WO2023075586A1 - Procédé et processus de décellularisation in vitro de tissu cardiovasculaire - Google Patents

Procédé et processus de décellularisation in vitro de tissu cardiovasculaire Download PDF

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WO2023075586A1
WO2023075586A1 PCT/MX2021/050071 MX2021050071W WO2023075586A1 WO 2023075586 A1 WO2023075586 A1 WO 2023075586A1 MX 2021050071 W MX2021050071 W MX 2021050071W WO 2023075586 A1 WO2023075586 A1 WO 2023075586A1
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tissue
solution
decellularization
cardiovascular
hypotonic
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PCT/MX2021/050071
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English (en)
Spanish (es)
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Beni CAMACHO PÉREZ
Juan Pablo AGUILAR ALEMÁN
Octavio Israel ROJAS GARCÍA
Omar PEZA CHÁVEZ
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Top Health, S.A.P.I. De C.V.
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Publication of WO2023075586A1 publication Critical patent/WO2023075586A1/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
    • 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

Definitions

  • the present invention is related to biotechnology and medical science in general, in particular it is related to methods and processes for decellularization of cardiovascular tissue and more specifically it refers to a method and process for in vitro decellularization of cardiovascular tissue using a model diffusion in gradients with hypotonic solutions
  • Aortic valve disease is a condition in which the valve between the heart's main pumping chamber (left ventricle) and the body's main artery (aorta) does not work properly.
  • Aortic valve disease may be present from birth (congenital heart disease) or may have other causes.
  • Aortic stenosis (frequent valvular abnormality, which generates an obstruction of the outflow of blood from the left ventricle to the aorta) is one of the most frequent valvulopathies worldwide with a significant prevalence, being a entity increasingly diagnosed in the elderly population who has a large degenerative component of valvular calcification and sometimes a substrate of underlying congenital heart disease with associated comorbidities (Genoveva Elva Henry Vera et al, 2018).
  • Bioprosthetic valves typically include a leaflet portion and a conduit portion, both generally made of biologic material and possibly a stent. While bioprosthetic valves have a number of advantages over mechanical valves, including a lower risk of complications from thrombus formation, they are associated with an increased risk of mineralization. This increased risk significantly limits the durability of the replacement valve (Black Kirby S and Goldstein Steven 2003).
  • surgical heart valve replacement may involve the implantation of one of three different types of prostheses; mechanical (synthetic), bioprosthetic (chemically fixed porcine valve or bovine pericardium), or human allograft.
  • mechanical synthetic
  • bioprosthetic chemically fixed porcine valve or bovine pericardium
  • human allograft These prostheses provide effective hemodynamic enhancement for the replacement of native aortic valves that are congenitally malformed or have been damaged by degenerative changes or disease resulting in aortic regurgitation or aortic stenosis. Criteria for an ideal prosthesis would include natural hemodynamics, long-term durability, low incidence of thromboembolic complications, absence of calcification, demonstrated lack of immunogenicity, and absence of inappropriate hyperplastic responses after implantation. Even in autotransplant situations, surgical manipulation of tissue, such as vein grafting, can itself be a stimulus for tissue hyperplasia and subsequent graft failure.
  • valve tissue of porcine or bovine origin are prepared from valve tissue of porcine or bovine origin. Because these are species immunologically discordant from man, they are rapidly rejected by the implant recipient despite the use of immunosuppressive drug therapy that would otherwise maintain an allograft. Significantly, these tissues are prone to hyperacute rejection by the recipient due to the presence in the recipient of preformed natural antibodies that recognize antigens on the surface of foreign cells, particularly those of the endothelial lining of heart valves and blood vessels. While bovine or porcine valve tissues are structurally and biomechanically appropriate for use in humans, the potential for such foreign tissue to stimulate immunological rejection in the recipient has in the past dictated treatments with chemical cross-linking agents such as glutaraldehyde.
  • tissue treatment reduces the stimulation of an immune response by the recipient to the foreign tissue and also stabilizes the collagen protein of the resulting non-viable valve tissue making it more resistant to degradation by proteolytic enzymes.
  • tissue grafts are not viable, there is no biosynthetic mechanism to repair the structural proteins degraded during tissue operation in the recipient.
  • tissue grafts tend to calcify over time, increasing the risk of structural damage and consequent failure. Although it occurs less frequently in relation to mechanical grafts.
  • organs such as kidneys have been allogeneically transplanted from one sibling to another in an effort to minimize immune-mediated reactions in the transplant recipient, which would result in organ rejection.
  • organs such as kidneys
  • immunosuppressive drugs Although the immune response to transplanted tissue or organs can be suppressed through the use of immunosuppressive drugs to minimize rejection, immunosuppressive therapy is general in nature. Therefore, immunosuppressive drugs also tend to suppress the immune response in general, reducing the transplant recipient's ability to fight infection.
  • the invention of said patent provides novel and advantageous processes for generating implant tissue suitable for implanting in humans.
  • the process of this invention generally relates to the treatment of xenogeneic or allogeneic tissue to generate a viable bioprosthesis that does not elicit an adverse immune response from the recipient upon implantation, and possesses the regenerative capabilities of allografts, while exhibiting only a propensity. Limited to calcify and little stimulation of thromboembolism.
  • a process that makes it possible to generate a substantially non-immunogenic tissue matrix suitable for subsequent processing in an implant tissue and that comprises the steps of: A. Eliminating the native cells by treating a tissue with components selected from the group consisting of enzymes and nucleases effective to inhibit subsequent native cell growth in the treated tissue and effective to limit the generation of new immunological sites in the tissue thereby producing a tissue matrix; B. Treating the tissue matrix with cell adhesion factor to promote subsequent attachment of cultured allogeneic or autologous cells to tissue matrix surfaces; and C. Repopulating the tissue matrix throughout the matrix with allogeneic or autologous cultured cells.
  • tissue undergoes a different decellularization process and, in the end, it must undergo a process of cell repopulation of the tissue matrix using cultured allogeneic or autologous cells.
  • patent US7318998B2 by Black Kirby S and Steven Goldstein dated March 24, 2003, which discloses a method for making tissues, including heart valves, resistant to mineralization or immunoreactivity by in vivo implementation while preserving the biomechanical properties of the tissue; it also provides a method of reducing the immunoreactivity of transplanted tissues that are not fixed by chemical or physical means, or combinations thereof, prior to implantation.
  • Said document also discloses a tissue decellularization method and in particular the tissue treatment method (heart valves, tendons and ligaments).
  • the method comprises exposing the tissue to a hypotonic solution in order to lyse the cells, then treating the tissue with a nuclease solution to remove nucleic acids and phosphorous-containing groups, which can bind to calcium, to avoid calcification. Finally, the tissue is transferred to an isotonic solution to maintain the intact tissue structure.
  • the cell lysis solution is a stepped gradient treatment so that the tissue adapts to changes in the concentration of the hypotonic solution (NaCI), taking advantage of the diffusion gradient and the transport phenomenon of the cellular remains to spread in a more controlled way to the solution, avoiding the rupture of the fibers of the extracellular matrix.
  • the decellularization efficiency of our invention is 99% compared to that of this document, which is 70%.
  • the decellularization process for a complete valve is carried out in a sterile 7 oz (207 mL) bottle and the decellularization process consists of tissue disinfection using a cocktail of antibiotics and antifungals such as netilmicin, lincomycin, cefotaxime, vancomycin, rifampin, fluconazole, amphotericin; cell lysis with a hypotonic solution; incubation of the tissue with a nuclease solution (DNAase and/or RNAase) and integrity of the tissue structure with base culture medium solution (Dulbecco's Modified Eagle Medium, DMEM), later terminal sterilization is performed by gamma irradiation, carbon dioxide ethylene, peracetic acid ⁇ beta-propio I actona, povidone iodide, UV irradiation in the presence or absence of photos
  • DMEM
  • cell lysis with hypotonic solution, tissue incubation with nucleases and isotonic solution treatment are carried out in a temperature range of 30°C to 40°C and at an atmosphere of 5% CO2.
  • the decellularization process for a complete valve is carried out in a sterile 7 oz (207 mL) bottle.
  • the volume of the decellularizing solutions is 80 mL and requires terminal sterilization by gamma irradiation, ethylene oxide, peracetic acid, beta-propio I actone, povidone iodide, UV irradiation in the presence or absence of photosensitizers. It has a decellularization efficiency of Another located document is the document EP1,698,356 A1 of Matsuda H et al.
  • a method to improve decellularization that, unlike our invention, consists of immersing a tissue in a solution containing an amphiphilic molecule in non-micellar form (for example, 1,2-epoxide polymer) and performing a radical reaction (for example, treatment selected from the group consisting of exposure to gamma irradiation, ultraviolet irradiation, a free radical supply source, ultrasound, electron beam irradiation, and X-ray irradiation) that is not used in our invention.
  • a radical reaction for example, treatment selected from the group consisting of exposure to gamma irradiation, ultraviolet irradiation, a free radical supply source, ultrasound, electron beam irradiation, and X-ray irradiation
  • the decellularization method consists of exposing the tissue in a solution with an antipathic molecule (polyethylene glycol), then exposing it to a phosphate buffered saline solution (also known by its acronym in English, PBS, phosphate buffered saline) with a cocktail of antibiotics. and antifungals, they are washed with PBS and then submerged in a PBS solution with DNase I and MgCh, the tissue is washed with PBS and finally preserved in PBS with antibiotics at 4°C. This methodology does not offer a 99% decellularization efficiency.
  • an antipathic molecule polyethylene glycol
  • EP2,431,063 by Taylor Dorris A and Ott Harald, dated August 28, 2006, which discloses a method and materials for decellularization of a solid organ and recellularization to thereby generate a solid organ that Comprising: providing a mammalian organ having an extracellular matrix and a substantially closed bed of vasculature, and cells embedded in the extracellular matrix, or providing a mammalian vascularized tissue having an extracellular matrix and vascular tree, and cells embedded in the extracellular matrix; cannulating said organ or tissue in one or more cavities, vessels and/or ducts, thus producing a cannulated organ or tissue; and perfusing the vasculature of said cannulated organ or vascularized tissue with a first cell disruption means through said one or more cannulations; wherein the entire vascular bed is brought into contact with the first means of cell disruption (which is an anionic detergent) and wherein said perfusion is multi-directed from each cavity, vessel and/or cannulated conduit, particularly comprising further
  • Tissue prepared according to the process of the invention exhibits physical and biological properties that make it particularly well suited for tissue grafting applications, as stated in the description.
  • An isolated ureter of animal or human origin is the tissue graft material to be subjected to a decellularization procedure.
  • Cell lysis is carried out with an aqueous hypotonic buffer solution or low ionic strength buffer, the decellularization solution may include other agents, such as protease inhibitors (EDTA).
  • EDTA protease inhibitors
  • Decellularization is preferably achieved by incubation of the tissue in a solution effective to lyse the native cells in the tissue.
  • the tissue is incubated (for example, at about 37°C) in sterile water (for example, for about 4 hours in the case of ureters), however, an aqueous hypotonic buffer or low ionic strength buffer can also be used.
  • the decellularization solution can include other agents, such as protease inhibitors (eg, chelating agents such as EDTA).
  • nucleases that can be used for digestion of cell native DNA and RNA include both exonucleases and endonucleases.
  • the nucleases are present in a buffer solution containing magnesium and calcium salts (eg, chloride salts).
  • the ionic strength and pH of the buffered solution, the temperature of the treatment, and the length of the treatment are selected to ensure the desired level of effective nuclease activity.
  • the buffer is preferably a Tris buffer at pH 7.6.
  • the nuclease cocktail contains about 0.1 pg/ml to 50 pg/ml, preferably 17 pg/ml, DNAse I , and about 0.1 pg/ml to 50 pg/ml, preferably 17 pg/ml, RNAse A.
  • Nuclease treatment can be effected at, for example, about 20°C to about 38°C, preferably 37°C, for approximately 1 to 36 hours. In the case of ureters, nuclease treatment for approximately 19 hours is typically sufficient.
  • the resulting tissue matrix can be treated (washed) to ensure removal of cell debris which may include cellular protein, cellular lipids, and cellular nucleic acid, as well as extracellular debris, such as extracellular soluble proteins, lipids and proteoglycans.
  • cell debris may include cellular protein, cellular lipids, and cellular nucleic acid, as well as extracellular debris, such as extracellular soluble proteins, lipids and proteoglycans.
  • the tissue can be incubated in a buffer (eg, PBS) or in a detergent solution such as a solution of TritonX-100 in water.
  • a buffer eg, PBS
  • a detergent solution such as a solution of TritonX-100 in water.
  • the cell lysis solution is a stepped gradient treatment so that the tissue adapts to changes in the concentration of the hypotonic solution (NaCI), taking advantage of the diffusion gradient and the transport phenomenon of the cellular remains to spread in a more controlled way to the solution, avoiding the rupture of the fibers of the extracellular matrix.
  • concentration of the hypotonic solution NaCI
  • Another important factor in our invention is the controlled conditions of the system, which allows the tissue to be preserved at body temperature (37°C), 95% relative humidity, and a 5% CO2 atmosphere to maintain neutral pH.
  • detergents Triton X-100
  • hypotonic solutions and/or detergents such as 100x triton, and SDS
  • the main objective of the present invention is to make available a methodology and process to decellularize cardiovascular tissue such as heart valves, great vessels, veins and peripheral arterial networks, which entails the use of a system that injects hypotonic solutions in "n" stepped gradient. , and nucleic acid degrading enzymes.
  • Another objective of the invention is to provide said method and process for decellularizing cardiovascular tissue that allows preserving the tissue at body temperature (37°C), a relative humidity of 95%, and an atmosphere of 5% CO2 to maintain neutral pH.
  • Another objective of the invention is to provide said methodology and process for decellularizing cardiovascular tissue, which also allows decellularization to be carried out simultaneously in an aortic valve and a pulmonary artery.
  • Another objective of the invention is to provide said methodology and process for decellularizing cardiovascular tissue, which also offers a stepped gradient treatment so that the tissue adapts to changes in the concentration of a hypotonic solution, taking advantage of the diffusion gradient and the bass of transport of the cellular remains spread in a more controlled way to the solution, avoiding the rupture of the fibers of the extracellular matrix.
  • Another objective of the invention is to provide said methodology and process for decellularizing cardiovascular tissue, which also avoids the denaturation of proteins caused by detergent solutions due to the unfolding of protein structures due to charge imbalance.
  • Another objective of the invention is to provide said methodology and process for decellularizing cardiovascular tissue, which also offers excellent quality of decellularized homografts, preservation of tissue architecture after the decellularization process, and successful recei - I u I a r i z a t i o No. of tissue.
  • Another objective of the invention is to provide said methodology and process for decellularizing cardiovascular tissue, which also makes it possible to obtain a decellularized aortic valve, pulmonary artery, and vascular structures with minimal adverse effects on their mechanical integrity.
  • tissue such as heart valves, pulmonary arteries, great vessels, veins, and peripheral arterial networks were subjected to a low concentration hypotonic solution (8-0 mM of NaCI) and it was found that it damaged the extracellular matrix of the tissue, causing the fragmentation of 40% of the fibers of the tissue. Consequently, it was possible to determine that the decellularization processes with hypotonic static solutions do not reach the disintegration of the cells and/or edemas can be generated in the intrace I u I ar zone. While with the use of detergent solutions, complete decellularization can be achieved, but compromising the denaturation of the anchoring proteins and growth factors that will promote adequate adoption of the graft and therefore cell proliferation and differentiation in the grafted patient. about the same.
  • hypotonic solutions cause edema and fail to complete complete decellularization of the tissue.
  • its functionality is validated by culturing primary human cells (fibroblasts and adipose tissue mesenchymal cells), which at the time of seeding did not use serum-added medium to promote cell recognition of intact anchoring proteins. in the tissue.
  • hypotonic solution and instead of subjecting it to static solutions, a system was devised that would allow the hypotonic solution to recirculate by passing through the tissue and surprisingly it was possible to identify that by varying the concentration of the hypotonic solution and making it present a flow dynamic, several of the problems detected in the previous experiments were resolved.
  • the methodology could be determined based on experimentation and the concentration gradients of the hypotonic solutions that offered better results in the decellularization process were found, together with the determination of the enzymatic treatment of the tissue for the elimination of cellular residues in the tissue and subsequent treatment with isotonic solutions to remove toxic residues and preserve the structural integrity of the tissue.
  • a chemical method is used, where the plasmatic membrane of the cells is solubilized to induce cell lysis through the action of hypotonic solutions. Its effectiveness lies in the fact that they break lipid interactions. However, it does not totally remove the cellular remnants of the tissue. Due to the above, it was necessary to complement the chemical method with an enzymatic treatment for the elimination of the remaining nucleic acids in the structure.
  • Nucleases such as endonucleases, catalyze the hydrolysis of the internal bonds of the ribonucleotide or deoxyribonucleotide chains, whereas exonucleases catalyze the hydrolysis of the terminal bonds of d e oxy r i b o n u c I e o t i d o or ribunonucleotides, allowing the degradation of DNA or RNA.
  • the method for in vitro decellularization of cardiovascular tissue consists of subjecting the cardiovascular tissue to a recirculation system of decellularizing hypotonic solutions in "n ”gradient in a stepwise manner with the aim of lysing the cells of the plasmatic membrane; but maintaining the extracellular matrix integrates; b) subjecting the cardiovascular tissue to a recirculation system of a solution of acid-degrading enzymes nucleases (nucleases) to remove cellular debris in tissue; and c) subjecting the cardiovascular tissue to a recirculation system of isotonic solutions to remove toxic residues and preserve the structural integrity of the tissue.
  • Said recirculation system is configured with means to preserve the tissue at body temperature between (36.1 to 37.2 °C) and preferably 37 °C, a relative humidity between (95 to 98%) and preferably 95%, and a atmosphere with a CO2 concentration of between (5 to 10%) and preferably a CO2 concentration of 5% to maintain neutral pH and in order to emulate the conditions of the human body and not damage the extracellular matrix.
  • Said hypotonic solutions are selected from NaCl or a solution composed of a buffer solution of 10 mM Tris (hydroxymethyl) aminomethane hydrochloride (Trizma-HCI) and 5 mM ethylenediaminetetraacetic acid also known as EDTA.
  • Tris (hydroxymethyl) aminomethane hydrochloride Trizma-HCI
  • EDTA ethylenediaminetetraacetic acid
  • nucleic acid degrading enzymes select from Deoxyribonucleases I (DNase I) and ribonuclease A (RNase A).
  • Said solution of degradative enzymes is defined by Tris (48 mM), MgCI 2 (2.88 mM), CaCI 2 (0.96 mM), DNase I (19 pg/mL) and RNase A (19.2 g/mL).
  • Said isotonic solution consists of a solution of base culture (Dulbecco's Modified Eagle Medium, DMEM).
  • the recirculation system for hypotonic solutions, recirculation of nucleic acid degrading enzymes, and isotonic solutions consists of a reactor defined by a closed and isolated reservoir with temperature control means, relative humidity control means; atmospheric CO2 concentration control means, preferably at 5%, to preserve the neutral pH and in order to emulate the conditions of the human body and not damage the extracellular matrix; configured to house a first container where the tissue to be treated is arranged with means for holding and supporting the tissue and conducting means for a solution selected from a hypotonic solution at different concentrations, sterile water, nucleic acid degrading enzymes and an isotonic solution , wherein said solution conduction means define at least one end to be connected to the upper end of a duct with or without a shunt inserted into at least one tissue to pass said solutions or to connect to the upper end of a second duct that opens close to the bottom of the first container to withdraw or discharge the solution that is recirculated by means of a recirculation pump to which said con
  • the process for decellularization of cardiovascular tissues consists of the following stages: a) Disinfect the tissue by subjecting it to a solution of 1 L of medium cell “Roswell Park Memorial Institute medium”, (better known by its acronym RPMI), with a cocktail of antibiotics and antifungals (50 pg/mL amphotericin B, 500 pg/mL vancomycin, 80 pg/mL gentamicin, 250 pg/mL cefuroxime , 240 pg/mL cefotaxime) for a period of (20 to 36 hours) and preferably 24 hours at a temperature between (6 to 8°C) and preferably at 4°C for tissue disinfection and inhibition of bacterial growth and fungi, preserving its viability.
  • RPMI medium cell
  • antibiotics and antifungals 50 pg/mL amphotericin B, 500 pg/mL vancomycin, 80 pg/mL gentamicin, 250 pg/mL cefuroxi
  • b) Decellularize the cardiovascular tissue in a reactor with a recirculation system of decellularizing hypotonic solutions, whose main objective is to eliminate cellular and nuclear material, minimizing the adverse effects on the mechanical integrity of the extracellular matrix of the tissue in a reactor with recirculation of the decellularizing solutions.
  • bi Subject the tissue to a hypotonic 60-40 mM NaCI solution. The solution is recirculated at a flow of between 2 to 8 L/h and preferably 4 L/h for 6 to 10 hours.
  • b.ii Replace the NaCI solution (60 to 40 mM) with a 35-20 mM NaCI solution.
  • the 35-20 mM NaCI solution is recirculated at a flow rate of between 2 and 8 L/h and preferably 4 L/h for 6 to 10 hours.
  • b.iii carry out a posteriori, a new change of the 35-20 mM NaCI solution for a 15-10 mM NaCI solution.
  • the 15-10 mM NaCl solution is recirculated at a flow rate of between 2 and 8 L/h and preferably 4 L/h for 24-30 hours.
  • b.iv again change the NaCI solution to 15-10 mM with sterile water.
  • the same procedure described above is followed.
  • the water is recirculated at a flow of between 2 to 8 L/h and preferably 4 L/h for 24-30 hours. Afterwards, the sterile water is removed.
  • bv Finally, recirculate 800 to 1000 mL of nucleic acid degrading enzyme solution to remove remaining nucleic acids.
  • the solution is recirculated for 24-30 hours at a flow of between 2 to 8 L/h and preferably 4 L/h. After this time, the nuclease solution is removed.
  • nucleases used are selected from Deoxyribonucleases I (DNase I) and ribonuclease A (RNase A).
  • DNase I Deoxyribonucleases I
  • RNase A ribonuclease A
  • the decellularization process is carried out simultaneously in an aortic valve and a pulmonary artery in a reactor with recirculation of the decellularizing solutions.
  • the decellularization process is capable of generating acellular three-dimensional structures, free of genetic material, without toxicity and, therefore, biocompatible.
  • Said isotonic solution consists of a base culture medium solution (Dulbecco's Modified Eagle Medium, DMEM).
  • DMEM Dulbecco's Modified Eagle Medium
  • Said solution of nucleic acid degrading enzymes is defined by Tris (48 mM), MgCh (2.88 mM), CaCh (0.96 mM), DNase I (19 pg/mL) and RNase A (19.2 ⁇ g/mL).
  • Said method and process allow having decellularized cardiovascular and vascular structures, which offer alternatives to allogeneic grafts with low/null immunohistological rejection, given the absence of genetic material and the presence of allogeneic proteins and structures.
  • the quality of the decellularization process reflected in the quality of the decellularized homograft, preservation of tissue architecture after the decellularization process, and the impact of successful tissue recellularization.
  • the quality of the decellularized homograft is assessed with histological analysis (absence of cell nuclei), quantification of residual DNA in the tissue ( ⁇ 50 ng DNA/g dry tissue), cell migration of human fibroblasts in the decellularized matrix (i.e. because the cell matrix preserves the signals of cell migration, proliferation and differentiation).
  • Figure 1 shows a tissue image illustrating a fragment of myocardium decellularized with a low concentration hypotonic solution (8-0 mM NaCI sodium chloride, showing 40% fragmentation of tissue fibers.
  • Figure 2 shows a graph of the gradual variation of the concentration of a hypotonic solution with which the tissue is treated for decellularization, in accordance with the present invention.
  • Figures 3 show images taken under a microscope at 20X of non-decellularized tissues of the myocardium, aortic arch, and aortic leaflet, respectively, showing that the structure and location of nuclei marked with arrows are preserved.
  • Figures 4 show images taken under a microscope at 20X of decellularized tissues of the myocardium, aortic arch, and aortic leaflet, respectively, showing that the structure is preserved, without localization of nuclei.
  • Figure 5 shows a diagram of the recirculation system for hypotonic solutions, recirculation of nucleic acid degrading enzymes, and isotonic solutions through the tissue to be treated, in accordance with one of the modalities of the system of the present invention.
  • Figure 6 shows a diagram of the recirculation system for hypotonic solutions, recirculation of nucleic acid degrading enzymes, and isotonic solutions through the tissue to be treated, in accordance with a second modality of the system of the present invention.
  • Figures 7A, 7B, 7C, 7D and 7E illustrate schematic diagrams of the recirculation system of decellularizing hypotonic solutions, through a heart valve and a pulmonary artery, simultaneously, where the variation of the concentration in gradients of these occurs. hypotonic solutions and changes of said hypotonic solution at a concentration of 60-40 mM NaCl to a concentration of 35-20 mM NaCl in the system.
  • Figures 8A, 8B, 8C, 8D and 8E illustrate schematic diagrams of the recirculation system of decellularizing hypotonic solutions through a heart valve and a pulmonary artery, simultaneously, where the variation of the concentration gradients of said solutions occurs. hypotonic solutions and changes of said hypotonic solution at a concentration of 35-20 mM NaCI to a concentration of 15-10 mM NaCI in the system.
  • Figures 9A, 9B, 9C, 9D and 9E illustrate schematic diagrams of the decellularizing hypotonic solution recirculation system through a heart valve and a pulmonary artery, simultaneously, where the change of the hypotonic solution occurs at a concentration of 15-10 mM NaCI per deionized water in the system.
  • Figure 1 shows an image of the tissue that illustrates a fragment of decellularized myocardium that underwent a decellularization process with a low concentration hypotonic solution (8-0 mM NaCI), showing a fragmentation of 40% of the cells. tissue fibers.
  • the present invention it was determined that using a method for in vitro decellularization of cardiovascular tissue such as heart valves, pulmonary arteries, great vessels, veins and peripheral arterial networks, using a stepwise concentration gradient of hypotonic solutions, since the The tissue gradually adapts to changes in the concentration of the hypotonic solution, taking advantage of the fact that the diffusion gradient and the phenomenon of transport of cell debris diffuse into the solution in a more controlled manner, avoiding the rupture of the extracellular matrix fibers.
  • the methodology of hypotonic solutions the denaturation caused by detergent solutions due to the unfolding of protein structures due to charge imbalance was avoided.
  • the method for in vitro decellularization of cardiovascular tissue consists of a) subjecting the cardiovascular tissue to a recirculation system of decellularizing hypotonic solutions in a stepped "n" gradient with the aim of lyse cells from the plasma membrane; but maintaining the extracellular matrix integrates; b) subjecting the cardiovascular tissue to a recirculating system of nucleic acid degrading enzymes (nucleases) to remove cellular debris in the tissue; and c) subjecting the cardiovascular tissue to a recirculation system of isotonic solutions to remove toxic residues and preserve the structural integrity of the tissue.
  • nucleic acid degrading enzymes nucleic acid degrading enzymes
  • the system of recirculation of hypotonic solutions, recirculation of acid degrading enzymes nucleic acid and isotonic solutions consists of a reactor defined by a closed and isolated reservoir (1) with temperature control means, relative humidity control means; atmospheric CO2 concentration control means, preferably at 5% (not shown), to maintain neutral pH, configured to house a container (2) where the tissue to be treated is placed (in this case, a heart valve (VC) is simultaneously treated ) and a pulmonary artery (PA)), with holding and support means (not shown) of the tissues and conduction means (3) of a solution selected from a hypotonic solution at different concentrations, sterile water, nucleic acid degrading enzymes and an isotonic solution, wherein said conduction means (3) of a solution are connected to a duct (4) with two shunts that are inserted into a heart valve (VC) and a pulmonary artery (AP) to pass said solutions; a duct (5) that opens close to the bottom of the first container (2) in which
  • the recirculation system for hypotonic solutions, recirculation of nucleic acid degrading enzymes, and isotonic solutions consists of a reactor defined by a closed and isolated reservoir (1) with temperature control means, control means relative humidity; means of atmospheric CO2 concentration control (not shown) preferably at 5%, to maintain neutral pH, configured to house a first container (2) where the tissue to be treated is placed (in this case a heart valve (VC) is treated simultaneously) and a pulmonary artery (PA)), with holding and support means (not shown) of the tissues and conduction means (3) of a solution selected from a hypotonic solution at different concentrations, sterile water, nucleic acid degrading enzymes and an isotonic solution, wherein said conduction means (3) of a solution define at least one end to be connected to the upper end of a duct (4) with two shunts inserted in the heart valve (VC) and in the pulmonary artery ( AP) to pass said solutions or to connect to the upper end of a second duct
  • the process for decellularization of cardiovascular tissues consists of the following stages: a) Disinfect the tissue by subjecting it to a 1L solution of cellular medium "Roswell Park Memorial Institute medium", (better known by its acronym RPMI), with a cocktail of antibiotics and antifungals (50 pg/mL amphotericin B, 500 pg/mL vancomycin, 80 pg/mL gentamicin, 250 pg/mL cefuroxime, 240 pg/mL cefotaxime) for a period of 20 to 36 hours and preferably 24 hours at a temperature between 6 to 8°C and preferably at 4°C for the disinfection of the tissue and inhibit the growth of bacteria and fungi, preserving its viability.
  • RPMI Roswell Park Memorial Institute medium
  • antibiotics and antifungals 50 pg/mL amphotericin B, 500 pg/mL vancomycin, 80 pg/mL gentamicin, 250 pg/
  • b) Decellularize the cardiovascular tissue in a reactor with a recirculation system of decellularizing hypotonic solutions, whose main objective is to eliminate cellular and nuclear material, minimizing the adverse effects on the mechanical integrity of the extracellular matrix of the tissue in a reactor with recirculation of the decellularizing solutions.
  • bi Submit the tissue to a hypotonic 60-40 mM NaCI solution. The solution is recirculated at a flow of between 2 to 8 L/h and preferably at 4 L/h for 6 to 10 hours.
  • b.ii Replace the NaCI solution (60 to 40 mM) with a 35-20 mM NaCI solution.
  • the 35-20 mM NaCI solution is recirculated at a flow rate of between 2 to 8 L/h and preferably 4 L/h for 6 to 10 hours.
  • b.iii Subsequently, change the 35-20 mM NaCI solution for a 15-10 mM NaCI solution. Following the same procedure described above.
  • the 15-10 mM NaCl solution is recirculated at a flow rate of between 2 to 8 L/h and preferably 4 L/h for 24-30 hours.
  • b.iv Change the NaCl solution again to 15-10 mM with sterile water.
  • the same procedure described above is followed.
  • the water is recirculated at a flow of between 2 to 8 L/h and preferably at 4 L/h for 24-30 hours. Afterwards, the sterile water is removed.
  • bv Finally, recirculate 800 to 1000 mL of a nuclease solution selected from Deoxyribonucleases I (DNase I) and Ribonuclease A (RNase A) to remove remaining nucleic acids.
  • DNase I Deoxyribonucleases I
  • RNase A Ribonuclease A
  • c) Recirculate a base culture medium solution (Dulbecco's Modified Eagle Medium, DMEM) at a flow of between 2 to 8 L/h and preferably 4 L/h for 10-7 days, changing the solution every 48 hours. for the preservation of the structural integrity of the tissue with the aim of removing nuclease residues.
  • DMEM Dulbecco's Modified Eagle Medium
  • FIGS. 7A, 7B, 7C, 7D and 7E schematic diagrams of the decellularizing hypotonic solution recirculation system through a heart valve and a pulmonary artery are illustrated simultaneously; where the variations of the concentration in gradients of said hypotonic solutions and the changes of said hypotonic solution at a concentration of 60-40 mM NaCl to a concentration of 35-20 mM NaCl in the system are given; noting the following: having started the treatment of the tissues, cardiac valve (VC) and in the pulmonary artery (PA), with recirculation of a hypotonic solution at a concentration of 60-40mM of NaCl, 500 mL of said hypotonic solution is withdrawn at a concentration of 60-40mM NaCl from the reactor vessel (2) by action of the recirculation pump (6) is discharged to the second vessel (7) ( Figure 7A).
  • VC cardiac valve
  • PA pulmonary artery
  • the concentration of the hypotonic solution is changed and 500 mL of this hypotonic solution at a concentration of 35-20 mM NaCl is added from the second container (7) and recirculated through the cardiac valve (VC) tissues and in the artery. lung (AP) by action of the recirculation pump (6) discharging into the container (2) ( Figure 7B).
  • the 1000 mL of the solution is removed from the container (2) and is discharged into the container (7) by action of the recirculation pump (6) ( Figure 7C).
  • the concentration of the hypotonic solution in the container (7) is changed and 1000 mL of the hypotonic solution at a concentration of 35-20 mM of NaCI are added, which is passed through the action of the recirculation pump (6) through the tissues heart valve (VC) and the pulmonary artery (AP) and are discharged into the first container (2) ( Figure 7D). Finally, in the same container (2) the concentration of the hypotonic solution is changed to a concentration of 35-20 mM NaCI and the solution is recirculated for 6 hours with a flow of 4L/h by action of the recirculation pump ( 6) ( Figure 7E). In all stages, the interior of the reservoir where the tissues are housed in the recirculation system maintains a temperature preferably of 37°C, a relative humidity p of 95%, and an atmosphere with a CO2 concentration of preferably 5%.
  • FIGS 8A, 8B, 8C, 8D and 8E they illustrate schematic diagrams of the recirculation system of decellularizing hypotonic solutions through a heart valve and a pulmonary artery, simultaneously, where the concentration variations occur in gradients. of said hypotonic solutions and changing said hypotonic solution at a concentration of 35-20 mM NaCI to a hypotonic solution at a concentration of 15-10 mM in the system; noting the following: 500 mL of the hypotonic solution at a concentration of 35-20mM NaCI is withdrawn from the reactor vessel (2) by action of the recirculation pump (6) ( Figure 8A).
  • the concentration is changed and 500 mL of the hypotonic solution at a concentration of 15-10 mM NaCI are added from the second container (7) and recirculated through the cardiac valve (VC) and pulmonary artery (AP) tissues. ) by action of the recirculation pump (6) discharging into the container (2) ( Figure 8B).
  • HE 1000 ml_ of the solution are removed from the container (2) and it is discharged into the container (7) by action of the recirculation pump (6) ( Figure 8C).
  • the concentration of the hypotonic solution in the container (7) is changed and 1000 mL of the hypotonic solution at a concentration of 15-10 mM of NaCI are added, which is passed through the action of the recirculation pump (6) through heart valve (VC) and pulmonary artery (PA) tissues and are discharged into the first container (2) ( Figure 8D). Finally, in the same container (2) the concentration of the hypotonic solution is changed to a concentration of 15-10 mM for 24 hours with a flow of 4L/h by action of the recirculation pump (6) ( Figure 8E). In all stages, the interior of the reservoir where the tissues are housed in the recirculation system maintains a temperature preferably of 37°C, a relative humidity preferably of 95% and an atmosphere with a CO2 concentration preferably of 5%.
  • FIGS 9A, 9B, 9C, 9D and 9E they illustrate schematic diagrams of the recirculation system of decellularizing hypotonic solutions through a heart valve and a pulmonary artery, simultaneously, where the change from the hypotonic solution to a concentration of 15-10 mM of NaCI by deionized water in the system, noting the following: 500 mL of the hypotonic solution with a concentration of 15-10 mM of NaCI is withdrawn from the vessel (2) of the reactor by action of the pump of recirculation (6) is discharged to the second container (7) ( Figure 9A).
  • the deionized water is recirculated for 24 hours with a flow of 4L/h by action of the recirculation pump (6) ( Figure 9E).
  • the interior of the reservoir where the tissues are housed in the recirculation system maintains a temperature preferably of 37°C, a relative humidity preferably of 95% and an atmosphere with a CO2 concentration preferably of 5%.

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Abstract

La présente invention concerne un procédé et un processus de décellularisation in vitro de tissu cardiovasculaire, tel que des valvules cardiaques, artères pulmonaires, grands vaisseaux, veines et réseaux artériels périphériques, ledit procédé étant caractérisé en ce qu'il consiste à a) soumettre le tissu cardiovasculaire à un système de recirculation de solutions hypotoniques de décellularisation en "n" gradient de concentration de manière échelonnée pour liser les cellules de la membrane plasmatique, mais en maintenant entière la matrice extracellulaire ; b) soumettre le tissu cardiovasculaire à un système de recirculation d'enzymes de dégradation des acides nucléiques pour éliminer des résidus cellulaires dans le tissu ; et c) soumettre le tissu cardiovasculaire à un système de recirculation de solutions isotoniques pour éliminer des résidus toxiques et préserver l'intégrité structurale du tissu.
PCT/MX2021/050071 2021-10-27 2021-10-29 Procédé et processus de décellularisation in vitro de tissu cardiovasculaire WO2023075586A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040076657A1 (en) * 1999-06-07 2004-04-22 Lifenet. Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants produced
US20110165676A1 (en) * 2009-11-06 2011-07-07 The Children's Mercy Hospital Method for decellularization
US20190374680A1 (en) * 2014-04-02 2019-12-12 Biotronik Ag Method for the preparation of biological tissue for dry use in an implant
US11077230B2 (en) * 2015-10-21 2021-08-03 Videregen Limited Decellularising tissue

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040076657A1 (en) * 1999-06-07 2004-04-22 Lifenet. Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants produced
US20110165676A1 (en) * 2009-11-06 2011-07-07 The Children's Mercy Hospital Method for decellularization
US20190374680A1 (en) * 2014-04-02 2019-12-12 Biotronik Ag Method for the preparation of biological tissue for dry use in an implant
US11077230B2 (en) * 2015-10-21 2021-08-03 Videregen Limited Decellularising tissue

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