WO1999041981A1 - Procede permettant de traiter et de conserver des tissus a base de collagene - Google Patents

Procede permettant de traiter et de conserver des tissus a base de collagene Download PDF

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Publication number
WO1999041981A1
WO1999041981A1 PCT/US1999/003667 US9903667W WO9941981A1 WO 1999041981 A1 WO1999041981 A1 WO 1999041981A1 US 9903667 W US9903667 W US 9903667W WO 9941981 A1 WO9941981 A1 WO 9941981A1
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Prior art keywords
tissue
solution
immersing
vsmd
detergent
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PCT/US1999/003667
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English (en)
Inventor
Stephen A. Livesey
Christopher L. Coleman
Lawrence E. Boerboom
Edward S. Griffey
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Lifecell Corporation
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Application filed by Lifecell Corporation filed Critical Lifecell Corporation
Priority to EP99908285A priority Critical patent/EP1056335A1/fr
Priority to AU27753/99A priority patent/AU752457B2/en
Priority to JP2000532008A priority patent/JP2002503678A/ja
Priority to CA002320887A priority patent/CA2320887A1/fr
Publication of WO1999041981A1 publication Critical patent/WO1999041981A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • 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
    • A61L27/3687—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 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents

Definitions

  • Tissue and organ transplantation is a rapidly growing therapeutic field as a result of improvements in surgical procedures, advancements in immunosuppressive drugs and increased knowledge of graft/host interaction.
  • modern tissue transplantation remains associated with complications including inflammation, degradation, scarring, contracture, calcification (hardening), occlusion and rejection.
  • calcification hardening
  • occlusion rejection
  • Autologous or self-derived human tissue is often used for transplant procedures. These procedures include coronary and peripheral vascular bypass surgeries, where a blood vessel, usually a vein, is harvested from some other area of the body and transplanted to correct obstructed blood flow through one or more critical arteries.
  • substitutes may be used, including man-made synthetic materials, animal-derived tissues and tissue products, or allogeneic human tissues donated from another individual (usually derived from cadavers).
  • Man-made implant materials include synthetic polymers (e.g. polytetrafluroethylene (PTFE), DACRONTM and GORETEXTM) sometimes formed into a tubular shape and used as a blood flow conduit for some peripheral arterial bypass procedures.
  • PTFE polytetrafluroethylene
  • DACRONTM DACRONTM
  • GORETEXTM GORETEXTM
  • man made materials such as plastics and carbonized metals, may be utilized for aortic heart valve replacement procedures.
  • Synthetic materials may be made with low immunogenicity but are subject to other limitations. In the case of mechanical heart valves, their hemodynamic characteristics necessitate life-long anticoagulant therapy. Synthetic vascular grafts, often used in above-the-knee peripheral vascular bypass procedures, are subjected to an even higher incidence of occlusion than autologous grafts. Indeed, the literature contains contraindications for the use of synthetic conduits for small diameter application (less than 6 mm diameter). In many cases, a preference is made for a biological implant which can be a processed animal tissue or allogeneic human tissue. Animal tissues (bovine or porcine) treated with chemicals may be used as replacements for defective human heart valves and for vascular grafts.
  • the concept in the chemical processing is to stabilize the structural protein and collagen matrix by cross-linking with glutaraldehyde or a similar cross-linking agent.
  • Chemical treatment also masks histocompatibility antigens and other antigenic determinants.
  • the human host should not recognize the implant as foreign and reduces or eliminates a specific immunorejection response.
  • the immune system may still recognize the implant as a foreign body resulting in encapsulation or overgrowth of the implant.
  • glutaraldehyde-treated tissues often do not allow in-migration of host cells which are necessary for biochemical and biomechanical homeostasis.
  • freeze-drying process results in a graft which elicits no significant rejection response as compared to fresh or cryopreserved allogeneic bone. It is believed that the freeze-dried bone following implant acts as a template, which is subsequently remodeled by the host. When the freeze-drying process has been applied to more complex tissues such as heart valves, the results have been reported as being unsatisfactory.
  • a study was conducted in which 15 allogeneic heart valves were processed by freeze-drying prior to transplantation. Most of the freeze-dried valves failed due to mechanical causes in the early post-graft interval. However, those freeze-dried valves which did not fail demonstrated prolonged functionality (up to 15 years).
  • the present invention is directed to a method of processing and preserving collagen based tissues for use as transplantable tissues.
  • the present invention is also directed to the product of the method of processing.
  • the process of preserving collagen-based tissues of the present invention includes: procuring the collagen-based tissue; treating the tissue in a first detergent solution; treating the tissue in an enzyme solution; treating the tissue in a cryopreservation solution; and, cryopreserving the tissue.
  • the process also includes treating the tissue so as to prevent or inhibit the molecular crosslinking of processed tissues via the Maillard reaction and the subsequent formation of advanced glycosylation end products; treating the tissue so as to prevent or inhibit the molecular crosslinking of processed tissues via reactive oxidative species of molecules; and treating the tissue so as to prevent or inhibit the molecular crosslinking of processed tissues via the formation and propagation of molecular free radicals.
  • the process may be applied to a collagen based tissue that is a heart valve or vascular conduits such as veins or arteries, nerve or nerve tissues, umbilical cord vessels, dura, dermis and the like.
  • the first detergent solution may be formulated so as to include one or more of t-octylphenoxypolyethoxyethanol (Triton X-100), n-octyl- ⁇ -D-glucopyranoside, deoxycholate, octanoic acid (caprylate), 3-[(3- Cholamidopropyl)dimethylammonio]-l-propane-sulfonate (CHAPS), ethylene diamine tetraacetate (EDTA), sodium chloride, and broad-spectrum antimicrobials in a physiological buffer solution.
  • a second detergent solution may alternatively be included in the process and such second detergent solution may be formulated from the same ingredients as the first 5 -
  • the enzyme solution may include one or more of DNase Type I, DNase Type II, RNase, phospholipase A, phospholipase C, and broad-spectrum antibiotics in a physiological buffer solution including broad spectrum antimicrobials.
  • the techniques and solution utilized may be selected from: depressed temperatures; use of non-reactive or non-reducing carbohydrates such as those that result from the molecular reduction of the carbonyl group of glucose and related compounds, and aminoguanidine; and combinations thereof.
  • the techniques used may include: depressed temperatures; inert atmosphere; and one or more of deferoxa ine mesylate, dimethyl sulfoxide (DMSO), catalase, superoxide dismutase, a-tocopherol, reduced glutathione, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; and combinations thereof.
  • DMSO dimethyl sulfoxide
  • the step of treating the tissue so as to prevent or inhibit propagation of molecular free radicals includes: using depressed temperatures; and one or more of dimethyl sulfoxide (DMSO), a-tocopherol, ascorbate, reduced glutathione, flavonoids, and inositol hexaphosphoric acid (phytic acid).
  • DMSO dimethyl sulfoxide
  • the cryopreservation solution utilized during cryopreservation may include non-reducing maltodextrin and ethylene diamine tetraacetate (EDTA) and a physiologic buffer solution.
  • the present invention is generally directed to a method of processing and preserving collagen based tissues, and in particular cardiovascular tissues and nerve tissues, for use as transplantable tissues.
  • Cardiovascular tissues included, but are not limited to, heart valves, arteries, veins and umbilical vessels.
  • Nerve tissue may include, central nervous tissues such as dura, as well as nerves and other collagen based tissues of the central and peripheral nervous systems.
  • the present invention is also directed to the product of the method of processing.
  • the process of the present invention generally includes procuring donor tissue from a donor source. Treating the donor tissue in a detergent solution, and treating in an enzyme solution so as to simultaneously remove harmful elements and give an acellular collagen matrix.
  • the process may include the use of several different detergent or enzyme solutions or alternatively the method may utilize a combination of detergent and enzyme solutions.
  • the process further includes protecting the matrix against non-enzymatic crosslinking due to the Maillard reaction or its subsequent end products, reactive oxidative molecular species, or molecular free radicals. This can be achieved by introducing non-participants and/or inhibitors to these reactions.
  • Preservation of the resulting acellular collagen matrix is carried out by cryopreservation, preferably by molecular distillation drying, to give a dried and hence preserved, acellular collagen matrix.
  • the resulting preserved, acellular collagen matrix may be rehydrated and used as a transplant alleviating the need for a conventional autologous, allogeneic tissues or a man-made synthetic transplant.
  • collagen based tissues may be processed using the principles of the present invention. That is to say, the processing and preserving of collagen based tissues such as cartilaginous tissues, such as tendons, ligaments, sinus tissue and the like; dura matter of the central nervous system, spinal cord, peripheral nerve, and other similar neurological tissues, dermis and other skin tissue, vascular tissues such as veins and arteries heart valves and the like, umbilical cord vessels, corneal tissue and other collagen containing tissue of the ocular system, periodontal tissues such as the gingiva and other such soft tissues that contain a collagen matrix are considered to be within the scope of the present invention.
  • cartilaginous tissues such as tendons, ligaments, sinus tissue and the like
  • vascular tissues such as veins and arteries heart valves and the like
  • umbilical cord vessels corneal tissue and other collagen containing tissue of the ocular system
  • a suitable donor source must be located for the procurement of the donor tissue.
  • This donor source may be either a human cadaver or an appropriately sized animal. Generally the donor should be in such condition that the tissues of interest have not undergone irreversible, adverse biochemical or mechanical changes.
  • the donor is a freshly killed pig which has not undergone scalding.
  • the donor is a human donor who has indicated the desire to be a tissue donor upon death. The donor tissue is dissected and removed from the donor. In addition to the tissue, a sample of the surrounding tissue should be taken for cryosection and testing. Typically the sample of surrounding tissue is frozen immediately after procurement. The donor tissue, should be procured such that there is sufficient geometry for transplantation.
  • the donor tissue may be placed into a stabilizing solution in circumstances in which the process of the present invention is not to be carried out soon after procurement.
  • Stabilizing solution may include cold (i.e. 4 °C) Roswell Park Memorial Institute (RPMI) solution or University of Wisconsin (UW) solution with additional antibiotics. Suitable antimircobial suchas cefoxitin, lincomycin, polymyxin, vancomycin and amphotericin.
  • the tissue in cold (i.e. 4 °C) stabilizing solution is shipped as soon as possible for processing as disclosed herein. Upon receipt, a second sample of the tissue is taken to determine its suitability for further processing.
  • the donor tissue prior to treatment is rinsed in a physiological saline solution to remove excess blood and tissue particles that may be present.
  • the donor tissue is then immersed in a first detergent solution.
  • the general purpose of this first detergent solution is to remove, as completely as possible, any residual blood elements which might contribute oxidative or enzymatic compounds which could damage the tissue.
  • the first detergent solution is formulated to contain a detergent and sodium chloride for solubilizing lipid membranes and proteins, and a divalent cation chelator to inhibit protease activity, in buffered water or culture medium.
  • the first detergent solution is comprised of 0.24 mM t-octylphenoxypolyethoxyethanol (Triton X-100), 25 mM ethylene diamine tetraacetate (EDTA), and 1M sodium chloride in a 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer.
  • the first detergent solutions may be formulated to include TX-100, sodium chloride, and EDTA in a Roswell Park Memorial Institute (RPMI) solution. Sufficient first detergent solution should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so as to promote the mixing and flow of the fluid - 8
  • the first detergent solution is drained from the donor tissue, with the solution being discarded.
  • VSMD vitrification solution maltodextrin
  • the components of the VSMD solution should include polymers of polyhydroxy compounds which may be of low browning (Maillard reaction) potential, chelators of divalent cations to inhibit proteolytic activity, water, and a suitable buffer for maintaining the desired pH.
  • the maltodextrin may be chemically modified to a dextrose equivalence (DE) less than 1 in order to prevent its participation in the Maillard reaction. This reaction, if unchecked, can result in non-enzymatic crosslinking of the matrix.
  • VSMD includes modified 75% maltodextrin, about 10 mM ethylene diamine tetraacetate (EDTA), water, and ⁇ -2-hydroxyethylpiperazine- ⁇ '-2-ethanesulfonic acid (HEPES).
  • the pH of the VSMD solution is preferably adjusted to about pH 7.4 to about pH 7.5. Sufficient VSMD should be used so that the donor tissue is completely immersed in the solution.
  • Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the VSMD Upon completion of the immersion step, the VSMD is drained from the donor tissue, with the solution being discarded. After completion of draining, the donor tissue is placed in a second detergent solution and gently agitated.
  • the second detergent solution is a solution that that disrupts and solubilizes cellular membranes and potentially antigenic components so that they may be washed free of the tissue.
  • the components of the second detergent solution include detergents and divalent cation chelators in buffered water or cell culture medium, plus broad range antimicrobials, antifungals, anti-oxidants and free radical scavengers.
  • the second detergent solution includes n-octyl- ⁇ -D-glucopyranoside, ethylene diamine tetraacetate (EDTA), deferoxamine, phytic acid, and aminoguanidine in degassed Roswell Park Memorial Institute (RPMI) solution. Roswell Park Memorial Institute (RPMI) solution is a commercially available cell culture medium.
  • the second detergent solution includes about 40 mM n-octyl- ⁇ -D-glucopyranoside, about 25 mM ethylene diamine tetraacetate (EDTA), about 10 mM deferoxamine, about 10 mM phytic acid, and about 100 mM aminoguanidine in degassed Roswell Park Memorial Institute (RPMI) solution at a pH of about 7.4 to 7.5.
  • the second detergent solution may contain antimicrobials to prevent transmission of bacterial or fungal infection.
  • Suitable antimicrobials include one or a combination of penicillins, aminoglycosides, cephalosporins, erythromycins, tetracyclines, polymyxins, antifungals, or others with suitable activity against expected organisms in the tissue procurement setting.
  • antimicrobials include lincomycin, polymyxin B sulfate, cefoxitin, vancomycin, and amphotericin B.
  • the second detergent solution includes 250 mM lincomycin, 1000 activity units/ml polymyin B sulfate, 0.5 M cefoxitin, 20 mM vancomycin, and 25 mM amphotericin B.
  • Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the duration and temperature of optimal treatment in the second detergent solution may vary with differing types of tissue, however, one skilled in the art should know how to systematically vary each parameter to obtain the optimum conditions.
  • the tissue may be trimmed to final specifications upon completion of the second detergent treatment.
  • the resulting treated tissue should be washed with sterile physiological salt solution, preferably at a pH the same as the detergent, and more preferably at a pH from about 7.4 to about 7.5.
  • the washed treated tissue is placed in an enzyme solution and is incubated for a suitable temperature and time.
  • the purpose of the enzyme solution is to disrupt and solubilize specific cellular components, such as the cell nucleus and cell phospholipids.
  • the enzyme solution of the present invention includes DNase I, deferoxamine, phytic acid, and aminoguanidine in a 20 mM HEPES buffered water.
  • the enzyme solution may also include phospholipase C to assist the clearance of phospholids.
  • agents with activity against non-enzymatic crosslinking of the tissue may also be included in the formulation of the enzyme solution, such as dimethyl sulfoxide (DMSO), catalase, superoxide dismutase, a-tocopherol, reduced glutathione, 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ascorbate, flavonoids, and inositol hexaphosphoric acid (phytic acid).
  • DMSO dimethyl sulfoxide
  • catalase superoxide dismutase
  • a-tocopherol reduced glutathione
  • 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid ascorbate
  • flavonoids flavonoids
  • inositol hexaphosphoric acid phytodecane
  • antimicrobials such as one or a combination of 10
  • the enzyme solution includes 150 activity units/mg DNase I, 10 mM deferoxamine, 100 mM phytic acid, and 100 mM aminoguanidine.
  • the enzyme solution also includes 250 mM lincomycin, 1000 activity units/ml polymyin B sulfate, 0.5 M cefoxitin, 20 mM vancomycin, and 25 mM amphotericin B as antimicrobials.
  • the donor tissue is preferably incubated in the enzyme solution at 37°C. Depressed temperatures may be used as well, however, to decrease the rate of reaction for non-enzymatic crosslinking within the tissue.
  • the enzyme solution is decanted and discarded. The tissue is washed three times with sterile degassed, physiological salt solution using fresh solution for each wash.
  • Cryopreservation When cell viability or tissue ultrastructure are to be preserved following cooling, two methods are generally available. The first is to ulfrarapidly cool the sample, resulting in the tissue fluids being vitrified, i.e., frozen in the absence of ice crystals. The second is to incorporate chemical additives to confer a degree of cryoprotection and allow vitification at slower cooling rates.
  • the chemicals range from naturally occurring cryoprotectants such as glycerol, proline, sugars, and alcohols to organic solvents such as dimethylsulfoxide (DMSO) to high molecular weight polymers such as polyvinylpyrrolidone (PVP), dextrin, maltodextrin and hydroxy ethyl starch (HES).
  • Vitrification of cells and tissues is limited by the rate at which the sample can be cooled and the insulating properties of the tissue itself. Due to physical limitations, one can only achieve vitrification of a thin layer of tissues using state of the art techniques. Thus chemical agents for cryoprotection and manipulating the cooling rate are utilized to cool and store biological samples without causing structural and functional damage.
  • cryoprotective compounds The cooling rate in the presence of cryoprotective compounds is a significant factor in freezing injury. Normally for cells, slow cooling is better than elevated cooling rates since the latter promotes intracellular ice formation. This occurs because there is insufficient time for - 11
  • cryoprotectant prevents intracellular ice formation.
  • tissue matrix samples there is a more direct correlation to the overall reduction in the degree of total ice crystal formation.
  • a source of damage to frozen tissue, other than freezing itself, is the osmotic and toxic effects of many of the cryoprotective agents.
  • the physicochemical effects of cryoprotectants are
  • cryoprotective compounds When used in mixtures, some cryoprotective compounds may counteract the toxicity of other cryoprotectants.
  • the factors affecting the cryoprotective nature of compounds are (a) chemical nature, (b) relative lack of toxicity, (c) molecular size and penetrating ability, and (d) interaction with other compounds in the mixture.
  • the tissue is incubated in a second vitrification solution maltodextrin (VSMD) for a second time.
  • the second VSMD may have the same formulation as previously disclosed above.
  • the second VSMD is formulated to comprise a 75% maltodextrin and the other previously disclosed components.
  • the second VSMD may utilize lower amounts of maltodextrin preferably about 35% maltodextrin may be used. Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the donor tissue is removed from the solution and incubated in fresh second VSMD for an additional minimum of four hours.
  • the tissue is drained and placed in TYVEK® pouches for cooling and drying. Cooling Parameters: With the tissue sealed in TYVEK® pouches, it is cryofrozen in a manner which prevents or limits the amount of ice crystal formation over the course of the drying process.
  • rapid cooling is considered essential to obtain the proper ice crystal blend.
  • a vitrification procedure is used which results in the formation of a substantial proportion of amorphous water in the biological sample. Regardless of the form of cooling that is used, it is believed that amorphous phase water, cubic ice crystals and hexagonal ice crystals are present in the final product.
  • the method of cooling has a distinct bearing on the distribution of ice crystal types found in the cooled cryosolution.
  • samples are cooled by an appropriate method such that ice crystal formation is below the degree that would cause damage to the sample. Once frozen, the sample is then stored below the transition temperature of the most unstable ice form. For amorphous ice, this is preferentially below - 160°C .
  • Drying Parameters The aim of controlled drying of a frozen biological sample by molecular distillation drying is to remove water from the sample without further mechanical or chemical damage occurring during the drying process. This involves avoiding, by use of appropriate drying conditions, two fundamental damaging events. The first is to remove water from ice crystalline phases without transition to larger more stable and more destructive crystals. The second is to remove water from solid but noncrystalline water or water-solute mixtures without melting or crystallization of these solid phases. This second component refers to water present in the amorphous condition, water together with solute in the eutectic or water together with a compound which binds and structures water and hence, prevents its crystallization during the freezing process. Hence, vitreous water can be of low energy and stability, as in ultrarapidly- cooled pure water, or high energy and stability, as that achieved with cryoprotective agents with intermediate rates of cooling.
  • the controlled drying processes must be able to remove each of these different states of water during the transition from one phase to another second phase and in less time than is required to complete the transition. This mode of drying, therefore, requires that several conditions be met.
  • the sample should be loaded into the dryer without temperature elevation above its lowest transition temperature. If elevation of temperature does occur, this should be over a short period of time such that no appreciable transition occurs. Ideally, loading occurs under liquid nitrogen at -190°C, well below the lowest discernible transition of -160°C for pure, ultrarapidly- cooled amorphous water.
  • a closed circuit refrigeration system may be sufficient to enable maintenance of the sample temperature below the onset of transition.
  • the sample should be exposed to vacuum and be in direct line of sight of the condenser surfaces.
  • the criteria for these are again determined by the nature of the water phases present in the sample.
  • the vacuum within the chamber during the drying of a particular phase should create a partial pressure of water at least equivalent to or less than the saturation vapor pressure of water in the phase to be removed.
  • This saturation vapor pressure is dependent on the nature of the water phase and its temperature.
  • the approximate saturation vapor pressures are 6 x 10 " mbar (-160°C) and 5 x 10 " mbar (-130°C), respectively.
  • transition times of amorphous to cubic ice in this same temperature range -160°C to -130°C, vary from 5 x 10 minutes to 5 minutes, drying will be very slow until temperatures of the order of -150°C to -140°C are reached requiring a vacuum of 5 x 10 " to 2 x 10 " mbar.
  • For cubic ice little if any drying will occur below its onset of transition at -130°C as its saturation vapor pressure will be of the order of one log lower than for amorphous water.
  • the saturation vapor pressure of cubic ice is approximately 5 x 10 " to 9 x 10 " mbar.
  • the transition times of cubic to hexagonal are 700 minutes and 109 minutes respectively.
  • the saturation vapor pressure therefore, determines the vacuum requirements for drying and can be applied to all water phases present. It is important to note that the same vacuum criteria are not applicable to all phases, but rather are phase-dependent.
  • the mean free path created be in excess of the distance between the sample and the condenser surface. Ideally, this should be a tenfold excess.
  • the condenser surface should be a lower temperature than the onset transition temperature of the phase of water being removed from the sample so that the saturation vapor pressure of water condensed on this surface during drying is considerably lower than that of the water phase within the sample. Ideally, this should be three orders of magnitude lower.
  • the temperature of the condenser surface should remain below the onset of transition of the least stable ice phase remaining to be removed. Ideally, the condenser should also be in line of sight of the sample.
  • the sample and sample holder should be heated so as to increase the mobility of water molecules and hence, cause their escape. This is an important component in the drying of a sample containing multiple phases or energy levels of water.
  • the temperature of the sample should be accurately known and the control of temperature and the rate of sample heating should be accurately controlled. This is to ensure that the drying of each phase of water in the sample is sequential.
  • N coefficient of evaporation
  • Ps saturation vapor pressure
  • M molecular weight of water
  • the rate of the amorphous to cubic transition is given by:
  • the sublimation rate and the transition rate will vary with temperature during this interval.
  • the rate of heating during this window Tj to T 2 must be such that sublimation occurs throughout the dimensions of the sample before transition at any particular temperature is completed.
  • the aim of controlled molecular distillation drying is achieved, i.e., the sequential removal of each phase of water under conditions appropriate to the properties of each phase without appreciable ice crystal growth, formation or melting of the particular phase.
  • samples are cooled by an appropriate method such that ice crystal formation is below the degree that would cause damage to the sample. This may be by immersion in liquid nitrogen, exposure to liquid nitrogen vapor, or other similar rapid freezing 16
  • the sample is then stored below the transition temperature of the most unstable ice form. For amorphous ice, this is preferentially below -160°C.
  • the sample is then loaded into a sample holder, precooled to -196°C and transferred into a molecular distillation dryer. The dryer chamber is then closed and sealed for vacuum integrity. To avoid recrystallization, the hydrated sample must remain below the transition temperature of the most unstable ice form throughout all manipulations.
  • the sample holder is then heated via a programmable heater microprocessor thermocouple loop. Heating programs are determined according to the nature of the sample.
  • a typical program for a sample containing amorphous, cubic and hexagonal ice is 10°C per hour from -180°C to -150°C, 1°C per hour from -150°C to -70°C, and 10°C per hour from -70°C to +20°C.
  • the sample Once dry, the sample must be physically or mechanically isolated from water on the condenser surface or any other source and stored in a closed container either under vacuum or dry inert gas.
  • the sample can be sealed inside an appropriate container within the vacuum chamber and unloaded for subsequent storage and packaging.
  • the sample In one configuration, the sample is contained within a glass vial and sealed with a butyl rubber lyophilization stopper at the end of cycle. More specific details of the operation of the molecular distillation dryer and the above process of drying is described in U.S. Patent No. 4,865,871. 5,336,616, the entire contents of which are hereby incorporated herein by reference.
  • collagen-based tissues may be processed using the principles of the present invention. That is to say, the processing and preserving of collagen based tissues, such as cartilaginous tissues such as tendons, ligaments, sinus tissue and the like; dura mater of the brain, spinal cord, peripheral nerve and other similar neurological tissues, - 17
  • dermis and other skin tissue vascular tissues such as veins and arteries, heart valves and the like, corneal tissue and other collagen containing tissue of the ocular system, periodontal tissues such as the gingiva and other such soft tissues that contain a collagen matrix are considered to be within the scope of the present invention.
  • vascular tissues such as veins and arteries, heart valves and the like
  • corneal tissue and other collagen containing tissue of the ocular system corneal tissue and other collagen containing tissue of the ocular system
  • periodontal tissues such as the gingiva and other such soft tissues that contain a collagen matrix
  • One of skill in the art should understand that depending on the size, shape, cellular structure and condition of the collagen based tissue upon harvest, the specific conditions, such as concentration, time, temperature and the like may be required. Such adjustments may be determined by routine variation of parameters disclosed herein and thus may be used to substantially achieve the results of the present invention.
  • the donor tissue is provided by a freshly killed pig which has not undergone scalding.
  • the heart valve should be dissected from the donor in a manner such that there is sufficient coronary and muscle skirt lengths to facilitate subsequent surgical implantation. If necessary the donor tissue may be placed in a stabilization fluid for transportation. However, in the present case this was not necessary.
  • the trimmed tissue is rinsed in a physiological saline solution to remove excess blood and tissue particles. It is then placed in the first detergent solution for about 1 hour at about 4 °C
  • the first detergent solution is comprised of about 0.24 mM t-octylphenoxypolyethoxyethanol (Triton X-100), about 25 mM ethylene diamine tetraacetate (EDTA), and about 1M sodium chloride in an about 20 mM N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid (RPMI) solution may be used as the first detergent solution.
  • RPMI Roswell Park Memorial Institute
  • the first detergent solution is drained from the donor tissue, with the first detergent solution being discarded.
  • the tissue is transferred to vitrification solution maltodextrin (VSMD) and the tissue is incubated in VSMD for about 1 to 4 hours.
  • the VSMD includes modified 75% maltodextrin, about 10 mM ethylene diamine tetraacetate (EDTA), water, and N- 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES).
  • the pH of the VSMD solution is preferably adjusted to about pH 7.4 to pH 7.5.
  • Sufficient VSMD should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the VSMD is drained from the donor tissue, with the solution being discarded.
  • the donor tissue is placed in a second detergent solution and gently agitated.
  • heart valves are agitated in the second detergent solution at room temperature for about 24 hours.
  • the second detergent solution includes about 40 mM n-octyl- ⁇ -D-glucopyranoside, about 25 mM ethylene diamine tetraacetate (EDTA), about 10 mM deferoxamine, about 10 mM phytic acid, and about 100 mM aminoguanidine in degassed Roswell Park Memorial Institute (RPMI) solution at a pH of about 7.4 to 7.5.
  • EDTA ethylene diamine tetraacetate
  • RPMI Roswell Park Memorial Institute
  • the second detergent solution may optionally include about 250 mM lincomycin, about 1000 activity units/ml polymyin B sulfate, about 0.5 M cefoxitin, about 20 mM vancomycin, and about 25 mM amphotericin B.
  • the resulting treated tissue is washed-with sterile physiological salt solution, preferably at a pH the same as the detergent, and more preferably at a pH from about 7.4 to about 7.5.
  • the washed treated tissue is placed in an enzyme solution that includes about 150 activity units/mg DNase Type I, about 10 mM deferoxamine, about 100 mM phytic acid, and about 100 mM aminoguanidine in a 20 mM HEPES buffered water.
  • the DNase solution also includes about 250 mM lincomycin, about 1000 activity units/ml polymyin B sulfate, 0.5 M cefoxitin, about 20 mM vancomycin, and about 25 mM amphotericin B as antimicrobials.
  • the heart valves are incubated in the enzyme solution for about 24 hours at about 37°C. Sufficient enzyme solution should be used so that the donor tissue 19
  • the tissue is incubated in a second VSMD for at least four hours.
  • the second VSMD is formulated so as to have the same composition and the first VSMD.
  • Sufficient VSMD should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the tissue is removed from the solution and incubated in fresh VSMD solution for an additional minimum of four hours.
  • the tissue is drained and placed in TYVEK® pouches.
  • tissue sealed in TYVEK® pouches With the tissue sealed in TYVEK® pouches, it is cryofrozen and freeze-dried in a manner which prevents or limits the amount of ice crystal formation as described above. The entire drying process takes place over the course of about three days. Typically this process involves cooling the sample below about -160 °C, placing the cooled sample under vacuum and warming as described above. A programmable temperature controller can be used during this process to ensure consistency and accuracy. Upon reaching room temperature the dried tissue sample is transferred and packaged to prevent contact with ambient moisture and under sterile conditions.
  • Example 2 Example 2.
  • the donor tissue is provided by a human donor.
  • a radial artery, saphenous vein or umbilical vessel should be dissected from the donor in a manner such that there is sufficient tissue to facilitate subsequent surgical implantation.
  • the donor tissue is placed in a stabilization fluid for transportation.
  • Stabilizing solution may include cold Roswell Park Memorial Institute (RPMI) solution or University of Wisconsin (UW) solution with additional antibiotics. Suitable antimicrobials include efoxitin, lincomycin, polymyxin, vancomycin, and amphotericin.
  • RPMI cold Roswell Park Memorial Institute
  • UW University of Wisconsin
  • Suitable antimicrobials include efoxitin, lincomycin, polymyxin, vancomycin, and amphotericin.
  • the tissue in cold stabilizing solution is shipped as soon as possible for - 20
  • a sample is taken of the tissue to determine its suitability for further processing.
  • the trimmed tissue is rinsed in a physiological saline solution to remove excess blood and tissue particles. It is then placed in the first detergent solution for about 1 hour at about 4 °C.
  • the first detergent solution is comprised of about 0.24 mM t-octylphenoxypolyethoxyethanol (Triton X-100), about 25 mM ethylene diamine tetraacetate (EDTA), and about 1M sodium chloride in a about 20 mM N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid (HEPES) buffer.
  • Triton X-100, sodium chloride, and EDTA in Roswell Park Memorial Institute (RPMI) solution may be used as the first detergent solution.
  • Sufficient first detergent solution should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so that all surfaces are well washed. Upon completion of the immersion step, the first detergent solution is drained from the donor tissue, with the solution being discarded.
  • the tissue is transferred to vitrification solution maltodextrin (VSMD) and the tissue is incubated in VSMD for approximately 1 to 4 hours.
  • the VSMD includes modified 75% maltodextrin, about 10 mM ethylene diamine tetraacetate (EDTA), water, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES).
  • the pH of the VSMD solution is preferably adjusted to about pH 7.4 to pH 7.5.
  • Sufficient VSMD should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the VSMD is drained from the donor tissue, with the solution being discarded.
  • the donor tissue is placed in a second detergent solution and gently agitated.
  • heart valves are agitated in the second detergent solution at room temperature for about 24 hours.
  • the second detergent solution includes about 40 mM n-octyl- ⁇ -D-glucopyranoside, about 25 mM ethylene diamine tetraacetate (EDTA), about 10 mM deferoxamine, about 10 mM phytic acid, and about 100 mM aminoguanidine in degassed Roswell Park Memorial Institute (RPMI) solution at a pH of about 7.4 to 7.5.
  • the second detergent solution may optionally include about 250 mM lincomycin, about 1000 activity 21
  • polymyin B sulfate about 0.5 M cefoxitin, about 20 mM vancomycin, and about 25 mM amphotericin B.
  • the resulting treated tissue is washed-with sterile physiological salt solution, preferably at a pH the same as the detergent, and more preferably at a pH from about 7.4 to about 7.5.
  • the washed treated tissue is placed in an enzyme solution that includes about 150 activity units/mg DNase Type I, about 10 mM deferoxamine, about 100 mM phytic acid, and about 100 mM aminoguanidine in a 20 mM HEPES buffered water.
  • the DNase solution also includes about 250 mM lincomycin, about 1000 activity units/ml polymyin B sulfate, 0.5 M cefoxitin, about 20 mM vancomycin, and about 25 mM amphotericin B as antimicrobials.
  • the heart valves are incubated in the enzyme solution for about 24 hours at about 37 °C.
  • Sufficient enzyme solution should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the enzyme solution is decanted and discarded. The tissue is washed three times with sterile degassed, physiological salt solution using fresh solution for each wash.
  • the tissue is incubated in a second VSMD for at least four hours.
  • the second VSMD is formulated so as to have the same composition and the first VSMD.
  • Sufficient VSMD should be used so that the donor tissue is completely immersed in the solution. Agitation should be provided so as to promote the mixing and flow of the fluid surrounding the tissue.
  • the tissue is removed from the solution and incubated in fresh VSMD solution for an additional minimum of four hours.
  • the tissue is drained and placed in TYVEK® pouches.
  • tissue sealed in TYVEK® pouches With the tissue sealed in TYVEK® pouches, it is cryofrozen and freeze-dried in a manner which prevents or limits the amount of ice crystal formation as described above. The entire drying process takes place over the course of about three days. Typically this process involves cooling the sample below about -160 °C, placing the cooled sample under vacuum and warming as described above. A programmable temperature controller can be used during this 22 -
  • Nerve tissue is procured and immediately placed into sterile Roswell Park Memorial Institute (RPMI) solution with or without antimicrobials including 240 mg/1 cefoxitin, 120 mg/1 lincomycin, 100 mg/1 polymyxin B, 30 mg/1 vancomycin and 25 mg/lamphotericin.
  • RPMI Roswell Park Memorial Institute
  • the tissue may remain in this solution for up to about 7 days provided the solution is changed approximately every 48 hours with fresh sterile solution. Ideally the processing of this tissue would begin within about 24 hours of procurement.
  • the transport solution (RPMI) is removed and replaced with the first decellularizing solution consisting of a sodium phosphate buffer (about 0.639% Na 2 HPO 4 and about 0.069 % NaH 2 PO 4 ) plus about 0.5 % Triton X-100 (n-octyl- ⁇ -D-glucopyranoside), about 1 M NaCl, about 10 mM EDTA.
  • This solution may optionally include antimicrobials such as cefoxitin, lincomycin, polymyxin, vancomycin, ans amphotericin.
  • the tissue is rinsed in this solution for about 5-30 minutes at room temperature with gentle agitation.
  • the solution is replaced with fresh solution and agitated for an additional about 15-24 hours.
  • tissue is removed from this solution and washed two times for about 15 minutes each in phosphate buffered saline containing about 10 mM EDTA. The tissue is then rinsed for about 5-30 minutes followed by approximately a 15-24 hour incubation at 37°C in a second decellularizing solution consisting of about 5.06 % n-octanoic acid (Caprylic acid) in a sodium phosphate buffer consisting of about 0.639% Na 2 HPO 4 and about 0.069 % NaH 2 PO 4 with or without antibiotics. Alternatively this second decellularizing solution may consist of about 2 % deoxycholate in about 10 mM HEPES buffer with or without antibiotics.
  • tissue is removed from this solution and washed two times for about 15 minutes each in phosphate buffered saline containing about 10 mM EDTA. The tissue is then incubated for about 15-24 hours at room temperature with agitation in a solution consisting of about 50 ug ml Dnase-I in about 0.9 % NaCl and about 10 mM MgCl 2 at a pH of about 7.4. The tissue is 23
  • Cryopreservation The tissue is then incubated for about 1-6 hours at room temperature with agitation in a solution consisting of about 35 % maltodextrin and about 10 mM EDTA in about 10 mM HEPES buffer. The tissue is then sealed in TYVEK pouches, cryofrozen and freeze-dried in a manner which prevents or limits the amount of ice crystal formation. The entire drying process takes place over the course of about three days. Typically this process involves cooling the sample below about -160 °C, placing the cooled sample under vacuum and warming as described above. A programmable temperature controller can be used during this process to ensure consistency and accuracy. Upon reaching room temperature the dried tissue sample is transferred and packaged to prevent contact with ambient moisture and under sterile conditions.
  • One such embodiment includes a process of preserving collagen-based tissues, the process including: procuring the collagen-based tissue; treating the tissue in a detergent solution; treating the tissue in an enzyme solution; treating the tissue via techniques and/or additives which prevent or inhibit the molecular crosslinking of processed tissues via the Maillard reaction and the subsequent formation of advanced glycosylation end products; treating the tissue via techniques and/or additives which prevent or inhibit the molecular crosslinking of processed tissues via reactive oxidative species of molecules; treating the tissue via techniques and/or additives which prevent or inhibit the molecular crosslinking of processed tissues via the formation and propagation of molecular free radicals; treating the tissue in a cryopreservation solution; and, cryopreserving the tissue.
  • the collagen based tissue is a heart valve.
  • the detergent solution includes one or more of t-octylphenoxypolyethoxyethanol (Triton X-100), n-octyl- ⁇ -D-glucopyranoside, deoxycholate, octanoic acid (caprylate), 3-[(3-Cholamidopropyl)dimethylammonio]-l-propane-sulfonate (CHAPS), ethylene diamine tetraacetate (EDTA), sodium chloride, and broad-spectrum antimicrobials in a physiological buffer solution.
  • the enzyme solution includes one or more of DNase Type I, DNase Type II, RNase, phospholipase A, 24 -
  • the techniques or inhibitors to molecular crosslinking via the Maillard reaction and the formation of advanced glycosylation end products includes depressed temperatures, use of non-reactive or non-reducing carbohydrates such as those that result from the molecular reduction of the carbonyl group of glucose and related compounds, and aminoguanidine.
  • Another embodiment includes techniques or inhibitors to molecular crosslinking via the reactive oxidative species which include depressed temperatures, inert atmosphere, and one or more of deferoxamine mesylate, dimethyl sulfoxide (DMSO), catalase, superoxide dismutase, a-tocopherol, reduced glutathione, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.
  • deferoxamine mesylate dimethyl sulfoxide (DMSO)
  • catalase catalase
  • superoxide dismutase a-tocopherol
  • glutathione reduced glutathione
  • 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
  • the techniques or inhibitors to molecular crosslinking via molecular free radicals includes depressed temperatures and one or more of dimethyl sulfoxide (DMSO), a-tocopherol, ascorbate, reduced glutathione, flavonoids, and inositol hexaphosphoric acid (phytic acid).
  • the cryopreservation solution preferably includes non-reducing maltodextrin and ethylene diamine tetraacetate (EDTA) in a physiologic buffer solution.
  • the product of the process is a preserved collagen based tissue that is suitable for transplantation after it has been rehydrated.
  • the product is a preserved heart valve that is suitable for transplantation once it has been rehydrated.
  • sapheous vein or other vascular graft that is suitable for transplantation after it has been rehydrated.
  • nerve or nervous tissue that is a preserved collagen base tissue that is suitable for transplantation after it has been rehydrated.

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Abstract

L'invention concerne un procédé permettant de conserver des tissus à base de collagène, consistant à obtenir le tissu à base de collagène; à traiter le tissu dans une solution détergente; à traiter le tissu dans une solution d'enzymes; à traiter le tissu de manière à empêcher ou à inhiber la réticulation moléculaire des tissus traités via la réaction de Maillard et la formation ultérieure de produits finaux de glycosylation avancée; à traiter le tissu de manière à empêcher ou à inhiber la réticulation moléculaire des tissus traités via des espèces moléculaires oxydatives réactives; à traiter le tissu de manière à empêcher ou à inhiber la réticulation moléculaire des tissus traités via la formation et la propagation de radicaux libres moléculaires; à traiter le tissu dans une solution de cryopréservation; et à réaliser la cryopréservation du tissu. Ce procédé peut être utilisé pour conserver différents types de tissu à base de collagène à savoir la valvule cardiaque, les greffons vasculaires notamment les veines et les artères, les vaisseaux ombilicaux, les nerfs, les tissus nerveux, la dure-mère, le derme et autres tissus semblables à base de collagène.
PCT/US1999/003667 1998-02-20 1999-02-19 Procede permettant de traiter et de conserver des tissus a base de collagene WO1999041981A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP99908285A EP1056335A1 (fr) 1998-02-20 1999-02-19 Procede permettant de traiter et de conserver des tissus a base de collagene
AU27753/99A AU752457B2 (en) 1998-02-20 1999-02-19 Method of processing and preserving collagen based tissues
JP2000532008A JP2002503678A (ja) 1998-02-20 1999-02-19 コラーゲンベース組織の処理および保存方法
CA002320887A CA2320887A1 (fr) 1998-02-20 1999-02-19 Procede permettant de traiter et de conserver des tissus a base de collagene

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US60/075,472 1998-02-20

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024244A2 (fr) * 2000-09-20 2002-03-28 Regeneration Technologies, Inc. Procede de preparation et de traitement de tissus de greffe
GB2375771A (en) * 2001-05-24 2002-11-27 Univ Leeds Decellularisation of tissue implant material
WO2003039610A1 (fr) * 2001-11-08 2003-05-15 Artiss Gmbh Procede de production d'une matrice tissulaire acelullarisee et matrice tissulaire obtenue a l'aide dudit procede
WO2004024170A1 (fr) * 2002-09-10 2004-03-25 Japan As Represented By President Of National Cardiovascular Center Procede de traitement d'un tissu biologique destine a la transplantation par application d'une pression hydrostatique ultra-elevee
WO2009044407A1 (fr) * 2007-10-03 2009-04-09 International Centre For Cardio Thoracic And Vascular Diseases (A Unit Of Frontier Lifeline Pvt. Ltd) Greffe vasculaire de petit diamètre obtenue à partir de veines saphènes de cadavre retraitées
US20110045566A1 (en) * 2008-03-24 2011-02-24 Zhichong Wang method for preparing the decellularized matrix
US9220259B2 (en) 2010-08-26 2015-12-29 Lifecell Corporation Passive methods for anti-microbial biological meshes
CN109497039A (zh) * 2018-10-29 2019-03-22 上海慧存医疗科技有限公司 脐带组织的冻存复苏方法和间充质干细胞的制备方法
WO2021173983A1 (fr) * 2020-02-28 2021-09-02 Somnio Global Holdings, Llc Procédés et matériaux de vitrification assistée par capillaire pour la conservation d'échantillons biologiques

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EP0128706A2 (fr) * 1983-06-10 1984-12-19 University Patents, Inc. Implants pour le corps de matrice extracellulaire et moyens et méthodes pour fabriquer et utiliser de tels implants
EP0564786A2 (fr) * 1992-02-12 1993-10-13 Lifecell Corporation Procédé pour le traitement et la conservation de tissus à base de collagène pour la transplantation
WO1995024873A1 (fr) * 1994-03-14 1995-09-21 Cryolife, Inc. Tissu traite pour une implantation et procedes pour sa preparation
WO1996032905A1 (fr) * 1995-04-19 1996-10-24 St. Jude Medical, Inc. Substrat matriciel destine a une prothese derivee d'un tissu biologique vivant et son procede de fabrication

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0128706A2 (fr) * 1983-06-10 1984-12-19 University Patents, Inc. Implants pour le corps de matrice extracellulaire et moyens et méthodes pour fabriquer et utiliser de tels implants
EP0564786A2 (fr) * 1992-02-12 1993-10-13 Lifecell Corporation Procédé pour le traitement et la conservation de tissus à base de collagène pour la transplantation
WO1995024873A1 (fr) * 1994-03-14 1995-09-21 Cryolife, Inc. Tissu traite pour une implantation et procedes pour sa preparation
WO1996032905A1 (fr) * 1995-04-19 1996-10-24 St. Jude Medical, Inc. Substrat matriciel destine a une prothese derivee d'un tissu biologique vivant et son procede de fabrication

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024244A3 (fr) * 2000-09-20 2003-05-30 Regeneration Tech Inc Procede de preparation et de traitement de tissus de greffe
WO2002024244A2 (fr) * 2000-09-20 2002-03-28 Regeneration Technologies, Inc. Procede de preparation et de traitement de tissus de greffe
US7354749B2 (en) 2001-05-24 2008-04-08 The University Leeds Decellularisation of matrices
GB2375771A (en) * 2001-05-24 2002-11-27 Univ Leeds Decellularisation of tissue implant material
WO2002096476A1 (fr) * 2001-05-24 2002-12-05 University Of Leeds Decellularisation de matrices
WO2003039610A1 (fr) * 2001-11-08 2003-05-15 Artiss Gmbh Procede de production d'une matrice tissulaire acelullarisee et matrice tissulaire obtenue a l'aide dudit procede
EP1317934A1 (fr) * 2001-11-08 2003-06-11 Artiss GmbH Procédé de préparation d'une matrice tissulaire acellularisée et la matrice tissulaire résultante
WO2004024170A1 (fr) * 2002-09-10 2004-03-25 Japan As Represented By President Of National Cardiovascular Center Procede de traitement d'un tissu biologique destine a la transplantation par application d'une pression hydrostatique ultra-elevee
WO2009044407A1 (fr) * 2007-10-03 2009-04-09 International Centre For Cardio Thoracic And Vascular Diseases (A Unit Of Frontier Lifeline Pvt. Ltd) Greffe vasculaire de petit diamètre obtenue à partir de veines saphènes de cadavre retraitées
US20110045566A1 (en) * 2008-03-24 2011-02-24 Zhichong Wang method for preparing the decellularized matrix
US9220259B2 (en) 2010-08-26 2015-12-29 Lifecell Corporation Passive methods for anti-microbial biological meshes
CN109497039A (zh) * 2018-10-29 2019-03-22 上海慧存医疗科技有限公司 脐带组织的冻存复苏方法和间充质干细胞的制备方法
WO2021173983A1 (fr) * 2020-02-28 2021-09-02 Somnio Global Holdings, Llc Procédés et matériaux de vitrification assistée par capillaire pour la conservation d'échantillons biologiques
CN115768261A (zh) * 2020-02-28 2023-03-07 乌普卡拉公司 用于保存生物样品的毛细管辅助玻璃化工艺和材料

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AU2775399A (en) 1999-09-06

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