Cell cultures, methods for evaluating cell cultures, methods for producing cell cultures, and markers for evaluating cartilage-like tissue formation characteristics.

Abstract

Provided is a cell culture suitable for use in the repair of a cartilage.　The present invention provides a cell culture having a chondroid tissue formation property, the cell culture comprising a cell mass in which the expression level of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106 and CD107b is equal to or lower than a threshold value for each of the cell surface markers, and / or the expression level of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a and CD90 is equal to or higher than a threshold value for each of the cell surface markers.

Description

[Technical Field]

[0001] This technology relates to cell cultures, methods for evaluating cell cultures, methods for producing cell cultures, and markers for evaluating cartilage-like tissue formation characteristics. More specifically, this technology relates to cell cultures containing a population of cells expressing a specific cell surface marker, methods for evaluating cell cultures based on a specific cell surface marker, methods for producing cell cultures containing a population of cells expressing a specific cell surface marker, and specific cell surface markers used for evaluating cartilage-like tissue formation characteristics. [Background technology]

[0002] Regarding the treatment of musculoskeletal disorders such as osteoarthritis, cartilage tissue therapy using tissue regeneration engineering technology is being employed. In this treatment, cultured chondrocytes or cartilage tissue created from chondrocytes can be transplanted to the affected area. Various transplant materials have been proposed to date.

[0003] For example, Patent Document 1 below describes "a transplant material for transplantation to a predetermined transplant site, wherein cells corresponding to the predetermined transplant site are held in a cell-holding carrier obtained by subjecting a tissue structure of the same type as the predetermined transplant site, obtained from somatic tissue, to an antigenicity suppression treatment while maintaining the shape of the tissue structure." (Claim 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2003-180819 [Overview of the project] [Problems that the invention aims to solve]

[0005] The primary objective of this invention is to provide a cell culture suitable for cartilage repair, and more particularly, a cell culture suitable for hyaline cartilage repair. [Means for solving the problem]

[0006] The inventors have found that cell cultures possessing specific characteristics are suitable for cartilage repair, particularly hyaline cartilage repair.

[0007] In other words, the present invention provides a condition in which the expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is less than or equal to a threshold for each cell surface marker, and / or The expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 is above the threshold for each cell surface marker. Including a population of cells, This invention provides a cell culture having characteristics that form cartilage-like tissue.

[0008] Furthermore, the present invention relates to a method for evaluating cell cultures, Regarding the cell population contained in the cell culture, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each surface marker, and / or Is the expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 equal to or greater than the threshold for each surface marker? Includes a determination step to determine, The aforementioned evaluation method is also provided.

[0009] Furthermore, the present invention is This includes a culture step in which cells are cultured to obtain a cell culture product. In the aforementioned culture step, Regarding the cell population contained in the aforementioned cell culture, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each cell surface marker, and / or The expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 is above the threshold for each cell surface marker culturing is performed so as to achieve this, A method for producing a cell culture having chondroid tissue formation characteristics is also provided.

[0010] Furthermore, the present invention at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b, and / or, at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 A marker for evaluating chondroid tissue formation characteristics including the same is also provided.

Advantages of the Invention

[0011] According to the present invention, a cell culture suitable for use in cartilage repair, particularly hyaline cartilage repair, is provided. Further, according to the present invention, it is also possible to evaluate the cartilage repair ability of a cell culture, particularly the hyaline cartilage repair ability.

Brief Description of the Drawings

[0012] [Figure 1] It is a flow chart of an experiment. [Figure 2] It is a graph showing the gene expression ratio of COL2A1 and COL1A1. [Figure 3] It is a figure showing the staining result. [Modes for carrying out the invention]

[0013] 1. Cell culture

[0014] The present invention provides a cell culture having cartilage-like tissue formation characteristics, and more particularly a cell culture having hyaline cartilage-like tissue formation characteristics. The cell population contained in the cell culture may have an expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b that is below a threshold for each cell surface marker, and / or an expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 that is above a threshold for each cell surface marker. By having these cell surface markers above or below a predetermined threshold, the cell culture is suitable for cartilage repair, and more particularly suitable for knee cartilage repair.

[0015] The cell culture of the present invention has the property of forming cartilage-like tissue, and more particularly, hyaline cartilage-like tissue. The cell culture of the present invention has the property of forming cartilage-like tissue, and more particularly, hyaline cartilage-like tissue, when transplanted into a human living organism, for example, when transplanted into the articular cartilage portion of the knee of a human living organism. In this specification, "cartilage-like tissue" may be tissue having components and / or functions equivalent to or similar to cartilage tissue (particularly knee articular cartilage tissue), and for example, tissue that expresses type II collagen in the same way as hyaline cartilage tissue.

[0016] In the present invention, preferably, the expression intensity of at least CD99 and / or GD2 among the cell surface markers is below a threshold for each of these cell surface markers. By having the expression intensity of CD99 and / or GD2 below a predetermined threshold, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Furthermore, in the present invention, preferably, the expression intensity of at least CD26 and / or CD73 among the cell surface markers is above a threshold for each of these cell surface markers. By having the expression intensity of CD26 and / or CD73 above a predetermined threshold, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Particularly preferable is that, among the cell surface markers, the expression intensity of at least CD99 and / or GD2 is below the threshold for each of these cell surface markers, and the expression intensity of at least CD26 and / or CD73 is above the threshold for each of these cell surface markers. When these four cell surfaces satisfy the criteria for the thresholds, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics.

[0017] Furthermore, in the present invention, preferably, the expression intensity of at least one, two, or three of the cell surface markers, CD26, CD73, and CD44, is above a certain threshold. By having the expression intensity of one, two, or three of the CD26, CD73, and CD44 above a predetermined threshold, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Particularly preferable is that the expression intensity of at least CD99 and / or GD2 among the cell surface markers is below the threshold for each of these cell surface markers, and the expression intensity of at least one, two, or three of the cell surface markers CD26, CD73, and CD44 is above the threshold for each of these cell surface markers. When these four cell surface markers satisfy the threshold criteria, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics.

[0018] In one preferred embodiment of the present invention, the expression intensity of at least CD44 among the cell surface markers is above a threshold for this cell surface marker. By having a CD44 expression intensity above a predetermined threshold, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Particularly preferable is that, among the cell surface markers, the expression intensity of at least CD99 and / or GD2 is below the threshold for each of these cell surface markers, and the expression intensity of at least CD44 is above the threshold for this cell surface marker. When these three cell surfaces satisfy the criteria for the thresholds, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics.

[0019] In the present invention, the expression intensity of cell surface markers may be the normalized mean fluorescence intensity (nMFI) measured by analyzing a cell population by flow cytometry. This normalized mean fluorescence intensity is suitable for evaluating the cartilage-like tissue formation characteristics, particularly the hyaline cartilage-like tissue formation characteristics, based on these cell surface markers. The normalized mean fluorescence intensity may be measured by the measurement method described in "5. Examples" below.

[0020] Preferably, the cell culture of the present invention satisfies at least one of the following conditions 1 to 10 and / or at least one of the following conditions 11 to 20, when the standardized mean fluorescence intensity measured when the cell population contained in the cell culture is analyzed by flow cytometry. Condition 1: When the cell population is labeled with an anti-CD166 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 240 or less, more preferably 220 or less, and even more preferably 200 or less. Condition 2: When the cell population is labeled with an anti-CD165 antibody conjugated with BB700, the standardized mean fluorescence intensity derived from BB700 is 132 or less, more preferably 120 or less, and even more preferably 110 or less. Condition 3: When the cell population is labeled with an anti-CD99 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.9 or less, more preferably 1.8 or less, and even more preferably 1.6 or less. Condition 4: When the cell population is labeled with an anti-GD2 antibody conjugated with BB700, the standardized mean fluorescence intensity derived from BB700 is 3.6 or less, more preferably 3.3 or less, and even more preferably 3.0 or less. Condition 5: When the cell population is labeled with an anti-STRO-1 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.4 or less, more preferably 1.3 or less, and even more preferably 1.2 or less. Condition 6: When the cell population is labeled with an anti-CD108 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 1.9 or less, more preferably 1.8 or less, and even more preferably 1.6 or less. Condition 7: When the cell population is labeled with an anti-CD164 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 3.0 or less, more preferably 2.8 or less, and even more preferably 2.5 or less. Condition 8: When the cell population is labeled with an anti-CD6 antibody conjugated to FITC, the standardized mean fluorescence intensity derived from the FITC is 1.4 or less, more preferably 1.3 or less, and even more preferably 1.2 or less. Condition 9: When the cell population is labeled with an anti-CD106 antibody conjugated to FITC, the standardized mean fluorescence intensity derived from the FITC is 2.4 or less, more preferably 2.2 or less, and even more preferably 2.0 or less. Condition 10: When the cell population is labeled with an anti-CD107b antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.0 or less, more preferably 1.9 or less, and even more preferably 1.7 or less. Condition 11: When the cell population is labeled with an anti-CD26 antibody conjugated to FITC, the standardized mean fluorescence intensity derived from the FITC is 2.3 or higher, more preferably 2.6 or higher, and even more preferably 2.9 or higher. Condition 12: When the cell population is labeled with an anti-CD73 antibody conjugated to FITC, the standardized mean fluorescence intensity derived from the FITC is 47 or higher, more preferably 53 or higher, and even more preferably 59 or higher. Condition 13: When the cell population is labeled with an anti-CD105 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 21 or higher, more preferably 24 or higher, and even more preferably 27 or higher. Condition 14: When the cell population is labeled with an anti-CD44 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 128 or higher, more preferably 145 or higher, and even more preferably 160 or higher. Condition 15: When the cell population is labeled with an anti-CD120a antibody conjugated with APC, the standardized mean fluorescence intensity derived from the APC is 24 or higher, more preferably 27 or higher, and even more preferably 30 or higher. Condition 16: When the cell population is labeled with an anti-CD201 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 18 or higher, more preferably 20 or higher, and even more preferably 23 or higher. Condition 17: When the cell population is labeled with an anti-EGFR antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 3.2 or higher, more preferably 3.6 or higher, and even more preferably 4.0 or higher. Condition 18: When the cell population is labeled with an anti-CD146 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.6 or higher, more preferably 1.8 or higher, and even more preferably 2.0 or higher. Condition 19: When the cell population is labeled with an anti-CD140a antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 5.6 or higher, more preferably 6.4 or higher, and even more preferably 7.0 or higher. Condition 20: When the cell population is labeled with an anti-CD90 antibody conjugated with APC, the standardized mean fluorescence intensity derived from the APC is 800 or higher, more preferably 900 or higher, and even more preferably 1000 or higher. By satisfying one or more of these conditions, the cell culture of the present invention has more desirable cartilage-like tissue formation characteristics, particularly hyaline cartilage-like tissue formation characteristics.

[0021] Particularly preferably, the standardized mean fluorescence intensity measured when the cell population is analyzed by flow cytometry satisfies condition 3 and / or 4, and / or condition 11 and / or 12. Even more preferably, the standardized mean fluorescence intensity measured when the cell population is analyzed by flow cytometry satisfies condition 3 and 4, and also satisfies condition 11 and / or 12. Satisfying one, two, three, or all four of these four conditions, and especially satisfying all four, brings particularly good cartilage-like tissue formation characteristics, particularly hyaline cartilage-like tissue formation characteristics, to the cell culture of the present invention.

[0022] In one embodiment of the present invention, the standardized mean fluorescence intensity measured when the cell population is analyzed by flow cytometry satisfies condition 3 and / or 4, and / or satisfies one, two, or three of conditions 11, 12, and 14. More preferably, the standardized mean fluorescence intensity measured when the cell population is analyzed by flow cytometry satisfies condition 3 and 4, and also satisfies conditions 11, 12, and 14. Satisfying one, two, three, four, or all five of these five conditions, and especially satisfying all four, brings about particularly good cartilage-like tissue formation characteristics, particularly hyaline cartilage-like tissue formation characteristics, in the cell culture of the present invention.

[0023] In one embodiment of the present invention, the standardized mean fluorescence intensity measured when the cell population is analyzed by flow cytometry satisfies conditions 3 and / or 4, and / or condition 14. More preferably, the standardized mean fluorescence intensity measured when the cell population is analyzed by flow cytometry satisfies conditions 3 and 4, and also satisfies condition 14. Satisfying one, two, or all three of these three conditions results in particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics, for the cell culture of the present invention.

[0024] (Shape of cell cultures)

[0025] According to one embodiment of the present invention, the cell culture may be in solid form. For example, the cell population contained in the cell culture may be organized into any shape. The solid cell culture may be, for example, sheet-like, granular, fibrous (thread-like), or network-like (mesh-like). According to other embodiments of the present invention, the cell culture does not have to be solid, but may be, for example, fluid. The non-solid cell culture may be, for example, fluid, liquid, or sol.

[0026] The cell culture of the present invention is preferably in the form of a sheet. The sheet-like cell culture is particularly suitable for cartilage tissue repair, and especially for hyaline cartilage tissue repair. When the cell culture is in the form of a sheet, the thickness of the sheet-like cell culture may be, for example, 4 μm to 5 mm, more particularly 4 μm to 3 mm, and more particularly 4 μm to 1 mm. The sheet-like cell culture may be a single sheet-like cell culture, or it may be a stack of multiple sheets (for example, 2 to 10 sheets, more particularly 2 to 8 sheets, and more particularly 2 to 5 sheets) of sheet-like cell cultures. More specifically, the thickness of the sheet-like cell culture may be, for example, 4 μm to 100 μm, preferably 4 μm to 70 μm, and more preferably 4 μm to 50 μm. The sheet-like cell culture of the present invention may be formed when cells have naturally layered during the cell proliferation process. The sheet-like cell culture of the present invention does not necessarily have to be formed by artificially stacking two or more separately manufactured sheet-like cell cultures and continuing the culture process.

[0027] (Cell culture derived from cartilage tissue)

[0028] In one embodiment of the present invention, the cell culture may be derived from cartilage tissue. That is, the cell culture may be a culture of cells derived from cartilage tissue, and in particular, the raw cells used in the culture to obtain the cell culture may be cartilage tissue-derived cells. Since the cell culture is obtained by artificial culture in vitro, it is not a natural product. The cartilage tissue-derived cells may be, for example, a plurality of cells obtained by separating cells contained in cartilage tissue from the cartilage matrix. For example, the cartilage tissue-derived cells may be a plurality of cells recovered by treating the cartilage tissue with an enzyme to release the cells in the cartilage tissue from the cartilage matrix, and then recovering the released cells by centrifugation.

[0029] The cell culture is preferably not derived from synovial membrane. That is, the cell culture preferably does not contain cells derived from synovial membrane. The cell culture is preferably not formed from a culture of synovial membrane-derived cells, nor from a culture of synovial cells. The cell culture of the present invention may preferably be a culture of only cells derived from cartilage tissue, and more particularly, a culture of only cells derived from hyaline cartilage tissue.

[0030] In the present invention, the cartilage tissue-derived cells may be derived from the cartilage tissue of an animal with polydactyly, or from the cartilage tissue of an animal with polylimbs. The animal is preferably a mammal, more preferably a primate, and even more preferably a human. The cartilage tissue may be obtained, for example, from tissue obtained during the excision of an extra finger. The tissue may be, for example, a part that does not appear white when photographed with an X-ray, that is, a part that appears black. Polydactyly may be of the distal phalanx type, middle phalanx type, or proximal phalanx type. The extra finger may be any finger, for example, the thumb or the little finger. If the extra finger (limb) to be harvested is wart-like and small, all of the harvested subcutaneous tissue may be used. If the animal is a human, there is no age limit for the human, but it may be, for example, 5 years old or younger, 3 years old or younger, or 2 years old or younger.

[0031] Examples of enzymes used in the aforementioned enzymatic treatment include collagenase, caseinase, clostrypain, trypsin, hyaluronidase, elastase, pronase, and dispase. Preferably, combinations of these enzymes can be used. An example of a preferred enzyme combination is, for example, collagenase, caseinase, clostrypain, and trypsin. Examples of enzyme preparations containing this combination include, but are not limited to, collagenase type I, collagenase type II, collagenase type III, collagenase type IV, and collagenase type V (all available from Fujifilm Wako Pure Chemical Industries, Ltd.). Another example of a preferred enzyme combination is, for example, a combination of collagenase and dispase or thermolysin. An example of an enzyme preparation containing this combination is, for example, liberase (available from Roche Diagnostics K.K.), but is not limited to this. Depending on the condition of the tissue, enzymatic treatment may be carried out stepwise using multiple types of enzymes. For example, isolation may be performed by treating the cells in this order with collagenase, caseinase, clostrypain, and trypsin. The conditions for enzyme treatment can be appropriately determined by those skilled in the art depending on the type of enzyme used and / or the state of the cartilage tissue. Enzyme treatment may be carried out at, for example, 30-50°C, preferably 33-45°C or 35-40°C, for, for example, 1-12 hours, preferably 2-5 hours. If the enzyme treatment temperature is too high, problems such as cell degeneration, a decrease in viable cells, decreased proliferative capacity, and inability to isolate may occur. Conversely, if the enzyme treatment temperature is too low, sufficient enzyme activity may not be achieved, and cell isolation may not be possible. Cell recovery can be achieved with high efficiency by applying physical stimulation during enzyme treatment.

[0032] The above enzymatic reaction can be stopped by diluting the cell suspension, in which the articular cartilage has been enzymatically treated, by washing. After the enzymatic reaction has stopped, the cell suspension can be separated into cell clumps and supernatant by centrifugation. With respect to liberase, the enzymatic reaction can be stopped by washing two or more times. The centrifugation may be carried out under conditions that allow for the collection of more cells smaller than 25 μm, particularly 20 μm or smaller, and 15 μm or larger. In order to collect more such cells, the centrifugation may be carried out at, for example, 1000 rpm or more, 1500 rpm or more, or 2000 rpm or more, for, for example 5 minutes or more, 7 minutes or more, or 10 minutes or more.

[0033] The cells derived from the cartilage tissue may be obtained by a so-called outgrowth method. The outgrowth method may include the steps of finely cutting the collected cartilage tissue, seeding the finely cut cartilage tissue pieces in a culture dish with a small amount of culture medium, and culturing them. Through this culturing, proliferated cells are generated from the cartilage tissue pieces. The generated cells are recovered by enzymatic treatment and centrifugation. The recovered cells can be used in the production of the cell sheet of the present invention.

[0034] The process of finely cutting the cartilage tissue can be carried out, for example, in a wet state. This process can be carried out, for example, by placing the tissue fragments and a small amount of culture medium in a 50 ml centrifuge tube and cutting them with Metzenbaum Scissors, SuperCut Tungsten Carbide 18cm Long Curve (World Precision Instruments). It is preferable to obtain the smallest possible cartilage tissue fragments. The culture medium for culturing the finely cut cartilage tissue fragments can be appropriately selected by those skilled in the art, but is preferably DMEM / F12 + 20% FBS + antibiotic (hereinafter also referred to as AB). After cell adhesion to the culture dish is confirmed after the start of culture, the medium may be preferably replaced with DMEM / F12 + 20% FBS + AB + ascorbic acid (hereinafter also referred to as AA). If the medium contains ascorbic acid from the start of culture, cell adhesion to the culture dish may be inhibited. Furthermore, the culture may be carried out under general culture conditions, for example, in an incubator at 37°C and 5% CO2. Culture may be carried out until subconfluence is achieved. Furthermore, the enzyme preparation used in the recovery of cells generated during culture may include, for example, trypsin and EDTA. Centrifugation can be performed as described above.

[0035] In the present invention, the cells derived from cartilage tissue may preferably include mesenchymal stem cells. In addition to mesenchymal stem cells, the cells derived from cartilage tissue may further include cells contained in cartilage tissue. That is, in the present invention, the cells derived from cartilage tissue may be a population of multiple types of cells, including mesenchymal stem cells. Examples of cells other than mesenchymal stem cells include, but are not limited to, chondrocytes and chondrocytes. The cell culture of the present invention is more suitable for cartilage repair because it is formed from a culture of cells derived from cartilage tissue.

[0036] (Cell cultures derived from stem cells)

[0037] In other embodiments of the present invention, the cell culture may be derived from stem cells. The stem cells may include pluripotent stem cells, embryonic stem cells, or somatic stem cells, and may include, for example, iPS cells.

[0038] In this embodiment, for example, pluripotent stem cells (particularly iPS cells) are differentiated by culturing them in a culture medium to obtain chondrocytes or chondrocyte-like cells, and the cell culture of the present invention is obtained by further culturing these chondrocytes or chondrocyte-like cells on a surface immobilized with a stimulus-responsive polymer, for example. The cell culture of the present invention can also be obtained by seeding pluripotent stem cells (particularly iPS cells) on a substrate immobilized with a stimulus-responsive polymer, differentiating them into chondrocytes or chondrocyte-like cells, and culturing them.

[0039] (Uses of cell cultures)

[0040] The cell culture of the present invention may be used for the repair of cartilage tissue, more preferably for the repair of knee cartilage tissue, and even more preferably for the repair of hyaline cartilage tissue of the knee. The cell culture of the present invention has cartilage-like tissue formation characteristics, and in particular hyaline cartilage-like tissue formation characteristics, and is therefore suitable for the aforementioned repair.

[0041] In the present invention, cartilage tissue repair includes, but is not limited to, treating cartilage tissue that is inflamed and / or damaged, reinforcing cartilage tissue, compensating for defects in cartilage tissue, and regenerating cartilage tissue. Furthermore, the cell cultures of the present invention may be used to prevent diseases related to cartilage tissue. The cell cultures of the present invention can be applied, for example, to cartilage or bone tissue that is diseased. Examples of diseases to which the cell cultures of the present invention may be applied include, but are not limited to, arthritis, arthropathy, cartilage damage, osteochondral damage, meniscal damage, and / or intervertebral disc degeneration.

[0042] The cell culture of the present invention is more particularly suitable for surgical procedures, and especially for surgical treatments, of cartilage tissue. Cartilage repair using the cell culture of the present invention can be performed, for example, by surgically exposing the cartilage portion that needs repair and applying the cell culture to the exposed portion. For example, a sheet-like cell culture of the present invention may be applied to the exposed area. The number and size of the sheet-like cell cultures to be applied can be appropriately determined by those skilled in the art, taking into consideration, for example, the condition of the area to be treated or the type of disease. Furthermore, preferably, the subchondral bone may be treated before the application of the sheet-like cell culture of the present invention so that bleeding from the subchondral bone is confirmed. This treatment may be carried out by a method known to those skilled in the art, for example, by microfracture or drilling. Performing this treatment further promotes the repair of cartilage by the sheet-like cell culture of the present invention. When applying the sheet-like cell culture of the present invention to the affected area, it may be joined or sutured using an adhesive usable in vivo. Alternatively, the sheet-like cell culture may simply be attached to the affected area without such joining or suturing.

[0043] When the cell culture of the present invention is a sheet-like cell culture, it may have a substrate on all surfaces of the sheet-like cell culture. Preferably, the substrate may contain fibronectin. When cells are cultured on a substrate, a substrate is usually produced between the cells and the substrate, and no substrate is produced on the side opposite the substrate, i.e., the part that does not come into contact with the substrate. The sheet-like cell culture of the present invention may have a substrate produced not only on the surface in contact with the substrate, but also on the surface that does not come into contact with the substrate. By having a substrate on all surfaces of the sheet-like cell culture, the sheet-like cell culture of the present invention may be more suitable for cartilage repair.

[0044] The sheet-like cell culture of the present invention preferably has a basement membrane-like protein formed between cells and the porous membrane during culture that is not destroyed by enzymes such as proteolytic enzymes such as dispase and trypsin. That is, the sheet-like cell culture of the present invention may have a basement membrane-like protein between cells and the porous membrane. In particular, the sheet-like cell culture of the present invention may have a basement membrane-like protein on one surface or both surfaces of the sheet-like cell culture. By having the basement membrane-like protein, the sheet-like cell culture may exhibit better cartilage repair ability.

[0045] The sheet-like cell culture of the present invention preferably has a cell density of 100×10 5 ~100×10 8 cells / cm 3 , more preferably 100×10 6 ~100×10 7 cells / cm 3 , more preferably 100×10 6 ~500×10 6 cells / cm 3 , even more preferably 200×10 6 ~300×10 6 cells / cm 3 and may be.

[0046] The cell culture of the present invention preferably does not contain artificial scaffold components. That is, the cell culture of the present invention may consist only of the cells in the culture and the components produced by the cells (as well as the medium components attached to the cell sheet). The cell culture of the present invention can preferably be transplanted into a patient without containing artificial scaffold components.

[0047] The cell culture of the present invention can be produced by the production method of the present invention described in 2. below. Therefore, for the production method of the cell culture of the present invention, refer to 2. below.

[0048] 2. Production method of cell culture

[0049] The present invention also provides a method for producing a cell culture having cartilage-like tissue-forming properties. The production method includes a culture step of culturing cells to obtain a cell culture. The production method may further include an analysis step of analyzing the expression intensity of cell surface markers with respect to the cell population contained in the cell culture, and a determination step of determining whether the cell culture has cartilage-like tissue-forming properties based on the expression intensity obtained in the analysis step. Each step will be described below.

[0050] 2-1.Culture process

[0051] In the culture step, with respect to the cell population contained in the cell culture, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each cell surface marker, and / or The expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 is above the threshold for each cell surface marker. The culture may be carried out in such a manner. The cell culture obtained by the aforementioned culture process has excellent cartilage-like tissue formation characteristics, particularly hyaline cartilage-like tissue formation characteristics. When transplanted into a human body, for example, particularly into the knee cartilage portion of a human body, the cell culture can form cartilage-like tissue, particularly hyaline cartilage-like tissue. In other words, the cell culture can exert a cartilage repair effect, particularly a hyaline cartilage repair effect. Furthermore, the cell culture exhibits functions similar to those of cartilage tissue.

[0052] The cells cultured in the culture step may be cartilage tissue-derived cells as described in 1. above, or stem cell-derived cells. For example, the raw material cells used for culture may be cartilage tissue-derived cells, and these cartilage tissue-derived cells may be cells obtained by culturing cells in cartilage tissue in DMEM / F12 containing FBS for at least two days. In a preferred embodiment of the present invention, the raw material cells used for culturing to obtain the cell culture may be prepared by a raw material cell preparation method comprising a first culture step of culturing cells in cartilage tissue in a culture medium to make them confluent, a dissociation step of separating the cell population that has become confluent in the first culture step from each other, and a second culture step of further culturing the cell population separated in the dissociation step in the same fresh culture medium as the first culture medium. The raw material cells prepared by this raw material cell preparation method are suitable for producing tissue cultures that have cartilage repair properties.

[0053] In the culture step, cells may be cultured in a culture medium containing a substrate having a surface on which a stimulus-responsive polymer is immobilized, for example, on the surface of a porous membrane having a surface on which a stimulus-responsive polymer is immobilized. By culturing cells in a culture medium containing a substrate (particularly a porous membrane) having a surface on which a stimulus-responsive polymer is immobilized, a cell culture having cartilage-like tissue-forming properties can be obtained, and the cell culture can be detached from the substrate without damaging it. The stimulus-responsive polymer may be, for example, a temperature-responsive polymer, a pH-responsive polymer, or a photoresponsive polymer, and more specifically, a polymer whose properties (e.g., hydration capacity) change in response to temperature stimulation (e.g., temperature change), pH stimulation (e.g., pH change), or light stimulation (e.g., light irradiation), respectively. The change in properties may be, for example, a change in properties that promotes the detachment of the culture from the substrate. For example, cells are seeded on a surface on which a stimulus-responsive polymer is immobilized, and the cells are cultured in a culture medium at a temperature range where the polymer's hydration ability is weak to form a cell culture. After the cell culture is formed, the temperature of the culture medium is changed to a temperature where the polymer's hydration ability is strong, thereby promoting the detachment of the cell culture from the surface. For example, the temperature range in which the culture takes place (i.e., the temperature range with weak hydration ability) may be, for example, 33°C to 40°C. The temperature for detachment (i.e., the temperature range with strong hydration ability) is lower than the above temperature range, for example, 31°C or lower. In one embodiment of this technology, the stimulus-responsive polymer is a temperature-responsive polymer. When the cells are cultured in a culture medium containing a substrate having a surface on which the temperature-responsive polymer is immobilized, for example, after culturing, by setting the temperature of the culture medium to be above the upper critical dissolution temperature or below the lower critical dissolution temperature of the temperature-responsive polymer, the surface changes from hydrophobic to hydrophilic, and as a result, separation between the culture and the porous membrane becomes easier. The culture in the manufacturing method of the present invention may be, for example, a two-dimensional culture (also called a planar culture) or a three-dimensional culture (for example, suspension culture or pellet culture).

[0054] When the porous membrane is used in the culture step described above, the cells may be in contact with the culture medium on the upper side of the porous membrane, and also in contact with the culture medium on the lower side of the porous membrane through the pores of the porous membrane. By culturing in this state, cells more suitable for cartilage repair can be obtained.

[0055] When the cell culture is detached from the substrate after the culture step, treatment with proteolytic enzymes such as dispase and trypsin is unnecessary. By utilizing the properties of the temperature-responsive polymer and changing the temperature of the culture medium, the cell culture can be detached from the substrate. Therefore, the cell culture produced by the manufacturing method of the present invention has the advantage of being able to be detached from the substrate without being damaged by the enzyme. Treatment with proteolytic enzymes leads to the degradation of desmosome structures between cells and basement membrane-like proteins between cells and the substrate, which can result in the cells in the cell culture becoming separated. On the other hand, cell cultures obtained by the production method of the present invention can be detached from the substrate by changing the temperature of the culture medium without treatment with proteolytic enzymes. As a result, the desmosome structure is preserved, and defects in the cell culture can be reduced. Furthermore, when cell cultures obtained by the production method of the present invention are detached from the substrate by changing the temperature of the culture medium, the basement membrane-like proteins are not destroyed by enzymes. Therefore, better adhesion to the affected tissue can be achieved during transplantation, enabling more efficient treatment. Dispase, a proteolytic enzyme, is known to be able to detach cell sheets while retaining 10-60% of the desmosome structure, but it destroys almost all of the basement membrane-like proteins, resulting in cell cultures with low strength. Cell cultures produced by the manufacturing method of the present invention can be detached from the substrate while retaining 80% or more of both the desmosome structure and the basement membrane-like protein.

[0056] The upper critical solution temperature or lower critical solution temperature of the temperature-responsive polymer used in the present invention is preferably 0°C to 80°C, more preferably 20°C to 50°C, and even more preferably 25°C to 45°C. If the upper critical solution temperature or lower critical solution temperature is too high, cells may die. If the upper critical solution temperature or lower critical solution temperature is too high, the cell proliferation rate may decrease or cells may die.

[0057] In the manufacturing method of the present invention, the temperature-responsive polymer may be either a homopolymer or a copolymer. The polymer may be, for example, a homopolymer of a (meth)acrylamide compound, an N-(or N,N-di)alkyl-substituted (meth)acrylamide derivative, or a vinyl ether derivative, or a copolymer of these monomers. The (meth)acrylamide compound may be, for example, acrylamide or methacrylamide. The N-alkyl-substituted (meth)acrylamide derivatives may be, for example, N-ethylacrylamide (lower critical solution temperature of the homopolymer is 72°C), Nn-propylacrylamide (21°C), Nn-propylmethacrylamide (27°C), N-isopropylacrylamide (32°C), N-isopropylmethacrylamide (43°C), N-cyclopropylacrylamide (45°C), N-cyclopropylmethacrylamide (60°C), N-ethoxyethylacrylamide (approximately 35°C), N-ethoxyethylmethacrylamide (approximately 45°C), N-tetrahydrofurfurylacrylamide (approximately 28°C), or N-tetrahydrofurfurylmethacrylamide (approximately 35°C). The N,N-dialkyl-substituted (meth)acrylamide derivative may be, for example, N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide (lower critical solution temperature of the homopolymer is 56°C), or N,N-diethylacrylamide (same, 32°C). The vinyl ether derivative may be, for example, methyl vinyl ether (with a lower critical solution temperature of 35°C for the homopolymer). In the present invention, the temperature-responsive polymer may be a copolymer of monomers other than those mentioned above, or it may be a polymer obtained by graft polymerization or copolymerization of polymers, or a mixture of polymers or copolymers. Furthermore, crosslinking may be performed to the extent that the inherent properties of the polymer are not impaired. The above polymers may be appropriately selected to select a temperature-responsive polymer having a critical dissolution temperature suitable for cultivation or exfoliation in the present invention, or to adjust the interaction between the porous membrane and the culture, or to adjust the hydrophilicity or hydrophobicity of the porous membrane surface. In a preferred embodiment, the temperature-responsive polymer is poly(N-isopropylacrylamide).

[0058] In the manufacturing method of the present invention, the amount of temperature-responsive polymer immobilized on the surface of the film is preferably 0.3 to 5.0 μg / cm³. 2More preferably 0.3 to 4.8 μg / cm³ 2 This is possible. Furthermore, the amount of temperature-responsive polymer immobilized on the surface of the membrane is preferably 0.3 to 1.5 μg / cm³, especially when the membrane is a porous membrane and culture is performed using a cell culture insert. 2 This is possible. Furthermore, if the film is not a porous film, the amount of temperature-responsive polymer immobilized on the film surface is preferably 1-2 μg / cm³. 2 This is possible. By having the amount of temperature-responsive polymer immobilized within this range, more efficient culture can be performed. If the amount of immobilized polymer is outside this range, cell cultures may not be formed or may not be produced efficiently. Furthermore, by having the amount of immobilized polymer within this range, the cell cultures can be more easily detached from the membrane.

[0059] In the manufacturing method of the present invention, preferably, the substrate is a porous membrane, and in the culture step, the cells are in contact with the culture medium on the upper side of the porous membrane and in contact with the culture medium on the lower side of the porous membrane through the pores of the porous membrane. By performing the culture in this state, the culture can be performed more efficiently.

[0060] In the manufacturing method of the present invention, the material of the membrane, particularly the porous membrane, may be, for example, polycarbonate, polyester, polyethylene terephthalate (PET), polystyrene, or polytetrafluoroethylene. Of these, PET is particularly preferred. By using a porous PET membrane, the cell cultures of the present invention can be prepared more efficiently.

[0061] A method for immobilizing a temperature-responsive polymer onto a porous film can be carried out, for example, by the method described in Japanese Patent Publication No. 2-211865. That is, the immobilization can be carried out by bonding the porous film and the temperature-responsive polymer by a chemical reaction or by bonding by physical interaction. These methods may be used in combination. In the method of bonding by the aforementioned chemical reaction, for example, electron beam irradiation (EB), gamma ray irradiation, ultraviolet irradiation, visible light irradiation, LED irradiation, plasma treatment, or corona treatment may be performed. Alternatively, the temperature-responsive polymer may be immobilized on the porous film by commonly used organic reactions such as radical reactions, anionic radical reactions, or cationic radical reactions. Furthermore, a block copolymer having a structure in which water-insoluble polymer segments and temperature-responsive polymer segments are bonded may be coated on the substrate surface as the temperature-responsive polymer component. Immobilization may also be performed by physical adsorption or hydrophobic means. In the method of bonding by physical interaction described above, a temperature-responsive polymer or a mixture of the polymer and any medium may be coated onto a porous film.

[0062] The culture medium used in the culture method of the present invention may be a medium that can be used for cell culture, particularly for mammalian cell culture, such as DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12). The medium may contain additives. Examples of additives include cell growth factors, hormones, binding proteins, cell adhesion factors, lipids, and other components.

[0063] Examples of the aforementioned cell growth factors include the TGF (Transforming Growth Factor) family, such as TGF-β, b-FGF, IGF, EGF (Epidermal Growth Factor), BMP (Bonemorphogenetic protein), Fibroblast growth factor receptor 3 (FGFR-3), Frizzled-related protein (FRZB), CDMP-1, Growth difference factor 5 (GDF-5), G-CSF (Granulocycyte Colony Sitimulating Factor), LIF (Leukemia Inhibitory Factor), interleukins, PDGF (Platelet-Derived Growth Factor), NGF (Nerve Growth Factor), and activin A, as well as the Wnt family, particularly Wnt-3a (Wingless-type MMTV integration site family, member Examples include 3A), but are not limited to these. The TGF family includes TGF-β1, TGF-β2, and TGF-β3.

[0064] Examples of the aforementioned hormones include, but are not limited to, insulin, transferrin, dexamethasone, estradiol, prolactin, glucagon, thyroxine, growth hormone, FSH (Follicle-Stimulating Hormone), LH (Leutenizing Hormone), glucocortinoids, and prostaglandins.

[0065] Examples of the aforementioned cell adhesion factors include, but are not limited to, collagen, collagen-like peptides, fibronectin, laminin, and vitronectin. Examples of collagen-like peptides include recombinant peptides in which RGD sequence-containing regions in collagen are linked. An example of such a recombinant peptide is cellnest (Fujifilm Corporation).

[0066] Examples of the aforementioned lipids include, but are not limited to, phospholipids and unsaturated fatty acids.

[0067] Other components that can be added to the culture medium include, but are not limited to, ascorbic acid, serum, insulin transferrin selenite (ITS), transferrin, sodium selenite, pyruvate, proline, albumin, lipoprotein, and certoplasmin. The serum can include, but is not limited to, fetal bovine serum (FBS) and human serum. In the production method of the present invention, the culture medium is preferably a medium containing FBS, and more particularly may be DMEM / F12 containing FBS. For more efficient culture, the FBS content may be preferably 1 to 30% by volume, more preferably 10 to 30% by volume, more preferably 12 to 28% by volume, and even more preferably 15 to 25% by volume relative to the total volume of the culture medium. Furthermore, in the manufacturing method of the present invention, the culture medium may be a serum-free medium. Since additives such as ITS can substitute for the function of serum, by including such additives in the culture medium, cultivation in the manufacturing method of the present invention can be carried out in a serum-free medium. In addition, by using a serum-free medium, the risks associated with using biological raw materials when transplanting to humans can be avoided.

[0068] Preferably, the culture medium used in the present invention may contain ascorbic acid at a concentration of, for example, 0.01 to 1 mg / mL, preferably 0.05 to 0.5 mg / mL, and more preferably 0.07 to 0.3 mg / mL relative to the volume of the medium, for better cell proliferation. Ascorbic acid promotes the production of articular cartilage-specific substrates from cultured cells and / or makes the phenotype expression of the cell culture more suitable for cartilage repair. That is, ascorbic acid can contribute to the regeneration of damaged articular cartilage with hyaline cartilage. If the ascorbic acid concentration is too high, the adhesion of cultured cells to the porous membrane may be hindered. If the ascorbic acid concentration is too low, the effect may not be achieved. In a preferred embodiment, the cell culture of the present invention is obtained by culturing cells in a culture medium, preferably in a culture medium containing FBS and / or ascorbic acid, and may be obtained by culturing cells in DMEM / F12 containing FBS and / or ascorbic acid.

[0069] In the manufacturing method of the present invention, the culture period on the cell membrane may be appropriately selected depending on the state of the culture, such as the state of phenotypic expression, but may be, for example, 10 to 20 days, preferably 11 to 18 days, and more preferably 12 to 16 days. Depending on the culture period, the cell culture may become more suitable for cartilage repair.

[0070] In the culture described above, the cell population may be in contact with the culture medium on the upper side of the porous membrane and in contact with the culture medium on the lower side of the porous membrane via the pores of the porous membrane. By culturing in this state, adhesion proteins can be produced not only on the adhesion surface of the cell sheet to the porous membrane, but also on the opposite side of the adhesion surface, the surface in contact with the upper culture medium. The cell sheet of the present invention may be more suitable for cartilage repair by having a substrate on both sides of the cell sheet. For culturing in the aforementioned state, a cell insert (also called a cell culture insert) or a culture vessel having a similar structure may be used. For example, a culture vessel having a cell insert on which a stimulus-responsive polymer (e.g., a temperature-responsive polymer) is immobilized on the porous membrane portion may be used in the manufacturing method of the present invention. The cell population may be cultured on the surface of the porous membrane on which the stimulus-responsive polymer (e.g., a temperature-responsive polymer) is immobilized. The immobilization of the stimulus-responsive polymer may be carried out by a method known to those skilled in the art, for example, by the method described in Japanese Patent Publication No. 2-211865. The cell insert on which the stimulus-responsive polymer is immobilized may be a commercially available one, for example, Thermo Scientific® Nunc® CC insert #140660, BD Falcon cell culture inserts #353090, #353490, 353102, #353091, #353092, and #353093 may be used.

[0071] In the manufacturing method of the present invention, the culture vessel may be appropriately selected by those skilled in the art, and examples include, but are not limited to, dishes, multiwell plates, flasks, and similar forms of containers.

[0072] The characteristics of the cell culture obtained by the manufacturing method of the present invention are as described in Section 1 above. Furthermore, the cells cultured in the culture step and the method for preparing them are as described in Section 1 above.

[0073] 2-2.Analysis process

[0074] The manufacturing method of the present invention preferably includes an analysis step of analyzing the expression intensity of cell surface markers of the cell population contained in the cell culture. Preferably, in the analysis, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b, and / or Expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 The following is measured. This measurement is preferably performed by flow cytometry. The flow cytometry measurement may be performed as described in "5. Examples" below.

[0075] In one embodiment of the present invention, the expression intensity of at least CD99 and / or GD2 is measured in the analysis step, and / or the expression intensity of at least CD26 and / or CD73 is measured. These four cell surface markers are particularly suitable for evaluating cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics.

[0076] In another embodiment of the present invention, the analysis step involves measuring the expression intensity of at least CD99 and / or GD2, and / or measuring the expression intensity of at least one, two, or three of CD26, CD73, and CD44. These five cell surface markers are particularly suitable for evaluating cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics.

[0077] In yet another embodiment of the present invention, the expression intensity of at least CD99 and / or GD2 is measured in the analysis step, and / or the expression intensity of at least CD44 is measured. These three cell surface markers are particularly suitable for evaluating cartilage-like tissue formation characteristics, in particular hyaline cartilage-like tissue formation characteristics.

[0078] 2-3. Judgment process

[0079] Preferably, the manufacturing method of the present invention may include a determination step of determining whether the cell culture has cartilage-like tissue formation characteristics (particularly whether it has hyaline cartilage-like tissue formation characteristics) based on the expression intensity of the cell surface marker obtained in the analysis step. In the determination step, it may be determined, based on the expression intensity of the cell surface markers obtained in the analysis step, whether the cell culture is suitable for the repair of cartilage tissue, particularly hyaline cartilage tissue, and more particularly knee hyaline cartilage tissue. In the determination step, preferably with respect to the cell population contained in the cell culture, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each surface marker, and / or Is the expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 equal to or greater than the threshold for each surface marker? This is determined.

[0080] Particularly preferably, in the determination step, if the standardized mean fluorescence intensity measured when the cell population contained in the cell culture is analyzed by flow cytometry satisfies at least one of the conditions 1 to 10 described in 1. above, and / or satisfies at least one of the conditions 11 to 20 described in 1. above, the cell culture may be determined to have cartilage-like tissue formation characteristics, for example, it may be determined to have hyaline cartilage-like tissue formation characteristics. Furthermore, in the determination step, if the standardized mean fluorescence intensity measured when the cell population contained in the cell culture is analyzed by flow cytometry satisfies at least one of the conditions 1 to 10 described in 1. above, and / or satisfies at least one of the conditions 11 to 20 described in 1. above, the cell culture may be determined to be suitable for the repair of cartilage tissue, particularly hyaline cartilage tissue, and more particularly knee hyaline cartilage tissue.

[0081] In the determination step described above, a cell culture determined to be suitable for cartilage repair may be used for transplantation into a human. That is, in the determination step described above, if the standardized mean fluorescence intensity measured when the cell population contained in the cell culture is analyzed by flow cytometry satisfies at least one of the conditions 1 to 10 described in 1. above, and / or satisfies at least one of the conditions 11 to 20 described in 1. above, it may be determined that the cell culture may be used for transplantation into a human knee.

[0082] 3. Evaluation Method

[0083] The present invention also provides a method for evaluating cell cultures. The evaluation method may include a determination step of determining whether the cell culture has cartilage-like tissue formation characteristics (particularly whether it has hyaline cartilage-like tissue formation characteristics) based on the expression intensity of cell surface markers of the cell population contained in the cell culture. Furthermore, in the determination step, it may be determined whether the cell culture is suitable for the repair of cartilage tissue, particularly hyaline cartilage tissue, and more particularly knee hyaline cartilage tissue, based on the expression intensity of the cell surface markers obtained in the analysis step described in 2 above. The aforementioned determination step may be the same as the determination step described in section 2 above. For example, the determination may be made based on whether the expression intensity of the specific cell surface markers listed in section 2 above, particularly the standardized mean intensity, satisfies the conditions defined for each cell surface marker. Furthermore, the evaluation method may further include an analysis step of analyzing the cell population by flow cytometry. The determination in the judgment step may be made based on the analysis results in the analysis step. The analysis step may be the same as the analysis step described in section 2 above.

[0084] The expression intensity of the specific cell surface markers mentioned above is an effective indicator for determining whether a cell culture has cartilage-like tissue formation characteristics (particularly hyaline cartilage-like tissue formation characteristics). Therefore, the evaluation method of the present invention enables appropriate evaluation of cartilage repair suitability, particularly hyaline cartilage repair suitability. Furthermore, since the evaluation method of the present invention determines suitability for cartilage repair based on the expression intensity of cell surface markers, it is possible to determine cartilage repair suitability without inducing cell cultures into cartilage-like tissue. Therefore, the time required for cartilage repair suitability can be reduced. Moreover, since the evaluation method of the present invention can be performed in a short time, it can be incorporated as one of the processes in the production of cell cultures for cartilage repair, thereby enabling the more reliable production of cell cultures with cartilage repair suitability. In addition, the evaluation method of the present invention can evaluate cartilage repair suitability at a lower cost compared to actually inducing cell cultures into cartilage-like tissue and evaluating cartilage repair suitability.

[0085] 4. Markers for evaluating cartilage-like tissue formation characteristics

[0086] The present invention provides a marker for evaluating cartilage-like tissue formation characteristics. The marker is At least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b, and / or It includes at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90. These cell surface markers are suitable for evaluating the cartilage-like tissue formation characteristics of cell cultures.

[0087] In one embodiment of the present invention, the marker for evaluating cartilage-like tissue formation characteristics comprises CD99 and / or GD2, and / or CD26 and / or CD73. These cell surface markers are particularly suitable for evaluating the cartilage-like tissue formation characteristics of cell cultures.

[0088] In another embodiment of the present invention, the marker for evaluating cartilage-like tissue formation characteristics of the present invention comprises CD99 and / or GD2, and / or one, two, or three of CD26, CD73, and CD44. These cell surface markers are particularly suitable for evaluating the cartilage-like tissue formation characteristics of cell cultures.

[0089] In yet another embodiment of the present invention, the marker for evaluating cartilage-like tissue formation characteristics of the present invention comprises CD99 and / or GD2 and / or CD44. These cell surface markers are particularly suitable for evaluating the cartilage-like tissue formation characteristics of cell cultures.

[0090] With respect to these cell surface markers, for example, the cartilage-like tissue formation characteristics of cell cultures can be evaluated by performing the analysis and determination steps described in sections 2 and 3 above.

[0091] Furthermore, the present invention also provides markers for evaluating cartilage repair suitability. These markers include cell surface markers described above in relation to markers for evaluating cartilage-like tissue formation characteristics.

[0092] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0093] 5. Example 1

[0094] We confirmed, as described below, that cell cultures containing cell populations expressing specific cell surface markers at specific expression levels possess cartilage-like tissue formation characteristics.

[0095] Specifically, in step 1 of Figure 1, multiple sheet-like cell cultures were prepared. Next, in step 2, the expression of cell surface markers was analyzed for these sheet-like cell cultures. For this expression analysis, the sheet-like cell cultures were isolated, stained with antibodies, and subjected to FACS analysis. Furthermore, in step 3, the cartilage-like tissue formation characteristics were evaluated. For this evaluation, three-dimensional culture of the sheet-like cell cultures and analysis of the expression ratios of COL2A1 and COL1A1 were performed. Next, in step 4, correlation analysis was performed using the results from steps 2 and 3. This correlation analysis confirmed that cell cultures containing cell populations expressing specific cell surface markers at specific expression levels possessed cartilage-like tissue formation characteristics. The details of steps 1-4 above are explained below.

[0096] 5-1. Step 1: Preparation of cell culture A total of 16 types of cartilage tissue fragments (cartilage derived from discarded tissue during polydactyly surgery) were prepared. Each of these cartilage tissue fragments was treated with enzymes (Collagenase Type-1 / Worthington CLS-1 or Liberase MNP-S / Roche) to isolate chondrocytes from each fragment. The isolated chondrocytes were seeded into culture inserts (UpCell® inserts, CellSeed Co., Ltd.). The seeding day was designated as day 0, and the culture medium was changed every 3-4 days. After 10-21 days, sheet-like cell cultures derived from chondrocytes in each cartilage tissue fragment were obtained.

[0097] 5-2. Step 2: Analysis of cell surface marker expression Each of the 16 sheet-like cell cultures obtained in Step 1 was washed with PBS and reacted with Accutase (Becton Dickinson) at room temperature for 15 minutes. After this reaction, each sheet-like cell culture was collected and the Accutase was removed. After removal, 0.25 mg / ml Collagenase P (Roche) was reacted at 37°C for 30 minutes to disperse the cells in each sheet-like cell culture. The dispersed cells were washed with PBS and suspended in 0.2% FBS / 5 mM EDTA / PBS to obtain suspensions. Antibodies against each of the 242 cell surface markers were added to the suspensions containing the cell populations obtained from each sheet-like cell culture, and the mixture was reacted on ice for 30 minutes. That is, 242 antibody-containing suspensions were obtained for each sheet-like cell culture. Table 1 below shows the antibodies against the cell surface markers that showed correlation in Step 4 described below.

[0098] In Table 1 below, "Fluorescent Dye" refers to the fluorescent dye used to label each antibody. "Target Surface Antigen" refers to the cell surface antigen targeted by each antibody. "Antibody Manufacturer" and "Antibody Lot Number" refer to the manufacturer and lot number (catalog number) of each antibody. Regarding the antibody manufacturers, BD is Becton Dickinson, SC is SANTA CRUZ BIOTECHNOLOGY, and BL is BioLegend.

[0099] [Table 1]

[0100] After the reaction on ice as described above, each cell population was washed with 0.2% FBS / 5mM EDTA / PBS, and the fluorescence intensity of each cell was measured using a flow cytometer (BD FACSVerse, Becton Dickinson). Analysis was performed using the analysis software Flowjo (Becton Dickinson). Single cells and live cell populations were gated using FSC and SSC, and the median fluorescence intensity for each fluorescently labeled antibody within each population was determined and normalized using the median fluorescence intensity of the unstained samples.

[0101] 5-3. Step 3 (Evaluation of cartilage-like tissue formation characteristics) A basic culture medium having the composition shown in Table 2 below was prepared.

[0102] [Table 2]

[0103] To the aforementioned basic medium, sodium pyruvate, dexamethasone, L-proline, insulin-transferrin-selenate, and TGF-β1 were added at the concentrations shown in Table 3 below to obtain induction medium.

[0104] [Table 3]

[0105] Each sheet cell culture was rolled into a spherical shape and placed in a low-adhesion 6-well plate containing the obtained induction medium, and cultured for 7 days under conditions of 5% CO2 and 37°C. The spherical tissue was harvested, RNA was extracted from the spherical tissue, and cDNA was synthesized from the RNA. The gene expression ratio of COL2A1 and COL1A1 was determined from the obtained cDNA using the qPCR method. The gene expression ratios for each of the 16 sheet cell cultures are shown in Figure 2. In Figure 2, the gene expression ratio of COL2A1 and COL1A1 in the sheet cell culture of donor ID 11 (11 on the horizontal axis) is shown as 1, and the gene expression ratios of the other sheet cell cultures are shown as relative values ​​to the gene expression ratio of the sheet cell culture of donor ID 11.

[0106] 5-4. Step 4 (Correlation Analysis) The gene expression ratio of the sheet cell culture from donor ID 11 is the same as that of sheet cell cultures created from cartilage discarded from total knee arthroplasty (hereinafter referred to as "TKA-derived sheet cell cultures") (Sato M et al., Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis., NPJ Regen Med, 4, 4, 2019.), which have been shown to possess cartilage-like tissue formation characteristics (particularly hyaline cartilage-like tissue formation characteristics). In other words, it can be used as a standard for cell cultures that possess cartilage-like tissue formation characteristics. Therefore, sheet cell cultures with a COL2A1 / COL1A1 gene expression ratio equal to or higher than that of donor ID 11 can be determined to possess cartilage-like tissue formation characteristics.

[0107] Pearson's product-moment correlation analysis was used to examine the correlation between 242 types of cell surface markers and the COL2A1 / COL1A1 gene expression ratio. As shown in Table 1, 20 types of cell surface markers were found to have a positive or negative correlation with the COL2A1 / COL1A1 gene expression ratio. Specifically, it was found that CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b have a negative correlation with the COL2A1 / COL1A1 gene expression ratio. Therefore, cell cultures containing cell populations with expression intensities below the threshold of these surface markers can be used as having cartilage-like tissue formation characteristics and are suitable for cartilage repair. Furthermore, it was found that CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 have a positive correlation with the COL2A1 / COL1A1 gene expression ratio. Therefore, cell cultures containing cell populations whose expression intensities of these surface markers are above a threshold can be used as having cartilage-like tissue formation characteristics and are suitable for cartilage repair.

[0108] Furthermore, among the 10 cell surface markers that negatively correlate with the COL2A1 / COL1A1 gene expression ratio, CD99 and GD2 showed stronger negative correlations (CD99: r = -0.430, GD2: r = -0.397) and exhibited a clear difference from the negative control. Therefore, cell cultures containing cell populations in which the expression levels of at least CD99 and / or GD2 are below the threshold for each of these cell surface markers can be used as having excellent cartilage-like tissue formation characteristics and are particularly suitable for cartilage repair (especially hyaline cartilage repair). Furthermore, among the 10 cell surface markers that show a positive correlation with the COL2A1 / COL1A1 gene expression ratio, CD26 and CD73 showed a stronger positive correlation (CD26: r=0.485, CD73: r=0.456) and exhibited a clear difference from the negative control. Therefore, cell cultures containing cell populations in which the expression intensity of at least CD26 and / or CD73 is above the threshold for each of these cell surface markers can be used as having excellent cartilage-like tissue formation characteristics and are particularly suitable for cartilage repair (especially hyaline cartilage repair).

[0109] Furthermore, regarding the 20 cell surface markers that have a positive or negative correlation with the COL2A1 / COL1A1 gene expression ratio, Table 4 below shows the nMFI of the sheet cell culture with the lowest COL2A1 / COL1A1 gene expression ratio among sheet cell cultures having a COL2A1 / COL1A1 gene expression ratio equal to or greater than that of TKA-derived sheet cell cultures.

[0110] [Table 4]

[0111] For example, with respect to the positively correlated cell surface marker CD26, the nMFI of the sheet cell culture with the lowest COL2A1 / COL1A1 gene expression ratio among sheet cell cultures having a COL2A1 / COL1A1 gene expression ratio equal to or higher than that of TKA-derived sheet cell cultures was 2.9, as shown in Table 4 above. Therefore, when a cell population in a sheet-like cell culture is labeled with an anti-CD26 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is, for example, 2.3 or higher, more preferably 2.6 or higher, and even more preferably 2.9 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0112] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD166 antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is, for example, 240 or less, more preferably 220 or less, and even more preferably 200 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0113] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD165 antibody conjugated to BB700, if the standardized mean fluorescence intensity derived from BB700 is 132 or less, more preferably 120 or less, and even more preferably 110 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0114] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD99 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 1.9 or less, more preferably 1.8 or less, and even more preferably 1.6 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0115] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-GD2 antibody conjugated to BB700, if the standardized mean fluorescence intensity derived from BB700 is 3.6 or less, more preferably 3.3 or less, and even more preferably 3.0 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0116] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-STRO-1 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 1.4 or less, more preferably 1.3 or less, and even more preferably 1.2 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0117] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD108 antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is 1.9 or less, more preferably 1.8 or less, and even more preferably 1.6 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0118] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD164 antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is 3.0 or less, more preferably 2.8 or less, and even more preferably 2.5 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0119] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD6 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 1.4 or less, more preferably 1.3 or less, and even more preferably 1.2 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0120] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD106 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 2.4 or less, more preferably 2.2 or less, and even more preferably 2.0 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0121] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD107b antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 2.0 or less, more preferably 1.9 or less, and even more preferably 1.7 or less, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0122] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD73 antibody conjugated to FITC, if the standardized mean fluorescence intensity derived from the FITC is 47 or higher, more preferably 53 or higher, and even more preferably 59 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0123] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD105 antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is 21 or higher, more preferably 24 or higher, and even more preferably 27 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0124] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD44 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 128 or higher, more preferably 145 or higher, and even more preferably 160 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0125] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD120a antibody conjugated with APC, if the standardized mean fluorescence intensity derived from the APC is 24 or higher, more preferably 27 or higher, and even more preferably 30 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0126] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD201 antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is 18 or higher, more preferably 20 or higher, and even more preferably 23 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0127] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-EGFR antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is 3.2 or higher, more preferably 3.6 or higher, and even more preferably 4.0 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0128] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD146 antibody conjugated with FITC, if the standardized mean fluorescence intensity derived from the FITC is 1.6 or higher, more preferably 1.8 or higher, and even more preferably 2.0 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0129] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD140a antibody conjugated with PE, if the standardized mean fluorescence intensity derived from the PE is 5.6 or higher, more preferably 6.4 or higher, and even more preferably 7.0 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0130] Similarly, when a cell population in a sheet-like cell culture is labeled with an anti-CD90 antibody conjugated with APC, if the standardized mean fluorescence intensity derived from the APC is 800 or higher, more preferably 900 or higher, and even more preferably 1000 or higher, then the sheet-like cell culture can be used as having cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0131] 6. Example 2

[0132] Regarding CD44, one of the cell surface markers mentioned in "5. Example 1" above, its usefulness in evaluating cartilage-like tissue formation characteristics was confirmed as described below.

[0133] Specifically, in step 2-1, multiple sheet-like cell cultures were prepared. Next, in step 2-2, CD44 expression analysis was performed on these multiple sheet-like cell cultures. In addition, in step 2-3, three-dimensional culture was performed on the multiple sheet-like cell cultures, and their cartilage-like tissue formation characteristics were confirmed by staining. The details of these steps and the results obtained are described below.

[0134] 6-1. Preparation of cell cultures (Step 2-1) As described in Step 1 of "5. Example 1" above, sheet-like cell cultures derived from chondrocytes in each of the three types of cartilage tissue fragments were obtained. The three obtained sheet-like cell cultures will be referred to below as "Sample No. 1," "Sample No. 2," and "Sample No. 3," respectively.

[0135] 6-2. CD44 expression analysis (Step 2-2) For each of the three sheet-like cell cultures obtained in Step 2-1, the degree of CD44 expression was analyzed by the following cell dispersion treatment and FCM measurement.

[0136] (1) Cell dispersion treatment

[0137] <Reagents> 20% FBS DMEM-F12 / AB / AA (hereinafter also referred to as "AA(+) medium") DPBS(-)(gibco:14190-250) Liberase TM TL Research Grade (sigma:54010200001) (hereinafter also referred to as "Liberase TL") Injectable water (Fujifilm Wako Pure Chemical Industries: 161-08247) 5.0g / l-Trypsin / 5.3mmol / l-EDTA Solution (Nacalai Tesque: 35556-44) (hereinafter also referred to as "Trypsin / EDTA") 0.4% Trypan Blue Stain (Invitrogen, T10282) STEM-CELLBANKER (ZENOAQ RESOURCE, CB045) (hereinafter also referred to as "cell banker")

[0138] <Instructions>

[0139] <0.65 Units / mL Liberase TL preparation> 1. Add 2 mL of injection water to Liberase TL and mix (hereinafter, the resulting mixture will also be referred to as "13 Units / mL Liberase TL"). Add 5.7 mL of AA(+) medium to a 2.50 mL tube. 3.13 Add 300 μL of Liberase TL (Units / mL) and mix (hereinafter, the resulting mixture will also be referred to as "0.65 Units / mL Liberase TL").

[0140] <Cell dispersion> 1. Prepare an appropriate number of cell sheets that have been cultured for 14 days. Dispense 3 ml / well of DPBS(+) into a 2.6-well plate. 3. Move the temperature-responsive insert (CellSeed: CS6301) to step 2. 4. Place 10 mL of Trypsin / EDTA into a 50 mL tube and warm it to 37°C. 5. Remove the culture medium from the insert and wash with 2 mL / well of DPBS(-). 6. Peel off the sheet and place it in the tube from step 4 (up to 4 sheets / tube is acceptable). Place in a 7.37℃ water bath and incubate for 30 minutes (shake the tube as needed). 8. Add 10 mL of AA(+) medium and stop the enzyme treatment. Centrifuge 9.350 × g for 5 minutes at 25°C. 10. Remove the supernatant. 11.0.65 Units / mL Add 3 mL of Liberase TL Place in a 12.37℃ water bath and incubate for 30 minutes (shake the tube as needed). Steps 13-16 are followed by step 17 once the cell clumps are gone. 13. If cell clumps remain, add 1 mL of 0.65 Units / mL Liberase TL. Place in a 14.37℃ water bath and incubate for 15 minutes (shake the tube as needed). 15. If cell clumps remain, add 1 mL of 0.65 Units / mL Liberase TL. Place in a 16.37℃ water bath and incubate for 15 minutes (shake the tube occasionally until the cell clumps disappear). 17. Once the cell clumps are gone, add the remaining 0.65 Units / mL Liberase TL. Pass the sample through an 18.40 μm cell strainer and collect it in a new 50 mL tube. 19. Rinse the 50 mL tube with 5 mL of AA(+) medium, pass it through the 40 μm cell strainer from step 18, and collect it in the 50 mL tube. Centrifuge 20.350 × g for 5 minutes at 25°C. 21. Remove the supernatant. 22. Resuspend in AA(+) medium. 23. Count the number of cells. Centrifuge at 25°C for 5 minutes at 24.350 × g. 25. Remove the supernatant. →Perform the following FCM measurements. →If you are not going to perform an FCM measurement immediately, store the product using the following method. 26. Suspend in cell banker. 27. Dispense into cryotubes. Store frozen at 28.-80°C. 29. Transfer to -150°C the following day or later for storage.

[0141] (2)FCM measurement

[0142] <Reagents> DPBS(-)(gibco:14190-250) 0.4% Trypan Blue Stain (Invitrogen, T10282)

[0143] <Antibody> NEG.CTRL IgG1(mouse)-FITC 100t CE (Beckman & Coulter: A07795) (hereinafter also referred to as "IgG1-FITC") MOUSE IgG1-APC, 100t CE (Beckman & Coulter: IM2475) (hereinafter also referred to as "IgG1-APC") Hu CD44 FITC G44-26 100Tst (BD Pharmingen: 559596) (hereinafter also referred to as "CD44-FITC")

[0144] <Instructions> 1. Cell suspension with live cells: 1 × 10 5 ~2×10 5 Dispense two 1.5 mL tubes of cells into each tube. 2. Add 1 mL of PBS(-) and suspend. Centrifuge 3.350 × g for 3 minutes at 4°C. 4. Remove the supernatant. 5. Add 1 mL of PBS(-) and suspend. Centrifuge 6.350 × g for 3 minutes at 4°C. 7. Remove the supernatant. 8. Add 50 μL of PBS(-) and suspend. 9. Add the antibody 5 μL / tube at a time and suspend. Tube 1: IgG1-FITC·IgG1-APC Tube 2: CD44-FITC 10. Incubate in a dark place at 4°C for 30-60 minutes. 11. Add 1 mL of PBS(-) and suspend. Centrifuge 12.350 × g for 3 minutes at 4°C. 13. Remove the supernatant. 14. Add 0.3 mL of PBS(-) and suspend. 15. Add to a 5 mL tube with a cell strainer. 16. Measure and analyze using BD FACSVerse.

[0145] The measurements and analyses were performed using BD FACSuite Software Application (Becton Dickinson). Specifically, the percentage of CD44 was calculated using a count of 10,000, with the control (IgG1-FITC) set to 1%. Here, "percentage of CD44" refers to the proportion of cells stained with anti-CD44 antibody, relative to the control (1%). The histogram range was set so that the control (IgG1-FITC) was 1% when overlaid, and the count (event) value at this time was in the range of 95 to 104.

[0146] 6-3. Three-dimensional culture and staining process (Steps 2-3) Each of the three sheet-like cell cultures obtained in Step 2-1 was subjected to the following three-dimensional culture and staining treatments.

[0147] (1) Three-dimensional culture treatment As described in step 3 of "5. Example 1" above, each sheet-like cell culture was rolled into a spherical shape and placed in a low-adhesion 6-well plate containing induction medium, and cultured for 7 days under conditions of 5% CO2 and 37°C. After the culture, the spherical tissues were harvested.

[0148] (2) Disinfection treatment The following safranin O staining treatment was performed on the spherical tissue in question.

[0149] <Reagents> Meyer Hematoxylin Solution (Fujifilm Wako Pure Chemical Industries, 131-09665) 0.08% Fast Green (Prepared Reagent) 1% aqueous acetate (reagent preparation) 0.1% Safranin O (Muto Chemical, 42262) 100% Ethanol (Fujifilm Wako Pure Chemical Industries, 054-00461) Xylene (Fujifilm Wako Pure Chemical Industries, 241-00096) Marinol 750cps (Muto Chemical, 20091)

[0150] <Procedure for preparing 1% acetate> 1. Mix acetic acid and purified water in a staining vat in a ratio of 1:99.

[0151] <Dyeing Procedure> 1. Rinse the microscope slide with water for 10 minutes. 2. While checking the degree of staining under a microscope, stain with hematoxylin (for about 10 seconds). 3. Rinse the microscope slide with water for 5 minutes. 4. Soak in 0.08% Fast Green for 5 minutes. Immerse in 5.1% acetic acid solution for 10 seconds. Soak in 6.0.1% safranin O for 5 minutes. Prepare three trays filled with 7.100% ethanol, and immerse the contents of each tray for approximately 10 seconds, repeating this process three times. 8. Prepare three trays containing xylene, and immerse each tray in the xylene while shaking it for about 10 seconds, repeating this process three times. 9. Place about two drops of Marinel on the coverslip and mix in the xylene. 10. Slowly place the slide over it. 11. Observe and photograph with a microscope.

[0152] 6-4. Results and Discussion The results of the expression analysis obtained in step 2-2 above are shown in Table 5 below. In Table 5 below, the unit is %. CD44 expression in the sheet cell cultures of sample No. 2 and sample No. 3 is stronger than that in sample No. 1.

[0153] [Table 5]

[0154] Furthermore, the staining results obtained in steps 2-3 above are shown in Figure 3.

[0155] As shown in Figure 3, the spherical tissues of samples No. 2 and No. 3 are stained more strongly with safranin O than the spherical tissue of sample No. 1. Stronger staining with safranin O in the spherical tissue indicates superior cartilage-like tissue formation characteristics. Therefore, the results shown in Figure 3 indicate that the sheet-like cell cultures of samples No. 2 and No. 3 have superior cartilage-like tissue formation characteristics compared to sample No. 1.

[0156] The results shown in Table 3 and Figure 3 indicate that stronger CD44 expression in sheet-like cell cultures is associated with superior cartilage-like tissue formation characteristics. Furthermore, it can be confirmed that CD44 is a useful indicator for evaluating the cartilage-like tissue formation characteristics of sheet-like cell cultures. In particular, from the perspective of the measurement and analysis methods described in "6-2. CD44 Expression Analysis (Step 2-2)" above, when the percentage of CD44 relative to the control (IgG1-FITC) is set at 1%, preferably 90% or more, and more preferably 92% or more, the sheet-like cell culture can be used as having particularly excellent cartilage-like tissue formation characteristics and is considered suitable for cartilage repair.

[0157] 6-5. Evaluation based on the expression ratio of COL2 and COL1

[0158] The superior cartilage-like tissue formation characteristics of sheet-like cell cultures when the expression level of CD44 is high can be confirmed from the gene expression ratio of COL2 and COL1 and the staining treatment, as described below.

[0159] First, a sheet-like cell culture derived from chondrocytes in one type of cartilage tissue piece was obtained from the cartilage tissue piece by following the procedure described in Step 1 of "5. Example 1" above.

[0160] The resulting sheet-like cell cultures showed almost no expression of COL2 and a high degree of expression of COL1. In other words, these sheet-like cell cultures were negative for type II collagen and positive for type I collagen. Such sheet-like cell cultures possess excellent cartilage-like tissue formation characteristics, as described in International Publication No. 2018 / 154813.

[0161] CD44 expression analysis was performed on the sheet-like cell culture as described in step 2-2 above. As a result, the CD44 expression level in the sheet-like cell culture was 94.95, which was similar to that of samples No. 2 and No. 3.

[0162] Based on these results, it was confirmed that sheet-like cell cultures exhibit superior cartilage-like tissue formation characteristics when the expression level of CD44 is high.

Claims

[Claim 1] A method for evaluating cell cultures, Regarding the cell population contained in the cell culture, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each surface marker, and / or Is the expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD120a, CD201, EGFR, CD146, and CD140a equal to or greater than the threshold for each surface marker? Includes a determination process, The process further includes an analysis step of analyzing the aforementioned cell population by flow cytometry, The determination in the determination step is made based on the analysis results in the analysis step. The expression intensity is the standardized mean fluorescence intensity measured by analyzing the cell population by flow cytometry, and the standardized mean fluorescence intensity satisfies at least one of the following conditions 1 to 10 and / or satisfies at least one of the following conditions 11 to 16. In the determination step, The expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each surface marker, and / or The expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD120a, CD201, EGFR, CD146, and CD140a is greater than or equal to the threshold for each cell surface marker. In such cases, the sheet-like cell culture is determined to have cartilage-like tissue formation characteristics. The aforementioned evaluation method. Condition 1: When the cell population is labeled with an anti-CD166 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 240 or less. Condition 2: When the cell population is labeled with an anti-CD165 antibody conjugated with BB700, the standardized mean fluorescence intensity derived from BB700 is 132 or less. Condition 3: When the cell population is labeled with an anti-CD99 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.9 or less. Condition 4: When the cell population is labeled with an anti-GD2 antibody conjugated with BB700, the standardized mean fluorescence intensity derived from BB700 is 3.6 or less. Condition 5: When the cell population is labeled with an anti-STRO-1 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.4 or less. Condition 6: When the cell population is labeled with an anti-CD108 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 1.9 or less. Condition 7: When the cell population is labeled with an anti-CD164 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 3.0 or less. Condition 8: When the cell population is labeled with an anti-CD6 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.4 or less. Condition 9: When the cell population is labeled with an anti-CD106 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.4 or less. Condition 10: When the cell population is labeled with an anti-CD107b antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.0 or less. Condition 11: When the cell population is labeled with an anti-CD26 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.3 or higher. Condition 12: When the cell population is labeled with an anti-CD120a antibody conjugated with APC, the standardized mean fluorescence intensity derived from the APC is 24 or higher. Condition 13: When the cell population is labeled with an anti-CD201 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 18 or higher. Condition 14: When the cell population is labeled with an anti-EGFR antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 3.2 or higher. Condition 15: When the cell population is labeled with an anti-CD146 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.6 or higher. Condition 16: When the cell population is labeled with an anti-CD140a antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 5.6 or higher.

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