JP2015047076A - Cell culture substrate, method for differentiation-inducing of osteoblasts using the same and method for producing osteoblasts - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently and potently inducing the differentiation of osteoblasts as compared with conventional methods, in differentiation-inducing of osteoblast cells from mesenchymal stem cells.SOLUTION: The method for differentiation-inducing of osteoblasts comprises a step of bringing a fish-derived collagen enzyme-treated using transglutaminase or a cell culture substrate containing it or the cell culture substrate containing the fish-derived collagen enzyme-treated by transglutaminase into contact with mesenchymal stem cells.

Description

  The present invention relates to a cell culture substrate, and an osteoblast differentiation induction method and an osteoblast production method using the same. According to the present invention, osteoblasts can be induced from mesenchymal stem cells and maintained for a long period of time.

  Osteoblasts are cells that form bone in bone tissue and produce and secrete proteins and the like to create a bone matrix. Specifically, osteoblasts secrete matrix proteins such as collagen in vivo and produce matrix vesicles called Matrix Vesicles there. Then, calcium phosphate is deposited around the matrix vesicles to complete the bone matrix, and the osteoblasts eventually become bone cells in the matrix.

  This osteoblast is differentiated from mesenchymal stem cells, and Non-Patent Document 1 discloses that differentiation can be induced by allowing dexamethasone, β-glycerophosphate, and ascorbic acid to act on mesenchymal stem cells. (Non-Patent Document 1). Patent Document 1 discloses that by using a compound having a Rho kinase inhibitory activity such as (+)-trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane and an osteoinductive factor in combination, It discloses that mesenchymal cells are induced to differentiate into osteoblasts. Furthermore, Patent Document 2 discloses that a calcium antagonist induces differentiation of mesenchymal cells into osteoblasts (Patent Document 2).

  However, the osteoblast differentiation-inducing activity, particularly the early differentiation-inducing activity by the above-mentioned compounds has not been sufficient. Further, dexamethasone and (+)-trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane are not originally present in the living body, and these are not present in the obtained osteoblasts. The effects of the synthesized compounds have not been fully investigated. Furthermore, when differentiating mesenchymal stem cells into osteoblasts, culture is performed using about 10% bovine fetal serum, etc., and pathogens of zoonotic diseases such as prions are present in bovine serum. The possibility of doing was also considered.

  In order to solve the above problems, it has been reported that osteoblasts can be induced to differentiate using biologically derived fish-derived collagen (Patent Document 4). This method is a method for efficiently and powerfully inducing differentiation of osteoblasts as compared with the conventional method, and in particular, early differentiation induction was possible. However, it was not sufficient in terms of maintaining differentiation induction into osteoblasts for a long period of time.

International Publication No. 2001/017562 International Publication No. 2006/123699 JP 2006-257014 A JP 2012-120529 A

“Science”, 1999, 284, p. 143-147

  The object of the present invention is to provide a method for inducing differentiation of osteoblasts efficiently and powerfully in comparison with conventional methods in inducing differentiation of osteoblasts from mesenchymal stem cells, Another object of the present invention is to provide a method capable of maintaining osteoblast differentiation for a long period. Furthermore, it is providing the osteoblast manufactured by the said manufacturing method.

As a result of earnest research on the osteoblast differentiation induction method or osteoblast production method capable of maintaining osteoblast differentiation induction for a long period of time, the present inventors have surprisingly been treated with transglutaminase. It has been found that by contacting fish-derived collagen with mesenchymal stem cells, the mesenchymal stem cells can be differentiated into osteoblasts more efficiently than the method described in Patent Document 4. . Furthermore, it has been found that this differentiation induction into osteoblasts can be maintained for a long time.
The present invention is based on these findings.
Therefore, the present invention
[1] Fish-derived collagen that has been enzymatically treated with transglutaminase,
[2] Collagen selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, and mixtures thereof The collagen derived from fish according to [1],
[3] The fish-derived collagen according to [1] or [2], wherein the collagen is fish carp-derived type I collagen,
[4] Any of [1] to [3], wherein the fish is selected from the group consisting of tilapia, gonzui, rabeo rojita, katra, carp, thunderfish, pirark, thailand, flounder, shark, sturgeon, and salmon Fish-derived collagen according to crab,
[5] A cell culture substrate containing the fish-derived collagen according to any one of [1] to [4],
[6] The cell culture substrate according to [5], which is for culturing mesenchymal stem cells.
[7] A cell culture kit comprising the fish-derived collagen according to any one of [1] to [4] and / or the cell culture substrate according to [5] or [6],
[8] An osteoblast differentiation inducing method comprising a step of contacting a cell culture substrate containing the fish-derived collagen according to any one of [1] to [4] and a mesenchymal stem cell,
[9] An osteoblast production method comprising a step of contacting a cell culture substrate containing the fish-derived collagen according to any one of [1] to [4] with a mesenchymal stem cell,
And [10] An osteoblast obtained by the method for producing osteoblast according to [9].

  According to the osteoblast differentiation inducing method or the osteoblast production method of the present invention, the mesenchymal stem cells are efficiently and powerfully compared with the osteoblast differentiation using only the fish-derived collagen. Can be differentiated into. In particular, differentiation induction into osteoblasts can be maintained for a long time.

Human bone marrow-derived mesenchymal stem cells (hMSCs), a cell culture plate coated with tilapia scale collagen treated with transglutaminase (Example 1: T & EC), a cell culture plate coated with tilapia scale collagen ( Comparative Example 1; TC), porcine collagen-coated cell culture plate (Comparative Example 3; PC), porcine collagen-coated cell culture plate treated with transglutaminase (Comparative Example 2; P & EC) and It is the figure which showed ALP activity at the time of culture | cultivating with the plate for cell cultures (Comparative example 4: TCPS) which is not coat | covered with collagen. It is the figure which showed the crosslinking degree of the tilapia scale collagen porous body which was enzyme-processed with transglutaminase. The results of studying the conditions of the amount of enzyme (a), temperature (b), and time (c) in the treatment with transglutaminase are shown. Electrons of tilapia scale porous body (a), telapia scale collagen porous body (b) treated with enzyme with transglutaminase, porcine collagen porous body (c), and porcine collagen porous body (d) enzymatically treated with transglutaminase It is a micrograph. Cell culture substrate of tilapia scale collagen porous material treated with transglutaminase (Example 6: ET), porcine collagen porous material enzymatically treated with transglutaminase (Comparative Example 5: EP), glutaraldehyde Of human bone marrow-derived mesenchymal stem cells (hMSC) cultured with tilapia scale collagen porous bodies cross-linked with (tissue) (Comparative Example 6: GA-T) and porcine collagen porous bodies cross-linked with glutaraldehyde (Comparative Example 7: GA-P) It is the figure which showed ALP activity. Cell culture substrate of tilapia scale collagen porous material enzyme-treated with transglutaminase (Example 7: ET / 1w and ET / 3w), or porcine collagen porous material enzyme-treated with transglutaminase (Comparative Example) FIG. 8 shows the expression of osteocalcin in human bone marrow-derived mesenchymal stem cells (hMSCs) cultured on a cell culture substrate of 8: EP / 1w and EP / 3w).

[1] Enzyme-treated fish-derived collagen The fish-derived collagen of the present invention is collagen that has been enzyme-treated with transglutaminase.

(Transglutaminase)
The transglutaminase used in the present invention is not limited as long as it can promote differentiation of osteoblasts by fish-derived collagen. The organism from which transglutaminase is derived is not limited, and includes vertebrates (mammals, birds, reptiles, amphibians, fish), invertebrates (squirts, crabs, insects, shellfish, squid, and nematodes), microorganisms (authentic) Bacteria, archaea, eukaryotes, algae, protists, fungi, and slime molds) or plant-derived transglutaminase can be used.
More specifically, mention of transglutaminase derived from human, mouse, rat, cow, horse, dog, cat, sheep, monkey, fruit fly, grasshopper, horseshoe crab, sea urchin, zebrafish, Escherichia coli, actinomycetes, or slime mold Can do.

  In humans, there are eight isozymes as transglutaminases: Factor XIII (molecular weight 85 kDa), TGase 1 (molecular weight 90-100 kDa), TGase 2 (molecular weight 76 kDa), TGase 3 (molecular weight 77 kDa), TGase 4 (molecular weight 77 kDa), TGase 5 (molecular weight 75 kDa), TGase 6 (molecular weight 79 kDa), or TGase 7 (molecular weight 80 kDa) have been reported. These transglutaminases can cross-link glutamic acid and lysine through a covalent bond. In the present invention, these human transglutaminases can be used without limitation. Moreover, the molecular weight of actinomycete-derived transglutaminase is 40 kDa, and glutamic acid and lysine can be cross-linked in the absence of calcium. In the present invention, transglutaminase derived from actinomycetes can also be used without limitation.

  The transglutaminase used in the present invention is not limited, but a transglutaminase that can cross-link glutamic acid and lysine by a covalent bond is preferable. It is presumed that collagen glutamic acid and lysine are covalently bound to induce differentiation of mesenchymal stem cells into osteoblasts, which can be maintained for a long time. However, the present invention is not limited by the above description.

(Fish-derived collagen)
The fish-derived collagen used for the culture substrate for inducing osteoblast differentiation of the present invention is not particularly limited. For example, the types of fish can include tilapia, gonzui, rabeo rojita, cutlery, carp, thunderfish, piraluk, thailand, flounder, shark, sturgeon (eg, wester sturgeon or amur sturgeon), and salmon. From the viewpoint of the denaturation temperature described below, fish that inhabit rivers, lakes, or seas with high water temperatures are preferred. Specific examples of such fish include fish of the genus Oreochromis, with tilapia being particularly preferred. Collagen with a relatively high denaturation temperature can be obtained from fish of the genus Oreochromis. For example, Nile tilapia (Oreochromis niloticus), which is cultivated for food in Japan and China, is easily available, and a large amount of collagen can be obtained. it can.
As collagen, molecular types of type I collagen to type VIII are known. The type of fish-derived collagen used in the culture substrate for inducing osteoblast differentiation of the present invention is not limited, but I, II, III, and V collagens that are fibrous collagens are preferred. Type I collagen can include tilapia scale collagen, and type II collagen can include sturgeon rostral cartilage or notochord collagen. The amino acid sequence of collagen has a primary structure in which glycine repeats every 3 residues. This repeated sequence is called a collagen-like sequence and is a characteristic of collagen protein.

  The part of the fish from which fish-derived collagen is obtained is not limited. For example, scales, skin, bones, cartilage, fins, organs and the like can be mentioned, but scales are preferred. This is because the scale has few lipids that cause fishy odor. Moreover, collagen derived from fish scales is more likely to be fibrillated than other collagens, and the fiber formation rate is significantly faster.

  As described above, the type of collagen is not limited, but fish type I collagen, for example, has three polypeptide chains with a molecular weight of about 100,000 to form a “triple helix structure”, and the molecular weight is About 300,000. It is shaped like one hard bar with a length of 300 nm and a diameter of 1.5 nm. It is the amino acid sequence of the polypeptide chain that forms the unique “triple helix structure” of fish type I collagen. The polypeptide chain is made up of a series of units “GXY” in which three amino acids are arranged. G often represents glycine, X is proline, and Y is often hydroxyproline. Hydroxyproline is not contained in ordinary proteins and is an amino acid unique to collagen, but it is believed that the triple helix structure is stabilized by hydrogen bonding between the hydroxyl group of hydroxyproline and hydrated water.

  When the temperature rises, the “triple helical structure” composed of three polypeptides is dissolved, and the three polypeptides are separated into gelatin. The change from collagen to gelatin is called denaturation. Once denaturation occurs, it is difficult to return to the “triple helix structure” even if the temperature is lowered again. The denaturation temperature of collagen is usually slightly higher than the inhabitant temperature of the organism from which the collagen is derived. Therefore, the denaturation temperature of fish scales inhabiting fish is not so high.

The fish-derived collagen used in the present invention is not limited by its denaturation temperature, but fish-derived collagen having a high denaturation temperature is preferred, specifically 20 ° C or higher, more preferably 25 ° C or higher, and 28 More preferably, it is 30 ° C. or higher. In the osteoblast differentiation inducing method of the present invention, the temperature at which fish-derived collagen and mesenchymal stem cells are brought into contact is often around 37 ° C., and gelatin in which the “triple helix structure” is destroyed is mesenchymal. This is because it is considered that differentiation induction of stem cells into osteoblasts does not occur.
However, even with fish-derived collagen having a denaturation temperature of less than 20 ° C., contact with mesenchymal stem cells at less than 20 ° C. allows the mesenchymal stem cells to become osteoblasts without causing collagen denaturation. Can be differentiated.

  The denaturation temperature of the collagen used in the present invention is increased stepwise by using a CD spectrometer (Jasco model 725 spectrometer) according to the method described in International Journal of Biological Macromolecules, 32, 199 (2003). Seek by letting That is, the collagen aqueous solution is diluted with dilute hydrochloric acid having a pH of 3, adjusted to 0.03 wt%, and placed in a quartz cell having an optical path length of 1 mm. The cell temperature is increased at 1 ° C./min, and the optical rotation at 221 nm is measured every 0.2 ° C. When the optical rotation at each temperature is plotted against temperature, a denaturation curve is obtained in which the value of optical rotation changes rapidly from the value of the collagen helix to the value of the random coil. The temperature that gives an intermediate value of the optical rotation values, that is, the temperature at which the helix (%) becomes 50% is defined as the denaturation temperature.

  The method for obtaining fish-derived collagen is not particularly limited as long as the triple helix structure of collagen is not destroyed. For example, it can be obtained by the obtaining method disclosed in Patent Document 3. Below, the manufacturing method of the collagen derived from a scale is demonstrated as a typical example.

  First, remove unnecessary items such as fish skin and sea bream from the collected scales with water. If necessary, adhere to the scale surface using alcohols such as methanol, ethanol and isopropanol, hydrophilic organic solvents such as acetone, surfactants, salts such as sodium chloride, alkaline solutions such as sodium hydroxide, etc. Substances that cause odor, such as proteins and lipids other than collagen, may be removed from the scales.

  Furthermore, in the decalcification treatment solution, the mixture is gently stirred using a stirring blade for 1 to 48 hours to remove the inorganic component (calcium phosphate) contained in the scale. The treatment solution only needs to be able to dissolve inorganic components, and may be an inorganic acid such as hydrochloric acid or phosphoric acid, an organic acid such as acetic acid or citric acid, or an aqueous solution such as ethylenediaminetetraacetic acid. Acetic acid is preferably used. The amount used is not particularly limited, and the scale after the decalcification is completed may be washed with water.

  The scales from which contaminants were removed in this manner were solubilized by gradual stirring using an agitating blade or the like in an acidic solution to which protease was added for 2 to 72 hours, thereby breaking the cross-links between collagen molecules. Collagen is extracted. The operation after this extraction step should be performed at a denaturation temperature or lower, preferably at a denaturation temperature of −5 ° C. or lower in order to prevent collagen denaturation.

  As the type of protease, pepsin, proctase, papain and the like having high activity in an acidic solution are preferably used. The pH of the solution may be in a range where the protease activity is high, and is generally about 2 to 5. The amount of protease used is not particularly limited, but is usually about 1 to 15% with respect to the dry weight of fish scales, and if the concentration is determined so that the scale can be uniformly stirred. Good. The acid to be used is not particularly limited, but is preferably selected from those having high safety for living organisms such as hydrochloric acid, acetic acid, citric acid and malic acid, and hydrochloric acid and acetic acid are particularly preferably used. By such a method, collagen (atelocollagen) in which non-helical regions (telopeptides) present at both ends of the collagen molecule are decomposed can be extracted.

  Collagen solubilized in this way is separated from unscaled fish scale residue by centrifugation, filtration or the like. The fish scale residue can be extracted again by solubilizing collagen by treating it in an acidic solution to which a protease is added. Therefore, the yield may be increased by repeating it about 2 to 4 times.

  The collagen solution thus obtained contains a protein other than protease, collagen, gelatin (denatured collagen) and the like, and thus is purified as necessary. As for the purification method, collagen is precipitated by adding a salt such as sodium chloride to the solubilized collagen solution and increasing the salt concentration. For example, sodium chloride is added to a solubilized collagen aqueous solution so as to have a final concentration of 1 M, and the mixture is allowed to stand for about 5 minutes to 24 hours, whereby collagen is precipitated.

  Precipitation can also be achieved by adding sodium hydroxide or the like to bring the pH to neutral or higher. For example, a sodium hydroxide solution is added until pH becomes about 7-9, and collagen is deposited by leaving still for about 5 minutes to 24 hours. Such collagen precipitation can be easily confirmed by the cloudiness of the solution.

  The precipitated collagen is collected by a general solid-liquid separation method such as centrifugation or filtration, and this is gently dissolved in an acidic solution and redissolved. For example, it may be gently stirred for about 1 to 48 hours in a hydrochloric acid solution having a pH of about 2 to 4. In this way, collagen can be purified, and the purity can be further increased by repetition. The salts used in the purification step can be removed by desalting against pure water using a dialysis membrane or the like.

  Since coexistence of bacteria and the like becomes a serious problem when cells are cultured, the obtained collagen solution is preferably used after being sterilized by a method that does not destroy the structure of collagen such as filtration sterilization.

(Enzyme treatment)
The enzyme treatment of collagen with transglutaminase is not particularly limited as long as transglutaminase comes into contact with collagen and can promote differentiation of osteoblasts by fish-derived collagen.

  As a 1st aspect, it can process by making transglutaminase and fish origin collagen contact in a buffer solution. The type of buffer solution, collagen concentration, enzyme amount, enzyme treatment temperature and enzyme treatment time in the first embodiment can be determined as appropriate depending on the transglutaminase used. For example, the following conditions are used. be able to.

  The buffer used is not limited as long as the enzyme action of transglutaminase is not completely inhibited. For example, hydrochloride, maleate, phosphate, CABS, piperidine, glycine, citrate , Malate, formate, succinate, acetate, propionate, piperazine, pyridine, cacodylate, succinate, MES, histidine, bis-tris, phosphate, ethanolamine, ADA, carbonate , ACES, PIPES, imidazole, bis-trispropane, BES, MOPS, HEPES, TES, MOPSO, MOBS, DIPSO, TAPSO, TEA, pyrophosphate, HEPPSO, POPSO, tricine, hydrazine, glycylglycine, TRIS, EPPS, Bicine, HEPBS, TAPS, AMPD, T BS, AMPSO, taurine, borate, CHES, glycine, ammonium hydroxide, and CAPSO, methylamine, piperazine, CAPS or buffer combinations of any of these.

The concentration of collagen in the buffer solution is not particularly limited, but is preferably 0.1% by weight to 5% by weight, more preferably 0.5% by weight to 2% by weight, and 1% by weight to 1.5%. More preferred is weight percent. If it is less than 0.05% by weight, the collagen may not be sufficiently crosslinked. If it exceeds 5% by weight, the crosslinking reaction may be insufficient.
The enzyme treatment time with transglutaminase is not particularly limited as long as the enzyme reaction occurs, but is preferably 5 minutes to 72 hours, more preferably 60 minutes to 48 hours, and still more preferably 8 hours to 24 hours.
The amount of the enzyme in the buffer solution is not particularly limited as long as the effect of the present invention is obtained. For example, 0.1 U to 100 U / mL is preferable, and 0.5 U to 50 U / mL is more preferable. 1U-10U / mL is still more preferable. For example, in the case of actinomycete-derived transglutaminase (Activa (registered trademark)), 0.1 U to 100 U / mL is preferable, 0.5 U to 50 U / mL is more preferable, and 1 U to 10 U / mL is still more preferable.
The temperature of the enzyme treatment is not particularly limited as long as the activity of the transglutaminase used is maintained, but is preferably 5 ° C to 65 ° C, more preferably 10 ° C to 50 ° C, and more preferably 15 ° C to More preferred is 45 ° C.

When using a petri dish or a plate as the material for the cell culture substrate and coating collagen that has been subjected to enzyme treatment with transglutaminase, the cell culture material is coated after the enzyme treatment in the buffer solution. May be. In this case, the enzyme treatment may be performed by increasing the concentration of collagen and transglutaminase, and the concentration may be diluted and coated on the cell culture material. In addition, the enzyme treatment in the buffer solution and the coating on the cell culture material can be performed simultaneously.
In addition, when a cell culture material is coated with fish-derived collagen and then subjected to an enzyme treatment with transglutaminase, the enzyme treatment may not be sufficient. Therefore, this aspect may be omitted.

As a second embodiment of the enzyme treatment, the collagen molded body can be subjected to an enzyme treatment in a buffer solution containing transglutaminase.
Examples of the collagen molded body include a collagen porous body, a collagen membrane, collagen fine particles, a collagen fiber by electron spinning, or a collagen dense body. Enzymatic treatment can be performed by immersing these collagen compacts in a buffer containing transglutaminase.
The type of buffer, the amount of enzyme, the temperature of enzyme treatment, and the time of enzyme treatment in the second aspect can be appropriately determined depending on the transglutaminase used. For example, the conditions described in the first aspect are It is possible to use.

[2] Cell culture substrate The cell culture substrate of the present invention contains fish-derived collagen that has been enzymatically treated with transglutaminase. The cell culture substrate of the present invention can be usefully used as a culture substrate for inducing osteoblast differentiation.
The cell culture substrate is not limited as long as it can be used for cell culture. For example, the cell culture material may be coated with fish-derived collagen that has been enzymatically treated with the transglutaminase of the present invention. In addition, collagen molded bodies such as collagen porous bodies, collagen membranes, fine particles, fibers by electron spinning, or collagen dense bodies themselves are used as cell culture substrates, and they are used as cell culture containers (for example, flasks, petri dishes, or multiculture plates). ) May be included.

(Material for cell culture)
The material for cell culture that coats the fish-derived collagen that has been enzymatically treated with the transglutaminase can be used without limitation as long as it is a material that is usually used for cell culture. As a material, glass, a porous body (for example, polycarbonate), or a plastic (for example, polystyrene or polypropylene) can be used, and for a shape, a flask, a petri dish, a multiculture plate (for example, 6-well multiculture) is used. Plates, 12-well multiculture plates, etc.), or shaped materials such as beads can be used. However, since the collagen solution is hydrophilic, a hydrophilic material substrate or a material having a hydrophilized surface is preferable.

(Collagen coating on the cell culture material)
The coating of fish-derived collagen that has been enzyme-treated with transglutaminase on the cell culture material may be performed after the enzyme treatment as described above, or may be performed simultaneously with the enzyme treatment.
The pH of the solution for dissolving collagen is preferably 2 to 5 which is lower than the isoelectric point of collagen. The acid source of the solution is not particularly limited, but is preferably selected from those that are highly safe for living organisms, such as hydrochloric acid, acetic acid, citric acid, or malic acid, and more preferably hydrochloric acid or acetic acid that vaporizes during drying. If the pH is too low, the structure of the collagen may be destroyed. If the pH is too high, the solubility may be reduced, resulting in thickening or gelation.

  Although the collagen concentration of the coating solution to be used is not particularly limited, it is generally 0.01 to 1% by weight, more preferably 0.03 to 0.3% by weight. When the concentration is low, the amount of collagen that can be applied to the cell culture material by one coating is reduced. Higher concentrations can increase the amount of collagen that coats the material, but only increase the thickness. The effect of the present invention is exerted mainly on the surface layer portion, so even if the thickness of the coated collagen increases, an effect commensurate with the amount of collagen cannot be expected.

  As a coating method, a collagen solution adjusted to an appropriate concentration using an acidic solution is poured onto the material for cell culture such as a dish so as not to be disturbed. Remove collagen. By drying this, the fibrillated collagen is uniformly coated on the substrate. This is a phenomenon peculiar to fish scale-derived collagen, and cannot be achieved by conventionally used collagen derived from mammals.

Since contamination with bacteria becomes a serious problem during cell culture, it is desirable to perform the collagen coating operation in a sterile environment such as a clean room or a clean bench using a clean sterile substrate. As needed, you may add the sterilization process which does not destroy the structure of collagen, such as ethylene oxide and UV irradiation.
Speaking of the temperature during coating and drying, it is carried out at a modification temperature or lower, preferably at a modification temperature of -5 ° C or lower. When the treatment is performed at a temperature higher than the denaturation temperature, the fibrosis function may be lost due to the denaturation of the collagen before the formation of the bonds between the base materials and the collagen fibers, and the characteristics as collagen may not be exhibited.
Although the drying time varies depending on the humidity and the like, it is dried in about 5 minutes to 2 hours under ventilation such as in a clean bench. When drying is insufficient, collagen fibrosis and adhesion with the cell culture material may be insufficient. When a cell culture solution is added, the effect of the present invention is achieved by re-dissolution or peeling of the coating. May not be fully demonstrated. If it is in a sufficiently dry and fibrotic state, it can exert its effect even after being stored for several months. Storage is preferably at a temperature lower than the denaturation temperature, preferably refrigerated storage.

(Collagen molded body)
The collagen molded body that can be used as the cell culture substrate of the present invention is not limited as long as it contains fish-derived collagen that has been enzymatically treated with transglutaminase. For example, a collagen porous body, collagen membrane, collagen Mention may be made of fine particles, collagen fibers by electron spinning, or collagen dense bodies. The enzyme treatment of the collagen molded body can be performed as described above.

(Cell culture vessel)
The cell culture container containing the collagen molded body as the cell culture substrate can also be used without limitation as long as it is a container normally used for cell culture. As a material, a glass, a porous body (for example, polycarbonate), or a plastic (for example, polystyrene or polypropylene) container can be used, and for a shape, a flask, a petri dish, a multiculture plate (for example, 6-well multiculture) Plates, 12-well multiculture plates, etc.) or containers in the form of beads or the like can be used.

[3] Cell culture kit and cell production apparatus The cell culture kit of the present invention comprises fish-derived collagen that has been subjected to enzyme treatment with transglutaminase and / or the cell culture substrate. The cell culture substrate of the present invention can be usefully used as a culture substrate for inducing osteoblast differentiation. The cell production apparatus of the present invention also includes a fish-derived collagen that has been subjected to an enzyme treatment with transglutaminase and / or the cell culture substrate. The cell production apparatus of the present invention can be usefully used particularly as a cell production apparatus for inducing osteoblast differentiation.

One embodiment of the cell culture kit of the present invention includes the cell culture substrate of the present invention. The cell culture substrate of the present invention is a cell culture material coated with the above-described fish-derived collagen that has been enzymatically treated with transglutaminase, or a collagen molding comprising a fish-derived collagen that has been enzymatically treated with transglutaminase Is the body.
Further, as another embodiment of the cell culture kit of the present invention, fish-derived collagen that has been subjected to enzyme treatment with transglutaminase can also be included. For example, the fish-derived collagen can be coated on a cell culture substrate and used as a culture substrate for inducing osteoblast differentiation.

In addition, the cell culture kit of the present invention may contain an osteoblast differentiation factor. Examples of osteoblast differentiation inducers include dexamethasone, immunosuppressive agents such as FK-506 or cyclosporine, bone morphogenetic proteins (BMP: Bone Morphogenic Proteins) such as BMP2, BMP4, BMP5, BMP6, BMP7 or BMP9, and bones such as TGFβ. Mention may be made of forming liquid factors. The osteoblast differentiation inducing kit of the present invention can contain one or more selected from these osteoblast differentiation inducing factors.
In addition, the cell culture kit of the present invention may include an instruction manual specifying that it is for inducing differentiation of osteoblasts from mesenchymal stem cells. The statement that it is for inducing differentiation of osteoblasts from mesenchymal stem cells may be attached to the container of the kit.

[4] Osteoblast differentiation inducing method The osteoblast differentiation inducing method of the present invention comprises a step of contacting a cell culture substrate containing fish-derived collagen that has been enzymatically treated with transglutaminase and a mesenchymal stem cell (hereinafter referred to as “the mesenchymal stem cell”). , Sometimes referred to as a collagen contact step). In the osteoblast differentiation inducing method of the present invention, the fish-derived collagen treated with the enzyme with the transglutaminase and the cell culture substrate can be used.

(Collagen contact process)
In the osteoblast differentiation induction method, the medium used for contacting the cell culture substrate and mesenchymal stem cells is not limited as long as it is a medium that can maintain mesenchymal stem cells, but is usually used for culturing mesenchymal stem cells. What is necessary is just to use the culture medium to be used. For example, a known medium such as a MEM medium, an α-MEM medium, or a DMEM medium can be appropriately selected and used according to the cells to be cultured.
The contact time is not limited as long as differentiation induction of mesenchymal stem cells into osteoblasts occurs, but the lower limit is substantially 10 minutes or more, preferably 1 hour or more, more preferably 1 More than a day. The upper limit of the contact time is not limited, but is 30 days or less, preferably 15 days or less, and more preferably 7 days or less.
The contact temperature can be appropriately determined according to the optimal culture temperature for mesenchymal stem cells. For example, in the case of mammalian mesenchymal stem cells, it is 30 ° C to 40 ° C, preferably 35 ° C to 37 ° C. However, it is also possible to contact at a temperature higher or lower than the culture temperature. For example, when fish-derived collagen with a low denaturation temperature is used, the cell culture substrate is contacted with the mesenchymal stem cells at a contact temperature lower than the culture temperature, and the mesenchyme is raised to the optimal culture temperature. It is also possible to culture stem cell.

Usually, osteoblast differentiation is induced in the presence of about 10 to 15% serum. In the osteoblast differentiation inducing method of the present invention, differentiation can be induced in the presence of about 10 to 15% serum, but bone can be obtained even in the presence of low-concentration serum or in the absence of serum. It is possible to induce blast differentiation. In order to eliminate the influence of various factors in serum when osteoblast differentiation is induced from mesenchymal stem cells, it is preferable to contact in the presence of a low concentration of serum. For example, the presence of 10% or less of serum is preferable, the presence of 5% or less of serum is more preferable, the presence of 3% or less of serum is more preferable, and the absence of serum is most preferable.
The serum that can be used in the induction of osteoblast differentiation is not particularly limited as long as it is mammalian serum, but serum from humans, cows, horses, sheep, goats, and the like can be used. However, when the mesenchymal stem cells are separated from the living body, it is most preferable to use the serum of the individual from which the mesenchymal stem cells are collected. By using the serum of the individual itself, it is possible to prevent contact with pathogens of zoonotic diseases that may be contained in the serum of cattle and the like.

(Osteoblast differentiation factor)
The osteoblast differentiation inducing method of the invention can include a step of bringing the mesenchymal stem cells into contact with an osteoblast differentiation inducing factor (hereinafter sometimes referred to as an osteoblast differentiation inducing factor contact step). The osteoblast differentiation inducing factor contact step may be performed simultaneously with the collagen contact step, or may be performed before or after the collagen contact step contact step. The osteoblast differentiation inducer contact step can be performed by adding an osteoblast differentiation inducer to a medium containing mesenchymal stem cells.
The osteoblast differentiation-inducing factor is not limited as long as it can induce differentiation of mesenchymal stem cells into osteoblasts. For example, an immunosuppressant such as dexamethasone, FK-506 or cyclosporine, BMP2, BMP4, BMP5 , BMP6, BMP7, BMP9 and other bone morphogenetic proteins (BMP: Bone Morphogenic Proteins) and TGFβ and other osteogenic fluid factors. By adding one or more selected from these osteoblast differentiation inducing factors to the medium, the osteoblast differentiation inducing factor contact step can be performed.
The concentration of the osteoblast differentiation inducing factor can be appropriately determined according to the type of osteoblast differentiation inducing factor used. For example, when dexamethasone is used, it can be used at a concentration of 1 to 100 nM, and 10 nM is particularly preferable.

The collagen contact step and the osteoblast differentiation inducing factor contact step in the osteoblast differentiation inducing method of the present invention may be performed simultaneously or separately. When performing each process separately, three processes may be performed separately, two processes may be performed simultaneously, and another process may be performed separately.
The order of performing each step is not particularly limited, but the collagen contact step strongly induces the initial differentiation of osteoblasts. Therefore, the osteoblast differentiation inducer contact step is performed after the collagen contact step. preferable.

(Mesenchymal stem cells)
The mesenchymal stem cells to be brought into contact with the cell culture substrate containing fish-derived collagen that has been enzyme-treated with transglutaminase are not limited as long as they can be induced to differentiate into medulloblasts. Mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth embryo-derived mesenchymal stem cells, auricular perichondrium-derived mesenchymal stem cells, peripheral blood-derived Examples include mesenchymal stem cells, cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cell-derived mesenchymal stem cells, or iPS cell-derived mesenchymal stem cells. The animal species from which the mesenchymal stem cells are derived is not particularly limited, and examples of mammals include humans, monkeys, dogs, cats, pigs, sheep, goats, cows, horses, rabbits, guinea pigs, rats, and mice. Can be mentioned. Birds can include chickens, quails, ducks, geese, ostriches and guinea fowls. Reptiles can include crocodiles, turtles and lizards. Amphibians include frogs and newts. Fish can include tilapia, Thailand, flounder, shark, and salmon. In addition, examples of invertebrates include crabs, shellfish, jellyfish, and shrimps.
The mesenchymal stem cells are cells capable of differentiating into cells belonging to the mesenchymal system such as osteoblasts, adipocytes, myocytes, or chondrocytes, and can constitute bone, blood vessels, or myocardium It will be.

  The mesenchymal stem cell may be a cell line, can be prepared according to a conventional method, and a commercially available cell can also be used. In addition, mesenchymal stem cells (eg, bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells) , Auricular perichondrium-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cells-derived mesenchymal stem cells, or iPS cell-derived mesenchymal stem cells) can be induced to differentiate into osteoblasts by the osteoblast differentiation inducing method of the present invention. In this case, the cells collected from the living body are preferably prepared by removing connective tissue and the like according to a conventional method. Furthermore, it is preferable to use primary cultured cells as cells collected from a living body. Although it is possible to use after passage, the number of passages is preferably small, preferably 10 times or less, more preferably 5 times or less, and most preferably 2 times or less.

[5] Osteoblast production method and osteoblast (culture process)
The medium in the osteoblast production method of the present invention is a mesenchymal stem cell to be used, for example, bone marrow mesenchymal stem cell, adipose tissue-derived mesenchymal stem cell, synovial tissue-derived mesenchymal stem cell, dental pulp-derived mesenchymal stem cell, tooth Embryonic-derived mesenchymal stem cells, auricular perichondrium-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cells A mesenchymal stem cell or an iPS cell-derived mesenchymal stem cell can be appropriately selected. For example, in the case of bone marrow mesenchymal stem cells, MEM medium, α-MEM medium, DMEM medium, or the like can be used. Moreover, you may add antibiotics etc. to the said culture medium.

The culture time is not limited as long as differentiation induction of mesenchymal stem cells into osteoblasts occurs, but it is 1 to 30 days, preferably 1 to 14 days, more preferably 3 to 10 days. Day.
The culture temperature can be appropriately determined according to the optimal culture temperature for mesenchymal stem cells, but in the case of mammalian mesenchymal stem cells, it is preferably 30 ° C to 40 ° C, more preferably 35 ° C to 37 ° C. However, when fish-derived collagen having a low denaturation temperature is used, it can be cultured at a temperature lower than the optimum temperature for culturing mesenchymal stem cells. The culture is preferably performed in the presence of 3 to 10% CO _{2, and} more preferably in the presence of 5% CO ₂ .

Usually, mesenchymal stem cells are cultured in the presence of about 10 to 15% serum to produce osteoblasts. In the osteoblast production method of the present invention, it is possible to produce osteoblasts in the presence of about 10 to 15% serum, but even in the presence of a low concentration of serum or in the absence of serum. It is possible to produce osteoblasts. In order to eliminate the influence of various factors in serum when producing osteoblasts from mesenchymal stem cells, it is preferable to produce them in the presence of a low concentration of serum. For example, the presence of 10% or less of serum is preferable, the presence of 5% or less of serum is more preferable, the presence of 3% or less of serum is more preferable, and the absence of serum is most preferable.
The serum that can be used in the production of osteoblasts is not particularly limited as long as it is mammalian serum, but serum from humans, cows, horses, sheep, goats, and the like can be used. However, when the mesenchymal stem cells are separated from the living body, it is most preferable to use the serum of the individual from which the mesenchymal stem cells are collected. By using the serum of the individual itself, it is possible to prevent contact with pathogens of zoonotic diseases that may be contained in the serum of cattle and the like.

(Osteoblast differentiation factor)
In the osteoblast production method of the present invention, an osteoblast differentiation inducing factor can be included in the culture medium. The osteoblast differentiation inducing factor may be added from the start of the culture or may be added during the culture.
The osteoblast differentiation-inducing factor is not limited as long as it can induce differentiation of mesenchymal stem cells into osteoblasts. For example, an immunosuppressant such as dexamethasone, FK-506 or cyclosporine, BMP2, BMP4, BMP5 , BMP6, BMP7, BMP9 and other bone morphogenetic proteins (BMP: Bone Morphogenic Proteins) and TGFβ and other osteogenic fluid factors. By adding one or more selected from these osteoblast differentiation inducing factors to the medium, the osteoblast differentiation inducing factor contact step can be performed.
The concentration of the osteoblast differentiation inducing factor can be appropriately determined according to the type of osteoblast differentiation inducing factor used. For example, when dexamethasone is used, it can be used at a concentration of 1 to 100 nM, and 10 nM is particularly preferable.

(Osteoblast)
The osteoblast of the present invention is an osteoblast that can be obtained by the production method of the present invention, and can perform bone formation in bone tissue. The osteoblasts are differentiated from mesenchymal stem cells into osteoprogenitor cells and further differentiated into osteoblasts. The cytoplasm of osteoblasts is basophil and has alkaline phosphatase activity. Furthermore, the osteoblasts of the present invention may have androgen and estrogen receptors. Androgens reduce osteoblast activity and estrogens stimulate osteoblasts.

<Action>
The mechanism by which the fish-derived collagen treated with the transglutaminase of the present invention can induce osteoblast differentiation has not been elucidated in detail, but can be considered as follows. However, the present invention is not limited by the following description.
The fish-derived collagen used in the present invention is excellent in orientation, but it is considered that the orientation is further improved by the transglutaminase treatment and the structure is maintained for a long time. In addition, fish-derived collagen is excellent in flexibility, and it is considered that the flexibility is improved by the treatment with transglutaminase or is maintained for a long time.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1
In this example, tilapia scale collagen was coated on a cell culture plate while being enzymatically treated with transglutaminase to prepare a cell culture substrate.
(A) Preparation of Cell Culture Substrate Tapia scale collagen (Cell Campus (registered trademark) AQ-03A; Taki Chemical) was diluted to 0.3% by weight with a 0.01M hydrochloric acid solution at pH 3. To this tilapia scale collagen solution, a transglutaminase derived from streptoverticillium (100 U / g: Activa (registered trademark) -GS; Ajinomoto) was mixed at 5 U / mL. 1 mL of the tilapia scale collagen solution to which transglutaminase was added was dispensed into a polystyrene 6-well plate for tissue culture. After leaving it at room temperature for 2 hours, it was washed with PBS to obtain a cell culture substrate.

(B) Culture of human bone marrow-derived mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza) were seeded on the cell culture plate at 1.0 × 10 ⁴ cells / well. As the medium, 10% FBS-added D-MEM was used and cultured at 37 ° C. in a 5% CO ₂ atmosphere for 24 hours.

(C) Measurement of alkaline phosphatase (ALP) activity In order to confirm the differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts, alkaline phosphatase was measured. The cultured cells were washed with PBS and stored at −80 ° C. until cell suspension. The cells were suspended in Lysis buffer (100 mM Tris-HCl (pH 7.5), 5 mM magnesium chloride, and 0.2% Triton X-100) and sonicated. The supernatant was collected by centrifugation at 6000 g for 10 minutes. Alkaline phosphatase activity was measured using a measurement kit (LabAssay ALP: Wako Pure Chemical Industries) according to the manufacturer's instructions. Absorbance was 450 nm. The results are shown as T & EC in FIG.

<< Comparative Example 1 >>
In this comparative example, tilapia scale collagen that was not enzyme-treated with transglutaminase was coated on a cell culture plate to produce a cell culture substrate.
The procedure of Example 1 was repeated except that no transglutaminase was used. The results are shown as TC in FIG.

<< Comparative Example 2 >>
In this comparative example, porcine collagen was used to coat a cell culture plate while performing enzyme treatment with transglutaminase to produce a cell culture substrate.
The procedure of Example 1 was repeated except that porcine collagen (Cellmatrix Type IC: Nitta Gelatin) was used instead of tilapia scale collagen. The results are shown as P & EC in FIG.

<< Comparative Example 3 >>
In this comparative example, porcine collagen that was not enzyme-treated with transglutaminase was coated on a cell culture plate to produce a cell culture substrate.
The operation of Comparative Example 1 was repeated except that no transglutaminase was used. The results are shown as PC in FIG.

<< Comparative Example 4 >>
In this comparative example, human bone marrow-derived mesenchymal stem cells were cultured using a cell culture plate not coated with collagen, and alkaline phosphatase activity was measured.
The procedures of (B) and (C) of Example 1 were repeated except that a cell culture plate not coated with collagen was used instead of the cell culture substrate coated with collagen. The result is shown as TCPS in FIG.

  As shown in FIG. 1, a cell culture plate not coated with collagen (Comparative Example 4: TCPS), a cell culture plate coated with porcine collagen (Comparative Example 3; PC), and enzyme treatment with transglutaminase The cultured porcine collagen-coated cell culture plate (Comparative Example 2; P & EC) had low alkaline phosphatase activity. However, in the cell culture plate coated with tilapia scale collagen (Comparative Example 1; TC), the ALP activity increased, and the cell culture plate coated with tilapia scale collagen treated with transglutaminase (Example) 1: T & EC), a marked increase in ALP activity was observed.

Example 2
In this example, a collagen porous body using tilapia scale collagen was produced, and the collagen porous body was treated with transglutaminase to prepare a cell culture substrate. Further, the amount of transglutaminase added was examined by measuring the degree of crosslinking by the TNBS method.
(A) Preparation of Cell Culture Substrate A 0.3% by weight tilapia scale collagen (Cell Campus (registered trademark) AQ-03A; Taki Chemical) solution was lyophilized for 48 hours. The lyophilized collagen porous body was added to 3 mL of 0.01 M acetic acid (pH 3) and mixed at 4 ° C. for 24 hours to obtain a 1 wt% tilapia scale collagen solution. The tilapia scale collagen solution was dispensed into a 24-well cell culture plate and freeze-dried to obtain a tilapia scale collagen sponge. The obtained tilapia scale collagen porous body was immersed in PBS containing 10% sodium chloride and subjected to negative pressure to remove air contained in the pores. The tilapia scale collagen porous body contains 1 U / mL, 3 U / mL, 5 U / mL, or 7 U / mL of transglutaminase derived from streptoverticillium (100 U / g: Activa (registered trademark) -GS; Ajinomoto) It was immersed in PBS containing 10% sodium chloride and left at 30 ° C. for 12 hours. After the treatment, the tilapia-scale collagen porous body was washed with PBS three times to obtain a porous cell culture substrate.

(B) Measurement of the degree of crosslinking The number of free amino groups was quantified by trinitrobenzene sulfonic acid (TNBS) absorption. The mass of the lyophilized collagen sponge was measured and placed in a 1.5 mL tube. 200 μL of 4% NaHCO ₃ and 200 μL of TNBS (Sigma) were added to each tube and left in the dark at 37 ° C. for 3 hours. Thereafter, 1 mL of 6M HCl was added to each tube and incubated at 90 ° C. for 20 minutes to hydrolyze and dissolve completely. Absorbance was measured at 345 nm.

  The result is shown in FIG. Transglutaminase showed the highest degree of crosslinking at 5 U / mL.

Example 3
In this example, in the same manner as in Example 2, a tilapia scale collagen porous material that was enzyme-treated with transglutaminase was produced, and the temperature of the enzyme treatment was examined.
The procedure of Example 2 was repeated except that the transglutaminase concentration was 5 U / mL and the enzyme treatment temperature was 30 ° C., 40 ° C., or 50 ° C.
The result is shown in FIG. The highest crosslinking degree was exhibited at a reaction temperature of 50 ° C.

Example 4
In this example, as in Example 2, a tilapia scale collagen porous material that was enzyme-treated with transglutaminase was produced, and the time for enzyme treatment was examined.
The procedure of Example 2 was repeated except that the concentration of transglutaminase was 5 U / mL and the enzyme treatment time was 4 hours, 8 hours, or 12 hours.
The result is shown in FIG. The reaction time of 12 hours showed the highest degree of crosslinking.

Example 5
In the present Example, the electron micrograph of the tilapia scale collagen porous body which was enzyme-treated with transglutaminase and the tilapia scale collagen porous body which was not enzyme-treated with transglutaminase were compared.
Using 5 U / mL transglutaminase, the operation of Example 2 (A) was repeated to obtain a tilapia scale collagen porous body treated with transglutaminase. Moreover, except not performing enzyme treatment, operation of Example 2 (A) was repeated and the tilapia scale collagen porous body which has not performed enzyme treatment with transglutaminase was obtained.
The obtained collagen porous body was observed with an electron microscope. The results are shown in FIG. 3a (non-enzymatic treatment) and FIG. 3b (enzymatic treatment). No deformation of the collagen wall or blockage of the pores was observed.

<< Comparative Example 4 >>
In this example, a comparison was made between electron microscopic images of porcine collagen porous bodies treated with transglutaminase and porcine collagen porous bodies not treated with transglutaminase.
Transglutaminase was repeated except that 5 U / mL transglutaminase was used and porcine collagen (Cellmatrix Type IC: Nitta Gelatin) was used instead of tilapia scale collagen. Porcine collagen porous material treated with enzyme was obtained. Similarly, except that the enzyme treatment was not performed, the procedure of Example 2 (A) was repeated to obtain a porcine collagen porous body that was not enzyme-treated with transglutaminase.
The obtained collagen porous body was observed with an electron microscope. The results are shown in FIG. 3c (non-enzyme treatment) and FIG. 3d (enzyme treatment). No deformation of the collagen wall or blockage of the pores was observed.

Example 6
In this example and Comparative Examples 5-7, the differentiation induction ability of osteoblasts was compared between a cell culture substrate treated with transglutaminase and a cell culture substrate chemically cross-linked with glutaraldehyde. In this example, a cell culture substrate of a tilapia scale collagen porous body that was enzymatically treated with transglutaminase was prepared.
Except for using 5 U / mL transglutaminase, the operation of Example 2 (A) was repeated to obtain a cell culture substrate (tilapia scale collagen porous body).

(B) Culture of human bone marrow-derived mesenchymal stem cells The obtained tilapia scale collagen porous body was sterilized with 70% ethanol for 20 minutes and washed 5 times with sterile PBS. Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza) were seeded at 1.0 × 10 ⁴ cells per porous cell culture substrate. The medium was cultured for 24 hours at 37 ° C. in a 5% CO ₂ atmosphere using D-MEM supplemented with 10% FBS and 1% antibiotic-antifungal agent.

(C) Measurement of alkaline phosphatase (ALP) activity In order to confirm the differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts, alkaline phosphatase was measured. The cultured cells were washed with PBS, and the cultured cells and the cell culture substrate (porous body) were collected. The collected cells and cell culture substrate were added to Lysis buffer (100 mM Tris-HCl (pH 7.5), 5 mM magnesium chloride, and 0.2% Triton X-100), and crushed with a ball mill. The supernatant was collected by centrifugation at 6000 g for 20 minutes. Alkaline phosphatase activity was measured using a measurement kit (LabAssay ALP: Wako Pure Chemical Industries) according to the manufacturer's instructions. Absorbance was 450 nm.
The results are shown as ET in FIG.

<< Comparative Example 5 >>
In this comparative example, a cell culture substrate of porcine collagen porous material treated with transglutaminase was produced.
The procedure of Example 6 was repeated except that porcine collagen (Cellmatrix Type IC: Nitta Gelatin) was used instead of tilapia scale collagen. The results are shown as E-P in FIG.

<< Comparative Example 6 >>
In this comparative example, a cell culture substrate of a tilapia scale collagen porous body cross-linked with glutaraldehyde was produced.
(A) Preparation of Cell Culture Substrate A 0.3% by weight tilapia scale collagen (Cell Campus (registered trademark) AQ-03A; Taki Chemical) solution was lyophilized for 48 hours. The lyophilized collagen porous body was added to 3 mL of 0.01 M acetic acid (pH 3) and mixed at 4 ° C. for 24 hours to obtain a 1 wt% tilapia scale collagen solution. The tilapia scale collagen solution was dispensed into a 24-well cell culture plate and freeze-dried to obtain a tilapia scale collagen porous body. The resulting tilapia scale collagen porous body was placed in a sealed container together with 2 mL of 25% glutaraldehyde solution and allowed to stand at 37 ° C. for 1 hour. Unreacted unreacted sites in the collagen porous body were blocked with 0.1 M glycine. The cell culture substrate was washed with PBS.
For the culture of human bone marrow-derived mesenchymal stem cells and the measurement of alkaline phosphatase (ALP) activity, the procedures of Example 6 (B) and (C) were repeated. The results are shown as GA-T in FIG.

<< Comparative Example 7 >>
In this comparative example, a cell culture substrate of a porcine collagen porous body cross-linked with glutaraldehyde was produced.
The procedure of Comparative Example 6 was repeated except that porcine collagen (Cellmatrix Type IC: Nitta Gelatin) was used instead of tilapia scale collagen. The results are shown as GA-P in FIG.

  As shown in FIG. 4, the cell culture substrate (Comparative Example 6: GA-T and Comparative Example 7: GA-P) crosslinked with glutaraldehyde did not show an increase in ALP activity. Also, no increase in ALP activity was observed in porcine collagen porous material (Comparative Example 5: E-P) treated with transglutaminase. However, cells cultured on a cell culture substrate of tilapia scale collagen porous body treated with enzyme with transglutaminase (Example 6: ET) showed high ALP activity, and differentiation into osteoblasts was confirmed. .

Example 7
In this example, the expression of osteocalcin in cultured cells cultured using a tilapia scale collagen porous body was examined. Osteocalcin is a protein expressed at a late stage in the differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts.
Using 5 U / mL transglutaminase, the operation of Example 2 (A) was repeated to obtain a cell culture substrate of tilapia scale collagen porous body.

(B) Culture of human bone marrow-derived mesenchymal stem cells The obtained tilapia scale collagen porous body was sterilized with 70% ethanol for 20 minutes and washed 5 times with sterile PBS. Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza) were seeded at 1.0 × 10 ⁴ cells per porous cell culture substrate. The medium was cultured for 24 hours at 37 ° C. in a 5% CO ₂ atmosphere using D-MEM supplemented with 10% FBS and 1% antibiotic-antifungal agent. Thereafter, the medium was replaced with D-MEM medium containing 3% FBS (added with 10 nM dexamethasone (Sigma), 50 μg / mL magnesium phosphate ascorbate (Wako Pure Chemical Industries), and 10 mM β-glycerophosphate (Sigma)). The cells were cultured for 1 day or 28 days at 37 ° C. in a 5% CO ₂ atmosphere. The medium was changed three times a week.

(C) Measurement of osteocalcin Osteocalcin was measured in order to confirm the differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts. Cultured cells were gently washed with PBS and placed in PBS containing 0.2% Triton X-100. The cells and cell culture substrate were crushed with a ball mill. A portion of the disrupted material was collected to measure the amount of DNA, and the remainder was mixed with an equal amount of 20% by volume formic acid and incubated at 4 ° C. for 2 days or longer. Centrifugation was performed at 14,000 rpm for 20 minutes at 4 ° C. Calcium contained in the supernatant was removed with a NAP-5 column (GE Healthcare), and the protein fraction was eluted with 10% formic acid. The buffer was exchanged for TE buffer using Vivaspin (5,000 MWCO). Osteocalcin was measured using an osteocalcin ELISA kit (Human Gla-Osteocalcin High Sensitive EIA (Takara Shuzo)) according to the manufacturer's instructions. The results are shown in FIG. 5 as ET / 1w and ET / 3w.

<< Comparative Example 8 >>
In this comparative example, the expression of osteocalcin in cultured cells cultured with porcine collagen was examined.
The procedure of Example 7 was repeated except that porcine collagen (Cellmatrix Type IC: Nitta Gelatin) was used instead of tilapia scale collagen. The results are shown in FIG. 5 as EP / 1w and EP / 3w.

  As shown in FIG. 5, cells cultured on a cell culture substrate (Example 7: ET / 1w and ET / 3w) of a tilapia scale collagen porous material treated with transglutaminase were treated with transglutaminase. Compared with the porcine collagen porous body treated with the enzyme (Comparative Example 8: EP / 1w and EP / 3w), high expression of osteocalcin was shown. In particular, it showed high expression after 3 weeks of culture, and was found to be a late stage of osteoblast differentiation induction.

  The fish-derived collagen treated with the transglutaminase of the present invention and the cell culture substrate using the same can be used for inducing differentiation of mesenchymal stem cells into osteoblasts. In addition, osteoblasts can be produced from mesenchymal stem cells using the osteoblast production method of the present invention, and can be effectively used for the treatment of bone diseases. In particular, differentiation induction into osteoblasts can be maintained for a long period of time, and an effective treatment method can be provided. Moreover, according to this invention, an osteoblast differentiation induction kit and an osteoblast production apparatus can be manufactured.

Claims (10)

  Fish-derived collagen that has been enzymatically treated with transglutaminase.   The collagen is collagen selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, and mixtures thereof. The fish-derived collagen according to claim 1.   The fish-derived collagen according to claim 1 or 2, wherein the collagen is fish carp-derived type I collagen.   4. The fish according to any one of claims 1 to 3, wherein the fish is selected from the group consisting of tilapia, gonzui, rabeo rojita, katra, carp, thunderfish, pirark, thailand, flounder, shark, sturgeon, and salmon. Fish-derived collagen.   The cell culture substratum containing the fish origin collagen as described in any one of Claims 1-4.   The cell culture substrate according to claim 5, which is used for culturing mesenchymal stem cells.   A cell culture kit comprising the fish-derived collagen according to any one of claims 1 to 4 and / or the cell culture substrate according to claim 5 or 6.   The osteoblast differentiation induction method including the process of contacting the cell culture substratum containing the fish origin collagen as described in any one of Claims 1-4, and a mesenchymal stem cell.   An osteoblast production method comprising a step of contacting a cell culture substrate containing the fish-derived collagen according to any one of claims 1 to 4 with a mesenchymal stem cell.   An osteoblast obtained by the osteoblast production method according to claim 9.

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