, maintaining normal tissue differentiation and regulating differentiation of cells in culture.
  • the increase in potential volume for cell growth in the three-dimensional system may allow the establishment of localized microenvironments conducive to cellular maturation.
  • the three-dimensional framework maximizes cell- cell interactions by allowing greater potential for movement of migratory cells.
  • Stromal cells comprising fibroblasts, with or without other stromal cells and elements described below, are inoculated onto the three-dimensional framework.
  • Human fibroblasts may be added to the culture prior to, during or subsequent to inoculation of other stromal cells.
  • the concentration of fibroblasts maintained in the cultures can be monitored and adjusted appropriately to optimize growth and to regulate scaffold colonization.
  • stromal cells that are genetically engineered to express and produce factors similar to those produced by cells of the heart valve, may be included in the inoculum. These cells could serve as a source of protein factor(s) in the culture. Preferably, the gene or coding sequence for factor(s) would be placed under the control of a regulated promoter, so that production of factor(s) in culture can be controlled.
  • the genetically engineered cells will be screened to select those cell types: 1) that bring about
  • Stromal tissue comprising dermal fibroblasts, cardiac fibroblasts and cells capable of producing collagen type I and III, elastin and other heart valve matrix proteins, for example, but not limited to
  • fibronectin and glycosaminoglycans are used to grow in vitro, transplantable tissue or bioprosthetic heart valves.
  • Stromal cells such as fibroblasts can be
  • Fetal and neonatal fibroblasts can be used to form a "generic" three-dimensional stromal tissue construct that will support the growth of a variety of different cells and/or tissues. Fibroblasts may be readily isolated by
  • tissue or organ which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art.
  • the tissue or organ can be disaggregated mechanically and/or treated with
  • Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include but are not limited to trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, pronase, etc.
  • Mechanical disruption can also be accomplished by a number of methods including, but not limited to the use of grinders, blenders, sieves, homogenizers, or pressure cells to name but a few.
  • the suspension can be fractionated into subpopulations from which the fibroblasts and/or other stromal cells and/or elements can be obtained. This also may be accomplished using standard techniques for cell separation including but not limited to cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential
  • centrifugation centrifugal elutriation (counter- streaming centrifugation), unit gravity separation, counter current distribution, electrophoresis and
  • the isolation of fibroblasts may, for example, be carried out as follows: fresh tissue samples are
  • HBSS Hanks balanced salt solution
  • Inoculation of the three-dimensional matrix with a high concentration of stromal cells e.g., approximately 10 6 to 5 x 10 7 cells/ml, will result in the establishment of the three-dimensional stromal construct in shorter periods of time.
  • the cultured cells are to be used for transplantation or implantation in vivo it is preferable to obtain the stromal cells from the patient's own tissues. However, it is also possible to use allogeneic compatible human cells, without significant rejection reactions following transplantation.
  • the growth of cells in the presence of the three-dimensional stromal support matrix may be further enhanced by adding to the matrix, or coating the matrix support with specific amino acids, proteins, glycoproteins, glycosaminoglycans, a cellular matrix, and/or other materials.
  • the three-dimensional matrix should be incubated in an appropriate nutrient medium.
  • Many commercially available media such as DMEM, RPMI 1640, Fisher's Iscove's, McCoy's, and the like may be suitable for use.
  • the three-dimensional stromal matrix be suspended or floated in the medium during the incubation period in order to maximize proliferative activity.
  • the container in this protocol is kept stable in the incubator, i.e., under static conditions (no circulating or flowing fluid).
  • the culture should be "fed” periodically to remove the spent media, depopulate released cells, and add fresh nutrients. The concentration of fibroblasts may be adjusted during these steps.
  • a bioreactor which is a closed system housing the three-dimensional framework inoculated with stromal cells.
  • a bioreactor reduces the possibility of contamination, maintains the cultures in recirculating, continuous culture medium and keeps the leaflets in a dynamic state by opening and closing them. More particularly, the U.S. patent
  • the stromal cells will attach and proliferate along the three-dimensional framework before beginning to migrate into the depths of the matrix.
  • One objective is to grow the cells to an appropriate degree which reflects the amount of stromal cells present in the in vivo tissue.
  • a second objective is to regulate the number of cells in the inoculum and/or their growth on the scaffold such that the amount of scaffold colonization can be controlled as desired, and reproducibly.
  • constructs should be of an appropriate size to allow the stromal cells to stretch across the openings.
  • stromal cells which stretch across the framework enhances the production of growth factors which are elaborated by the stromal cells, and hence will support long term cultures. For example, if the openings are too small, the stromal cells may rapidly achieve confluence but be unable to easily exit from the mesh; trapped cells may exhibit contact inhibition and cease production of the appropriate factors necessary to support proliferation and maintain long term cultures. If the openings are too large, the stromal cells may be unable to stretch across the opening; this will also decrease stromal cell production of the appropriate factors necessary to support proliferation and maintain long term cultures.
  • openings ranging from about 150 ⁇ m to about 220 ⁇ m will work satisfactory. However, depending upon the three-dimensional structure and intricacy of the framework, other sizes may work equally well. In fact, any shape or structure that allows the stromal cells to stretch and continue to replicate and grow for lengthy time periods will work in accordance with the invention.
  • the human dermal fibroblasts exhibit a varied affinity for the different types of porcine tissue matrices.
  • the greatest fibroblast colonization occurs when using a porcine matrix that is detergent and/or enzyme extracted. Additionally, the amount of fibroblast colonization in the porcine tissue correlates with time.
  • collagen types I and III are preferably deposited in the initial matrix.
  • the appropriate isotypes or subclass that are capable of activating complement, and which define particular collagen type.
  • These antibodies and complement can be used to negatively select the fibroblasts which express the desired collagen type.
  • the stroma used to inoculate the matrix can be a mixture of cells which synthesize the appropriate collagen types desired. The distribution and origins of the five types of
  • proliferating cells may be released from the framework. These released cells may stick to the walls of the culture vessel where they may continue to
  • the culture system could be agitated to prevent the released cells from adhering, or instead of periodically feeding the cultures, the culture system could be set up so that fresh media continuously flows through the system.
  • the flow rate could be adjusted to both maximize proliferation within the three-dimensional culture, and to wash out and remove cells released from the matrix, so that they will not adhere to the walls of the vessel and grow to confluence.
  • the biological heart valves produced in the three- dimensional culture system of the invention can be used in the treatment of aortic stenosis, aortic
  • Aortic stenosis is the obstruction to flow across the aortic valve during left ventricular systolic
  • ejection It can be caused by a congenital unicuspid or bicuspid valve, rheumatic fever, or degenerative
  • Aortic regurgitation is the diastolic flow of blood from the aorta into the left ventricle. It is caused by incompetent closure of the aortic valve which results from intrinsic disease of the cusp or from diseases affecting the aorta. Acquired intrinsic diseases of the aortic valve are either rheumatic or from bacterial origin. In the Marfan syndrome the primary basis for aortic insufficiency usually resides in the aorta, but there may be prolapse of the aortic cusps due to
  • myxomatous changes Infrequent changes are seen with rheumatoid arthritis, systemic lupus erythematosus, and trauma. Oh, W.M.C., Taylor, T.R. and Olsen, E.G.J., 1974, Br. Heart J. 36:413. Patients with chronic aortic regurgitation who are symptomatic are advised to have surgery. The type of operation used depends primarily on the etiology. In patients with diseases limited to the valve, the operation is essentially as described above, for aortic stenosis.
  • Mitral stenosis designates resistance to flow through the mitral apparatus during diastolic filling of the left ventricle. Resistance to diastolic flow across the mitral valve can result from rheumatic valvulitis, congenital stenosis, thrombus formation, atrial myxoma, bacterial vegetations, and calcification in the valve, as well as in the annulus. The decision to intervene surgically in patients with mitral stenosis is based on the anticipated necessity of valve replacement versus valve reconstruction therapy.
  • Mitral regurgitation occurs when contraction of the left ventricle ejects blood into left atrium as a result of abnormalities in the mitral valve apparatus.
  • Acute mitral regurgitation can be created from mechanical disruption of the chordae tendineae, rupture of the papillary muscle, or perforation of the leaflet.
  • Rheumatic fever, mitral valve prolapse and coronary artery disease such as left ventricular dilation, calcified mitral annulus, heritable disorders (Marfan syndrome, Ehlers-Danlos, osteogenesis), congenital heart disease, systemic lupus erythematosus, rupture of
  • papillary muscle and perforation of leaflet are the predominant mechanisms for the incompetence of the mitral valve.
  • Replacement of the mitral valve, valve components and/or other affected parts such as the chordae is required in cases of rheumatic involvement leading to severe mitral regurgitation, mitral stenosis with loss of pliability of the leaflets, and various other causes of mitral regurgitation, such as infective endocarditis, and in some cases in chronic heart disease. Calcification and immobility of the leaflets are also indications for valve replacement.
  • Pulmonary stenosis is created by obstruction to systolic flow across the valve and is most commonly congenital. It generally leads to pulmonary embosis
  • Pulmonary valve replacement may be performed for acquired conditions such as carcinoid heart disease and infective endocarditis.
  • Tricuspid regurgitation develops when the tricuspid valve allows blood to enter the right atrium during right ventricular contraction.
  • Tricuspid stenosis represents obstruction to diastolic flow across the valve during diastolic filling of the right ventricle.
  • the main cause of tricuspid and mitral regurgitation is the rupture of one or more of the elements of the tensor apparatus, with disruption of the papillary muscle and rupture of the chordae tendineae. Replacement is necessary if the changes in the leaflets and subvalvular mechanism are advanced, or if severe regurgitation cannot be relieved by annulopolasty.
  • Multivalvular disease indicates obstruction and/or incompetence of the aortic, mitral, and tricuspid valves.
  • Rheumatic fever, connective tissue diseases, Marfan syndrome, calcification of the mitral valve in the aging patient and bacterial endocarditis remain important causes in combined disease of the mitral and aortic valves.
  • both valves are generally repressed by surgery.
  • Artificial valve disease includes any abnormality of a surgically implanted device to replace a diseased cardiac valve. Artificial valve disease can result from prosthetic dysfunction, thrombus formation, infection, fibrosis, or calcification. Roberts, W.C., 1973, Prog. Cardiovasc. Dis. 15:539. Congestive heart failure due to mechanical valve dysfunction is the major indication for replacement of a mechanical artificial valve.
  • the second most common operation performed in adults is replacement of the aortic or mitral valve.
  • the valves produced in accordance with the invention may be
  • aortic valve transplanted using similar, if not the same surgical techniques, well known to those skilled in the art.
  • the procedure for the replacement of the aortic valve is performed through a median sternum-splitting incision.
  • a vascular clamp is placed across the distal ascending aorta.
  • a sump suction cannula is placed in the left atrium through an incision in the right superior pulmonary vein to
  • Coronary artery perfusion usually is not necessary for single-valve replacement, provided it can be
  • either portions of the culture or the entire three-dimensional culture could be implanted, depending upon the type of tissue involved.
  • three-dimensional heart valve cultures can be maintained in vitro for long periods. Section of tissues or the entire three- dimensional tissue structure can be transplanted in vivo in patients needing new heart valves.
  • Three-dimensional tissue culture implants may, according to the inventions, be used to replace or augment existing tissue, to introduce new or altered tissue, to modify artificial prostheses, or to join together biological tissues or structures.
  • the three-dimensional cultures may be used in vitro to screen a wide variety of compounds, for effectiveness and cytotoxicity of pharmaceutical agents,
  • growth/regulatory factors natural and modified blood products, anticoagulants, clotting agents or anti- calcification agents, etc.
  • the cultures are maintained in vitro and exposed to the compound to be tested.
  • the activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture. This may readily be assessed by vital staining techniques.
  • the effect of growth/regulatory factors may be assessed by analyzing the cellular content of the matrix, e.g., by total cell counts, and differential cell counts. This may be accomplished using standard
  • cytological and/or histological techniques including the use of immunocytochemical techniques employing antibodies that define type-specific cellular antigens.
  • the effect of various drugs on normal cells cultured in the three- dimensional system may be assessed.
  • the three-dimensional culture system of the invention may afford a vehicle for introducing genes and gene products in vivo to assist or improve the results of the transplantation and/or for use in gene therapies.
  • the stromal cells can be genetically
  • the stromal cells can be genetically engineered to express TPA, streptokinase or urokinase to reduce the risk of clotting.
  • the stromal cells can be engineered to express anti-inflammatory gene products, for example, peptides or polypeptides corresponding to the idiotype of neutralizing antibodies for TNF, IL-2, or other inflammatory cytokines.
  • the cells are engineered to express such gene products transiently and/or under inducible control during the post-operative recovery period, or as a chimeric fusion protein anchored to the stromal cells, for example, a chimeric molecule composed of an intracellular and/or transmembrane domain of a receptor or receptor-like molecule, fused to the gene product as the extracellular domain.
  • the stromal cells could be genetically engineered to express a gene for which a patient is deficient, or which would exert a therapeutic effect, e.g., HDL, apolipoprotein E, etc.
  • the genes of interest engineered into the stromal cells need to be related to heart disease.
  • the stromal cells can be engineered to express gene products that are carried by the blood; e.g., cerebredase, adenosine deaminase, ⁇ -1- antitrypsin.
  • pulmonary valve can be used to deliver gene products such as ⁇ -1-antitrypsin to the lungs; in such an approach, constitutive expression of the gene product is preferred.
  • the stromal cells can be engineered using a
  • recombinant DNA construct containing the gene used to transform or transfect a host cell which is cloned and then clonally expanded in the three-dimensional culture system.
  • the three-dimensional culture which expresses the active gene product could be implanted into an individual who is deficient for that product.
  • genes that prevent or ameliorate symptoms of various types of valvular heart diseases may be
  • genes involved in preventing the following pathological conditions may be down-regulated, for example: thrombus formation, inflammatory reactions, and fibrosis and calcification of the valves.
  • the activity of gene products may be diminished, leading to the manifestations of some or all of the above pathological conditions and eventual development of symptoms of valvular disease.
  • the level of gene activity may be increased by either
  • Target gene refers to a gene involved in valvular disease in a manner by which
  • modulation of the level of target gene expression or of target gene product activity may act to ameliorate symptoms of valvular disease.
  • patients may be treated by gene replacement therapy during the post-recovery period after
  • Heart valve constructs or sheets may be designed specifically to meet the requirements of an individual patient, for example, the stromal cells may be genetically engineered to regulate one or more genes; or the regulation of gene expression may be transient or long-term; or the gene activity may be non-inducible or inducible.
  • one or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein product with target gene function may be inserted into human cells that populate the three-dimensional constructs using either non-inducible vectors including, but are not limited to, adenovirus, adeno-associated virus, and retrovirus vectors, or inducible promoters, including metallothionein, or heat shock protein, in addition to other particles that introduce DNA into cells, such as liposomes or direct DNA injection or in gold particles.
  • the gene encoding the human complement regulatory protein which prevents rejection of the graft by the host, may be inserted into human fibroblasts.
  • the three-dimensional cultures containing such genetically engineered stromal cells e.g., either mixtures of stromal cells each expressing a different desired gene product, or a stromal cell engineered to express several specific genes are then implanted into the patient to allow for the amelioration of the symptoms of valvular disease.
  • the gene expression may be under the control of a non-inducible (i.e., constitutive) or inducible promoter.
  • the level of gene expression and the type of gene regulated can be controlled depending upon the treatment modality being followed for an individual patient.
  • the use of the three-dimensional culture in gene therapy has a number of advantages. Firstly, since the culture comprises eukaryotic cells, the gene product will be properly expressed and processed in culture to form an active product. Secondly, gene therapy techniques are useful only if the number of transfected cells can be substantially enhanced to be of clinical value,
  • the three-dimensional cultures of the invention allow for expansion of the number of transfected cells and amplification (via cell division) of transfected cells.
  • a variety of methods may be used to obtain the constitutive or transient expression of gene products engineered into the stromal cells.
  • the transkaryotic implantation technique described by Seldon, R.F., et al., 1987, Science 236:714-718 can be used.
  • Transkaryotic suggests that the nuclei of the implanted cells have been altered by the addition of DNA sequences by stable or transient transfection.
  • the cells can be engineered using any of the variety of vectors including, but not limited to, intergating viral vectors, e.g., retrovirus vector or adeno-associated viral vectors, or non-integrating replicating vectors, e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors.
  • intergating viral vectors e.g., retrovirus vector or adeno-associated viral vectors
  • non-integrating replicating vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g
  • the expression control elements used should allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo.
  • the promoter chosen would depend, in part upon the type of tissue and cells cultured. Cells and tissues which are capable of secreting proteins (e.g., those
  • Hosts cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow in an enriched media, and then are switched to a
  • recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which, in turn, can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which express the gene protein product.
  • any promoter may be used to drive the expression of the inserted gene.
  • viral promoters include but are not limited to the CMV promoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus, elastin gene promoter and ⁇ -globin. If transient expression is desired, such constitutive promoters are preferably used in a non- integrating and/or replication-defective vector.
  • inducible promoters could be used to drive the expression of the inserted gene when necessary.
  • inducible promoters include, but are not limited to, metallothionein and heat shock protein.
  • transcriptional control regions that exhibit tissue specificity for connective tissues which have been described and could be used, include but are not limited to: elastin or elastase I gene control region which is active in pancreatic acinar cells (Swit et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515). The deposition of elastin is correlated with specific physiological and
  • atrioventricular valve cusps are initially thick and fleshy in an embryo, and later in the development are transformed into thin and fibrous cusps.
  • elastin deposition appears to be coordinated with changes in arterial pressure and mechanical activity.
  • Animals that contain valves and ligamental structures that are elastic contain elastin.
  • elastin-synthesizing cells are attached to elastin through cell-surface receptors, the synthesis of additional elastin and other matrix proteins may be influenced by exposure to stress or mechanical forces in the tissue (for example, the constant movement of the construct in the bioreactor) or other factors that influence cellular shape.
  • TPA tumor necrosis factor
  • streptokinase urokinase activity
  • urokinase activity can bring about amelioration of platelet aggregation, blood coagulation or
  • thromboembolism This activity is maintained for a limited time only, for example, to prevent potential complications that generally develop during the early phase after valve implantation, such as, platelet aggregation, blood clotting, coagulation or
  • engineered cells are implanted into an individual, the presence of the anti-inflammatory gene products, for example, peptides or polypeptides corresponding to the idiotype of neutralizing antibodies for TNF, IL-2, or other inflammatory cytokines, can bring about
  • the stromal cells used in the three-dimensional culture system of the invention may be genetically engineered to "knock out" expression of factors or surface antigens that promote clotting or rejection at the implant site. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below. "Negative modulation”, as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
  • the expression of a gene native to stromal cell can be reduced or knocked out using a number of techniques, for example, expression may be inhibited by inactivating the gene completely (commonly termed "knockout") using the
  • an exon encoding an important region of the protein is interrupted by a positive
  • selectable marker for example neo
  • a gene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene. By using a construct with two regions of homology to the target gene that are far apart in the genome, the sequences intervening the two regions can be deleted. Mombaerts, P., et al., 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084-3087.
  • Antisense and ribozyme molecules which inhibit expression of the target gene can also be used in
  • antisense RNA for example, antisense RNA
  • HLA histocompatibility gene complexes
  • fibrinogen v Willebrands factor
  • factor V any cell surface molecule that binds to the platelet a2B ⁇ -3 receptor can be knocked out in the stromal cells to reduce the risk of clot formation at the valve.
  • MHC class II molecules can be knocked out in order to reduce the risk of rejection of the graft.
  • the three-dimensional culture system could be used in vitro to produce biological products in high yield.
  • a cell which naturally produces large quantities of a particular biological product e.g., a growth factor, regulatory factor, peptide hormone, antibody, etc.
  • a host cell genetically engineered to produce a foreign gene product could be clonally expanded using the three-dimensional culture system in vitro. If the transformed cell excretes the gene product into the nutrient medium, the product may be readily isolated from the spent or conditioned medium using standard separation techniques (e.g., HPLC, column chromatography,
  • the gene product is isolated (e.g., by HPLC column chromatography, electrophoresis, etc.) from the outflow of spent or conditioned media.
  • stromal cells such as fibroblasts upon decellularized heart valves in vitro, in a system designed to mimic physiologic conditions in vivo.
  • the cells replicated in this system synthesize proteins similar to those produced by the normal aortic wall and leaflet cells.
  • Heart valve extracellular matrix is composed mainly of elastin and collagen types I and III.
  • the following example describes a method for growing transplantable or bioprosthetic heart valve tissue in culture by
  • Porcine aortic walls and leaflets were washed with phosphate buffered saline and used fresh or after being frozen at -20°C to -70°C in sterile water or after detergent and/or enzyme extraction or any aforementioned tissue in combination with sterilization techniques as described in U.S. Patent No. 4,776,85. See Section 5.1.
  • Dermal foreskin fibroblasts were cultured in vitro by routine procedures. Fibroblasts used in the studies were in their eighth passage at the time of seeding to the porcine tissues.
  • Porcine aortic leaflets and walls were seeded in eight well dishes with 1x10 5 human dermal fibroblasts and cultured for one day. The aortic walls and leaflets were transferred into new well dishes and grown for an
  • the eight cultures were made up of: (1) previously frozen leaflet seeded with human fibroblasts; (2) previously frozen wall seeded with human fibroblasts; (3) previously frozen leaflet without seeding; (4) previously frozen wall without seeding; (5) fresh leaflet seeded with human fibroblasts; (6) fresh wall seeded with human fibroblasts; (7) fresh leaflet without seeding; and (8) fresh wall without seeding.
  • the cultures were labeled with [ 35 S]-methionine and [ 35 S]- cysteine (Tran 35 S-Label, ICN) for four hours.
  • the samples were boiled in Laemmli sample buffer containing ⁇ -mercaptoethanol, fractionated by SDS polyacrylamide gel electrophoresis (SDS ⁇ PAGE), and analyzed by
  • porcine leaflet or wall tissues were housed in a multi-well dish (one piece of tissue/well) as described above in Section 6.1.2.
  • the human dermal fibroblasts were suspended in a nutrient-rich growth medium and were seeded onto the specific types of porcine leaflet or wall tissues such as: 1) frozen leaflets and walls; 2) electron beamed leaflets; 3) detergent and/or enzyme extracted leaflets and walls; and 4) detergent and/or enzyme extracted + electron beamed leaflets.
  • Each culture dish was maintained at 37°C in a sterile, static culture (no media flow) environment.
  • These human fibroblast-porcine tissue composites which grow in the tissue culture dish are referred to as heart valve constructs.
  • the constructs were analyzed for: a) Cell Distribution
  • MTT Assay This assay is used to assess the viability of cells after growing on the porcine matrix. Metabolically active (living) fibroblasts convert the MTT substrate (0.5mg/ml) into an insoluble purple precipitant within the cells. The purple precipitant can be
  • the MTT reaction can be quantified by measuring the optical density with a spectrophotometer (540nm) after extraction in isopropanol as described in Triglia, D., et al., 1991, Toxic, in Vitro 5:573-578.
  • Glucose consumption As an indicator of fibroblast viability, nutrient consumption (glucose) and metabolic waste products (lactate) contained in the tissue construct are measured as described in
  • Thymidine [ 3 H-thy] Incorporation Radioactive thymidine (10 ⁇ Ci) is added to the nutrient-rich media during the 24-72 hr culture of tissue constructs. When fibroblasts in the constructs divide to produce additional cells, some of the 3 H-thy becomes incorporated in the DNA of the cells. Excess, non-incorporated 3 H-thy is removed after washing the labelled constructs in 1% triton-X-100 for 2 hr and rinsing in PBS. The
  • incorporated 3 H-thy can be measured using a scintillation counter.
  • BrdU Incorporation An alternative method for measuring fibroblast proliferation in tissue constructs is to add 5-bromodeoxyuridine (BrdU) to the culture media.
  • BrdU is a non-radioactive, thymidine analog which incorporates into newly synthesized DNA of dividing fibroblasts. Tissues are incubated in BrdU-containing media for 24-48 hr. The fibroblasts containing BrdU can be visualized in the histology sections of the tissue constructs using a monoclonal antibody to the BrdU, followed by an enzyme-chromogen detection system using the Zymed Kit. (ZYMED Laboratories, Inc. San Francisco, Ca). d) Protein Assays
  • radiolabelled amino acids which are added to the nutrient-rich media during the tissue culture process.
  • the radiolabelled amino acids are incorporated into newly synthesized proteins in the tissue constructs and can be measured using a
  • Proline is a major amino acid constituent of the collagen proteins in the tissue constructs. The amount of radioactive proline
  • the labelled tissues can then be digested in laemeli sample buffer under reducing ( ⁇ -mercaptoethanol) conditions and separated by SDS-PAGE. Specific proteins can be quantified by western blotting. e) Protein Immunohistochemistry
  • This method detects specific proteins in a
  • histological section of tissue using monoclonal or polyclonal antibodies The antibodies used specifically detect human proteins and react with: 1) human
  • fibroblasts prolyl-4-hydroxylase
  • elastin fibers in valve wall and leaflet tissue
  • human tenascin matrix glycoprotein
  • a second antibody which recognizes the primary antibody is conjugated to an enzyme-chromogen visualization system.
  • Porcine leaflets were glued (medical grade
  • cyanoacrylate either along one surface (immobilizing the tissue) or on one edge (allowing some movement) to a polycarbonate cassette sterilized by electron beam radiation (E-beam).
  • E-beam electron beam radiation
  • Human fibroblasts were seeded dynamically (5 ml/min flow rate) on these tissues arid cultured for three days. The tissues were excised from the cassette and labeled with [ 35 S] -methionine and [ 35 S]- cysteine for four hours. Tissues were boiled in Laemmli sample buffer, insoluble material was pelleted, and supernatants were fractionated by SDS*PAGE and visualized by autoradiography.
  • Lanes 5 through 8 describe corresponding results obtained with fresh, unfrozen leaflets and walls.
  • Lanes 7 and 8 containing unseeded fresh, unfrozen aortic leaflets and walls, respectively, demonstrated protein synthesis by
  • Protein production measured as collagen synthesis ( 3 H-proline labeling) indicated that the human dermal fibroblasts were producing collagen and some proteins that are present in porcine leaflets ( 33 S- cysteine/methionine labeling) (Figure 5).
  • Fibroblasts produced human tenascin to supplement the existing porcine scaffolding ( Figure 6). 6.2.3. COLONIZATION OF AORTIC WALLS AND
  • Lanes 1 and 2 (Fig. 7) containing samples of porcine leaflets that were glued along their entire surface, and cultured under dynamic flow had no appreciable staining.
  • Lanes 3 and 4 show porcine leaflets that were glued on one edge, with an appreciable amount of radioactivity was incorporated after growth for three days post seeding; when porcine leaflets were glued along an entire surface, minimal [ 3S S] was incorporated into protein.
  • An unseeded, E-beam sterilized leaflet used as a control (lane 5) showed no incorporation of radioactivity.
  • porcine aortic leaflets and walls can be statically or dynamically seeded with human fibroblasts. These human fibroblasts attach and colonize the aortic leaflet and wall scaffolds, and remain metabolically active by secreting extracellular matrix molecules.
  • the matrix seeded under dynamic, pulstile flow conditions had greater and more uniform fibroblast attachment that the matrix grown under static conditions, as shown by using the MTT assay (an indicator of cell viability as

Abstract

L'invention concerne un procédé qui permet de faire croître différents tissus et cellules dans des cultures tridimensionnelles in vitro en utilisant des fibroblastes humains dans des milieux de culture. Des cellules du stroma, comprenant notamment des fibroblastes dermiques et cardiaques humains, sont inoculés et croissent sur une grille ou structure tridimensionnellle. Ces fibroblastes humains secrètent des protéines de matrice humaine destinées aà compléter et remplacer la matrice porcine existante composée de valvules cardiaques ou de parois et valves aortiques privées de leurs cellules et prenant la forme de reconstructions tridimensionnelles dotées d'espaces intersticiels reliés par les cellules du stroma. Le tissu du stroma vivant ainsi formé fournit un support ainsi que des facteurs de croissance et de régulation nécessaires pour assurer la viabilité et la prolifération à long terme des cellules mises en culture et/ou implantées in vivo. Lorsqu'elles croissent sur cette structure tridimensionnelle, les cellules qui prolifèrent deviennent matures et se différencient nettement pour former des composants de tissus analogues à leur équivalents in vivo.
PCT/US1995/011395 1994-09-12 1995-09-08 Cultures tridimensionnelles de cellules humaines sur des structures de valvules cardiaques, et leurs utilisations WO1996008213A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU35855/95A AU700911B2 (en) 1994-09-12 1995-09-08 Three-dimensional human cell cultures on cardiac valve frameworks and their uses
JP8510252A JPH10511563A (ja) 1994-09-12 1995-09-08 心臓弁枠組み上での三次元ヒト細胞培養物およびその使用
EP95933062A EP0781116A4 (fr) 1994-09-12 1995-09-08 Cultures tridimensionnelles de cellules humaines sur des structures de valvules cardiaques, et leurs utilisations
NZ293419A NZ293419A (en) 1994-09-12 1995-09-08 Stromal cell-coated heart valves, production thereof
KR1019970701618A KR970705951A (ko) 1994-09-12 1995-09-08 심장 판막 구조에서 3차원 사람 세포 배양물과 이의 용도(three-dimensional human cell cultures on cardiac valve franeworks and their uses)

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US30406294A 1994-09-12 1994-09-12
US48816595A 1995-06-07 1995-06-07
US488,165 1995-06-07
US304,062 1995-06-07

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KR (1) KR970705951A (fr)
AU (1) AU700911B2 (fr)
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WO1998004300A1 (fr) * 1996-07-31 1998-02-05 St. Jude Medical, Inc. Utilisation de micro-organismes pour le traitement de tissus bioprothetiques
WO1999000152A2 (fr) * 1997-06-27 1999-01-07 Augustinus Bader Transplant biosynthetique et son procede de production
WO1998049972A3 (fr) * 1997-05-02 1999-04-08 St Jude Medical Traitement differentiel de dispositifs de prothese
WO2000061204A1 (fr) * 1999-04-12 2000-10-19 Advanced Tissue Sciences, Inc. Tissu du stroma tridimensionnel
WO2001003750A1 (fr) * 1999-07-09 2001-01-18 Advanced Tissue Sciences, Inc. Appareil revetu d'une matrice extracellulaire naturellement secretee par l'homme
WO2001030274A1 (fr) * 1999-10-22 2001-05-03 Gunze Limited Valve cardiaque mecanique et procede de production
US6284284B1 (en) 1995-06-06 2001-09-04 Advanced Tissue Sciences, Inc. Compositions and methods for production and use of an injectable naturally secreted extracellular matrix
WO2002040076A1 (fr) * 2000-11-17 2002-05-23 Autotissue Gmbh Procede et dispositif pour produire des protheses biologiques
US6432712B1 (en) * 1999-11-22 2002-08-13 Bioscience Consultants, Llc Transplantable recellularized and reendothelialized vascular tissue graft
WO2003043674A1 (fr) * 2001-11-16 2003-05-30 Children's Medical Center Corporation ¨ Augmentation de la fonction d'un organe
US6652583B2 (en) 2000-04-07 2003-11-25 Rhode Island Hospital Cardiac valve replacement
EP1579827A2 (fr) 2003-10-24 2005-09-28 DEUTSCHE INSTITUTE FÜR TEXTIL- UND FASERFORSCHUNG STUTTGART Stiftung des öffentlichen Rechts Implant cardiovasculaire, methode et dispositif de fabrication
WO2006102063A2 (fr) * 2005-03-19 2006-09-28 Cook Biotech Incorporated Implants prothetiques comprenant un materiau composite a matrice extracellulaire
EP1782849A2 (fr) * 1999-04-12 2007-05-09 Theregen, Inc. Tissu stromal tridimensionnel
US7569076B2 (en) 1997-10-31 2009-08-04 Children's Medical Center Corporation Bladder reconstruction
US7795027B2 (en) 2003-09-04 2010-09-14 Cook Biotech Incorporated Extracellular matrix composite materials, and manufacture and use thereof
US7915038B2 (en) 2000-05-29 2011-03-29 Augustinus Bader Method for producing a recipient-specific tissue transplant or tissue implant
US20120009677A1 (en) * 2000-05-29 2012-01-12 Augustinus Bader Method for producing a bio-artificial transplant
US8372433B2 (en) 2007-01-18 2013-02-12 Gunze Limited Substrate for culture of cardiovascular tissue
US8470520B2 (en) 2005-08-26 2013-06-25 Regents Of The University Of Minnesota Decellularization and recellularization of organs and tissues
US8748142B2 (en) 1999-09-09 2014-06-10 Gunze Limited Culture of cardiovascular cells on a matrix and method for regenerating cardiovascular tissue
US9051550B2 (en) 2009-04-09 2015-06-09 Arizona Board Of Regents, On Behalf Of The University Of Arizona Cellular seeding and co-culture of a three dimensional fibroblast construct
US9290738B2 (en) 2012-06-13 2016-03-22 Miromatrix Medical Inc. Methods of decellularizing bone
US9534203B2 (en) 2007-06-08 2017-01-03 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
US9580688B2 (en) 2007-06-08 2017-02-28 Wake Forest University Health Sciences Kidney structures and methods of forming the same
US9643153B2 (en) 2014-05-19 2017-05-09 Evonik Degussa Gmbh Membrane-supported catalyst removal in the epoxidation of cyclic unsaturated C12 compounds, for example cyclododecene (CDEN)
US9877822B2 (en) 2012-04-24 2018-01-30 Biostage, Inc. Engineered tissue scaffolds and supports therefor
US9968446B2 (en) 2011-03-23 2018-05-15 The Regents Of The University Of California Tubular scaffold for fabrication of heart valves
US10233420B2 (en) 2010-09-01 2019-03-19 Regents Of The University Of Minnesota Methods of recellularizing a tissue or organ for improved transplantability
US10590391B2 (en) 2007-06-08 2020-03-17 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
US11278643B2 (en) 2016-09-06 2022-03-22 Mayo Foundation For Medical Education And Research Use of resected liver serum for whole liver-engineering
US11452797B2 (en) 2013-03-15 2022-09-27 Miromatrix Medical Inc. Use of perfusion decellularized liver for islet cell recellularization
CN115177789A (zh) * 2022-07-01 2022-10-14 天津市康婷生物工程集团有限公司 透明质酸在制备促进瓣膜间质细胞分泌胶原蛋白、糖胺聚糖或弹性蛋白的产品中的应用
US11890395B2 (en) 2017-06-16 2024-02-06 Avery Therapeutics, Inc. Three dimensional tissue compositions and methods of use

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EP2085054A1 (fr) * 2008-02-01 2009-08-05 Technische Universiteit Eindhoven Procédé de fabrication de construction créée par tissu
US10016461B2 (en) 2012-12-03 2018-07-10 The Regents Of The University Of California Apparatus and process for growing a heart valve in three-dimensions
DE102014209413A1 (de) 2014-05-19 2015-11-19 Evonik Degussa Gmbh Membrangestützte Katalysatorabtrennung bei der Epoxidierung von Fettsäurealkylestern

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Cited By (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6284284B1 (en) 1995-06-06 2001-09-04 Advanced Tissue Sciences, Inc. Compositions and methods for production and use of an injectable naturally secreted extracellular matrix
US6121041A (en) * 1996-07-31 2000-09-19 St. Jude Medical, Inc. Use of microorganisms for decellularizing bioprosthetic tissue
WO1998004300A1 (fr) * 1996-07-31 1998-02-05 St. Jude Medical, Inc. Utilisation de micro-organismes pour le traitement de tissus bioprothetiques
US6531310B1 (en) 1996-07-31 2003-03-11 St. Jude Medical, Inc. Use of microorganisms for processing bioprosthetic tissue
US6132473A (en) * 1997-05-02 2000-10-17 St. Jude Medical, Inc. Differential treatment of prosthetic devices
WO1998049972A3 (fr) * 1997-05-02 1999-04-08 St Jude Medical Traitement differentiel de dispositifs de prothese
US6497725B2 (en) 1997-05-02 2002-12-24 St. Jude Medical, Inc. Differential treatment of prosthetic devices
US6206917B1 (en) 1997-05-02 2001-03-27 St. Jude Medical, Inc. Differential treatment of prosthetic devices
WO1999000152A3 (fr) * 1997-06-27 1999-05-27 Augustinus Bader Transplant biosynthetique et son procede de production
WO1999000152A2 (fr) * 1997-06-27 1999-01-07 Augustinus Bader Transplant biosynthetique et son procede de production
US7569076B2 (en) 1997-10-31 2009-08-04 Children's Medical Center Corporation Bladder reconstruction
US8128707B2 (en) 1997-10-31 2012-03-06 Children's Medical Center Corporation Bladder reconstruction
EP1782849A2 (fr) * 1999-04-12 2007-05-09 Theregen, Inc. Tissu stromal tridimensionnel
WO2000061204A1 (fr) * 1999-04-12 2000-10-19 Advanced Tissue Sciences, Inc. Tissu du stroma tridimensionnel
US8128924B2 (en) 1999-04-12 2012-03-06 Theregen, Inc. Methods for using a three-dimensional stromal tissue to promote angiogenesis
AU777853B2 (en) * 1999-04-12 2004-11-04 Theregen, Inc. Three-dimensional stromal tissue
EP1782849A3 (fr) * 1999-04-12 2007-05-23 Theregen, Inc. Tissu stromal tridimensionnel
AU777853C (en) * 1999-04-12 2005-08-11 Theregen, Inc. Three-dimensional stromal tissue
WO2001003750A1 (fr) * 1999-07-09 2001-01-18 Advanced Tissue Sciences, Inc. Appareil revetu d'une matrice extracellulaire naturellement secretee par l'homme
US8748142B2 (en) 1999-09-09 2014-06-10 Gunze Limited Culture of cardiovascular cells on a matrix and method for regenerating cardiovascular tissue
US6875230B1 (en) 1999-10-22 2005-04-05 Gunze Limited Mechanical heart valve and production method thereof
WO2001030274A1 (fr) * 1999-10-22 2001-05-03 Gunze Limited Valve cardiaque mecanique et procede de production
US7179287B2 (en) 1999-11-22 2007-02-20 Bioscience Consultants Bioreactor mediated recellularization of natural and tissue engineered vascular grafts
US6432712B1 (en) * 1999-11-22 2002-08-13 Bioscience Consultants, Llc Transplantable recellularized and reendothelialized vascular tissue graft
US6652583B2 (en) 2000-04-07 2003-11-25 Rhode Island Hospital Cardiac valve replacement
US7915038B2 (en) 2000-05-29 2011-03-29 Augustinus Bader Method for producing a recipient-specific tissue transplant or tissue implant
US20120009677A1 (en) * 2000-05-29 2012-01-12 Augustinus Bader Method for producing a bio-artificial transplant
WO2002040076A1 (fr) * 2000-11-17 2002-05-23 Autotissue Gmbh Procede et dispositif pour produire des protheses biologiques
WO2003043674A1 (fr) * 2001-11-16 2003-05-30 Children's Medical Center Corporation ¨ Augmentation de la fonction d'un organe
US9186435B2 (en) 2003-09-04 2015-11-17 Cook Biotech, Incorporated Extracellular matrix composite materials, and manufacture and use thereof
US7795027B2 (en) 2003-09-04 2010-09-14 Cook Biotech Incorporated Extracellular matrix composite materials, and manufacture and use thereof
EP1579827A3 (fr) * 2003-10-24 2007-12-12 DEUTSCHE INSTITUTE FÜR TEXTIL- UND FASERFORSCHUNG STUTTGART Stiftung des öffentlichen Rechts Implant cardiovasculaire, méthode et dispositif de fabrication
EP1579827A2 (fr) 2003-10-24 2005-09-28 DEUTSCHE INSTITUTE FÜR TEXTIL- UND FASERFORSCHUNG STUTTGART Stiftung des öffentlichen Rechts Implant cardiovasculaire, methode et dispositif de fabrication
WO2006102063A2 (fr) * 2005-03-19 2006-09-28 Cook Biotech Incorporated Implants prothetiques comprenant un materiau composite a matrice extracellulaire
US8454678B2 (en) 2005-03-19 2013-06-04 Cook Biotech Incorporated Prosthetic implants including ECM composite material
WO2006102063A3 (fr) * 2005-03-19 2007-03-08 Cook Biotech Inc Implants prothetiques comprenant un materiau composite a matrice extracellulaire
US8470520B2 (en) 2005-08-26 2013-06-25 Regents Of The University Of Minnesota Decellularization and recellularization of organs and tissues
US10441609B2 (en) 2005-08-26 2019-10-15 Miromatrix Medical Inc. Decellularization and recellularization of solid organs
US10220056B2 (en) 2005-08-26 2019-03-05 Miromatrix Medical, Inc. Decellularization and recellularization of solid organs
US8372433B2 (en) 2007-01-18 2013-02-12 Gunze Limited Substrate for culture of cardiovascular tissue
US9534203B2 (en) 2007-06-08 2017-01-03 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
US9580688B2 (en) 2007-06-08 2017-02-28 Wake Forest University Health Sciences Kidney structures and methods of forming the same
US10590391B2 (en) 2007-06-08 2020-03-17 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
US9051550B2 (en) 2009-04-09 2015-06-09 Arizona Board Of Regents, On Behalf Of The University Of Arizona Cellular seeding and co-culture of a three dimensional fibroblast construct
US11345894B2 (en) 2009-04-09 2022-05-31 Arizona Board Of Regents On Behalf Of The University Of Arizona Cellular seeding and co-culture of a three dimensional fibroblast construct
US9976123B2 (en) 2009-04-09 2018-05-22 Arizona Board Of Regents On Behalf Of The University Of Arizona Cellular seeding and co-culture of a three dimensional fibroblast construct
US11414644B2 (en) 2010-09-01 2022-08-16 Regents Of The University Of Minnesota Methods of recellularizing a tissue or organ for improved transplantability
US10233420B2 (en) 2010-09-01 2019-03-19 Regents Of The University Of Minnesota Methods of recellularizing a tissue or organ for improved transplantability
US9968446B2 (en) 2011-03-23 2018-05-15 The Regents Of The University Of California Tubular scaffold for fabrication of heart valves
US9877822B2 (en) 2012-04-24 2018-01-30 Biostage, Inc. Engineered tissue scaffolds and supports therefor
US9290738B2 (en) 2012-06-13 2016-03-22 Miromatrix Medical Inc. Methods of decellularizing bone
US11452797B2 (en) 2013-03-15 2022-09-27 Miromatrix Medical Inc. Use of perfusion decellularized liver for islet cell recellularization
US9643153B2 (en) 2014-05-19 2017-05-09 Evonik Degussa Gmbh Membrane-supported catalyst removal in the epoxidation of cyclic unsaturated C12 compounds, for example cyclododecene (CDEN)
US11278643B2 (en) 2016-09-06 2022-03-22 Mayo Foundation For Medical Education And Research Use of resected liver serum for whole liver-engineering
US11890395B2 (en) 2017-06-16 2024-02-06 Avery Therapeutics, Inc. Three dimensional tissue compositions and methods of use
CN115177789A (zh) * 2022-07-01 2022-10-14 天津市康婷生物工程集团有限公司 透明质酸在制备促进瓣膜间质细胞分泌胶原蛋白、糖胺聚糖或弹性蛋白的产品中的应用

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NZ293419A (en) 1998-11-25
IL115261A0 (en) 1995-12-31
AU3585595A (en) 1996-03-29
CA2199810A1 (fr) 1996-03-21
AU700911B2 (en) 1999-01-14
KR970705951A (ko) 1997-11-03
EP0781116A4 (fr) 1999-08-25
EP0781116A1 (fr) 1997-07-02
JPH10511563A (ja) 1998-11-10

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