Cellular spheroid from subperichondral regeneration blastema cells of perichondrium for cartilage restoration

Abstract

FIELD: biotechnology.SUBSTANCE: transplant for restoring cartilage in a subject. The invention includes cellular spheroids based on autologous perichondrium cells of a subject.EFFECT: production of a transplant based on cellular spheroids possessing pronounced regenerative potential and the properties of regenerative blastema cells for the effective restoration of hyaline cartilage.1 cl, 1 ex

Description

[0001] Technical field

[0002] The invention relates to the field of biomedicine, namely to regenerative medicine and tissue engineering.

[0003] Technology Level

[0004] The development of scaffold-free tissue engineering is linked to the improvement of regenerative medicine cell products—individual standardized organ building blocks, including cellular spheroids, organoids, and assemblies. These organ building blocks contain living cells and possess the ability to self-assemble and self-organize both in cell culture and in vivo. Standard building blocks enable the efficient creation of larger tissue-engineered constructs based on the principles of a modular approach known in tissue engineering, which involves the assembly of both small cell-filled modules and larger structures designed to fill post-traumatic defects and stimulate reparative regeneration.

[0005] The perichondrium is a dense, vascularized connective tissue membrane covering the cartilage of growing bones, the hyaline cartilage of the costals, the cartilages of the larynx, and so on. Articular cartilage lacks a perichondrium. The perichondrium serves for the growth and repair of cartilage tissue.

[0006] It is known that connective tissue membranes of bones and cartilages of the skeleton are capable of forming regenerative blastemas, which ensure, for example, bone restitution after complete subperiosteal enucleation of the diaphyses of the tibia bones, and such a high regenerative capacity of skeletal organs was observed in experiments on different species of birds and mammals, which were carried out under the supervision of Professor A. N. Studitsky (Runikhin, Yu. A. Sources of restoration of whole tubular bones after complete subperiosteal enucleation [Text]: (Experimental - morphological study): Abstract of dis. for the degree of candidate of biological sciences / USSR Academy of Sciences. Institute of Evolutionary Morphology and Ecology of Animals named after A. N. Severtsov. - Moscow: [b. i.], 1968. - 24 p.).

[0007] The restoration of skeletal fragments after subperiosteal and subperichondral enucleation of bone and cartilage in humans is documented in clinical practice. A 42-year-old man required craniofacial reconstruction in the Department of Maxillofacial Surgery due to trauma. A portion of the right sixth rib (8 cm) and adjacent costal cartilage (1 cm) were harvested for free reconstruction, preserving the integrity of their periosteum and perichondrium. At 6 months postoperatively, three-dimensional reconstructed CT imaging revealed bone regeneration, cartilage regeneration, and costochondral joint regeneration (Srour MK, Fogel JL, Yamaguchi CT, Montgomery AR, Izuhara AK, Misakian AL, Lam S, Lakeland DL, Urata MM, Lee JS, Mariani FV. Natural large-scale regeneration of rib cartilage in a mouse model. J Bone Miner Res. 2015 Feb;30(2):297-308. doi: 10.1002 / jbmr.2326. PMID: 25142306; PMCID: PMC8253918).

[0008] In vivo bioreactor technology is known, which is based on manipulating an intentionally created space inside the body so that it serves as an “in vivo bioreactor,” in which the construction of new growing tissue is achieved by inducing a therapeutic response within the bioreactor space (Stevens MM, Marini RP, Schaefer D, Aronson J, Langer R, Shastri VP. In vivo engineering of organs: the bone bioreactor. Proc Natl Acad Sci USA. 2005 Aug 9; 102(32): 11450-5. doi: 10.1073 / pnas.0504705102. Epub 2005 Jul 29. PMID: 16055556; PMCID: PMC1183576). The authors achieved controlled manipulation of the periosteal space using a procedure they termed hydraulic periosteal elevation. The periosteum was punctured with a syringe needle, bevel side up, and separated from the bone by simultaneously injecting fluid (Ringer's solution) between the cambium and bone and slowly advancing the needle with an oscillating motion, peeling the periosteum away.After creating a space of the desired geometry, it was filled with a hyaluronic acid-based gel. This procedure allowed the creation of a space over the tibia of skeletally mature New Zealand White rabbits, into which 200 mm of hyaluronic acid-based gel was then reproducibly placed. 3The space created by hydraulic periosteal elevation was filled with gel and Ringer's solution was sequentially filled with gel, also using a syringe. Hyaluronan (Genzyme) was chemically modified to contain aldehyde groups (HA-ALD) by reaction with sodium periodate and hydrazide groups (HA-ADH) by reaction with adipic dihydrazide. Hyaluronic acid hydrogels were prepared by mixing equal volumes of 2% (w / v) aqueous solutions of HA-ALD and HA-ADH. Suramin (Bayer, Leverkusen, Germany) was included in free (0.4 mol / liter) and liposome-encapsulated (0.4 mol / liter) forms where indicated during gelation. Liposomes containing suramin were prepared using a previously described method (Kim S, Turker MS, Chi EY, Sela S, Martin GM. Preparation of multivesicular liposomes. Biochim Biophys Acta. 1983 Mar 9;728(3):339-48. doi: 10.1016 / 0005-2736(83)90504-7. PMID: 6824663).This hydrogel was used to induce perichondrium regenerative blastema.

[0009] The authors discovered the formation of cartilage regenerate in the in vivo bioreactor space. The rate of cartilage formation in the in vivo bioreactor space is noteworthy. Cartilage regenerate was detected as early as 10 days after the introduction of hyaluronic acid gel containing suramin into the bioreactor.

[0010] Cartilaginous and osteochondral defects remain a major challenge in modern medicine and tissue engineering of the musculoskeletal system. Significant damage to articular cartilage often requires removal of the entire joint and its replacement with an endoprosthesis, with all the known complications and disadvantages of endoprosthetics. This problem is particularly pressing because articular cartilage damage often progresses, leading to degradation of the articular cartilage and osteoarthritis (OA). Numerous attempts have been made to develop effective tissue-engineered constructs for filling cartilage defects. Matrix-associated chondrocyte implantation (MACI) is a well-known technology. The disadvantages of culturing any cells on matrices (biodegradable carriers) are well known, and they also apply to tissue-engineered cartilage constructs.Cells are unevenly distributed within the matrix due to gradients in oxygen and carbon dioxide partial pressures, as well as variable nutrient availability and metabolite removal rates depending on the depth of cell placement within the scaffold, which is immersed in the culture medium. The scaffold is difficult to bond to the recipient surface. Current autologous chondrocyte transplantation technologies require destruction of the healthy, non-load-bearing articular surface to harvest cartilage fragments for chondrocyte isolation in a cell laboratory and the production of chondrocyte grafts in the form of cellular spheroids. Therefore, additional arthroscopic surgery and anesthesia are necessary. Scaffold-free technologies for cartilage restoration using chondrospheres are also known. Three-dimensional cultivation of chondrocytes in the absence of a substrate suitable for cellular adhesion leads to the formation of organ-specific cellular spheroids—chondrospheres.Chondrospheres, like the vast majority of cellular spheroids, have a unique ability to tissue fusion, are capable of merging with each other to form larger spheroids and assemblies, and also of spreading on the surface of adhesive plastic and the recipient bed.

[0011] A method for the in vitro production of three-dimensional living cartilage-like microtissues chondrospheres and their use as a transplant material is known (US 7887843 B2 Method for in vitro production of three-dimentional vital cartilage tissue and use thereof as transplant material (co.don® AG, Teltow, DE). This method involves obtaining cellular spheroids from cultured articular cartilage chondrocytes in 96-well plates made of non-adhesive plastic. An additional technological step of working with the cellular material is required - mutual fusion of spheroids in a 24-well plate to increase the size of the spheroids, which indicates a limited potential for independent growth of spheroids obtained in this way. The resulting spheroids are used for partial replacement (filling at a density of 3 to 70 spheroids per cm2) of defects in the articular surfaces of bones in order to form new cartilage in the damaged area.Prolonged exposure of chondrocytes to 3D culture can lead to cell death, particularly in the center of the spheroids. Articular cartilage contains a single population of differentiated cells—chondrocytes—whose proliferative potential is limited. Also known from the prior art is a method for producing spheroids from cartilage cells in agarose microplates containing 81 wells with a diameter of 800 μm or 256 wells with a diameter of 300 μm (Stuart MP, Matsui RAM, Santos MFS, Cortes I, Azevedo MS, Silva KR, Beatrici A, Leite PEC, Falagan-Lotsch P, Granjeiro JM, Mironov V, Baptista LS. Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel. Stem Cells Int. 2017;2017:7053465. doi: 10.1155 / 2017 / 7053465. Epub 2017 Dec 20. PMID: 29527227; PMCID: PMC5750468).Spheroids were formed from cultured chondroprogenitor cells of the nasal septum in chondrogenic differentiation medium (DMEM, human albumin 1.25 μg / ml, ascorbic acid 50 μg / ml, insulin 6.25 μg / ml, penicillin 100 U / ml, streptomycin 100 μg / ml, insulin-transferrin-selenite (single concentration)). The seeding density was 2 million cells per microplate. Spheroids obtained by this method are characterized by the presence of a fibrillar extracellular matrix - collagen type 2. Also, a tendency to growth (an increase in diameter relative to the original) was observed in large-diameter spheroids (obtained in an 81-well plate).A significant drawback of this method is the limited amount of source material that can be obtained from nasal septal cartilage for cell isolation without creating a noticeable cosmetic defect. This necessitates prolonged cell culture to accumulate the required cell biomass. During spheroid formation, gradual cell death occurs, and the spheroids shrink in size. Thus, despite existing methods for cartilage restoration, they all have a number of drawbacks and limitations, necessitating the development and creation of new methods and approaches to address this issue.

[0012] The closest analogue of the present invention is a known method for producing cellular spheroids from the perichondrium (Patent No. 2731314 C1 Russian Federation, IPC A61F 2 / 30, C12N 5 / 07. Method for producing cellular spheroids for cartilage restoration: No. 2019134905: declared 10 / 30 / 2019: published 09 / 01 / 2020 / A. V. Kovalev, S. A. Rodionov; applicant LIMITED LIABILITY COMPANY "INTERNATIONAL CENTER FOR MEDICAL RESEARCH AND DEVELOPMENT" (LLC "MC MIIR"). The cambial layer of the perichondrium contains cambial elements, is involved in growth, reparative and physiological regeneration of cartilage, but in the normal state it is weakly expressed and contains a relatively small number of chondrogenic cells; with age, it can almost completely disappear.

[0013] Disclosure of invention

[0014] The objective of the present invention is to develop a new transplant based on cellular spheroids from autologous cells of the subperichondral regeneration blastema formed at the border of the perichondrium exfoliated from the costal cartilage and a hyaluronic acid-based gel in vivo of a bioreactor installed under the perichondrium of the costal cartilage, for the effective restoration of hyaline cartilage.

[0015] The technical result of this invention is the production of a transplant based on cellular spheroids possessing pronounced regenerative potential and the properties of regenerative blastema cells for the effective restoration of hyaline cartilage.

[0016] In particular embodiments of the invention, the cartilage is articular cartilage, cartilage of the nose, larynx, trachea, and bronchi. In particular embodiments, the subject is a human. In particular embodiments, the spheroid size is 250-700 μm.

[0017] In particular embodiments of the invention, cellular spheroids are cellular spheroids based on cultured cells of the subperichondral regenerative blastema of a subject.

[0018] In particular embodiments of the invention, cellular spheroids based on autologous cells of the subperichondral regenerative blastema of a subject.

[0019] In particular embodiments of the invention, the subject is a human being.

[0020] The technical result of the present invention is also achieved by using cells of the subperichondral regeneration blastema of the perichondrium of the subject's own costal cartilage to obtain a transplant in the form of cellular spheroids.

[0021] The technical result of the present invention is also achieved through the in vivo use of a hyaluronic acid hydrogel injected subchondrally into the costal cartilage. The hydrogel is obtained by mixing equal volumes of 2% (w / v) aqueous solutions of HA-ALD and HA-ADH. Suramin (Bayer, Leverkusen, Germany) was included in free (0.4 mol / liter) form.

[0022] Definitions and Terms Various terms related to the objects of the present invention are used above and also in the description and claims. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art.

[0023] In the description of this invention, the terms "includes" and "including" are interpreted to mean "includes, among other things." These terms are not intended to be construed as meaning "consists solely of." In the description of this invention, the term "tissue" refers to a system of cells and non-cellular structures that share a common structure, in some cases a common origin, and are specialized to perform specific functions.

[0024] The term "blastema" in this document refers to a heterogeneous cell mass that, through migration and proliferation, is temporarily formed at the site of injury and undergoes morphogenesis to form the missing organ.

[0025] The term "perichondrium" (perichondrium) refers to the connective tissue sheath surrounding cartilage. Perichondrium cells differentiate into cartilage cells simultaneously with the formation of the dense, homogeneous ground substance of cartilage.

[0026] Subperichondral regenerative blastema is a heterogeneous cellular mass forming beneath the perichondrium, containing chondrogenic cells. The source of this mass is the hypertrophied perichondrium, activated by trauma and deprived of direct contact with bone. The detached perichondrium of the costal cartilage can form a subperiosteal regenerative blastema (blastoma subperichondral regenerate) between the 3rd and 8th days after in vivo placement of a bioreactor with a hyaluronic acid-based gel on the costal cartilage.

[0027] Sub-, Pod- (Sub-) - a prefix denoting located below, under something.

[0028] In Vivo Bioreactor - An intentionally created space within the body so that it serves as a "tissue-engineered bioreactor" in which neotissue engineering is achieved by inducing a therapeutic regenerative response from the surrounding tissues of the individual within the space confined by the device.

[0029] Aggrecan is a polyanion containing about 8000 negatively charged sulfate and carboxylate groups per mole and at a concentration of about 50 mgmL-1 (~25 μM) in tissues, which attracts mobile counterions and creates a large Donnan osmotic pressure, drawing water into the cartilage matrix, which elongates the collagen network until the collagen tension balances the tissue edema pressure.

[0030] The term “regenerative potential” in this document represents the ability of tissue-engineered constructs to initiate reparative or transplantative regeneration, to be a source of cellular regeneration, to support reparative regeneration with the achievement of a positive result - complete or partial restoration of a damaged organ.

[0031] Detailed disclosure of the invention

[0032] Spheroids are a well-known type of organ building block. They are dense, three-dimensional, rounded cellular aggregates capable of tissue fusion and can act as a source of regeneration under certain experimental conditions. However, the regenerative potential and optimal application of cellular spheroids for regenerative medicine purposes are determined by the spheroid's cellular composition, the conditions of its biofabrication, and its size.

[0033] Examples of epimorphic regeneration indicate the extremely important role of the blastema in organ regeneration, including cartilage regeneration.

[0034] According to the present invention, subperichondral regenerative blastema cells are used as a source of blastema cells for cell spheroid transplants. Growth of such a blastema can be induced in the recipient's own body. This requires the creation of an in vivo cartilage bioreactor using a known method on the subject's costal cartilage.

[0035] In an experiment on 8-month-old skeletally mature male Californian rabbits, periosteum manipulation techniques were used, namely the well-known method of hydraulic elevation of the periosteum (Marini RP, Stevens MM, Langer R, Shastri VP. Hydraulic elevation of the periosteum: a novel technique for periosteal harvest. J Invest Surg. 2004 Jul-Aug;17(4):229-33. doi: 10.1080 / 08941930490472073. PMID: 15371165.) for hydraulically lifting the perichondrium of the costal cartilage. For this purpose, surgical access to the outer surface of the costal cartilage was performed in an operating room to expose the perichondrium. For this, a 5 / 8 inch, 25 gauge needle was bent at a 45° angle at half its length and attached to a 3 ml syringe filled with Ringer's solution.The perichondrium was punctured with a needle, with the beveled side of the needle pointing upward. The perichondrium was separated from the costal cartilage by injecting Ringer's solution between the inner cambium layer of the perichondrium and the adjacent costal cartilage. By injecting Ringer's solution under the perichondrium and advancing the needle tip, a space was gradually created beneath the perichondrium. A thicker needle was then used to inject hyaluronic acid-based gel into the space between the perichondrium and the costal cartilage until it was fully distended (dimensions of the in vivo bioreactor: length 3 cm, width 0.4 cm, height above the costal cartilage plane 4 mm). The holes in the perichondrium were sutured with absorbable sutures to prevent gel leakage, and the incision itself was closed layer by layer with surgical sutures.

[0036] Preparation of hyaluronic acid gel with the addition of suramin was carried out by a known method (Stevens MM, Marini RP, Schaefer D, Aronson J, Langer R, Shastri VP. In vivo engineering of organs: the bone bioreactor. Proc Natl Acad Sci USA. 2005 Aug 9;102(32):11450-5. doi: 10.1073 / pnas.0504705102. Epub 2005 Jul 29. PMID: 16055556; PMCID: PMC1183576).

[0037] Unlike the known methods of using a bone in vivo bioreactor, we have found that at the border of the exfoliated costal cartilage and the gel of a known in vivo bioreactor, a subperichondral regenerative blastema develops in rabbits within 4-7 days, the cells of which we use in laboratory conditions after isolation or during isolation and subsequent cultivation for aggregation into cellular spheroids.

[0038] Induction of subperichondral regeneration blastema occurs using an in vivo bioreactor with hyaluronic acid gel.

[0039] First Operation

[0040] To extract subperichondral regenerative blastema cells from the perichondrium, a second surgical procedure is performed. The perichondrium is completely excised with the regenerative blastema over the hyaluronic acid-based gel in an in vivo bioreactor. The need for two consecutive surgical procedures, 4-5 days apart, is somewhat inconvenient for the patient; however, these procedures can be performed on an outpatient basis under local anesthesia.

[0041] The perichondrium is laid out on a sterile dissection table, and while it is constantly moistened, the cellular mass is scraped from the inner surface of the perichondrium. On the donor surface of the costal cartilage, the excised periosteum completely regenerates after some time.

[0042] The isolated perichondrium portion containing the blastema was subjected to further mechanical and enzymatic disaggregation (using collagenase type 1). After centrifugation and resuspension, the isolated blastema cells were seeded into culture flasks with a treated surface. To maintain the growth of the primary cell culture and at all stages of passaging, culture flasks with a non-ventilated lid and a treated surface were used, as well as a culture nutrient medium of the following composition: DMEM / P12 450 ml, L-glutamine 292 mg, 50 ml of thrombolysate, penicillin 100 U / ml, streptomycin 100 μg / ml (at 100% humidity above the layer of nutrient medium inside the culture flask and a gas mixture for filling the culture flask consisting of 5% CO2, 10% oxygen and 85% nitrogen), the replacement of the nutrient medium in the flask is carried out at least once every 3 days.

[0043] Subculturing of a monolayer culture of subperichondral regeneration blastema cells is carried out until the third to fifth passage. After the final passage, the cells are removed from the plastic flasks with trypsin / EDTA. The resulting cell suspension is washed to remove trypsin / EDTA, including with DMEM medium supplemented with 10% autologous rabbit serum, followed by centrifugation and transferred to 81-well agarose plates with a well diameter of 800 µm at a concentration of up to 1.4 million cells per plate. Cell spheroids are formed from the cultured cells in the agarose wells. Cell aggregation and spheroid maturation occur under these 3D culture conditions for no more than 7 days.

[0044] For use, cell spheroids are collected from the plates and transferred to a test tube, where they settle to the bottom without further centrifugation. The cell spheroids are then transferred using an applicator with a needle lumen of at least 1 mm. 2 .

[0045] Implementation of the invention

[0046] Example 1

[0047] In an experiment on 8-month-old male Californian rabbits, an experimental surgical approach to the outer surface of the costal cartilage was performed under operating conditions. The perichondrium was exposed. Using a syringe and needle and Ringer's solution, the perichondrium was hydraulically lifted. The subperichondrium space was then filled with hyaluronic acid-based gel, forming an in vivo bioreactor measuring 3 cm in length, spanning the entire width of the costal cartilage, and 4 mm in height above the costal cartilage surface. The opening in the perichondrium was additionally sutured with absorbable sutures to prevent gel leakage from beneath the perichondrium, and the wound was sutured layer by layer. The preparation of hyaluronic acid-based gel was carried out using a known method (Stevens MM, Marini RP, Schaefer D, Aronson J, Langer R, Shastri VP. In vivo engineering of organs: the bone bioreactor. Proc Natl Acad Sci USA. 2005 Aug 9;102(32):11450-5. doi: 10.1073 / pnas.0504705102.Epub 2005 Jul 29. PMID: 16055556; PMCID: PMC1183576).

[0048] Five days after in vivo bioreactor placement, the perichondrium containing the developed subperichondral regenerative blastema is removed from the rabbits. The perichondrium is stretched on a sterile dissection table, and under sterile conditions, the cellular mass is scraped from the inner surface of the perichondrium under constant moisture. The isolated and partially crushed portion of the perichondrium containing the regenerative blastema undergoes further mechanical and enzymatic disaggregation (using collagenase type 1). After centrifugation and resuspension, the isolated cells are seeded into culture flasks with an adhesive surface.To maintain the growth of the primary cell culture and at all stages of passaging, culture flasks with a non-ventilated lid and a treated surface were used; the same composition of the culture nutrient medium was used: DMEM / P12 450 ml, L-glutamine 292 mg, 50 ml of rabbit thrombolysate, penicillin 100 U / ml, streptomycin 100 μg / ml (at 100% humidity above the nutrient medium layer inside the culture flask, a ready-made gas mixture was used to fill the culture flask consisting of 5% CO2, 10% oxygen and 85% nitrogen), the nutrient medium and gas mixture in the culture flask were replaced at least once every 3 days. Subcultivation of the monolayer culture of regenerative blastema cells formed under the perichondrium was carried out up to the 4th passage. After the last passage, cells were removed from the surface of plastic flasks with trypsin / EDTA.The resulting cell suspension was washed from trypsin / EDTA using DMEM medium with 10% autologous serum, followed by centrifugation and transferred to 81-well agarose plates with a well diameter of 800 µm at a concentration of up to 1.4 million cells per plate. 3D cell cultivation was performed in the wells, during the initial stages of which cell aggregation occurred with the formation of cell spheroids from regeneration blastema cells. The number of cells in one spheroid was at least 8,000 cells. The production of cell spheroids using 3D agarose Petri Dish®, cell aggregation, and maturation of cell spheroids in agarose wells were carried out taking into account the recommendations of the silicone mold manufacturer (MicroTissues Inc.®).

[0049] 3D cell culture growth, cell aggregation, and maturation of cell spheroids occurred in agarose wells for 5 days. For transplantation, cell spheroids were collected from the agarose wells and transferred to a test tube, where they settled to the bottom without further centrifugation. The spheroids were then transferred using a special applicator with a needle lumen of at least 1 mm. 2 .

[0050] In California rabbits, a 3-cm-long segmental costal cartilage defect was surgically created in the operating room using a scalpel and pliers. The cartilage was removed along with the perichondrium. A polymethylmethacrylate spacer, identical in shape to the removed costal cartilage segment, was placed in place of the cartilaginous defect. After 10 days, the spacer was surgically removed, allowing a foreign body membrane to form around it, acting as a biological chamber. The empty space was filled with cellular spheroids from regenerative blastema cells. Morphological and histological examination of the regenerated tissue was performed 4 months after the cellular spheroid transplantation. Before the histological examination of the cartilage regenerate, microCT of the chest fragment with the restored costal cartilage was performed, followed by digital 3D reconstruction of the resulting series of images.

[0051] Such cartilaginous defects of the costal cartilages are considered critical and never spontaneously regenerate if the perichondrium is removed. It has been found that cellular spheroids from perichondrium blastema cells provide complete and adequate regeneration of the lost costal cartilage fragment, restoring the normal anatomy of this experimentally damaged portion of the chest.

[0052] Example 2

[0053] Nine 8-month-old male Californian rabbits underwent surgical access to the costal cartilage and placement of the in vivo bioreactor described above. A regenerative blastema was grown subchondrally in the presence of the in vivo bioreactor containing a hyaluronic acid-based hydrogel. Mechanical and enzymatic disaggregation of the blastema resulted in the isolation of cells in suspension, which were subsequently used for 3D culturing in 3D Petri Dish® agarose wells, bypassing the 2D culturing step in culture flasks. Self-assembly of 3D microtissue—cell spheroids from subperiosteal regenerative blastema cells—was carried out in an agarose well for 4 days. In a culture nutrient medium of the following composition: DMEM / P12 450 ml, L-glutamine 292 mg, 50 ml rabbit thrombolysate, penicillin 100 U / ml, streptomycin 100 μg / ml, the nutrient medium in agarose 3D Petri Dish® was replaced at least once every 2 days.The cultivation of cell spheroids was carried out in multi-gas incubators maintaining the composition of the gas environment in the inner chamber: 5% CO2, 10% oxygen and 85% nitrogen.

[0054] Then the cell spheroids were transferred to culture flasks with a non-adhesive surface for further cultivation; the culture supplement MSCgo Chondrogenic Differentiation Supplement Mix (Biological Industries) was added to the culture nutrient medium of the above-described composition; then the cell spheroids from blastema cells were cultivated for 12 days in a periodically changed nutrient medium of this composition in culture flasks with a non-adhesive surface.

[0055] The cellular spheroids were then subjected to histochemical and immunohistochemical studies to detect cartilage markers—type II collagen and aggrecan. Expression of these markers indicates the possibility of chondrogenic differentiation of regenerative blastema cells.

Claims

A transplant for the restoration of a subject's cartilage, comprising cellular spheroids based on autologous cells of the subject's perichondrium, characterized in that the spheroids consist of cells of the subperichondral regenerative blastema of the subject's perichondrium, formed under the action of an in vivo bioreactor, in the form of a hyaluronic acid hydrogel introduced under the perichondrium of the costal cartilage, obtained by mixing equal volumes of 2 vol.% aqueous solutions of HA-ALD and HA-ADH and suramin, wherein the number of cells in the spheroid is 8,000 pieces.