Method of in vitro chondrocyte and cartilage culture to obtain material for treatment of articular cartilage defects

Abstract

To provide a new method for treating human or animal tissue such as cartilage, particularly damaged tissue, more specifically, a method to obtain material for treating the damaged tissue, for example any cartilage damage, in particular as a result of traumatic or degenerative cartilage damage.SOLUTION: A method for producing a cartilage tissue implant in vitro, characterized by the following features: i) providing at least a first and a second fragment of cartilage tissue, each having at least one edge; ii) placing the fragments in a culture medium such that the at least one edge of the first fragment is spaced 1 cm or less apart from the at least one edge of the second fragment; iii) adding a suspension of chondrocytes to the fragments; and iv) cultivating the fragments and chondrocytes under cell culture conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for treating human or animal tissues such as cartilage, in particular damaged tissue. More particularly, the present invention relates to a method for obtaining materials for treating damaged tissue, for example tissue damaged as a result of any cartilage injury, in particular traumatic or degenerative cartilage injury. [Background technology]

[0002] Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain and functional impairment due to the progressive and irreversible loss of articular cartilage. Osteoarthritis is the fifth leading cause of disability in the US population, after cardiovascular, cerebrovascular, and respiratory diseases. The main risk factor for OA is aging. Additional contributing factors include obesity, joint injury, and strenuous physical activity, such as competitive sports.

[0003] The number of diagnosed cases of OA is still increasing in many countries; for example, in the United States, there was an increase of approximately 6 million new cases between 1995 and 2005 (Lawrence RC, Felson DT, Helmick CG, et al., Estimates of the prevalence of arthritis and other rheumatic conditions in the United States.Part II.Arthritis Rheum.2008;58(1):26~35;Lawrence RC, Helmick CG, Arnett FC, et al. Estimates of The prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum. 1998;41:778-799). At the same time, the number of people suffering from degenerative and rheumatic diseases in the general population is increasing. By 2030, the number of diagnosed cases of degenerative disease in the United States is expected to reach 67 million, or 25% of the adult population, and of these, 25 million, or 9.3% of the adult population, will experience activity limitations (Hootman JM, Helmick CG. Projections of US prevalence of arthritis and associated activity limitations. Arthritis Rheum. 2006;54:226-229). These trends result in increasing levels of negative impact on quality of life and significantly increased costs to the health care system.

[0004] Nearly all patients undergoing surgical treatment for widespread degenerative joint disease, particularly in the knee, hip, shoulder, and ankle, experience a period in which destruction of the cartilaginous joint surface is limited to a single site before progressing to generalized joint disease. This is the reason for the slow spread of the degenerative process throughout the joint. With increased patient access to specialized diagnostic tools such as MRI and arthroscopy, the time required to detect inflammatory lesions has decreased. Currently, osteoarthritis is not diagnosed at the level of radiological changes visible on X-rays; it usually involves a significant portion of the joint and requires treatment with joint replacement. However, due to the lack of appropriate tools for treating small to moderate articular cartilage damage, patients still cannot expect to avoid arthroplasty. Therefore, the next stage of treatment is partial or surface arthroplasty, which unfortunately cannot guarantee complete recovery without repeat surgery.

[0005] All endoprostheses still have problems with patient mobility, physical compatibility, and postoperative complications. It does not prevent morbidity. Patients' physical abilities after endoprosthesis replacement are significantly reduced over a three-month period, and patients rarely regain full ability. Patient satisfaction levels, as measured by the degree of pain reduction and improved function of the operated limb, vary widely. The main postoperative problems with arthroplasty include pain level, limb function, and acceptance of the effects of the surgery itself. Arthrectomy represents definitive destruction of the joint; the next step can only be ever more extensive tissue removal and implantation of an ever larger graft, usually leading to permanent disability after the second or third surgery.

[0006] One newly developed method in joint treatment is autologous cultured chondrocyte implantation (ACI), which involves harvesting a fragment of the patient's cartilage from an undamaged part of the joint, isolating chondrocytes, growing them in vitro, and then implanting the chondrocyte suspension into the site of cartilage damage.

[0007] However, currently available methods do not allow for obtaining tissue with the appropriate structure and biological characteristics that closely resemble those of native tissue. Available methods, including the implantation of chondrocytes supported in a collagen matrix, involve long periods of treatment and recovery. Such methods often fail with a high failure rate of over 50%. This is due to the fact that the defect is not replaced with a homogenous tissue, but is only partially replaced with tissue components. The cost of the procedure is very high. The cost-benefit ratio of the treatment does not appear to be favorable for the patient.

[0008] Known methods of in vitro chondrocyte culture for use in autologous transplantation, i.e., cell growth in culture and administration of suspension cultures to joints, do not guarantee joint repair and are expensive and of limited effectiveness compared to traditional surgical methods. Reconstructing tissue in vivo is difficult. Therefore, known methods are primarily applicable to the treatment of minor injuries. The present invention addresses the above-mentioned problems.

[0009] In available chondrocyte cultures, it is not possible for the expanded cells to transform into cartilage tissue because it is difficult to create intercellular connections, which require time, ideal conditions for cell nutrition, and mechanical constraints. Summary of the Invention [Problem to be solved by the invention]

[0010] For the above reasons, new and effective therapeutic solutions are needed. The inventors have discovered an efficient method in the field of transplantation of materials obtained from cell cultures, such as cell culture-based, more particularly autologous chondrocyte transplantation. In particular, the present invention provides a method for the in vitro culture of chondrocytes, which can be used for the above transplantation methods at an early stage of the disease, thus reducing the risk of the need for surgery and therefore post-operative complications; moreover, the discovered method reduces the burden on the healthcare system compared to prior art methods. [Means for solving the problem]

[0011] In a first aspect, the present invention provides a method for producing a cartilage tissue graft in vitro, comprising the following features: i) providing at least first and second pieces of cartilage tissue, each having at least one margin; ii) placing the fragments in a culture medium such that at least one edge of the first fragment is spaced no more than 1 cm or less from at least one edge of the second fragment; iii) adding a suspension of chondrocytes to the fragments; and iv) culturing the fragments and chondrocytes under cell culture conditions The present invention provides a method for producing a cartilage tissue graft in vitro, the method comprising:

[0012] In a second aspect, the present invention relates to a tissue graft obtainable by the method of the first aspect for use in tissue repair in a patient in need thereof. In a third aspect, the present invention relates to a tissue graft obtainable by the method according to the first aspect for use in the treatment of damaged tissue in a patient with a disease.

[0013] Below, the contents of the drawings contained in this specification are described, in which connection reference is also made to the detailed description of the invention above and / or below. [Brief explanation of the drawings]

[0014] [Figure 1] Examples of preferred divisions and configurations: Figure 1a) is a diagram of a linear division; Figure 1b) is a diagram of a straight division; Figure 1c) is a diagram of an arched division; Figure 1d) is a diagram of angular and geometric tissue fragments, and combinations thereof. [Figure 2] An example of an unfavourable fragment arrangement - edges not complementary along the entire length of the joining division, with large spaces between the tissue edges. Disadvantages: more difficult to fill spaces, lower mechanical resistance. [Figure 3]Microscopic images of tissue piece culture in Medium 1. Microscopic images of tissue piece culture in Medium 1 containing 4.5 g / L glucose, 10% FBS, and 2 mM glutamine—visualization of new tissue formation over time. Figure 3A is an image of two tissue piece fragments positioned as close as possible to each other along a border approximately 1 cm long on day 1 of culture (culture baseline, image recorded on day 1). A clearly visible empty space is visible between the contacting surfaces of the two tissue pieces. Figure 3B is an image of two tissue piece fragments positioned as close as possible to each other along a border approximately 1 cm long on day 17 of culture in Medium 1 supplemented with 4.5 g / L glucose, 10% FBS, and 2 mM glutamine, and a visible tissue fill formed between the contacting surfaces of the two tissue pieces. Figure 3C shows two tissue fragments in close proximity along a 1 cm separation after 52 days of culture in Medium 1 supplemented with 4.5 g / L glucose, 10% FBS, and 2 mM glutamine, with visible tissue filling formed between the contacting surfaces of the two tissue fragments. Figure 3D shows two tissue fragments in close proximity along a 1 cm separation after 52 days of culture in Medium 1 supplemented with 4.5 g / L glucose, 10% FBS, and 2 mM glutamine, with visible tissue filling formed between the contacting surfaces of the two tissue fragments. The new tissue formed after 52 days of culture has clearly repaired the empty space at the junction of the two tissue fragments that was present on Day 1 of culture. Significant color matching of the old and new tissue at one end of the contacting surfaces of the two tissue fragments is visible. Images were taken using 10x magnification (A, B, C) and 40x magnification (D). [Figure 4]Microscopic images of tissue fragments cultured in a mixture of Medium 1 and Medium 2. Microscopic images of tissue fragment cultures in a mixture of Medium 1 and Medium 2—visualization of the formation of new tissue bonds over time. Figure 4A shows an image of two tissue fragments positioned as close as possible to each other along a 0.6 cm separation on day 1 of culture (image recording the culture baseline). A clearly visible empty space is visible between the contacting surfaces of the two tissue fragments. Figure 4B shows an image of two tissue fragments positioned as close as possible to each other along a separation of approximately 0.6 cm on day 19 of culture in a mixture of Medium 1 and Medium 2 supplemented with 4.5 g / L glucose, 10% FBS, 2 mM glutamine, and additives: IGF-I and TGF at 0.01% concentrations, and a visible tissue bond formed between the contacting surfaces of the two tissue fragments. New tissue is visible both on the surface of one of the adjacent fragments and between the tissue fragments. The resulting bond has a fibrous nature, binding the two contacting surfaces together. Images were taken using 10x magnification. [Figure 5]Microscopic images of tissue fragments (fragment 1 and 2) cultured in Medium 1. Microscopic images of tissue fragment cultures (fragment 1 and 2) in Medium 1 - visualization of the formation of new tissue connections over time. Figure 5A shows an image of two tissue fragments (fragment 1 and 2) positioned as close as possible to each other along a 1 cm long separation on day 1 of culture (image recording the culture baseline). There is an empty space between the first tissue fragment, located closest to the edge of the plate, and the adjacent tissue fragment. The first fragment was gently cut at the edge of the contacting surface to allow it to be identified in the next stage of the experiment (fragment 1 is at the bottom of the image). Figure 5B shows an image of two tissue fragments (first and second) and visible tissue bonds formed between approximately 1 cm of contacting surfaces of the two tissue fragments after 27 days of culture in Medium 1 supplemented with 4.5 g / L glucose, 2% FBS, 2 mM glutamine, and 0.01% chondroitin. Figure 5C shows an image of two tissue fragments (first and second) and visible tissue bonds formed between approximately 1 cm of contacting surfaces of the two tissue fragments after 27 days of culture in Medium 1 supplemented with 4.5 g / L glucose, 2% FBS, 2 mM glutamine, and 0.01% chondroitin. Fibrillar bonds form between the contacting surfaces of the tissue fragments. After several days of culture, new tissue forms between the contacting surfaces of the tissue fragments, permanently connecting the two tissue fragments. The same type of bonding was observed between the second and third pieces, and between the third and fourth pieces, respectively. Images [A, B] were taken at 10x magnification, and image [C] was taken at 40x magnification. DETAILED DESCRIPTION OF THE INVENTION

[0015] Before the present invention is described in detail below, it is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0016] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," edited by Leuenberger, H.G.W., Nagel, B., and Klbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland; and in "Pharmaceutical Substances: Syntheses, Patents, Applications," by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals," edited by Susan Budavari et al., CRC Press, 1996; and United States Pharmacopeia-25 / National Formulary-20, published by United States Pharmaceutical Convention, Inc., Rockville, Md., 2001.

[0017] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise," and variations such as "comprises" and "comprising," will be understood to refer to the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps. In the following sections, various aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or group of aspects, unless clearly contradictory. In particular, any feature indicated as optional, preferred, or advantageous may be combined with any other feature or group of features indicated as optional, preferred, or advantageous.

[0018] Below, various elements of the present invention are described. These elements are listed with specific embodiments. However, it should be understood that these elements can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments in which the explicitly described embodiments are combined with any number of disclosed and / or preferred elements. Furthermore, any permutation or combination of all elements described herein should be considered disclosed by the description herein, unless the context dictates otherwise. definition Below are definitions of some terms frequently used herein, which have their respective defined and preferred meanings in each instance of their use in the remainder of the specification.

[0019] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "cell culture" or "cell culture conditions" refers to a process in which cells are grown under controlled conditions outside the cells' natural environment. Specifically, it refers to the growth of cells derived from multicellular eukaryotic organisms, particularly animal cells (see also the definition of cells below). Typical growth conditions include temperatures between 35°C and 38°C, 3-7% CO2, and relative humidity above 80%, preferably 90-99%. Cell cultures of the present invention may include cells and tissues. The terms "cell culture medium," "culture medium," or simply "medium" refer to a liquid or gel for supporting the survival or growth of cells and / or tissues, particularly cells derived from multicellular eukaryotic organisms, particularly animal cells and / or tissues. Such media contain all nutrients necessary to support the survival or growth of such cells and / or tissues for at least 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. The term "cell culture medium composition" refers to the components that a cell culture medium contains. Such components are not necessarily all nutrients but may serve other purposes, such as adjusting cell culture conditions, e.g., pH or osmolality, within a desired range. Furthermore, not all of the components are necessarily required to support cell survival or growth or recombinant protein production; some may be dispensable but still improve the medium's ability to support cell survival or growth or recombinant protein production. The cell culture medium composition can be a dry powder composition, a liquid composition, or a solid (e.g., gel or agar) composition. In certain embodiments, the composition is sterile, i.e., free of all living organisms and / or infectious / replicative agents, such as fungi, bacteria, protozoa, parasites, viruses, abnormal proteins such as prions, etc.

[0020] The cell culture medium may contain salts, amino acids, additional components such as galactose, dextran sulfate, spemidine, beta-glycerol phosphate, and adenine, as well as buffers. The salts may be selected from the group including calcium chloride, sodium phosphate, ammonium iron(III) citrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium selenite, zinc sulfate, and sodium chloride. The amino acids may be selected from the group including arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, penicillamine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, alanine, aspartic acid, citrulline, cystine, glycine, and ornithine. The vitamins may be selected from the group including L-ascorbate, D-(+) biotin, D-calcium pantothenate, choline chloride, folic acid, myo-inositol, nicotinamide, pyridoxine, riboflavin, thiamine, and vitamin B12. Additional components may be selected from the group including dextrose, glutathione, hypoxanthine, lipoic acid, ethanolamine, uridine, spermine, pluronic F68, putrescine, thymidine, sodium pyruvate, galactose, dextran sulfate, spermidine, beta-glycerol phosphate, and adenine. The buffer may be selected from the group including HEPES, MOPS, MES, BES, MOPSO, ACES, TAPS, bicine, and tricine. Furthermore, the medium may contain growth factors, for example, contained in human and / or calf serum, preferably FBS. Alternatively, the medium may be serum-free.

[0021] The term "free of" refers to a medium lacking certain components, which are often components of cell culture media in the art. Generally, the term refers to the fact that these components were not intentionally made part of the composition, including the possibility of contamination due to, for example, handling of the medium, its container, or tools for handling the medium or its container. In particular, the term excludes all measurable trace amounts of these components, as measured, for example, by state-of-the-art methods, such as chromatography, especially high performance liquid chromatography (HPLC).

[0022] The term "serum" refers to the blood fraction containing electrolytes, antibodies, antigens, hormones, and exogenous substances, as well as all other substances contained in blood (excluding blood cells, clotting factors, and all other proteins involved in blood coagulation or clotting, which form part of a blood clot). In particular, the term refers to serum commonly used in cell culture applications, such as cow, chicken, goat, human, sheep, pig, or rabbit serum. In particular, the term refers to undefined cell culture components (to the extent that their composition and / or the concentrations of their components are unspecified or unknown). Thus, the exclusion does not exclude any of the components of the medium compositions of the present invention, particularly those identified herein (even though serum may contain one or more of these components).

[0023] As used herein, a "monolayer culture" is a type of culture in which cells are grown in a single layer in a flask or petri dish containing culture medium. A "suspension culture" is a type of culture in which cells are maintained evenly distributed in suspension in the culture medium. The term "protein" refers to a biological molecule comprising one or more polypeptides. A polypeptide is a single linear polymeric chain of amino acids linked by peptide bonds, e.g., at least 50, 60, 70, 80, 90, or 100 amino acids in length.

[0024] As used herein, "treat," "treating," "treatment" or "treatment of" a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in individuals who previously had the disorder; and (e) limiting or preventing the recurrence of symptoms in individuals who previously exhibited symptoms of the disorder.

[0025] As used herein, a "cell" or "tissue" can be an animal or human cell or tissue. An animal cell or tissue can be a cell or tissue of a primate, mouse, rat, rabbit, dog, cat, hamster, cow, insect, etc. A cell can be a suspension cell or an adherent cell. A suspension cell is a cell that can naturally survive in suspension (i.e., not attached to a surface) or that can survive in suspension culture. Adherent cells are cells that have been modified to grow at a higher density than the adhesion conditions allow, for example, to allow adhesion. Adherent cells are cells that require a surface, such as tissue culture plastic or microcarriers, which can be coated with extracellular matrix components (such as collagen and laminin) to enhance adhesion and provide other signals necessary for proliferation and differentiation. In one embodiment, the adherent cells are monolayer cells. For example, the cells are selected from the group consisting of chondrocytes, hybridoma cells, primary epithelial cells, endothelial cells, keratinocytes, monocytes / macrophages, lymphocytes, hematopoietic stem cells, fibroblasts, and hepatocytes. More particularly, the cells can be chondrocytes selected from the group consisting of xenogeneic chondrocytes, allogeneic chondrocytes, and autologous chondrocytes. In particular, the cells are autologous chondrocytes. In particular, the tissue is cartilage tissue, more particularly hyaline cartilage tissue.

[0026] As used herein, a "tissue fragment" is a piece of tissue. In the context of the present invention, tissue fragments that are "complementary" in shape to one another refer to fragments that, because of their shape, can fit together like a lock and key, or in other words, like pieces of a puzzle. "Complementary" shapes refer to edges of tissue fragments that fit together particularly well because they are parallel.

[0027] As used herein, a "graft" or "tissue graft" is a biomedical tissue used in organ transplantation. It is manufactured to replace lost biological structures, support damaged biological structures, or augment existing biological structures. The surface of the graft that contacts the body may be made of biomedical materials such as titanium, silicone, or apatite. In one embodiment of the present invention, the tissue graft is made of tissue obtained by culturing cells with tissue, where the cells are preferably chondrocytes and the tissue is preferably cartilage. Aspects and Preferred Embodiments of the Invention.

[0028] In the following, the various aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or group of aspects, unless clearly inconsistent. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or group of features indicated as being preferred or advantageous.

[0029] In the research leading to the present invention, it has surprisingly been shown that tissue grafts can be produced by an in vitro method, which method involves culturing cartilage and chondrocytes and can be effectively used for transplantation methods at an early stage of the disease, thus reducing the risk of the need for surgery and therefore reducing post-operative complications, and furthermore the discovered method reduces the burden on the healthcare system compared to prior art methods. In a first aspect, the present invention provides a method for producing cartilage tissue grafts in vitro, comprising the following features: i) providing at least first and second pieces of cartilage tissue, each having at least one margin; ii) placing the fragments in a culture medium such that at least one edge of the first fragment is spaced no more than 1 cm or less from at least one edge of the second fragment; iii) adding a suspension of chondrocytes to the fragments; and iv) culturing the fragments and chondrocytes under cell culture conditions The present invention provides a method for producing a cartilage tissue graft in vitro, the method comprising:

[0030] The term "margin" in reference to a fragment of cartilage tissue refers to the outer boundary of the fragment of cartilage tissue. The margin may be the natural outer boundary of the cartilage tissue, or it may be the result of cartilage tissue being isolated, for example by cutting with a scalpel, in which case the margin is an artificial margin. The margin may be of natural origin along its entire length, since it is present around the entire cartilage fragment (in which case the "fragment" is not actually a fragment, but a piece of cartilage tissue, since it occurs in nature). Alternatively, the margin may be partly natural and partly artificial, or the margin may be entirely artificial. The latter may be the case when a fragment of cartilage is cut from a larger piece of cartilage tissue. This may be the case when a fragment is cut from a larger piece of cartilage tissue during a surgical procedure, or when a larger piece of cartilage can be cut into two or more fragments after surgical resection. The cartilage tissue may be removed from the patient who will subsequently be treated (autologous cartilage), or the cartilage tissue may be derived from a different person - donor - (xenogeneic cartilage). Therefore, the term "margin" is also used in the context of the present invention to refer to a portion of the margin of a fragment of cartilage tissue. Preferably, the portions of the margins of two fragments placed adjacent to each other have corresponding shapes. For example, the portions of both margins are straight, or one curves inward and the other curves outward.

[0031] As noted above, the term "cartilage tissue fragment" can refer to a piece of cartilage tissue as it occurs naturally, or to a piece cut into smaller pieces than its natural counterpart. The cartilage fragment may have its natural thickness, preferably between 0.01 mm and 10 mm, more preferably between 0.1 and 6 mm. In some cases, it may be preferable to cut a thicker piece of natural cartilage tissue into thinner cartilage slices. Such cartilage fragments preferably have a thickness between 0.1 mm and 5 mm, more preferably between 0.1 and 4 mm, and more preferably between 1 and 3 mm. It is further preferred that the thickness of the fragment be essentially uniform throughout the fragment.

[0032] The preferred cartilage source that can be used in the method of the present invention is a xenogeneic or autologous source, preferably an autologous source. Preferably, the source is any joint, more preferably an injured joint of the person to be treated. Preferably, in this case, the tissue source is a healthy tissue source.

[0033] In step ii), the space between at least one edge of the first piece and at least one edge of the second piece is 0.9 cm, 0.8 cm, 0.7 cm, 0.6 cm, 0.5 cm, 0.4 cm, 0.3 cm, 0.2 cm, 0.1 cm. Preferably, the space is 0.5 cm or less, preferably 0.1 cm or less, and preferably there is no space.

[0034] In step iii), the chondrocytes are xenogeneic, allogeneic or autologous chondrocytes. Preferably, the chondrocytes are autologous chondrocytes. In a preferred embodiment of the first aspect, preferably when the edges of the segments are in contact with each other, the edges of the segments are in contact along a line of contact, preferably in one of the configurations shown in Figure 1. Preferably, the line of contact has a length of at least 0.1 cm, more preferably at least 0.5 cm, more preferably at least 1 cm.

[0035] In preferred embodiments, at least three, more preferably at least four, at least five or at least six fragments are spaced as indicated above for the first and second fragments, in this way larger cartilage sheets can be generated from several fragments.

[0036] In a preferred embodiment, the medium comprises one or more salts, amino acids, additional components such as galactose, dextran sulfate, spemidine, beta-glycerolphosphat and adenine, and a buffer.

[0037] The salt may be selected from the group including calcium chloride, sodium phosphate, ammonium ferric citrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium selenite, zinc sulfate, and sodium chloride.

[0038] The amino acid may be selected from the group including arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, penicillamine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, alanine, aspartic acid, citrulline, cystine, glycine, and ornithine.

[0039] The vitamins may be selected from the group including L-ascorbate, D-(+) biotin, D-calcium pantothenate, choline chloride, folic acid, myo-inositol, nicotinamide, pyridoxine, riboflavin, thiamine, and vitamin B12.

[0040] Further components may be selected from the group including dextrose, glutathione, hypoxanthine, lipoic acid, ethanolamine, uridine, spermine, pluronic F68, putrescine, thymidine, sodium pyruvate, galactose, dextran sulfate, spermidine, beta-glycerolphosphat, and adenine.

[0041] The buffering agent may be selected from the group including HEPES, MOPS, MES, BES, MOPSO, ACES, TAPS, bicine, and tricine. Furthermore, the medium may contain growth factors, for example, contained in human and / or bovine serum, preferably FBS. Alternatively, the medium may be serum-free. In a preferred embodiment, the medium further contains insulin growth factor (IGF) and / or transforming growth factor (TGF), and optionally chondroitin and / or insulin. Preferably, the medium contains at least 0.01 wt% IGF and / or at least 0.01 wt% TGF, and optionally at least 0.01 wt% chondroitin and / or at least 0.1 wt% insulin. Preferably, the medium contains at least 0.01 wt% IGF, at least 0.01 wt% TGF, at least 0.01 wt% chondroitin, and at least 0.1 wt% insulin.

[0042] In a preferred embodiment, after step (ii), the fragments are cultured in culture medium for at least 14 days before adding the chondrocyte suspension in step (iii). Preferably, the fragments are cultured for 14 to 18 days, preferably 18 to 25 days, preferably 25 to 30 days, preferably 30 to 42 days, and up to at least 5 months.

[0043] In a preferred embodiment, in step (iii) of the method of the first aspect, chondrocytes are added to the culture medium to a final concentration of at least 4,000, preferably 4,000 to 10,000, preferably 10,000 to 40,000, preferably 16,000 to 40,000 chondrocytes per ml of culture medium. Preferably, the final concentration of chondrocytes added to the culture medium in step (iii) of the method of the first aspect is 16,000 chondrocytes per ml of culture medium.

[0044] In a preferred embodiment, the chondrocyte suspension is obtained by enzymatic digestion, preferably by digestion with a protease. Preferably, the enzymatic digestion is carried out with collagenase, trypsin, hyaluronidase, and / or tosyl-lysylchloromethane, more preferably with collagenase, even more preferably with collagenase II.

[0045] In a preferred embodiment, the chondrocyte suspension is cultured in an adherent monolayer prior to suspension. Preferably, the culture temperature is between 35 and 38°C and / or the CO2 concentration is between 4 and 6%, more preferably the temperature is 37°C and the CO2 concentration is 5%.

[0046] In a preferred embodiment, one edge of the first fragment is complementary to one edge of the second fragment, for example, one edge of the first fragment fits into one edge of the second fragment, similar to a lock and key model. The shape of the fragments may be oval, round, square, rectangular or bulky, preferably oval. The shape of at least one edge of the fragments may be curved or straight, preferably straight.

[0047] In a second aspect, the present invention provides a tissue graft obtainable by the method according to the first aspect of the invention for use in tissue repair in a patient in need thereof, preferably wherein the tissue to be repaired is damaged tissue, more preferably damaged cartilage tissue.

[0048] In a third aspect, the present invention provides a tissue graft obtainable by the method according to the first aspect of the present invention for use in treating damaged tissue in a patient with a disease. Preferably, the disease includes joint diseases such as osteoarthritis, rheumatoid arthritis, spondyloarthritis, juvenile idiopathic arthritis, lupus, gout, and bursitis. Preferably, the disease is osteoarthritis.

[0049] In a preferred embodiment, the treatment involves the implantation of a tissue graft at the defect site. In other words, as a result of experimental studies, a method of hyaline chondrocyte culture has been developed to obtain materials for the treatment of cartilage conditions. A method has been developed that involves the simultaneous use of natural tissue fragments in the culture, where the natural tissue fragments are combined in vitro into larger pieces and a chondrocyte suspension that acts as an adhesive to bind the tissue fragments together.

[0050] Therefore, in order to develop a method for performing chondrocyte culture, well-designed experimental studies were carried out, in which, among other things, different cell densities, lengths of contact surfaces of the bonded fragments, different types of additives and culture media were tested to create intercellular bonds that are not easily broken.

[0051] According to the present invention, cells grow and divide while attached to a surface, e.g., a plate, in a culture medium. Only when the cells are detached from the surface and passaged are they placed in suspension, and once suspended in the medium used to flood the tissue.

[0052] The present invention relates to a method for the in vitro culture of cells and tissue fragments, allowing for the production of cartilage tissue in a form closer to its natural state than the cell suspensions or cells placed in a matrix known in the art. The culture of cells and fragments of hyaline cartilage is carried out in vitro, and the resulting material in the form of tissue is then implanted into the defect site. The method involves harvesting smaller fragments of normal cartilage tissue from a low- or no-load area in a patient's joint and then placing them in a medium suitable for cell / tissue culture, where at least two, preferably at least 3-10, tissue fragments-pieces are placed in close proximity, i.e., with at least one edge of each fragment in contact with the edge of another fragment, so that during culture, the smaller fragments combine to form smaller pieces with a larger surface area. The parallel edges of the at least two, preferably at least 3-10, tissue fragments are matched according to their shape, allowing the pieces to join by joining the matched edges along the contact points, with the entire fragments or edges being parallel.

[0053] According to the present invention, a method for in vitro chondrocyte culture to obtain a material for treating articular cartilage defects comprises the steps of cutting the obtained hyaline cartilage into at least two tissue fragments and placing these fragments side by side on a culture plate in a culture medium containing at least 2 mM glutamine, at least 2% FBS, and at least 4.5 g / L glucose. The method comprises placing tissue fragments on a plate, the fragments having matching shapes along at least one dividing line of each fragment's edge, such that after the fragments are placed close to each other, the space between the at least two tissue fragments is as small as possible and the area of the culture plate filled with the tissue fragments is as small as possible. The edges are adjusted so that during culture, each fragment will join with another fragment along at least one dividing line. A chondrocyte suspension is added to the culture, and the tissue fragments are cultured with the chondrocytes. The culture medium is exchanged by adding chondrocytes to the culture medium for a predetermined period of time, and the cells are cultured until the tissue fragments join at least a portion of the dividing line where they contact the other fragment. Preferably, the dividing line between the at least two joined tissue fragments is a minimum of 0.1 cm, and most preferably at least 0.5 cm. Preferably, the tissue fragments should have an oval-like shape. Preferably, the shape of the tissue fragments is selected so that the tissue fragments adhere very tightly to each other along the dividing line during culture.

[0054] Preferably, the culture medium contains additives at concentrations of at least 0.01% IGF and / or 0.01% TGF and / or 0.01% chondroitin and / or at least 0.1% insulin. Preferably, the culture medium contains 55-75 mg / L sodium pyruvate and sodium bicarbonate, 2 mM to 4 mM glutamine, 2% to 10% FBS, and at least 4.5 g / L glucose and ethanolamine and / or glutathione and / or ascorbic acid and / or insulin and / or transferrin and / or copper sulfate and / or manganese chloride.

[0055] Preferably, the tissue fragments themselves are cultured in culture medium, preferably for at least 14 days, before the chondrocytes are added to the culture. Preferably, the chondrocyte suspension is added to the tissue fragments in an amount such that there are at least 40,000 chondrocytes per 2.5 ml of culture medium. Preferably, the minimum chondrocyte concentration for culturing two fragments with abutting lengths of 0.1 cm to 1 cm is 40,000 chondrocytes per 2.5 ml of medium.

[0056] Preferably, the cultured chondrocytes are obtained by enzymatic digestion, after which the required number of cells are obtained by culturing the chondrocytes in a monolayer in adherent culture. Preferably, the cells are cultured at 37°C and 5% CO2.

[0057] During experimental studies, it was established that the minimum abutting distance along the joining edges, i.e., the abutting distance must be at least 0.1 cm long. The edges can be more closely joined along parallel abutments. The abutting distance can be a fragment of the tissue fragment's edge or the entire edge of the tissue fragment. The edges to be joined should be straight or rounded. During culture, the fragments are combined using a chondrocyte suspension culture obtained from the same donor tissue. Cartilage contains both chondrocytes and extracellular matrix. It is important to select the shape of the tissue fragments so that the space between the joined edges of the cartilage fragments is as small as possible; the edges or fragments of the edges should contact each other along a abutment of at least 0.1 cm. Matching of the abutting edges of the fragments should be achieved at the latest by placing them in close proximity on the culture plate before adding the chondrocytes.

[0058] This allows for the production of not only cells but also hyaline cartilage through culture. Although the resulting tissue may be characterized by weak mechanical strength or low maturity at the junctions of cultured tissue fragments, the resulting tissue has a solid structure provided by intercellular junctions.

[0059] According to the invention, the procedure includes: 1. Collection of hyaline cartilage from the patient; 2. Cut the tissue into suitable pieces; 3. Enzymatic digestion of some of the pieces to obtain chondrocytes and establish cell culture - chondrocyte culture; 4. Place the remaining pieces (undigested) in culture medium - culture of tissue fragments to which chondrocytes are added; 5. After obtaining an adequate amount of chondrocytes in culture, the tissue and cell fragments are combined in culture to obtain cells for further use, while the cells allow the binding of undigested tissue particles. 6. The suspended chondrocytes are added to the culture plate containing the tissue fragments.

[0060] The suspended cells are positioned within the grooves to support fusion of at least two edges of at least two tissue fragments, and / or the cells adhere to the cell culture plate, proliferate, and release factors that promote fusion of the cartilage fragments with each other.

[0061] During combined culture, the undigested fragment-cultured tissue fragments are placed close to each other with the edges shaped along the borders so that the space between the borders is as small as possible, i.e., so that the bordering edges of the tissue fragments can be joined.

[0062] It is important to select the shape of the fragment-contacting edges so that the space between the edges of the cartilage fragments that come into contact with each other is as small as possible. Matching of the border shapes should be done at the latest when the fragments are placed in close proximity on the culture plate before adding the suspended chondrocytes.

[0063] The present invention was developed as a result of long-term experimental research. Examples 1-3 show attempts to culture cells to obtain hyaline cartilage material for the reconstruction of damaged articular cartilage. The present invention is explained using preparation examples and figures. Figure 1 shows the principle of joining tissue fragments, A-B show the joining line, and C shows the shape of the tissue fragments. Figure 2 shows the principle of joining tissue fragments and shows the improper shape of the tissue fragments. Figures 3-5 - Microscope images.

[0064] Examples of unfavorable joints are all kinds of arc-straight lines and geometric joints with various angles, where the spaces between the pieces are relatively wide and there are no adjacent parts with edges of at least 0.1 cm length of the pieces parallel to one another. They are shown in Figure 2. Figure 2 shows an example of an unfavorable arrangement of pieces - the edges are not complementary along the entire length of the adjacent part and there are wide spaces between the edges. Disadvantages: it is more difficult to fill the spaces and the mechanical resistance is low. [Example]

[0065] Example 1 Attempt to culture only hyaline cartilage tissue fragments according to the present invention To obtain material for the treatment of articular cartilage defects, an in vitro method was used to create a uniform flap of cartilage tissue. Clinical material was initially obtained from an adult human in the form of hyaline cartilage tissue fragments harvested from the least weight-bearing part of the knee joint. The tissue was harvested from a location that places minimal strain on the patient's body. This tissue should be healthy. Therefore, the shape of the harvested flap or cartilage flap may vary. One or more cartilage fragments can be harvested. One or more fragments can be divided into smaller pieces by cutting across or along the cartilage-bone boundary. If a large fragment of articular cartilage was harvested, it was then cut into smaller pieces under sterile conditions.

[0066] Immediately after the material was collected from the patient, it was placed in a sterile container filled with physiological saline (0.90% NaCl). The material was transferred to a 50 ml tube so that the tissue was completely immersed in the solution. The cartilage was then cut into thin, up to 3 mm thick, slices of various shapes and sizes less than 1 cm in length, width, or thickness.

[0067] The obtained tissue fragments were transferred to the cell culture laboratory in sterile 50 ml tubes, fully immersed in saline (0.90% NaCl). The tissue transportation was carried out under sterile conditions at 2-8°C. All manipulations performed on the tissue were carried out under laminar flow conditions. After preparing pieces of the appropriate size, the obtained material served as the starting substrate for obtaining the final tissue flaps.

[0068] The final tissue flap pieces for the treatment of articular cartilage defects were to be formed on a matrix of tissue slices, appropriately positioned relative to each other on the surface of a culture plate and cultured under appropriate conditions and culture medium composition.

[0069] The key step was to properly shape the borders of the tissue sections so that they were as close as possible to each other on the culture plate by joining the tissue fragments along their borders.

[0070] Culture tests were performed using two to four tissue fragments in contact with each other along at least one linear division—abutting edges 0.1 to 1 cm in length. As mentioned above, the shapes were matched according to the border-joined edges of at least two tissue fragments-pieces. The maximum length / width and the maximum number of fragments-pieces were determined according to the size of the culture plate.

[0071] From all the tissue pieces collected, it was necessary to select as many pieces (at least two) as possible that would fit the surface of the wells of the culture plate. If the previously prepared piece system described above could not be used due to mismatches between the edges of adjacent pieces, the material was cut after collection from the patient to give it the desired shape.

[0072] At this stage of tissue culture, where the formation of new tissue fragments is planned based on previously harvested and prepared tissue fragments, we hypothesized that it is crucial to place at least two tissue fragments next to each other. These fragments should be positioned so that the contact edges of the joined fragments are as long as possible and conform to the shape of the adjacent fragment / groups of fragments, thereby minimizing the amount of space filled in the culture plate. This effect can be achieved both at the stage of tissue harvesting from the patient by harvesting appropriately shaped fragments, and later in vitro in the laboratory. The conformity of the edges—their adjacent positioning—ensures that at least one edge of one tissue fragment is in contact with the edge of the other, thereby minimizing the volume of empty space between the cartilage fragments, facilitating the filling of the inter-edge area with the chondrocyte suspension, and ensuring the mechanical resistance of the newly obtained tissue fragments at the joining site.

[0073] During the culture, it was observed whether the tissue flap-sliver-increased its area and whether the intermarginal space was filled. A suitable tissue sliver, approximately 1 mm x 1.2 cm x 0.7 cm, was transferred to a well of a 6-well sterile cell culture plate filled with 2.5 ml of cell culture medium 2. This medium contains calcium chloride anhydrous, copper sulfate pentahydrate, ferrous sulfate heptahydrate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium hydrogen phosphate anhydrous, zinc sulfate heptahydrate, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride, L-glutamic acid, L-glutamine, glycine, L-histidine trihydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine dibasic dihydrate, L-valerate, L-threon ... The medium contained 1x concentrated Glutamax® additive solution, folic acid, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucan ozone, hypoxanthine, linoleic acid, phenol red, putrescine hydrochloride, monosodium pyruvate, and thymidine. Cells were cultured at 37°C and 5% CO2 for 7 days. The junction between the two tissue fragments, their viability, and proliferation were monitored using 10x and 40x optical magnification. Periodically, before each subsequent passage and medium change, tissue transformation: junction, viability, and proliferation were observed using an inverted optical microscope at 10x and 40x magnification. The culture medium and additives were changed weekly.

[0074] For each subsequent medium change, 1) Removal of old medium 2) Gently transfer the tissue fragment to a new culture plate. 3) Addition of fresh medium Including.

[0075] During the culture, it was observed whether the tissue flap - the small piece - increased its area and whether the space between the edges was filled. The culture of the tissue fragments alone (without the chondrocyte suspension) was carried out according to the present invention, and after 5 months, no bonding was observed between pairs of cartilage tissue fragments placed in one well. There was also no growth of new tissue on the surface of the old tissue.

[0076] Example 2 Attempt to culture only cartilage tissue fragments according to the present invention The culture procedure was the same as that described in Example 1.

[0077] Compatible tissue pieces were transferred to each well of a 6-well sterile cell culture plate filled with 2.5 ml of cell culture medium 2 supplemented with a 1x concentrated solution of Glutamax additive and glucose [4.5 g / l].

[0078] After five months of culture, no bonding was observed between pairs of cartilage fragments placed in a single well, and no new tissue grew on the surface of the old tissue. Example 3 Attempt to culture only cartilage tissue fragments according to the present invention The culture procedure was carried out as described in Example 1, except that compatible tissue slices were transferred to 6-well sterile cell culture plates filled with 2.5 ml of cell culture medium 1. This medium contained calcium chloride anhydrous, ferrous nitrate, potassium nitrate chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, disodium L-tyrosine dihydrate, L-valine, D-calcium pantothenate, choline chloride, folic acid, as well as inositol, niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt at a high glucose concentration [4.5 g / L].

[0079] After five months of culture, no bonding was observed between pairs of cartilage fragments placed in a single well, and no new tissue grew on the surface of the old tissue. Example 4 Overview of the method according to the present invention Cartilage tissue using in vitro methods to obtain materials for the treatment of articular cartilage defects To create a uniform skin flap, clinical material was initially obtained from an adult human in the form of hyaline cartilage tissue fragments harvested from the knee joint. The hyaline cartilage should preferably be harvested from the treated joint (which could be the knee, hip, ankle, shoulder, or any other joint). If the treated joint is small or the hyaline cartilage is difficult to extract, it can be harvested from another joint. It should always be the site with the least weight-bearing. The tissue is harvested from a location that places the patient at a low physical load. This tissue should be healthy. Therefore, the shape of the harvested skin or cartilage flap may vary. One or more cartilage fragments can be harvested. One or more fragments can be divided into smaller pieces by cutting across or along the cartilage-bone boundary.

[0080] If a large piece of articular cartilage was harvested, it was then cut into smaller pieces under sterile conditions. Immediately after the material was collected from the patient, it was transferred to a sterile 50 ml tube filled with saline (0.90% NaCl) so that the tissue was completely immersed in the solution. The cartilage was then cut into thin, maximum 3 mm pieces of various shapes and sizes less than 1 cm in length / width. (This applies both to the fragments that will form the main material for obtaining the final tissue fragments and to the fragments that will result in the formation of free chondrocytes.)

[0081] The preferred thickness / length / width of the fragments were: 1 mm x 0.5 cm x 0.5 cm, 1 mm x 0.6 cm x 0.6 cm, 1 mm x 0.7 cm x 0.7 cm, 1 mm x 0.8 cm x 0.8 cm, 1 mm x 0.9 cm x 0.9 cm, 1 mm x 1 cm x 1 cm, 2 mm x 1 cm x 1 cm, and 3 mm x 1 cm x 1 cm. The resulting tissue fragments, approximately 1 mm x 1 cm x 1 cm, were placed completely immersed in saline (0.90% NaCl) in a sterile 50 ml tube and transported to the cell culture laboratory. The tissue was transported under sterile conditions at 2-8°C.

[0082] The size and shape of the joined flap fragments are not critical. It is important to prepare the flap fragments so that the margins are joined along a junction of at least 0.1 cm in length, with the space between the joined edges of at least two tissue fragments as small as possible. The size of the fragment itself also depends on the size and shape of the defect and the size and shape of the healthy cartilage harvested to obtain the "bioprosthesis" in the form of a reconstituted fragment of cartilage tissue according to the present invention. The junction was at least 0.1 cm in length. All manipulations with tissue or cells were performed under laminar flow conditions. After preparing fragments of appropriate size, all fragments were divided into two parts. One of them was used to obtain a chondrocyte suspension, i.e., a mixture that served as a tissue building material in cell culture with the tissue fragments. The other constituted the fragments for culturing the tissue flap fragments and the main material for obtaining the final flap of reconstituted tissue.

[0083] The final tissue flap pieces for the treatment of articular cartilage defects were obtained according to the following procedure: neatly shaped tissue slices were placed against each other on the surface of the wells of a culture plate and combined in a "tissue-cell" culture using a chondrocyte suspension.

[0084] 1) Preparation of cartilage tissue fragments for further culturing of the tissue fragments. The key step is the proper selection of tissue pieces so that their edges allow them to be positioned as close as possible to each other on the culture plate. The tissue fragments must be in contact with each other along at least one linear section. There must be at least two tissue fragments. Minimum length of tissue fragments of any shape: minimum 0.1 cm, preferably at least 0.5 cm. Minimum width of tissue fragments of any shape: at least 0.1 cm, preferably at least 0.5 cm. Thickness of tissue: at least 1 mm, and no more than 4 mm.

[0085] As mentioned above, the shape is matched according to the borders of at least two tissue fragments-pieces, joined along the borders-joining line. The length of the borders should be at least 0.1 cm long, preferably a minimum of 0.5 cm long, regardless of whether the line is straight or curved, because this length refers to the border-joining line, not the straight line. The maximum length / width and number of fragments-pieces depend on the size of the culture plate and the therapeutic purpose.

[0086] From all the tissue pieces collected, it was necessary to select as many pieces (at least two) as possible that would fit the surface of the wells of the culture plate. If the previously prepared piece system described above could not be used due to mismatches between the edges of adjacent pieces, the material was cut after collection from the patient to give it the desired shape.

[0087] At this stage of tissue culture, where the formation of new tissue fragments was planned based on previously harvested and prepared tissue fragments, it was crucial to place a minimum of two tissue fragments next to each other. These fragments should be positioned so that the borders of the joined fragments are as long as possible (at least 0.1 cm) and conform to the adjacent fragments / groups of fragments to minimize the empty space between the cartilage fragments and thereby minimize the volume of the filled space in the culture plate. This effect can be achieved both at the stage of tissue harvesting from the patient by harvesting appropriately shaped fragments and later in vitro in the laboratory. The borders' conformity—their adjacent positioning—ensures that at least one border or its bifurcation of one tissue fragment contacts the border of the other tissue fragment, thereby minimizing the volume of empty space between the cartilage fragments, facilitating the filling of the inter-border area with the chondrocyte suspension, and ensuring the mechanical resistance of the newly obtained tissue fragment at the joining site. Examples of possible border shapes and their joining methods are shown in Figure 1, although the examples are not exhaustive. During cultivation, it is important to obtain and join a minimum of two tissue fragments immediately adjacent to each other along a borderline separation, with a minimum length of separation of 0.1 cm for both straight and curved separations. This value applies to the length of the borderline section of the entire border of the fragment or tissue fragment, regardless of its shape.

[0088] FIG. 1 depicts the edges of a whole tissue fragment or tissue fragment-piece, showing the junction of matching edges by approximating them along the borders. According to the present invention, it is important to adjust the shape of the tissue fragments by planning the path of the adjacent parallel edges - the joining site of at least two tissue fragments. This shape is present in all drawings and has straight edges. This shape can also be partially straight and partially arched. Thus, the tissue fragments can have any shape. The path of the edges should be compatible and parallel to both tissue fragments that are joined along the bordering line so that both tissue fragments can be joined along these edges. The entire bordering line, or the joining site of at least two tissue fragments, of the tissue fragments joined along the bordering line is called the bordering line.

[0089] The principle is that the length of the abutting division of the joined tissue margins - the length of part or all of the fragment edges along the abutting division - the junction of the tissue fragments should be at least 0.1 cm long - the abutting division length. Figure 1 shows examples of abutting division shapes: A - straight division, B - arched division, and examples of shapes of joined tissue fragments, C - angular and geometric tissue fragments, and combinations thereof.

[0090] Examples of undesirable connections are all kinds of arc-straight lines and geometric connections with various angles, where the spaces between the pieces are relatively large and there are no tangent divisions with edges of at least 0.1 cm of the pieces parallel to one another. These are shown in Figure 2. Figure 2 shows an example of an unfavourable fragment arrangement - the edges are not complementary along the entire length of the joining part and there are large spaces between the edges. Disadvantages: more difficult to fill the spaces, lower mechanical resistance.

[0091] 2) Pre-culture of tissue fragments only As described above, selected or appropriately trimmed tissue fragments were transferred using tweezers to sterile 1-, 6-, or 12-well cell culture plates. The selected or appropriately trimmed tissue fragments were placed in each well pre-filled with 10 ml, 2.5 ml, or 1.25 ml of cell culture medium 1 (also known as DMEM) with 4.5 g / L glucose, medium 2 (also known as F12) supplemented with a 1x concentrated solution of Glutamax additive, or a 1:1 mixture of medium 1 and medium 2 supplemented with 2%, 4%, 5%, or 10% FBS serum and 2 mM to 4 mM glutamine.

[0092] In this example, culture medium 1, known by the trade name DMEM, was used, which contained anhydrous calcium chloride, ferric nitrate hydrate, potassium chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, disodium L-tyrosine dihydrate, L-valine, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, and 4-(2-hydroxyethyl)piperazine-monosodium salt-ethanesulfone supplemented with glucose.

[0093] In this example, culture medium 2 was used, which contains calcium anhydrous, copper sulfate pentahydrate, ferrous sulfate heptahydrate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium dibasic anhydrous, zinc sulfate heptahydrate, L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride, L-glutamic acid, L-glutamine, glycine, L-histidine trihydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline ... It contained phosphorus, L-serine, L-threonine, L-tryptophan, L-tyrosine dibasic dihydrate, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucose, hypoxanthine, linoleic acid, phenol red, putrescine hydrochloride, monosodium pyruvate, and thymidine supplemented with 1x concentrated Glutamax additive solution.

[0094] In this example, the effect of a 1:1 mixture of both media was also confirmed. Preferably, the medium contains a minimum of 4.0 g / l glucose, preferably 4.5 g / l glucose, a minimum of 10% FBS and a minimum of 2 mM glutamine.

[0095] Preferably, the culture medium should contain 55-75 mg / L sodium pyruvate and sodium bicarbonate, 2 mM-4 mM glutamine, 2%-10% FBS, and at least 4.5 g / L glucose, as well as ethanolamine, glutathione, ascorbic acid, insulin, transferrin, copper sulfate, and manganese chloride.

[0096] As an example, a minimum of two tissue flap pieces approximately 1 mm x 1 cm x 1 cm in size are placed in each well of the culture plate, and along one of their edges, along their maximum length or a fragment of their edge, they are separated by a contiguous separation of at least 0.1 cm in length. The fragments were placed along the periphery of the tissue fragments, with the fragments adhering to each other. Multiple experiments were performed using two to four fragments with a 0.1-, 0.5-, or 1-cm separation. The cartilage fragments were incubated at 37°C and 5% CO2 for 7 days. After this period, microscopic observations were performed and the medium was replaced with fresh medium. For the 6-well plates used in this experiment, the volume of fresh medium was 2.5 ml, replaced every 7 days. An inverted microscope was used to assess the viability of the tissue fragments, the microbial purity of the culture, and the possibility of the emergence of new cellular structures resulting from the division of cells present in the tissue. The junction between the two tissue fragments, as well as the viability and proliferation of the tissue fragments, were monitored using optical magnifications of 10x and 40x.

[0097] The tissue fragments were cultured (dissociated culture - adherent culture) until a sufficient number of chondrocytes was obtained, and then the tissue fragments were overflowed with a suspension of the expanded chondrocytes. In this experiment, the culture lasted for 17 days. This tissue fragment culture required a kind of incubation, aiming to keep the tissue fragments in an active state, ready for the next step, which was already carried out in the appropriate culture with isolated chondrocytes.

[0098] 3) Obtaining a chondrocyte suspension and initial adherent culture of chondrocytes alone Obtaining and culturing chondrocytes and their expansion can be carried out using known methods.

[0099] After selecting the tissue fragments that would serve as the main material for the future new tissue flap, the remaining fragments (accounting for 15% to 50% of the weight of all fragments) were used for chondrocyte isolation to obtain a chondrocyte suspension. The shape of the tissue fragments is not critical for obtaining chondrocytes. For this purpose, the tissue for enzymatic digestion was further cut into smaller pieces (3 mm x 3 mm x 3 mm or less) using a scalpel. The material thus prepared was treated with collagenase II.

[0100] The resulting tissue fragments were transferred to a sterile tube with a volume of 15 to 50 ml. In this case, a 50 ml tube was used. The tissue fragments were rinsed three times with 1x concentrated phosphate-buffered saline (1x PBS). The enzyme digestion time and amount were adjusted to match the mass of the tissue fragments and to ensure adequate cell viability after the reaction. In this study, the following tissue-to-enzyme weight ratio ranges were used: 1 g:1 mg, 1 g:2 mg, 1 g:3 mg, and 1 g:4 mg. Digestion reactions were performed in culture medium 1, known by the trade name DMEM, containing anhydrous calcium chloride, ferric nitrate hydrate, potassium chloride, magnesium sulfate anhydrous, sodium chloride, sodium bicarbonate, potassium phosphate monobasic monohydrate, D-glucose, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, disodium L-tyrosine dihydrate, L-valine, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, and 4-(2-hydroxyethyl)piperazine sodium salt 1-ethanesulfone with added glucose, or culture medium 2, containing anhydrous calcium chloride, ferric nitrate hydrate, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, disodium L-tyrosine dihydrate, L-valine, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, and 4-(2-hydroxyethyl)piperazine sodium salt 1-ethanesulfone with added glucose. Calcium, Copper Sulfate Pentahydrate, Ferrous Sulfate Heptahydrate, Magnesium Chloride, Potassium Chloride, Sodium Bicarbonate, Sodium Chloride, Sodium Hydrogen Phosphate Anhydrous, Zinc Sulfate Heptahydrate, L-Alanine, L-Arginine Hydrochloride, L-Asparagine Monohydrate, L-Aspartic Acid, L-Cysteine Hydrochloride, L-Glutamic Acid, L-Glutamine, Glycine, L-Histidine Trihydrochloride Monohydrate, L-Isoleucine, L-Leucine, L-Lysine Salt Acid salt, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine dibasic dihydrate, L-valine, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, D-glucose, hypoxanthine, phenolic linoleic acid red, protease inhibitor. Threcin hydrochloride, monosodium pyruvate, thymidine, and 1x concentrated Glutamax additive solution], or in a mixture of the above two culture media in a 1:1 ratio and supplemented with glucose.

[0101] The tissue was placed in medium containing the appropriate concentration of collagenase II in a ratio of 3 ml medium:0.1 g tissue, or 3 ml medium:0.2 g tissue, or 3 ml medium:0.3 g tissue, or 3 ml medium:0.5 g tissue. The mass of the tissue (in grams) is the total mass of the tissue pieces used. Pieces were digested up to a total of 0.5 g.

[0102] Digestion was performed on pieces weighing 0.5 g in total. Digestion was performed in a cell culture incubator at 37°C and 5% CO2. The reaction was continued for a minimum of 16 hours until the tissue was completely digested. After digestion, the supernatant containing the isolated chondrocytes was removed and centrifuged at 300 × g for a minimum of 10 minutes at room temperature. The resulting cell pellet, which contained between 0.3 million and 1.5 million chondrocytes per gram of tissue, was rinsed three times in 1x concentrated PBS. The amount of tissue used for digestion typically averages approximately 0.1-0.2 g. Finally, for digestion observation and qualitative and quantitative assessment, the cell pellet was resuspended in a minimum of 1 ml of Medium 1 with 4.5 g / L glucose, Medium 2 supplemented with 1x concentrated Glutamax additive solution, or a 1:1 mixture of Medium 1 and Medium 2 containing 2%, 4%, 5%, or 10% FBS and a minimum of 2 mM glutamine solution. After digestion, cells were assessed for good viability (qualitative assessment) and for complete digestion of the tissue fragments (quantitative assessment). Cell viability (minimum 80% viability was considered positive), cell shape, and overall condition (cells with visible round nuclei were considered good) were qualitatively assessed. Cells with a viability of 80% or more were seeded into 25 cm or 75 cm culture flasks at a density of 150,000 cells / 2 ml, 200,000 cells / 2 ml, or 250,000 cells / 2 ml. During culture, cells were observed every other day using an inverted optical microscope to assess proliferation rate and determine the timing of cell passage, which is closely related to the level of confluence. The culture medium was changed twice weekly. When cells reached adequate confluence (maximum 85%), they were trypsinized with 2 ml of 0.05% (w / v) trypsin / EDTA in PBS for 5 minutes at 37°C. Trypsinization was inhibited by adding Medium 1 supplemented with 4.5 g / L glucose, Medium 2 supplemented with 1x concentrated Glutamax additive solution, or a 1:1 mixture of Medium 1 and Medium 2 containing 2%, 4%, 5%, or 10% FBS in a volume 1x, 1.5x, or 2x the volume of the trypsin / EDTA solution used. Cell number and viability were determined using a microscope and trypan blue solution according to standard procedures.

[0103] During chondrocyte culture, cell proliferation and viability were primarily monitored. Cultures were continued until they reached 85% confluence and a minimum of 80% cell viability. The goal of this culture was to grow enough cells for a subsequent culture in which tissue fragments were combined with chondrocytes, i.e., "tissue flooding." In this case, the chondrocyte suspension served as an adhesive to hold the tissue fragments together. Some of the cells were used for the tissue "flooding," and another portion was used to establish another culture so that the cellular material would continue to be available.

[0104] The cells intended for use in stimulating the connections between pieces of cartilage tissue described in point a) were left in continuous culture. The culture of chondrocytes for cell expansion purposes is always performed in monolayer-adherent culture. When the cells are detached from the surface of the plate and washing and centrifugation steps are performed, the cell suspension and suspension of chondrocytes in the target buffer and the spillage of the flap tissue pieces during the combined culture are discussed.

[0105] 4) Combined culture of tissue fragments and chondrocyte suspension according to point 3 Assuming that two tissue pieces of approximately 1 mm x 1 cm x 1 cm size are used, it is important to add a minimum of 40,000 chondrocytes to a well containing 2.5 ml of culture medium. The maximum concentration of chondrocytes for culturing two pieces with a 0.1 cm contact length is 8000 chondrocytes per 2.5 ml of medium.

[0106] Cartilage tissue pieces cultured in Medium 1 with 4.5 g / l glucose, or Medium 2 supplemented with 1x concentrated Glutamax additive solution, or a 1:1 ratio of Medium 1 and Medium 2 as above, supplemented with 2%, 4%, 5%, or 10% FBS, a minimum of 2 mM glutamine, and a minimum of 0.01% IGF-1 and / or a minimum of 0.01% TGF-β1 and / or a minimum of 0.01% chondroitin and / or a minimum of 0.1% insulin, were "flooded" with chondrocytes obtained from the culture after digestion (minimum viability ≥ 85%).

[0107] It is important that the medium contains a minimum of 4.5 g / l glucose, a minimum of 10% FBS and a minimum of 2 mM glutamine. Preferably, the culture medium should contain 55-75 mg / L sodium pyruvate and sodium bicarbonate, 2 mM to 4 mM glutamine, 2% to 10% FBS, and at least 4.5 g / L glucose, as well as ethanolamine, glutathione, ascorbic acid, insulin, transferrin, copper sulfate, manganese chloride, and a minimum of 0.01% IGF-1 and / or a minimum of 0.01% TGFβ1 and / or a minimum of 0.01% chondroitin and / or a minimum of 0.1% insulin.

[0108] After centrifugation and prior to the quantitative and qualitative assessment described above, cells were suspended in Medium 1 with 4.5 g / L glucose, Medium 2 supplemented with 1x concentrated Glutamax additive solution, or a 1:1 ratio of Medium 1 and Medium 2, at a minimum of 500 μl per 40,000 cells. This was then added to wells containing a minimum of two tissue fragments. It is important that the culture medium contain 55 mg / L to 75 mg / L sodium pyruvate, sodium bicarbonate, 2 mM to 4 mM glutamine, 2% to 10% FBS, and 4.5 g / L to 5.0 g / L glucose.

[0109] The resulting cultures, in which the tissue fragments and chondrocytes were combined, were cultured in a cell culture incubator at 37°C and 5% CO2 for a minimum of 12 weeks until new tissue bonds were formed between the tissue fragments. The process of "flooding" the tissue with a suspension containing a fixed amount of chondrocytes each time was repeated once a week. The indicated frequency of medium replacement was determined based on experience gained in tissue culture. The tissue fragments were gently transferred to new wells of a 6-well plate before the addition of chondrocytes. The medium was also replaced at that time. For 6-well plates, the medium was adjusted to a final volume of 2.5 ml, including additives.

[0110] Tissue transformation: attachment, viability, and proliferation were observed regularly using an inverted light microscope at 10x and 40x magnification, each time prior to "flooding" the tissue with subsequent cells. Culture medium and additives were changed weekly.

[0111] After each subsequent "flooding" with new chondrocytes, the following procedure was performed. 1) Removal of old medium 2) Gently transfer tissue slices to a new culture plate 3) Addition of fresh medium 4) The pieces are flooded with cells (the number of cells mentioned above, and the medium in which they are suspended - per volume of fresh medium just prepared) - as above.

[0112] A few days after the initiation of the test culture, visible changes were observed in the cartilage tissue. New tissue connections were observed between the old tissue fragments. After several weeks, the gaps between the old pieces of tissue were filled with new tissue and overgrown. New tissue flap fragments were obtained from the cartilage fragments and free chondrocytes that were regularly supplied to the culture. One new tissue flap fragment obtained from the two initial tissue fragments had a "groove" in the center, presumably representing new tissue formed by the participation of chondrocytes that were regularly supplied to the culture in suspension. The tissue flap measured approximately 1 mm x 2.1 cm x 1 cm. It was possible to transfer it from one well to another without the need for two tweezers and without risk of damage. As shown in Figure 3, the new connections between the initial tissue fragments were slightly lighter in color compared to the old tissue. Inverted microscopy images of a cell-tissue culture using two tissue fragments with abutting divisions were taken on the day of culture initiation (Figure 3A), 17 days (Figure 3B), and 52 days (Figure 3C, D).

[0113] As described above, a separate chondrocyte suspension culture was run simultaneously throughout the appropriate culture period, in which the tissue pieces and cell suspension were combined. The cell suspension culture was necessary to maintain a continuous source of cellular material used to "flood" the tissue pieces. Observations were made at 40x magnification.

[0114] Figure 3 shows microscopic images of tissue piece cultures in Medium 1, described above, with 4.5 g / l glucose, 10% FBS, and 2 mM glutamine—visualization of new tissue formation over time. A: Image of two tissue piece fragments as close as possible to each other along a border about 1 cm long, on day 1 of culture (culture baseline, image recorded on day 1). Clearly visible empty space between the contacting surfaces of the two tissue pieces. B: Image of two tissue piece fragments adhering to each other along a border about 1 cm long, and visible tissue filling formed between the contacting surfaces of the two tissue pieces, on day 17 of culture in Medium 1 supplemented with 4.5 g / l glucose, 10% FBS, and 2 mM glutamine. C, D: Images of two tissue fragments adhering to each other along a ~1 cm seam, and visible tissue filling formed between the contacting surfaces of the two tissue fragments, after 52 days of culture in Medium 1 supplemented with 4.5 g / L glucose, 10% FBS, and 2 mM glutamine. The new tissue formed after 52 days of culture has clearly repaired and left a mark the empty space at the junction of the two tissue fragments that was present at day 1 of culture. There is a significant color matching of the old and new tissue at one end of the contacting surfaces of the two tissue fragments. Images were taken using 10x magnification (A, B, C) and 40x magnification (D).

[0115] Example 5 Variations in application of the invention The cultivation is carried out as described in Example 4 under the following conditions, with some modifications:

[0116] 1) Obtaining cartilage tissue fragments for further culture. Eight small pieces of oval-shaped tissue were obtained, each cut on one side to maximize the contact area between two adjacent pieces. The resulting pieces were thin, only about 1 mm thick. They were about 0.9 cm long and 0.5-0.6 cm wide.

[0117] 2) Pre-culture of tissue fragments only The selected and appropriately trimmed pieces were transferred to a 1-well or 6-well sterile cell culture plate using tweezers. Two selected tissue pieces, approximately 1 mm thick, 0.9 cm long, and 0.6 cm wide, were placed in wells pre-filled with 2.5 ml of Medium 1 and Medium 2 with 4.5 g / l glucose, 10% FBS serum, 55 mg / l sodium pyruvate, and 2 mM glutamine. The connecting parts were straight. Media 1 and 2 were as described in the previous examples.

[0118] Two tissue pieces per well were placed in three wells of a culture plate, arranged so that one edge (the bordering edge) of each piece, approximately 0.6 cm in length, was attached as closely as possible to the edge of the other piece. These were biological replicates of the same test. The cartilage was incubated at 37°C and 5% CO2 for 7 days. The culture medium was changed every 7 days, and each time an inverted microscope was used to assess the viability of the tissue pieces, the microbial purity of the culture, and to analyze the possible appearance of new cellular structures resulting from the division of cells present in the tissue. Optical magnifications of 10x and 40x were used. Observations were made before the medium change.

[0119] 3) Obtaining and culturing chondrocyte suspensions The other two pieces (approximately 1 mm × 0.9 cm × 0.5 cm) were used for chondrocyte isolation, which together accounted for approximately 25% of the total piece weight. For this purpose, the tissue for enzymatic digestion was further cut into smaller pieces using a scalpel (approximately 2 mm × 2 mm × 2 mm in size). The material thus prepared was treated with collagenase II. The resulting tissue pieces were transferred to a 30 ml sterile tube. The tissue pieces were rinsed three times with 1x concentrated phosphate-buffered saline (1x PBS). In this study, the following tissue-to-enzyme weight ratio was used: 1 g:1 mg. The digestion reaction was carried out under the following conditions: in a mixture of Medium 1 and Medium 2 containing the appropriate amount of collagenase II, i.e., 0.2 mg, with the addition of 4.5 g / l glucose (i.e., 2 ml of medium per 0.3 g of tissue). The mass of the tissue (unit: grams) was the sum of the masses of the tissue pieces used. Digestion was carried out in a cell culture incubator at 37°C and 5% CO2. The reaction was allowed to proceed overnight for 20 hours. After digestion, the supernatant containing the isolated chondrocytes was collected and centrifuged at 300 × g for 10 minutes at room temperature (21°C). The resulting cell pellet containing chondrocytes was rinsed three times with 1x concentrated PBS. Finally, the cell pellet was resuspended in 1 ml of a mixture of Medium 1 and Medium 2 supplemented with 4.5 g / L glucose, 10% FBS, and 2 mM glutamine for observation and qualitative evaluation (mainly evaluation of cell viability and cell condition after digestion—e.g., morphology was periodically evaluated) as well as quantitative digestion. A chondrocyte suspension containing 490,000 viable cells with a viability of 98% was obtained. The resulting cells were then seeded at a density of 10,000 cells / cm2. The cells were seeded into 6-well plates and 25 cm2 culture flasks.

[0120] The cells were left in continued culture for use in stimulating connections between pieces of cartilage tissue. 4) Combined Culture - Tissue Fragment and Chondrocyte Suspension Cartilage tissue fragments grown in a mixture of Medium 1 and Medium 2 supplemented with 4.5 g / L glucose, 10% FBS, 2 mM glutamine, and 55 mg / L pyruvate, as described in point 2 of this example, were "flooded" with chondrocytes obtained as a result of the digestion (survival rate ranging from 85% to 92%). After centrifugation and prior to the quantitative and qualitative evaluation described above (point 3 of this example), the cells were suspended in a mixture of Medium 1 and Medium 2 supplemented with 4.5 g / L glucose in a final volume of 1,000 μl, and a suspension containing 80,000 cells was added to each well containing two tissue fragments. The additives were then added. In this test, the additives were IGF-1 and TGF, and the final concentration in the wells for both additives reached 0.01%.

[0121] The resulting cultures of combined tissue fragments and chondrocytes were cultured in a cell culture incubator at 37°C and 5% CO2 for 5 weeks until new tissue bonds were formed between the tissue fragments. The process of "flooding" the tissue with a suspension containing a fixed amount of chondrocytes (80,000 cells per 2.5 ml of medium per well containing two tissue fragments) was repeated weekly (every 7 days). The tissue fragments were observed before tissue transfer and the addition of fresh medium and chondrocytes (as described in Example 4). The tissue fragments were gently transferred to new wells of a 6-well plate before the addition of chondrocytes.

[0122] Visible changes in the cartilage tissue were observed 7 days after the initiation of the experimental culture. As shown in Figure 4, tissue connections between old tissue fragments were confirmed after 19 days. Figures 4A and 4B show inverted microscopy images taken on the day of culture initiation (Figure 4A) and 19 days after the initiation of cell-tissue culture (Figure 4B) using two tissue fragments with a 0.6 cm abutment. The new connections between the fragments appeared as fibers connecting the fragments. After 5 weeks, the gap between the old tissue fragments had been filled with new tissue overgrowth. The junction between the two tissue fragments was filled with new cellular connections. The new tissue between the cartilage fragments took on a "scar"-like appearance. In microscopic images, the new tissue was lighter than the original cartilage fragments. New tissue flaps were obtained from the cartilage fragments and free chondrocytes that were periodically added to the culture. A new tissue flap measuring approximately 1 mm x 1.8 cm x 0.6 cm was obtained.

[0123] Figure 4 shows microscopic images of tissue fragment cultures in a mixture of media 1 and 2—visualization of the formation of new tissue bonds over time. A: Image of two tissue fragments positioned as close as possible to each other along a 0.6 cm long separation on day 1 of culture (image recording the culture baseline). A clearly visible empty space is visible between the contacting surfaces of the two tissue fragments. B: Image of two tissue fragments adhering to each other along a separation of approximately 0.6 cm on day 19 of culture in a mixture of media 1 and 2 supplemented with 4.5 g / l glucose, 10% FBS, 2 mM glutamine, and additives: IGF-I and TGF at 0.01% concentrations, and a visible tissue bond formed between the contacting surfaces of the two tissue fragments. New tissue is visible both on the surface of one of the adjacent fragments and between the tissue fragments. The resulting bond has a fibrous nature, binding the two contacting surfaces together. Images were taken using 10x magnification.

[0124] Example 6 Variations in application of the invention The culture is carried out as described in Examples 4 and 5, with the following differences.

[0125] 1) Obtaining cartilage tissue fragments for further culture: Each flap was cut on two sides (longest side) to obtain 10 oval-shaped tissue pieces. The resulting pieces were approximately 3 mm thick. They were approximately 1 cm long and 0.2 cm wide.

[0126] 2) Pre-culture of tissue fragments only: The selected and appropriately trimmed pieces were transferred to a 1-well or 6-well sterile cell culture plate using tweezers. Four selected tissue pieces, each approximately 3 mm thick, 1 cm long (the length of the contact surface of each piece, the shape of the surface was almost linear to maximize the contact area), and 0.2 cm wide, were placed in a well pre-filled with 1.25 ml of a mixture of culture media 1 and 2 supplemented with 4.5 g / l glucose, 2% FBS serum, and 2 mM glutamine.

[0127] The contact gap between each pair was approximately 1 cm. Four tissue pieces per well were placed in three wells of a 12-well culture plate, positioned so that one edge (the bordering edge) of each piece, approximately 1 cm in length, was attached as tightly as possible to the edge of another piece.

[0128] 3) Obtaining and culturing chondrocyte suspensions: In this study, the following tissue to enzyme weight ratio was used: 1 g:2 mg. The digestion reaction was carried out under the following conditions: in a mixture of Medium 1 and Medium 2 containing an appropriate amount of collagenase II, i.e., 0.4 mg, with the addition of 4.5 g / l glucose (ratio of 3 ml of medium per 0.3 g of tissue - i.e., 2 ml). The reaction was carried out for 14 hours. Finally, observation and qualitative evaluation (mainly To assess cell viability and cell condition after digestion (e.g., cell shape was periodically assessed) and for quantitative digestion, the cell pellet was resuspended in 1 ml of a mixture of Medium 1 and Medium 2 supplemented with 4.5 g / L glucose, 2% FBS, and 2 mM glutamine. A chondrocyte suspension containing 420,000 viable cells with a viability of 92% was obtained.

[0129] 4) Combined Culture - Tissue Fragment and Chondrocyte Suspension Cartilage tissue fragments, grown in a mixture of Medium 1 and Medium 2 supplemented with 4.5 g / l glucose, 2% FBS, and 2 mM glutamine, as described in point 2 of this example, were "flooded" with chondrocytes obtained after digestion (survival rates ranging from 85% to 92%). An appropriate volume of a suspension containing 80,000 cells / 1.25 ml medium was added to the well containing the tissue fragments. An additive was then added. In this test, the additive was chondroitin, reaching a final concentration of 0.01% in the medium in the wells.

[0130] The resulting cultures, in which tissue fragments and chondrocytes were combined, were incubated in a cell culture incubator at 37°C and 5% CO2 for a total of 8 weeks until new tissue bonds were formed between the used tissue fragments. The process of "flooding" the tissue with a suspension containing a certain amount of chondrocytes (80,000 cells per well containing two tissue fragments) was repeated weekly (every 7 days). The tissue fragments were observed before tissue transfer and the addition of fresh medium and chondrocytes (as described in Example 4). The tissue fragments were gently transferred to new wells of a 12-well plate before the addition of chondrocytes.

[0131] Visible changes in the cartilage tissue were observed 14 days after the initiation of the test culture. As shown in Figure 5, tissue integration between the old tissue fragments was confirmed after 19 days using four tissue fragments with a 1 cm abutment. Inverted microscopy images taken on the day of culture initiation (Figure 5A) and 7 days after the initiation of cell-tissue culture (Figure 5B, C) were taken using four tissue fragments with a 1 cm abutment.

[0132] The new connections between the pieces appeared as fibers holding the pieces together. After 7 weeks, the gaps between the old pieces of tissue were filled with new tissue and overgrown. The junctions between the two pieces of tissue were filled with new cellular connections. A new tissue flap was obtained, formed from the cartilage pieces and free chondrocytes that were periodically fed into the culture. A new tissue flap measuring approximately 3 mm x 1 cm x 0.8 cm was obtained.

[0133] Figure 5 shows microscopic images of tissue fragment cultures (first and second) in Medium 1—visualization of the formation of new tissue bonds over time. A: Image of two tissue fragments (first and second) positioned as close as possible to each other along a 1 cm-long separation on day 1 of culture (image recording the culture baseline). There is an open space between the first tissue fragment, located closest to the edge of the plate, and the adjacent tissue fragment. The first fragment was gently cut along the edge of the contacting surface to allow identification in the next stage of the experiment (the first fragment is at the bottom of the image). B, C: Images of two tissue fragments (first and second) and visible tissue bonds formed between the approximately 1 cm-long contacting surface of the two tissue fragments on day 27 of culture in Medium 1 supplemented with 4.5 g / L glucose, 2% FBS, 2 mM glutamine, and 0.01% chondroitin. Fibrous bonds form between the contacting surfaces of the tissue fragments. After several days of incubation, new tissue forms between the contacting tissue pieces, permanently connecting the two pieces. The same type of bond was observed between the second and third pieces, and between the third and fourth pieces, respectively. Images [A, B] were taken at 10x magnification, and image [C] was taken at 40x magnification.

[0134] Example 7 Variations in application of the invention The culture is carried out as described in Example 5, with the following differences.

[0135] 1) Obtaining cartilage tissue fragments for further culture: Five rectangular pieces of tissue were obtained. The pieces obtained were approximately 2 mm thick. They were approximately 0.5 cm long and 0.4 cm wide.

[0136] 2) Pre-culture of tissue fragments only: The selected and appropriately trimmed pieces were transferred to a 1-well or 6-well sterile cell culture plate using tweezers. Three selected tissue pieces, approximately 2 mm thick, 0.5 cm long, and 0.4 cm wide (length of contact surface), were placed in a well pre-filled with 2.5 ml of culture medium 2 containing 4% FBS serum pyruvate and 2 mM glutamine. The tissue pieces were oval in shape, with two of the three pieces having convex contact surfaces and one piece having two concave surfaces. All contact surfaces were compatible with each other; that is, they were as close as possible to each other.

[0137] Three tissue pieces per well were placed in three wells of a 6-well culture plate, positioned so that one edge (the bordering edge) of each piece, approximately 0.4 cm in length, was attached as tightly as possible to the edge of another piece.

[0138] 3) Obtaining and culturing chondrocyte suspensions: In this study, the following tissue-to-enzyme weight ratio was used: 1 g:4 mg. The digestion reaction was carried out under the following conditions: a mixture of Medium 1 and Medium 2 containing 4.5 g / L glucose (6 ml of medium per 0.3 g of tissue, i.e., 4 ml) and an appropriate amount of collagenase II, i.e., 0.8 mg. The reaction was carried out for 8 hours. Finally, for observation and qualitative evaluation (mainly evaluation of cell viability and cell condition after digestion—e.g., morphology was periodically evaluated) as well as quantitative digestion, the cell pellet was resuspended in 1 ml of Medium 2 supplemented with 4.5 g / L glucose, 4% FBS, and 2 mM glutamine. A chondrocyte suspension containing 320,000 viable cells with a viability of 86% was obtained.

[0139] 4) Combined Culture - Tissue Fragment and Chondrocyte Suspension Cartilage tissue fragments grown in Medium 2, supplemented with 4.5 g / l glucose, 4% FBS, and 2 mM glutamine, as described in point 2 of this example, were "flooded" with chondrocytes obtained after digestion (survival rates ranging from 85% to 92%). An appropriate volume of a suspension containing 60,000 cells was added to the well containing the tissue fragments. An additive was then added. In this test, the additive was insulin, reaching a final concentration of 0.2% in the medium in the wells.

[0140] The resulting cultures of combined tissue fragments and chondrocytes were cultured in a cell culture incubator at 37°C and 5% CO2 for 11 weeks until new tissue bonds were formed between the tissue fragments. The process of "flooding" the tissue with a suspension containing a fixed amount of chondrocytes (60,000 cells per well containing three tissue fragments) was repeated weekly (every 7 days). The tissue fragments were observed before tissue transfer and the addition of fresh medium and chondrocytes (as described in Example 4). The tissue fragments were gently transferred to new wells of a 6-well plate before the addition of chondrocytes.

[0141] Twenty-one days after the start of the test culture, visible changes were observed in the cartilage tissue. After 43 days, new tissue connections were observed between the old tissue fragments in all tissue fragments, i.e., between the first and second fragments, and between the second and third fragments. After 11 weeks, the gaps between the old pieces of tissue had been filled with new tissue overgrowth. The junctions between the three tissue fragments were filled with new cellular connections. A flap of new tissue was obtained, approximately 2 mm x 1.5 cm x 0.4 cm, formed from the cartilage fragments and free chondrocytes that were periodically supplied to the culture. m of new tissue flap was obtained.

[0142] The present invention further relates to the following items: 1. A method for in vitro chondrocyte culture to obtain material for treating articular cartilage defects, comprising the steps of: dissecting the obtained hyaline cartilage into at least two tissue fragments; then, placing the tissue fragments side by side on a culture plate in a culture medium containing at least 2 mM glutamine, at least 2% FBS, and at least 4.5 g / L glucose, adjusting the shape of the tissue fragments along at least one marginal dividing line of each fragment so that the space between two similar tissue fragments is as small as possible after the fragments are placed close together and so that the space in the culture plate containing the tissue fragments is as small as possible; adding a chondrocyte suspension to the culture to join the fragments along at least one border dividing line during culture; continuing cell culture of the chondrocyte-loaded tissue fragments; periodically changing the culture medium; and adding chondrocytes to the culture medium. The culture is continued until the tissue fragments form a joint along at least the border dividing line.

[0143] 2. The method according to item 1, characterized in that the culture medium contains additives at a concentration of at least 0.01% IGF and / or 0.01% TGF and / or 0.01% chondroitin and / or at least 0.1% insulin.

[0144] 3. The method according to item 1 or 2, characterized in that the culture medium contains 55 to 75 mg / L sodium pyruvate and sodium bicarbonate, 2 mM to 4 mM glutamine, 2% to 10% FBS, and at least 4.5 g / L glucose and ethanolamine and / or glutathione and / or ascorbic acid and / or insulin and / or transferrin and / or copper sulfate and / or manganese chloride.

[0145] 4. The method according to item 1 or 2 or 3, characterized in that the tissue fragments are cultured alone in the medium, preferably for at least 14 days, before the chondrocytes are added to the culture. 5. The method according to items 1 to 4, characterized by the step of adding a chondrocyte suspension to the tissue fragments in an amount such that there are at least 40,000 chondrocytes per 2.5 ml of culture medium.

[0146] 6. The method according to item 1, 4 or 5, characterized in that the minimum concentration of chondrocytes for culturing two fragments with abutting sections of 0.1 cm to 1 cm in length is 40,000 chondrocytes per 2.5 ml of medium.

[0147] 7. The method according to item 1, characterized in that the cultured chondrocytes are obtained by enzymatic digestion, after which the required number of cells is obtained by culturing the chondrocytes in a monolayer in adherent culture.

[0148] 8. The method of item 1, characterized by culturing the cells at an elevated temperature of 37°C and 5% CO2. 9. The method according to any one of items 1 to 8, characterized in that the separation of the junction between the at least two joined tissue fragments is a minimum of 0.1 cm, most preferably at least 0.5 cm.

[0149] 10. The method according to any one of items 1 to 19, characterized in that the tissue fragments have an oval-like shape. 11. The shapes of the tissue fragments are very similar to each other along the divisions where the tissue fragments meet in culture. 11. The method according to any one of items 1 to 10, characterized in that the adhesive is selected to adhere tightly.

Claims

1. 1. A method for producing a cartilage tissue graft in vitro, comprising the steps of: i) providing at least first and second pieces of cartilage tissue, each having at least one margin; ii) placing the first fragment in culture medium such that at least one edge of the first fragment is spaced no more than 1 cm or less from at least one edge of the second fragment; iii) adding a suspension of chondrocytes to the fragments; and iv) culturing the fragments and chondrocytes under cell culture conditions A method for producing a cartilage tissue graft in vitro, comprising:

2. 2. The method of claim 1, wherein the edges of the segments are in contact with one another along a border, the border preferably having a length of at least 0.1 cm, more preferably at least 0.5 cm.

3. 3. The method of claim 1 or 2, wherein the culture medium comprises a medium containing one or more salts, amino acids, additional components such as galactose, dextran sulfate, spermidine, beta-glycerolphosphate and adenine, and a buffer.

4. 4. The method according to any one of claims 1 to 3, wherein the culture medium further comprises growth factors, e.g. contained in human and / or bovine serum, preferably FBS, and preferably wherein the culture medium is serum-free.

5. 5. The method of claim 1, wherein the culture medium comprises IGF and / or TGF, preferably a minimum of 0.01% IGF, 0.01% TGF.

6. 6. The method of any one of claims 1 to 5, wherein after step (ii) the fragments are cultured in the culture medium for at least 14 days before adding a suspension of chondrocytes in step (iii).

7. 7. The method according to claims 1 to 6, wherein in step (iii) the chondrocytes are added to the culture medium to a final concentration of at least 4,000, preferably 4,000 to 40,000, preferably 10,000 to 40,000, most preferably 20,000 to 40,000 chondrocytes per ml of culture medium.

8. 8. The method according to any one of claims 1 to 7, wherein the suspension of chondrocytes is obtained by enzymatic digestion, preferably by digestion with a protease.

9. 9. The method of claim 1, wherein the chondrocytes are cultured in an adherent monolayer before being suspended.

10. The incubation temperature is between 35 and 38°C, preferably 37°C, and / or CO ２ The method according to any one of claims 1 to 9, wherein the concentration is between 4 and 6%, preferably 5%.

11. 11. The method of claim 1, wherein one edge of the first piece is complementary in shape to one edge of the second piece.

12. A tissue graft obtainable by the method according to any one of claims 1 to 11 for use in repairing tissue in a patient in need thereof, preferably in the tissue to be repaired. The tissue to be treated is damaged tissue, more preferably damaged cartilage tissue.

13. 12. A tissue graft obtainable by the method of any of claims 1 to 11 for use in treating damaged tissue in a patient with a disease, said disease comprising joint diseases such as osteoarthritis, rheumatoid arthritis, spondyloarthritis, juvenile idiopathic arthritis, lupus, gout, and bursitis.

14. The tissue graft of claim 13 , wherein the treatment comprises transplantation of the tissue at a defect site.

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