Cell sheet fragments, syringe containing the cell sheet fragments, method for manufacturing the cell sheet fragments, and method for using the same.

Abstract

To provide cell sheet pieces suitable for minimally invasive therapies.SOLUTION: The present invention provides cell sheet pieces formed from a culture of cells, which are capable of passing through a syringe needle. The invention also provides a method for producing small cell sheet pieces formed from cell cultures. The method comprises culturing cells on a surface of an instrument on which a stimulus-responsive polymer is immobilized and a subsection is formed to obtain the cell sheet pieces.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cell sheet fragments, syringes containing said cell sheet fragments, a method for manufacturing said cell sheet fragments, and a method for using said cell sheet fragments. [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 Initiative] [Problems that the invention aims to solve]

[0005] In treatments using transplant materials, it is sometimes necessary to employ highly invasive methods, such as exposing the affected area through incision and transplanting under direct visualization.

[0006] Therefore, the main objective of the present invention is to provide cell sheet fragments suitable for minimally invasive treatment. [Means for solving the problem]

[0007] In other words, the present invention provides a cell sheet fragment formed from a cell culture that can pass through an injection needle. The aforementioned injection needle may be an 18-gauge or thinner injection needle. The aforementioned cell sheet fragment has an area of ​​20 mm². 2 The following are possible: The aforementioned cell sheet fragments may be used for the repair of cartilage tissue. The aforementioned cells may originate from cartilage tissue. The aforementioned cartilage tissue may belong to an animal with polydactyly (extra fingers / toes). The aforementioned cells may be derived from stem cells. The aforementioned stem cells may include pluripotent stem cells, embryonic stem cells, or somatic stem cells. The aforementioned stem cells may include iPS cells.

[0008] Furthermore, the present invention also provides a syringe containing the aforementioned cell sheet fragments.

[0009] The present invention also provides a method for producing cell sheet fragments formed from a cell culture, comprising culturing cells on the surface of a device having a surface on which a stimulus-responsive polymer is immobilized and which has small compartments formed thereon, in order to obtain the cell sheet fragments. The aforementioned small section has an area of ​​20 mm². 2 The following are possible:

[0010] Furthermore, the present invention also provides a method for administering the cell sheet fragments to animals by injection. [Effects of the Invention]

[0011] The present invention provides cell sheet fragments suitable for minimally invasive treatment. [Brief explanation of the drawing]

[0012] [Figure 1] This graph shows the results of cell count and cell viability measurements. [Figure 2]It is a graph showing the measurement results of the secretion amount of the liquid factor. [Figure 3] It is a graph showing the quantitative analysis results of cartilage-related gene expression. [Figure 4] It is a graph showing the analysis results of cell surface markers. [Figure 5] It is a photograph showing the evaluation results of tissue sections. [Figure 6] It is a graph showing the measurement results of cell viability after injection administration.

Mode for Carrying Out the Invention

[0013] 1. Cell sheet piece

[0014] The cell sheet piece of the present invention can pass through an injection needle. Whether the cell sheet piece can pass through the injection needle can be confirmed using a liquid containing the cell sheet piece. Specifically, the syringe equipped with an injection needle and a plunger is filled with the liquid containing the cell sheet piece, and the plunger is pushed to confirm whether the cell sheet piece is ejected from the injection needle. The cell sheet piece ejected from the injection needle can be confirmed to be able to pass through the injection needle.

[0015] The above liquid may be appropriately prepared by those skilled in the art, but preferably, it can be prepared so that the cell viability of the cell sheet piece in the liquid is high. The cell viability of the cell sheet piece in the liquid can preferably be 80% or more. That is, the cell viability of the cell sheet piece of the present invention before passing through the injection needle can preferably be 80% or more. The cell viability of the cell sheet piece of the present invention can be measured by trypan blue assay.

[0016] The cell sheet fragments of the present invention can be used for treatment by injection because they can pass through an injection needle. In conventional treatments using cell sheets, the affected area is often exposed by surgical procedure, and the cell sheet is then applied to the exposed area. Such conventional treatments are highly invasive and place a significant burden on the patient. On the other hand, treatment performed by administering the cell sheet fragments of the present invention by injection is less invasive than the conventional treatments described above, and can reduce the burden on the patient. In other words, the cell sheet fragments of the present invention are suitable for minimally invasive treatments.

[0017] The above-mentioned injection needle may preferably have a thickness of 18 gauge or less. That is, the above-mentioned injection needle may preferably have an outer diameter of 1.2 mm or less. The cell sheet fragments of the present invention are more susceptible to damage the thinner the injection needle is when passing through it. Therefore, the above-mentioned injection needle may preferably have a thickness of 23 gauge or more. That is, the above-mentioned injection needle may preferably have an outer diameter of 0.6 mm or more.

[0018] It is preferable that the cell viability of the cell sheet fragments of the present invention does not decrease significantly immediately after passing through the injection needle compared to the cell viability before passing through the needle. The difference in cell viability before and immediately after passing through the injection needle may be, for example, 10 points or less. Furthermore, the difference in cell viability of the cell sheet fragments of the present invention 24 hours after passing through the injection needle may also be, for example, 10 points or less. The smaller the difference in cell viability before and after passing through the injection needle, the less susceptible the cell sheet fragments of the present invention are to the effects of injection. Such cell sheet fragments are more suitable for administration by injection.

[0019] The cell sheet fragments of the present invention may preferably have a cell viability of 80% or more immediately after passing through the injection needle. Furthermore, the cell sheet fragments of the present invention may preferably have a cell viability of 80% or more 24 hours after passing through the injection needle.

[0020] The cell sheet fragments of the present invention are sheet-shaped fragments, specifically fragments that have a two-dimensional surface and a thickness that is small compared to the surface area.

[0021] The smaller the area of ​​the cell sheet fragment, the easier it is for the injection needle to pass through. Therefore, the area of ​​the cell sheet fragment of the present invention is preferably 20 mm². 2 More preferably 15mm 2 More preferably, 10 mm 2 The following are possible. On the other hand, if the area is too small, it may be difficult to form the cell culture into a sheet shape. Therefore, the area is preferably 0.02 mm. 2 That is all that is possible.

[0022] The cell sheet fragments of the present invention are formed by culturing cells on equipment, as described later, and are recovered by detaching them from the equipment. These cell sheet fragments may be shrinkable. In this case, the apparent area of ​​the cell sheet fragment may differ between when it is on the equipment and when it is detached from the equipment. If the cell sheet fragments are shrinkable, the area of ​​the cell sheet fragment detached from the equipment can be measured when it is placed on a plane.

[0023] Furthermore, shrinkable cell sheet fragments may shrink and take on a clump-like shape after being detached from the equipment. Even in this case, the cell sheet fragments of the present invention can be confirmed to be sheet-shaped by spreading them out and arranging them on a plane. For example, spheroids formed by three-dimensional cell culture are clump-like but do not take on a sheet shape, and are therefore distinguished from the cell sheet fragments of the present invention. In other words, the cell sheet fragments of the present invention do not contain spheroids.

[0024] In one embodiment of the present invention, the cell sheet fragments may preferably be used for the repair of cartilage tissue. Conventional cell sheets for cartilage tissue repair are often administered by highly invasive surgical procedures. On the other hand, the cell sheet fragments for cartilage tissue repair of the present invention can be administered by injection, thus enabling a less invasive treatment than conventional cell sheets.

[0025] 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 sheet fragments of the present invention may be used to prevent diseases related to cartilage tissue. The cell sheet fragments of the present invention can be administered, for example, by injection to diseased cartilage or bone tissue. Examples of diseases to which the cell sheet fragments 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.

[0026] The cell sheet fragments of the present invention are formed from a cell culture. The cells used to form the cell sheet fragments are preferably animal cells. The animal is more preferably a mammal, even more preferably a primate, and particularly preferably a human.

[0027] In one embodiment of the present invention, the cells used to form the cell sheet fragments may be derived from cartilage tissue. That is, the cell sheet fragments of the present invention may be formed from a culture of cartilage tissue-derived cells. This makes the cell sheet fragments of the present invention more suitable for the repair of cartilage tissue.

[0028] The cells derived from the cartilage tissue mentioned above may be, for example, multiple cells obtained by separating cells contained in the cartilage tissue from the cartilage tissue. For example, these cartilage tissue-derived cells may be multiple cells recovered by treating the cartilage tissue with an enzyme to release cells from the cartilage tissue, and then recovering the released cells by centrifugation.

[0029] In the present invention, the cartilage tissue may be from an animal with polydactyly (or polytodactyly), or from an animal with polylimbs. In the present invention, "an animal with polydactyly (or polytodactyly)" means an animal that has polydactyly and / or polytodactyly (an animal that has an extra finger and / or extra toe). The animal is preferably a mammal, more preferably a primate, and even more preferably a human. The cartilage tissue may be taken from tissue obtained, for example, during the excision of an extra finger or toe. 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 (or polytodactyly) may be of the distal phalanx type, the middle phalanx type, or the proximal phalanx type. The extra finger or toe may be any finger or toe, for example, the thumb or the little finger. If the extra finger (toe) to be harvested is small and wart-like, all of the harvested subcutaneous tissue may be used. If the animal is human, for more efficient cultivation, the age of the human may preferably be 5 years or younger, more preferably 3 years or younger, even more preferably 2 years or younger, and even more preferably 1 year or younger.

[0030] Examples of enzymes used in the above-mentioned enzymatic treatment include collagenase, caseinase, clostripine, 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, clostripine, 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. 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.

[0031] 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 aggregates and supernatant by centrifugation. In the case of 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, especially 20 μm or smaller, and 15 μm or larger. 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.

[0032] The cells derived from the cartilage tissue described above 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 to manufacture the cell sheet pieces of the present invention.

[0033] The process of finely cutting the cartilage tissue described above can be carried out, for example, in a moist 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 above 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.

[0034] 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.

[0035] In other embodiments of the present invention, the cells used to form the cell sheet fragments may be derived from stem cells. These stem cells may include pluripotent stem cells, embryonic stem cells, or somatic stem cells, and may include, for example, iPS cells.

[0036] In this embodiment, for example, pluripotent stem cells (particularly iPS cells) can be differentiated by culturing them in a culture medium to obtain chondrocytes or chondrocyte-like cells, and these chondrocytes or chondrocyte-like cells can be further cultured on a surface immobilized with a temperature-responsive polymer to obtain the cell sheet fragments of the present invention. For example, pluripotent stem cells (particularly iPS cells) can also be seeded on equipment immobilized with a temperature-responsive polymer, differentiated into chondrocytes or chondrocyte-like cells, and cultured to obtain the cell sheet fragments of the present invention.

[0037] The cell sheet fragments of the present invention can be manufactured by the manufacturing method of the present invention described in section 3 below. Therefore, please refer to section 3 below for the manufacturing method of the cell sheet fragments of the present invention.

[0038] 2. Syringe containing a cell sheet fragment

[0039] The present invention also provides a syringe containing a cell sheet fragment. The characteristics of the cell sheet fragment are as described in section 1 above. The syringe of the present invention may contain a liquid containing the cell sheet fragment. Preferably, the syringe of the present invention may be a medical syringe containing an injectable agent containing the cell sheet fragment.

[0040] The syringe of the present invention comprises a needle and a plunger. The diameter of the needle may be appropriately selected by those skilled in the art, but it may be a needle of the diameter described in 1. above.

[0041] 3. Method for producing cell sheet fragments

[0042] The present invention also provides a method for producing cell sheet fragments. This method includes culturing cells on a surface of an apparatus having a surface on which a stimulus-responsive polymer is immobilized and which has small compartments formed thereon, in order to obtain the cell sheet fragments. The cell sheet fragments provided by the production method of the present invention may preferably be used for the repair of cartilage tissue. That is, the production method of the present invention may preferably be a method for producing cell sheet fragments for cartilage tissue repair.

[0043] The cells cultured in the manufacturing method of the present invention 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 the 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.

[0044] In the manufacturing method of the present invention, cells may be cultured on a surface of a device having a surface on which a stimulus-responsive polymer is immobilized. By culturing cells in a culture medium containing the device having a surface on which a stimulus-responsive polymer is immobilized, the culture (i.e., cell sheet fragments) on the surface can be detached from the device after culturing 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) are changed by 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 device.

[0045] In one embodiment of the present invention, the stimulus-responsive polymer is a temperature-responsive polymer. When the cells are cultured in a culture medium containing equipment having a surface on which the temperature-responsive polymer is immobilized, for example, after culturing, by raising the temperature of the culture medium to above the upper critical solution temperature or below the lower critical solution temperature of the temperature-responsive polymer, the surface changes from hydrophobic to hydrophilic, and as a result, the cell sheet fragments and the equipment become easier to separate. The culture in the manufacturing method of the present invention may preferably be a two-dimensional culture (also called a planar culture).

[0046] When detaching the cell sheet fragments from the equipment after the culture process, 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 sheet fragments can be detached from the equipment. Therefore, the cell sheet fragments produced by the manufacturing method of the present invention have the advantage of being able to be detached from the equipment without being damaged by such enzymes.

[0047] Treatment with proteolytic enzymes leads to the degradation of desmosome structures between cells and basement membrane-like proteins between cells and equipment, which can result in the cells in the cell culture separating into individual cells. On the other hand, cell sheet fragments obtained by the manufacturing method of the present invention can be detached from equipment 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 sheet fragments can be reduced. Furthermore, when cell sheet fragments obtained by the manufacturing method of the present invention are detached from equipment by changing the temperature of the culture medium, the basement membrane-like proteins are not destroyed by enzymes. Therefore, they can act more effectively on the affected tissue during treatment, enabling efficient treatment.

[0048] While dispase, a proteolytic enzyme, is known to be able to detach cell sheets while retaining 10-60% of the desmosome structure, it almost completely destroys the basement membrane-like protein, resulting in a weak cell culture. Cell sheet fragments produced by the manufacturing method of the present invention can be detached from equipment while retaining 80% or more of both the desmosome structure and the basement membrane-like protein.

[0049] The upper critical solution temperature or lower critical solution temperature of the temperature-responsive polymer used in the present invention is preferably 0 to 80°C, more preferably 20 to 50°C, and even more preferably 25 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 low, the cell proliferation rate may decrease or cells may die.

[0050] 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.

[0051] The above (meth)acrylamide compound may be, for example, acrylamide or methacrylamide.

[0052] The above 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).

[0053] The above N,N-dialkyl-substituted (meth)acrylamide derivatives 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).

[0054] The vinyl ether derivative mentioned above may be, for example, methyl vinyl ether (with a lower critical solution temperature of 35°C for the homopolymer).

[0055] 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.

[0056] The above polymers may be appropriately selected to select a temperature-responsive polymer having a critical solution temperature suitable for culturing or exfoliation in the present invention, or to adjust the interaction between the equipment and the culture, or to adjust the hydrophilicity or hydrophobicity of the surface of the equipment.

[0057] 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 equipment is preferably 0.3 to 5.0 μg / cm³. 2 More preferably 0.3 to 4.8 μg / cm³ 2 This is possible. By having the amount of temperature-responsive polymer immobilized within this range, more efficient culture can be performed than described above. If the amount of immobilized polymer is outside this range, cell sheet fragments may not be formed or may not be produced efficiently. Furthermore, by having the amount of immobilized polymer within this range, the cell sheet fragments can be more easily detached from the equipment.

[0059] In the manufacturing method of the present invention, the form of the equipment is not particularly limited, but examples include dishes, multiplates, flasks, cell inserts, etc.

[0060] The method of immobilizing the temperature-responsive polymer on the surface of the device can be carried out by, for example, the method described in Japanese Patent Laid-Open No. 2-211865. That is, the immobilization can be carried out by a method of bonding the device and the temperature-responsive polymer by a chemical reaction or a method of bonding them by a physical interaction. These methods may be used in combination.

[0061] In the method of bonding by the above chemical reaction, for example, electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, visible light irradiation, LED irradiation, plasma treatment, or corona treatment can be carried out. Alternatively, the temperature-responsive polymer may be immobilized on the device by a generally used organic reaction such as a radical reaction, an anion radical reaction, or a cation radical reaction. Further, a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded may be coated on the surface of the device as the temperature-responsive polymer component. Immobilization by physical adsorption or hydrophobicity may also be carried out.

[0062] In the method of bonding by the above physical interaction, the temperature-responsive polymer or a mixture of the polymer and an arbitrary medium can be applied to the device.

[0063] In the production method of the present invention, the cells are cultured on the surface of the device having a surface on which small compartments are formed. By culturing the cells on the surface on which the small compartments are formed, the cell culture can be formed into sheet-shaped small pieces. The area of the small compartment is preferably 20 mm 2 or less, more preferably 15 mm 2 or less, and even more preferably 10 mm 2 or less. On the other hand, if the area is too small, it may be difficult to form the cell culture into a sheet shape. Therefore, the area is preferably 0.02 mm 2 or more.

[0064] The equipment on which the above-mentioned temperature-responsive polymer is immobilized and on which the above-mentioned small compartments are formed may be commercially available, for example, RepCell® (Cellseed Co., Ltd.), a temperature-responsive culture dish with a grid wall on the culture surface, can be used.

[0065] 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.

[0066] Examples of the above-mentioned cell growth factors include, but are not limited to, TGF-β, b-FGF, IGF, EGF (Epidermal Growth Factor), BMP (Bone morphogenetic protein), Fibroblast growth factor receptor 3 (FGFR-3), Frizzled-related protein (FRZB), CDMP-1, Growth differentiation factor 5 (GDF-5), G-CSF (Granulocyte Colony Stimulating Factor), LIF (Leukemia Inhibitory Factor), interleukins, PDGF (Platelet-Derived Growth Factor), NGF (Nerve Growth Factor), the TGF (Transforming Growth Factor) family such as activin A, the Wnt family, and especially Wnt-3a (Wingless-type MMTV integration site family, member 3A). The TGF family includes TGF-β1, TGF-β2, and TGF-β3.

[0067] Examples of the above-mentioned hormones include, but are not limited to, insulin, transferrin, dexamethasone, estradiol, prolactin, glucagon, thyroxine, growth hormone, FSH (Follicle Stimulating Hormone), LH (Luteinizing Hormone), glucocorticoids, and prostaglandins.

[0068] Examples of the cell adhesion factors mentioned above 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).

[0069] Examples of the lipids mentioned above include, but are not limited to, phospholipids and unsaturated fatty acids.

[0070] 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 ceruloplasmin. 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.

[0071] Furthermore, in the manufacturing method of the present invention, the culture medium may be a serum-free medium. Additives such as ITS can substitute for the function of serum, so 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 administering to humans can be avoided.

[0072] When producing cell sheet fragments for cartilage tissue repair, the culture medium used in the present invention may preferably 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 culture medium, for better cell proliferation. Ascorbic acid promotes the production of articular cartilage-specific matrix from the cultured cells and / or the phenotype expression of the cell sheet fragments may become more suitable for cartilage tissue repair. That is, ascorbic acid may contribute to the regeneration of damaged articular cartilage with hyaline cartilage. If the ascorbic acid concentration is too high, the adhesion of the cultured cells to the equipment may be hindered. If the ascorbic acid concentration is too low, the effect may not be achieved.

[0073] In a preferred embodiment, the cell sheet fragments of the present invention are obtained by culturing cells in a culture medium, preferably in a culture medium containing FBS and / or ascorbic acid, and may be obtained, for example, by culturing cells in DMEM / F12 containing FBS and / or ascorbic acid.

[0074] In the manufacturing method of the present invention, the culture period on the cell apparatus 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 sheet fragments may become more suitable for cartilage repair.

[0075] The characteristics of the cell sheet fragments obtained by the manufacturing method of the present invention are as described in Section 1 above. Furthermore, the cells cultured in the manufacturing method of the present invention and the method for preparing them are as described in Section 1 above.

[0076] 4. How to use cell sheet fragments

[0077] The present invention also provides a method for administering cell sheet fragments to animals by injection. The cell sheet fragments are those described in 1. above. In the method of the present invention, the cell sheet fragments may be administered to animals using a syringe containing the cell sheet fragments described in 2. above. The animals are more preferably mammals, even more preferably primates, and particularly preferably humans.

[0078] The method of the present invention may be a method for repairing cartilage tissue. Because the cell sheet fragments can be administered by injection, the method of the present invention enables a less invasive treatment. In the method of the present invention, cartilage tissue repair includes, but is not limited to, treating inflamed and / or damaged cartilage tissue, reinforcing cartilage tissue, compensating for defects in cartilage tissue, and regenerating cartilage tissue. Furthermore, the present invention may be a method for preventing diseases related to cartilage tissue. In the method of the present invention, the cell sheet fragments can be administered, for example, by injection to diseased cartilage or bone tissue. Examples of diseases to which the method of the present invention is applicable include, but are not limited to, arthritis, arthropathy, cartilage damage, osteochondral damage, meniscal damage, and / or intervertebral disc degeneration. [Examples]

[0079] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0080] <Test Example 1> (Preparation of cell sheet fragments) A temperature-responsive culture dish (RepCell®, CellSeed Co., Ltd.) with a 3mm x 3mm grid wall on the culture surface, and a temperature-responsive culture insert (UpCell® insert, CellSeed Co., Ltd.) were used to collect 1 x 10⁻¹ chondrocytes isolated from the cartilage tissue of polydactyly patients. 4 pieces / cm 2 The cells were seeded at the specified density. The chondrocytes were cultured for two weeks. After allowing each culture medium to stand at room temperature for at least 30 minutes, the cell sheet fragments of the example (group M) were collected from the temperature-responsive culture dish, and the cell sheet of the comparative example (group S) was collected from the temperature-responsive culture insert. The cell sheet (group S) is a conventional cell sheet used in articular cartilage treatment.

[0081] (Evaluation of cell sheet fragments) Cell sheet fragments (Group M) and cell sheets (Group S) were evaluated based on the following criteria. • Measurement of cell count and cell viability • Measurement of humoral factor secretion levels by ELISA method (melanoma inhibitory activity (MIA) and TGF-β1) • Quantitative analysis of cartilage-related gene expression using qPCR (COL1A1, COL2A1, COL10A1, ACAN, SOX9, RUNX2, and MMP3) • Analysis of cell surface markers (CD29, CD31, CD44, CD45, CD73, CD81, CD90, and CD105) • Evaluation of tissue sections (staining with HE, safranin O, and toluidine blue) The results of each evaluation are shown in Figures 1 to 5.

[0082] As shown in Figure 1, there were no significant differences in cell number or cell viability between cell sheet fragments (group M) and cell sheets (group S). As shown in Figure 2, there were no significant differences in the secretion of humoral factors between cell sheet fragments (group M) and cell sheets (group S). As shown in Figure 3, there were no significant differences in the expression levels of cartilage-related genes between cell sheet fragments (group M) and cell sheets (group S), except for COL2A1. In cell sheet fragments (group M), the expression level of COL2A1 was lower than in cell sheets (group S). This is thought to be partly due to the fact that cell sheet fragments (group M) were cultured in flat culture dishes rather than inserts. As shown in Figure 4, there were no significant differences in cell surface markers between cell sheet fragments (group M) and cell sheets (group S). As shown in Figure 5, stratification of chondrocytes was observed in both cell sheet fragments (group M) and cell sheets (group S), but both safranin O and toluidine blue staining were poor.

[0083] (Verification of the effects of injection administration) To investigate the effect of injection on cell viability, the above cell sheet fragments (group M) were divided into the following four groups, and a test was conducted comparing cell viability immediately after injection, 4 hours later, and 24 hours later. • Non-injection control group 18 gauge needle group 23 gauge needle group • Syringe group (no needle, syringe only) After culturing for two weeks and allowing to stand at room temperature for at least 30 minutes, the cell sheet fragments (group M) were collected from the temperature-responsive culture dish. The cell sheet fragments (group M) were suspended in 3 mL of culture medium. For the injection administration group, the fragments were slowly drawn into a 5 mL syringe and then passed through an 18-gauge needle, a 23-gauge needle, or a syringe. Cell viability was measured by the trypan blue assay at 0 hours (immediately after injection), 4 hours, and 24 hours after injection. In addition, the cell viability of the cell sheet (group S) was measured at 0 hours (immediately after detachment) and 24 hours after detachment from the temperature-responsive culture insert. The results of the cell viability measurement are shown in Figure 6.

[0084] In the above test, the cell sheet fragments (group M) were able to pass through 18-gauge and 23-gauge injection needles. As shown in Figure 6, the cell viability was over 90% in all four groups at each time point. Although the cell viability tended to decrease over time, no significant differences were observed among the four groups at each time point (immediately after administration: p=0.748, 4 hours later: p=0.987, 24 hours later: p=0.994). From these results, it was confirmed that the cell sheet fragments of the present invention are less affected by injection administration.

[0085] <Test Example 2> A study was conducted to compare the cell sheet fragments of the present invention with conventional cell sheets and to verify the therapeutic effect of the cell sheet fragments on articular cartilage in a non-traumatic knee arthritis model. Chondrocytes derived from polydactyly were seeded in a conventional temperature-responsive culture dish and in a temperature-responsive culture dish (RepCell®, CellSeed Co., Ltd.) equipped with a 3mm x 3mm grid wall on the culture surface. Cell sheets were then prepared as a comparative example and as small cell sheet fragments as in the example. Cell number, cell viability, cell surface marker expression, COL2A1 / COL1A1 expression ratio, and humoral factor secretion were measured to compare the sheet characteristics. In the arthritis model, 18 F344 / NJcl-rnu / rnu cattle (8 weeks old) received intra-articular administration of 0.2 mg / 30 μL of monoiodoacetic acid (MIA) into their right knee, while the 6 control cattle received intra-articular administration of 30 μL of saline into their right knee. Patients were randomly assigned to one of three groups after MIA administration: a non-transplant group (Group A), a cell sheet transplant group (Group B), or a cell sheet fragment transplant group (Group C) (n=6 in each group). Four weeks after MIA administration, Group B received cell sheet transplantation to their right knee, while Group C received cell sheet fragments suspended in 30 μL of saline and injected into the right knee joint using a 23-gauge needle. Histological evaluation (OARSI score) was performed eight weeks after MIA administration.

[0086] As a result, there were no significant differences in the sheet characteristic evaluation items between cell sheets and cell sheet fragments. There were also no significant differences in the COL2A1 / COL1A1 expression ratio, melanoma inhibitory activity, and secretion levels of TGF-β1, ESM1, MCP-1, DKK-1, MMP-13, and MMP-3, which are used to evaluate the effectiveness of the sheets. The OARSI score on the femoral side was 11.0±3.0 in group A, 3.0±2.0 in group B, and 3.2±1.8 in group C. There was a significant improvement between group A and group B, and between group A and group C (P<0.01), but no significant difference between group B and group C.

[0087] These results suggest that the sheet properties of conventional cell sheets and the cell sheet fragments of the present invention are equivalent, and that the cell sheet fragments of the present invention, which can be transplanted with less invasiveness, may have the same therapeutic effect on articular cartilage as conventional cell sheets.

Claims

1. An injectable implant comprising a cell sheet fragment with an area of ​​20 mm² or less and 0.02 mm² or more, formed from a cell culture derived from cartilage tissue of an animal with polydactyly (polytodactyly), and passable through an 18-gauge or thinner injection needle. The aforementioned cell sheet fragment is a fragment that extends as a two-dimensional surface, and the aforementioned cell sheet fragment is not a spheroid formed by three-dimensional culture of cells. For the repair of cartilage tissue in animals, it is used to administer the injection into the cartilage tissue of the animal by passing the injection needle through the cartilage tissue without making an incision, into the joint. The aforementioned injectable agent for transplantation.

2. A transplant syringe formed from a cell culture derived from cartilage tissue of an animal with polydactyly (polytodactyly), which contains a cell sheet fragment with an area of ​​20 mm² or less and 0.02 mm² or more, through which an 18-gauge or thinner injection needle can pass, The aforementioned cell sheet fragment is a fragment that extends as a two-dimensional surface, and the aforementioned cell sheet fragment is not a spheroid formed by three-dimensional culture of cells. The aforementioned cell sheet fragments are used for intra-articular administration by injection through the injection needle without incision into the cartilage tissue of an animal, for the purpose of repairing the cartilage tissue of the animal. The aforementioned syringe for transplantation.

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