Culture method of mesenchymal stem cells

Abstract

The present invention addresses the problem of providing a novel technique for simply culturing mesenchymal stem cells from extracted hair. In order to solve the above-mentioned problem, the present invention provides a method for culturing mesenchymal stem cells, which comprises a culture step for culturing, in a culture solution, hair that has been extracted and contains hair bulbs covered with genuine hair root sheath tissue.

Description

Technical Field

[0001] The invention relates to a method for culturing mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSC), also known as mesenchymal stromal cells, are a type of adult stem cells found in bone marrow, adipose tissue, placenta, umbilical cord, ovarian membrane, hair follicles, etc. Mesenchymal stem cells are pluripotent cells that have the ability to differentiate into bone, cartilage, and fat cells, and because they have a low risk of cancer, they are being studied as a source of cell therapy and regenerative medicine that replaces artificial pluripotent stem cells.

[0003] Mesenchymal stem cells are usually obtained from tissues by invasive methods. For example, bone marrow mesenchymal stem cells (BM-MSCs) are obtained from the iliac crest by femoral puncture, and adipose-derived mesenchymal stem cells (AD-MSCs) are obtained from subcutaneous fat by liposuction and lipectomy. Dermal papilla cells, which are mesenchymal stem cells of hair follicles, are obtained from excised skin tissue.

[0004] In addition, mesenchymal stem cells can also be isolated and cultured using samples obtained by non-invasive methods. For example, Patent Document 1 discloses a method for isolating and culturing mesenchymal stem cells by removing the hair bulb from plucked hair and culturing the obtained hair in the bulge region of the hair follicle on a Transwell membrane. Prior art documents Patent Literature

[0005] Patent Document 1: International Publication No. 2021 / 009246 Summary of the invention Problems to be Solved by the Invention

[0006] Despite the above-mentioned prior art, an object of the present invention is to provide a new technology for easily culturing mesenchymal stem cells from plucked hair. Means of solving problems

[0007] The present invention for solving the above-mentioned problems is a method for culturing mesenchymal stem cells, the method comprising a culturing step of culturing hair obtained by plucking and containing a hair bulb covered with a dermal root sheath tissue in a culture medium. According to the present invention, mesenchymal stem cells can be obtained by using the hair bulb portion of plucked hair. In addition, since culture samples can be easily obtained by a non-invasive method such as plucking hair, the burden on donor individuals when obtaining samples can be reduced.

[0008] In a preferred embodiment of the present invention, the mesenchymal stem cells are derived from the hair bulb. By proliferating mesenchymal stem cells from the vicinity of the hair bulb, mesenchymal stem cells can be efficiently obtained.

[0009] In a preferred embodiment of the present invention, the culturing step includes a static culturing step of statically culturing the hair while replenishing a fresh culture medium without replacing the entire amount of the culture medium. By including the static culture step, the proliferation of hair-derived mesenchymal stem cells can be promoted.

[0010] In a preferred embodiment of the present invention, the culturing time in the static culturing step is 6 days or longer from the start of the static culturing. By setting the static culture time within the above range, the proliferation of mesenchymal stem cells after static culture can be promoted.

[0011] In a preferred embodiment of the present invention, the culture method includes a step of contacting the obtained hair with a disinfectant for less than 1 minute before the culture step. By performing the above steps, the adhesion of the sample hair to the culture medium can be improved, so that mesenchymal stem cells can be efficiently obtained from the vicinity of the hair bulb.

[0012] In a preferred embodiment of the present invention, the culture method includes a step of sowing one hair per well in a culture vessel before the culture step. The present invention including the above steps can proliferate a sufficient amount of mesenchymal stem cells from one hair.

[0013] In a preferred embodiment of the present invention, the culture method does not include a degradation step of degrading components of the extracellular matrix in the hair before the culture step. By not including the above-mentioned degradation step, damage to cells can be reduced, so that mesenchymal stem cells can be efficiently obtained from the vicinity of the hair bulb. In addition, compared with conventional methods including the above-mentioned degradation step, the workload of sample processing can be saved and the process can be simplified.

[0014] In a preferred embodiment of the present invention, the degrading step is a step of treating the hair with collagenase. By not performing collagenase treatment before the culture step, mesenchymal stem cells can be efficiently obtained from the vicinity of the hair bulb, and the process can be further simplified.

[0015] In a preferred embodiment of the present invention, in the culturing step, the mesenchymal stem cells are subjected to adhesion culture.

[0016] In a preferred embodiment of the present invention, the adhesion culture is performed on a culture surface coated with laminin or a fragment thereof. By the above method, mesenchymal stem cells can be efficiently proliferated.

[0017] In a preferred embodiment of the present invention, the culture medium contains L-ascorbic acid, selenium, transferrin and insulin. By using the above culture medium, mesenchymal stem cells can be efficiently proliferated.

[0018] In a preferred embodiment of the present invention, the culture medium contains basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF). By using the above culture medium, mesenchymal stem cells can be efficiently proliferated.

[0019] The present invention relates to a method for producing mesenchymal stem cells, which comprises the above-mentioned method for culturing mesenchymal stem cells. The present invention also relates to mesenchymal stem cells obtained by the above-mentioned mesenchymal stem cell culture method. In a preferred embodiment of the present invention, the mesenchymal stem cells have the ability to differentiate into one or more cells selected from osteoblasts, adipocytes and chondrocytes.

[0020] The present invention also provides a method for producing differentiated cells, which comprises: a step of obtaining a cell group containing mesenchymal stem cells by the above-mentioned mesenchymal stem cell culture method; and a step of culturing the cell group containing mesenchymal stem cells in a differentiation induction medium to obtain a cell group containing one or more differentiated cells selected from osteoblasts, adipocytes and chondrocytes. The present invention also relates to differentiated cells produced by the above-mentioned method for producing differentiated cells.

[0021] In addition, the present invention for solving the above-mentioned problems is as follows. [1] A method for culturing mesenchymal stem cells, the method comprising a culturing step of culturing hair in a culture medium, wherein the hair is obtained by plucking and contains a hair bulb covered by dermal root sheath tissue. [2] The culture method according to [1], wherein the mesenchymal stem cells are derived from the hair bulb. [3] The culture method according to [1] or [2], wherein the culture step includes a static culture step of statically culturing the hair while replenishing a fresh culture medium without replacing the entire amount of the culture medium. [4] The culture method according to [3], wherein the culture time in the static culture step is 6 days or more from the start of the static culture. [5] The culture method according to any one of [1] to [4], wherein, before the culture step, the culture method includes a step of contacting the obtained hair with a disinfectant for less than 1 minute before culture. [6] The culture method according to any one of [1] to [5], wherein, before the culture step, the culture method includes a step of sowing one hair per well in a culture vessel. [7] The culture method according to any one of [1] to [6], wherein the culture method does not include a degradation step of degrading components of the extracellular matrix in the hair before the culture step. [8] The culture method according to [7], wherein the degradation step is a step of treating the hair with collagenase. [9] The culture method according to any one of [1] to [8], wherein in the culture step, the mesenchymal stem cells are subjected to adhesion culture.

[10] The culture method according to [9], wherein the adhesion culture is performed on a culture surface coated with laminin or a fragment thereof.

[11] The culture method according to any one of [1] to

[10] , wherein the culture solution contains L-ascorbic acid, selenium, transferrin and insulin.

[12] The culture method according to any one of [1] to

[11] , wherein the culture medium contains basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF).

[13] A method for producing mesenchymal stem cells, the method comprising the culturing method according to any one of [1] to

[12] .

[14] A mesenchymal stem cell obtained by the culture method described in any one of [1] to

[12] .

[15] The mesenchymal stem cells according to

[14] , wherein the mesenchymal stem cells have the ability to differentiate into one or more cells selected from osteoblasts, adipocytes and chondrocytes.

[16] A method for producing differentiated cells, the method comprising: A step of obtaining a cell population containing mesenchymal stem cells by the culture method according to any one of [1] to

[12] ; A step of culturing a cell population containing mesenchymal stem cells in a differentiation induction medium to obtain a cell population containing one or more differentiated cells selected from osteoblasts, adipocytes, and chondrocytes.

[17] A differentiated cell produced by the production method described in

[16] . Effects of the Invention

[0022] According to the present invention, mesenchymal stem cells can be efficiently obtained from hair obtained from any individual, and differentiated cells such as adipocytes can be produced using the mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [ Figure 1 ] is a process diagram for illustrating the steps of a method for culturing mesenchymal stem cells according to an embodiment of the present invention. [ Figure 2 ] is a bright field photograph of mesenchymal stem cells proliferating near the hair bulb of plucked hair in Example 1. [ Figure 3 ] is a bright field photograph showing the state of keratinocytes proliferating from the vicinity of the outer root sheath attached to the plucked hair in Example 1. [ Figure 4 ] is a bright field photograph showing the results of Alizarin red staining of the hair-derived mesenchymal stem cells (DS-MSC), adipose tissue-derived mesenchymal stem cells (AD-MSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and umbilical cord mesenchymal stem cells (UC-MSC) cultured in Example 1 after culturing them in an osteoblast induction medium in Example 2. "+" indicates culturing in a differentiation induction medium, and "-" indicates culturing in an undifferentiation maintenance medium. [ Figure 5 ] is a bright field photograph showing the results of oil red staining after culturing the hair-derived mesenchymal stem cells (DS-MSC), adipose tissue-derived mesenchymal stem cells (AD-MSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and umbilical cord mesenchymal stem cells (UC-MSC) cultured in Example 1 in an adipocyte induction medium in Example 2. "+" indicates culturing in a differentiation induction medium, and "-" indicates culturing in an undifferentiation maintenance medium. [ Figure 6 ] is a graph showing the results of analyzing the relative expression of the chondrocyte marker gene (SOX9) to the expression of the housekeeping gene GAPDH after culturing the hair-derived mesenchymal stem cells (DS-MSC) and bone marrow-derived mesenchymal stem cells (BM-MSC) cultured in Example 1 in a chondrocyte induction medium in Example 2. "+" indicates that the cells were cultured in a differentiation induction medium, and "-" indicates that the cells were cultured in an undifferentiated maintenance medium, and the expression level of the cells cultured in the undifferentiated maintenance medium was used as a reference (1). [ Figure 7] is a graph showing the results of analyzing the relative expression of the chondrocyte marker gene (ACAN) to the expression of the housekeeping gene GAPDH after culturing the hair-derived mesenchymal stem cells (DS-MSC) and bone marrow-derived mesenchymal stem cells (BM-MSC) cultured in Example 1 in a chondrocyte induction medium in Example 2. "+" indicates cells cultured in a differentiation induction medium, and "-" indicates cells cultured in an undifferentiated maintenance medium, with the expression level of the cells cultured in the undifferentiated maintenance medium being used as a reference (1). [ Figure 8 ] is a graph showing the results of analyzing the mesenchymal stem cell positive markers (CD105, CD73 or CD90) of the hair-derived mesenchymal stem cells (DS-MSCs) cultured in Example 1 by fluorescence activated cell sorting (FACS) in Example 3. DETAILED DESCRIPTION

[0024] <Method for culturing mesenchymal stem cells> Next, refer to Figure 1 Preferred embodiments of the method for culturing mesenchymal stem cells of the present invention will be described. In addition, the present invention is not limited to the embodiment, and the design can be changed appropriately.

[0025] Figure 1 The method shown includes: an acquisition step S1 of acquiring hair including a hair bulb covered with dermal root sheath tissue from an arbitrary individual; a sterilization step S2 of sterilizing the acquired hair; and a cultivation step S3 of culturing the hair.

[0026] Here, the "hair bulb" in the present invention refers to the bulge of the hair located deep inside the hair in the skin. The dermal papilla exists in the center of the hair bulb. The dermal papilla cells constituting the dermal papilla are one of the well-known mesenchymal stem cells.

[0027] In addition, the "dermal sheath (DS) tissue" in the present invention refers to one or more layers of dermal tissue surrounding the outermost layer of the hair follicle, and is a tissue surrounding the epithelial outer root sheath. The dermal root sheath is connected to the hair papilla at the lowest end of the hair bulb.

[0028] Hereinafter, the details of each step of the acquisition step S1, the disinfection step S2, and the cultivation step S3 of the present invention will be described.

[0029] (1) Obtaining process S1 The hair used in the present invention is obtained by plucking it from any individual, preferably from a mammal (eg, human, mouse, monkey, cow, pig, rat, dog, etc.) In the present invention, the hair is preferably obtained from a human. By pulling out the hair, we can obtain a sample of the part of the dermal root sheath tissue attached to the hair bulb. In addition, since we use hair that can be obtained by a non-invasive method, we can reduce the burden of collecting samples from donor individuals.

[0030] There is no particular limitation on the tissue individual (specifically, mammalian individual) from which hair is collected. For example, when mesenchymal stem cells are used as a cell source for screening to evaluate the cosmetic effect, drug sensitivity, and the presence or absence of side effects of the donor individual, hair is obtained from the donor himself or another person who has the same gene polymorphism related to the donor's drug sensitivity, etc. In addition, when used for transplantation to a patient, hair is preferably obtained from a donor (e.g., a patient) or another person with the same or substantially the same HLA type as the donor. In this way, the risk of rejection can be avoided, thereby making the obtained mesenchymal stem cells suitable for regenerative medicine. Here, the type of HLA is "substantially the same" means that when mesenchymal stem cells obtained by using immunosuppressants, etc. are transplanted into a donor individual, the type of HLA is consistent with the degree to which the transplanted cells can survive. For example, the case where HLA (for example, three loci of HLA-A, HLA-B and HLA-DR) are the same, etc.

[0031] In the present invention, the number of hairs to be plucked out is at least 1, preferably 2 or more, and more preferably 5 or more. Specifically, 1 to 10 hairs are preferably used. Then, the lower part of the hair collected from the donor individual is cut to obtain the hair bulb covered by the dermal hair root sheath tissue. The length of the hair including the obtained hair bulb is preferably 0.3 to 5 mm.

[0032] The present invention may further include a step of plucking the area to which the outer root sheath of the hair is attached to obtain only the hair bulb. By including this step, only mesenchymal stem cells proliferating from the vicinity of the hair bulb can be efficiently obtained.

[0033] Although the embodiment including the acquisition step S1 of acquiring hair from a donor individual has been described, in the present invention, it is also preferable to adopt a form in which hair acquired in advance by plucking is optionally subjected to the following sterilization step S2 and then subjected to the culturing step S3.

[0034] (2) Disinfection process S2 In the disinfection step S2, the obtained hair is brought into contact with a disinfectant before the culture step S3. The method of bringing the hair into contact with the disinfectant is not particularly limited, but a preferred method is to immerse the hair in the disinfectant. The contact time with the disinfectant is preferably less than 1 minute, more preferably less than 30 seconds, further preferably less than 10 seconds, and particularly preferably less than 5 seconds. By keeping the contact time with the disinfectant within the above range, damage to the hair can be reduced and the adhesion between the hair and the culture vessel can be improved, thereby efficiently proliferating mesenchymal stem cells.

[0035] The disinfectant used in the present invention is preferably a solution to which an antibiotic is added. The preferred antibiotic is one or more selected from a cell wall synthesis inhibitor, a protein synthesis inhibitor, and a cell membrane function inhibitor. More preferably, one or more antibiotics are selected from a cell wall synthesis inhibitor, a protein synthesis inhibitor, and a cell membrane function inhibitor.

[0036] The cell wall synthesis inhibitor is preferably a β-lactam antibiotic. Examples of the β-lactam cell wall synthesis inhibitor include penicillin, ampicillin, carbenicillin sodium, and the like. In the present invention, penicillin is preferably used. Preferred examples of the protein synthesis inhibitor include aminoglycoside antibiotics. Examples of the aminoglycoside protein synthesis inhibitor include kanamycin, gentamicin, streptomycin, neomycin, hygromycin, and tobramycin. In the present invention, streptomycin is preferably used. As the cell membrane function inhibitor, polyene antibiotics are preferred. Examples of polyene cell membrane function inhibitors include amphotericin B, nystatin, natamycin, and the like. In the present invention, amphotericin B is particularly preferably used. The disinfectant solution according to the present invention is preferably a solution containing the antibiotics penicillin, streptomycin and amphotericin B.

[0037] (3) Cultivation step S3 In the culture step S3, after the disinfection step S2 is performed, the obtained hair is cultured in a culture solution. In this way, mesenchymal stem cells can be obtained from the hair bulb.

[0038] The culture medium used for culturing hair may be any known culture medium for culturing mesenchymal stem cells, and preferably includes DMEM culture medium, DMEM / F12 culture medium, Essential 8, AmnioMAX TM C-100, StemPro MSC SFM, etc.

[0039] In addition, the culture medium used in the culture step S3 is preferably a culture medium containing L-ascorbic acid, selenium, transferrin, and insulin. Such a culture medium includes Essential 8. Essential 8 culture medium is a DMEM / F12 culture medium containing L-ascorbic acid, selenium, insulin, NaHCO as additives. 3 , transferrin, bFGF and TGFβ family signaling pathway active substances (TGFβ1 or Nodal) in the culture medium. By using such a culture medium, mesenchymal stem cells can be efficiently proliferated.

[0040] The culture solution used in the culture step S3 preferably contains basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF). By including the above factors, mesenchymal stem cells can be efficiently obtained while suppressing cell differentiation. In addition, by particularly including basic fibroblast growth factor (bFGF), the adhesion of the proliferating mesenchymal stem cells can be improved. In a preferred embodiment, the first culture medium is a culture medium prepared by adding 5 to 15 ng / mL (more preferably 10 ng / mL) of bFGF and 5 to 15 ng / mL (more preferably 10 ng / mL) of EGF to Essential 8.

[0041] The culture medium used in the culture step S3 preferably contains serum. The serum is not particularly limited as long as it is derived from an animal, but is preferably derived from mammals, and specific examples thereof include fetal bovine serum and human serum.

[0042] The calcium concentration of the culture solution used in the culture step S3 is preferably 0.2 mM or more, more preferably 0.5 mM or more, more preferably 0.8 mM or more, and even more preferably 1.0 mM or more. The culture medium used in the culturing step S3 may have a calcium concentration of 4.0 mM or less, 3.5 mM or less, or 3.0 mM or less. Specifically, the calcium concentration of the culture solution used in the culture step S3 is preferably 0.2 to 4 mM, more preferably 0.5 to 3.5 mM, and even more preferably 1.0 to 3.5 mM. By using a medium having the above-mentioned calcium concentration, the proliferation of cells other than mesenchymal stem cells, such as keratinocytes, can be suppressed.

[0043] It is preferred to culture hair as an adherent culture using a culture vessel to which cells can adhere. The appropriate culture vessel used when performing adhesion culture can be appropriately selected according to the culture scale, culture conditions, and culture period. As the culture vessel with cell adhesion, there can be cited culture vessels that are artificially treated to improve the adhesion between the culture vessel surface and the cells, specifically including culture vessels with processed surfaces and culture vessels with coating agents applied inside.

[0044] In the present invention, it is preferred to culture hair using a culture device coated with a coating agent. Examples of coating agents include extracellular matrices such as laminin, endonuclein, collagen, gelatin, vitronectin, Synthemax (Corning), and Matrigel, and polymers such as polylysine and polyornithine. In the present invention, an extracellular matrix is ​​preferably used, and laminin or a fragment thereof is preferably used.

[0045] Preferred laminins to be used include laminin α5β1γ1 (laminin 511), laminin α5β2γ1 (laminin 521), laminin α4β1γ1 (laminin 411), laminin α2β1γ1 (laminin 211) and fragments thereof, and more preferably laminin 511 or laminin 521. Recombinant laminins and laminin fragments may also be used.

[0046] The laminin or laminin fragment described above preferably has an integrin binding site. Furthermore, it is more preferred that the laminin fragments form heterotrimers. The type of integrin bound by laminin or laminin fragment is preferably integrin α6β1, integrin α6β4, integrin α3β1 or integrin α7β1, more preferably integrin α6β1. The use of laminin or laminin fragment bound to integrin α6β1 can promote the proliferation of mesenchymal stem cells. The laminin fragment having binding activity to these integrins preferably includes a laminin fragment derived from at least one selected from the group consisting of laminin 511, laminin 521, laminin 411, and laminin 211, more preferably a laminin fragment derived from laminin 511 or laminin 521, and still more preferably a laminin fragment derived from laminin 511. Such a laminin fragment preferably includes an E8 fragment obtained by digesting laminin with elastase, or a recombinant E8 fragment. In the present invention, laminin 511E8 is more preferably used. As laminin 511E8, commercially available products such as iMatrix-511 (manufactured by Nippi Co., Ltd., Japan) can be used.

[0047] In addition, specific forms of culture vessels include, for example, flasks, tissue culture flasks, culture dishes (plates), tissue culture plates, multi-well culture dishes, microplates, microwell plates, multi-well plates, chamber slides, culture dishes, test tubes, trays, culture bags, microcarriers, microbeads, stacked plates, spinner flasks or roller bottles. The culture vessel used is preferably a 12-well plate or a 24-well plate for cell culture, and more preferably a 24-well plate.

[0048] Preferably, one hair per well is seeded onto the plate. According to the present invention, a sufficient amount of mesenchymal stem cells can be efficiently proliferated from one hair (one sample), so there is no need to sow multiple hairs in one well, and compared with the existing method of Patent Document 1, there is an advantage that only a smaller amount of hair needs to be prepared.

[0049] The amount of culture medium used during seeding is preferably sufficient to submerge the entire excised hair tissue and to keep the liquid level 1 mm or less above the entire tissue. By using the above amount of culture medium during seeding, the hair can be attached to the plate.

[0050] In the culture step S3, the mesenchymal stem cells are cultured by replenishing and replacing the culture medium in full. Here, in the culture step S3, it is preferred that the obtained hair is first subjected to static culture. The static culture step S31 for statically culturing the hair will be described in detail below.

[0051] In the static culture step S31, the seeded hair is preferably cultured in a static state without replacing the culture medium in full, and the culture medium is replenished with a fresh culture medium without replacing the culture medium in full. The number of times the culture medium is replenished is preferably 1 to 4 times, more preferably 2 to 3 times, and even more preferably 3 times.

[0052] The amount of the culture medium added in the static culture step S31 is preferably such that the entire excised hair is immersed and the liquid level is 2 mm or less above the entire hair, and more preferably 1.5 mm or less. In addition, the amount of the medium to be replenished is preferably 0.5 to 3 times, more preferably 1 to 3 times, and even more preferably 1 to 2 times, of the amount of the medium at the time of hair seeding. When the number of times of replenishing the medium is 3 or more, the amount of the medium replenished after the third time is preferably greater than the amount of the medium replenished from the first to the second time. In this case, the amount of the medium replenished from the third time onwards is preferably 1.2 to 3 times, more preferably 1.5 to 2.5 times, and even more preferably 2 to 2.5 times the amount of the medium replenished from the first to the second time. In addition, the amount of the medium replenished from the second time onwards is preferably such that the entire excised hair tissue is immersed and the liquid surface is located 1 to 2 mm above the entire tissue, and more preferably 1 to 1.5 mm.

[0053] As a specific form of the static culture step S31, a preferred example is that the culture medium is replenished three times, with the culture medium being replenished once between 20 and 24 hours and once between 100 and 120 hours after hair seeding with an amount of culture medium equal to (1 times) that at the time of seeding, and replenished once between 140 and 170 hours after hair seeding with an amount of culture medium twice that at the time of seeding.

[0054] The culture time of the static culture step S31 is preferably 4 days or more, more preferably 6 days or more, and even more preferably 8 days or more. The total culture time of the static culture step S31 refers to the time from the day of hair seeding to the time when the culture medium is completely replaced.

[0055] By performing such a static culture step S31, the proliferation of mesenchymal stem cells derived from the hair bulb can be promoted.

[0056] In the culturing step S3, it is preferred that after the static culturing step S31, the medium is supplemented and / or completely replaced under the above-mentioned conditions according to the cell proliferation level. Preferably, the medium is supplemented and / or replaced in full until the proliferating cells reach 50% to 80% confluence. The culture time for supplementing and replacing the medium after the static culture step S31 is preferably 7 to 28 days, more preferably 14 to 21 days.

[0057] In the culturing step S3, the cells are preferably subcultured before the cells become overcrowded in the expansion area of ​​the cells proliferating from the hair. For example, when culturing in a 24-well plate (well bottom diameter 15 to 16 mm), when the cells proliferating from the hair reach 50 to 80% confluence, it is preferable to perform subculture and further culture the cells. The culture medium used for subculturing is DMEM, DMEM / F12, Essential 8, AmnioMAX TM C-100 (Gibco), StemPro MSC SFM (Gibco), StemFit For MSC (Ajinomoto) and other culture media can be used appropriately, and Essential 8 or StemFit For MSC can be preferably used in the present invention. In addition, the culture medium used for subculturing preferably contains L-ascorbic acid, selenium, transferrin and insulin. In addition, the culture medium used for subculturing preferably contains basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF).

[0058] The calcium concentration of the culture medium used for subculturing is preferably 0.2 mM or more, more preferably 0.5 mM or more, more preferably 0.8 mM or more, and even more preferably 1.0 mM or more. Specifically, the calcium concentration of the culture solution used for subculturing is preferably 0.2 to 4 mM, more preferably 0.5 to 3.5 mM, and further preferably 1.0 to 3.5 mM. If hair with the outer root sheath attached is directly cultured, repeated passages using a culture medium having a calcium concentration within the above range will eliminate proliferating keratinocyte-like cells, thereby allowing mesenchymal stem cells to be isolated and cultured.

[0059] Furthermore, the present invention preferably does not include a degradation step of degrading components of the extracellular matrix in the hair before the culturing step S3. In the existing methods, as shown in Patent Document 1, when culturing mesenchymal stem cells from hair, the extracellular matrix such as collagen fibers attached to the sample is usually degraded to promote the proliferation of target cells. On the other hand, the present invention makes it possible to obtain mesenchymal stem cells by proliferating mesenchymal stem cells from the vicinity of the hair bulb without degrading the extracellular matrix. As a result, according to the present invention, the workload of sample processing can be saved compared to the existing methods, and mesenchymal stem cells can be efficiently obtained from a small amount of sample.

[0060] In the present invention, it is more preferred that the step of treating the hair with collagenase is not included before the culture step S3. Since collagenase treatment does not degrade collagen, damage to the sample is minimized and mesenchymal stem cells can be efficiently proliferated from the vicinity of the hair bulb. Furthermore, the present invention may be configured not to include a step of treating the hair with collagenase and / or dispase before the culturing step S3.

[0061] According to the present invention, a sufficient amount of mesenchymal stem cells can be obtained from a single hair obtained from a donor individual. That is, mesenchymal stem cells can be efficiently proliferated with a smaller number of samples compared to the conventional culture method using hair disclosed in Patent Document 1. Therefore, the burden of obtaining hair from a donor individual can be reduced, and the time required to obtain a required amount of mesenchymal stem cells can be shortened.

[0062] The present invention also provides a method for producing mesenchymal stem cells, the method comprising the above-mentioned method for culturing mesenchymal stem cells. The present invention also relates to mesenchymal stem cells obtained by the above-mentioned method for culturing mesenchymal stem cells.

[0063] The mesenchymal stem cells of the present invention are preferably positive for one or more (more preferably two or more, and even more preferably three or more) mesenchymal stem cell markers selected from CD105, CD73, CD90, and CD44. In addition, it is preferred that the expression of mesenchymal stem cell markers is positive and the expression of molecules not observed in mesenchymal stem cells is negative. Examples of molecules not observed to be expressed in mesenchymal stem cells include CD45, CD34, CD14, CD11b, CD79a, CD19, and HLA-DR.

[0064] In addition, the mesenchymal stem cells according to the present invention have the ability to differentiate into mesodermal cells. The mesodermal cells differentiated from the mesenchymal stem cells of the present invention include osteoblasts, adipocytes, chondrocytes, osteocytes, cardiomyocytes, tendon cells, dental pulp cells, odontoblasts, etc., preferably osteoblasts, adipocytes and chondrocytes, etc. The mesenchymal stem cells obtained in the present invention preferably have the ability to differentiate into one or more cells selected from osteoblasts, adipocytes and chondrocytes, and more preferably have the ability to differentiate into osteoblasts, adipocytes and chondrocytes.

[0065] The mesenchymal stem cells obtained according to the present invention can be used in a variety of ways, such as pharmaceutical raw materials and screening materials. For example, existing differentiation induction methods can be used to produce cells such as osteocytes and adipocytes from mesenchymal stem cells. In addition, differentiated cells can also be used to perform in vitro screening of the efficacy and toxicity of candidate drug compounds. In addition, by culturing mesenchymal stem cells from the hair of a donor individual and differentiating the obtained mesenchymal stem cells into skin tissue including hair follicle tissue, the skin tissue can be used to confirm the effectiveness of drugs, cosmetics, etc.

[0066] <Method for producing differentiated cells> The present invention also relates to a method for producing differentiated cells, which comprises: a step of obtaining a cell group containing mesenchymal stem cells by the above-mentioned mesenchymal stem cell culture method of the present invention; and a step of culturing the cell group containing mesenchymal stem cells in a differentiation induction medium to obtain a cell group containing one or more differentiated cells selected from osteoblasts, adipocytes and chondrocytes.

[0067] In a preferred embodiment, the present invention is a method for producing osteoblasts, wherein the differentiation induction medium is an osteoblast induction medium, and the differentiated cells are osteoblasts. In another preferred embodiment, the present invention is a method for producing adipocytes, wherein the differentiation induction medium is an adipocyte induction medium, and the differentiated cells are adipocytes. In another preferred embodiment, the present invention is a method for producing chondrocytes, wherein the differentiation induction medium is a chondrocyte induction medium, and the differentiated cells are chondrocytes.

[0068] The osteoblasts of the present invention include not only mature osteoblasts but also immature osteoblasts such as osteoprogenitor cells and pre-osteoblasts. Osteoblast differentiation can be confirmed by alizarin red staining. Alizarin red can detect osteoblasts by staining calcium deposited in cells that have undergone bone differentiation or mineralization. It can also be confirmed by alkaline phosphatase (ALP) staining.

[0069] The adipocytes according to the present invention include brown adipocytes, leukocytes, beige adipocytes and precursors thereof. Differentiation into adipocytes can be confirmed by staining with Oil Red, a nonpolar, lipid-soluble azo dye that, when in contact with adipocytes, dissolves in the solvent of intracellular lipids and stains fat droplets. In addition, differentiation into adipocytes can be confirmed by measuring triglycerides in cells.

[0070] The chondrocytes of the present invention include cells that produce extracellular matrix such as collagen constituting cartilage and its precursors. Differentiation into chondrocytes can be confirmed by staining with Alcian blue. Alcian blue forms ionic bonds with the carboxyl and sulfate groups of acidic mucopolysaccharides, and can stain acidic mucopolysaccharides such as chondroitin sulfate and hyaluronic acid contained in chondrocytes. In addition, differentiation into chondrocytes can be confirmed by confirming the expression of chondrocyte markers such as SOX9 and ACAN.

[0071] The induction of mesoderm cell differentiation can be carried out using existing techniques. For example, a known basal medium to which various differentiation inducers are appropriately added can be used for induction. Examples of basal medium include DMEM medium and the like.

[0072] The osteoblast differentiation induction medium preferably contains one or more components selected from ascorbic acid, ascorbic acid-2-phosphate, β-glycerophosphate, dexamethasone, and proteins involved in bone formation (for example, bone morphogenetic protein (BMP)-2, BMP-4, insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, parathyroid hormone (PTH), Wnts, etc.).

[0073] The adipocyte differentiation inducing medium preferably contains one or more components selected from the group consisting of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX).

[0074] The chondrocyte differentiation induction medium preferably contains one or more components selected from BMP-2, BMP-6, TGF-β3, dexamethasone, ascorbic acid, L-ascorbic acid 2-phosphate, dexamethasone, insulin, insulin-like growth factor (IGF), L-proline, and pyruvate.

[0075] By the method for producing differentiated cells of the present invention, cells differentiated into prescribed cells such as osteoblasts, adipocytes, chondrocytes, etc. can be obtained. That is, the present invention may be in the form of differentiated cells (including cell groups) produced by the above-mentioned method for producing differentiated cells. In addition, the cells to be obtained may be cell clusters.

[0076] The obtained cells can be further differentiated into various tissues (bone tissue, cartilage tissue, adipose tissue, etc.), and thus can be suitably used as materials for regenerative medicine. Example

[0077] Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

[0078] <Example 1> Cultivation of mesenchymal stem cells (1) Reagents used (a) Antibiotic mixture Transfer 247 ml of HBSS to a sterile container, and add 2.5 ml of penicillin-streptomycin mixed solution (final concentration of 100 U / ml penicillin, 100 mg / ml streptomycin) and 250 μL of amphotericin B solution (final concentration of 250 ng / ml). (b) Mesenchymal stem cell culture medium (DS-MSC culture medium) 45 mL of Essential 8Flex medium (Gibco, supplemented) was transferred to a 50 mL tube, and 0.5 mL of penicillin-streptomycin mixed solution (final concentration of 100 U / ml penicillin, 100 μg / ml streptomycin) and 5 ml of FBS were added. Thereafter, bFGF (Reprocell) and EGF (epithelial cell growth factor (EGF), human, Fujifilm and Kozen Chemical) were added to a final concentration of 10 ng / ml, respectively. (c) iMatrix coating 5 μL of iMatrix-511 (Nippi) dilution was added to 1 mL of DPBS (- / -) and added to the bottom of a 24-well cell culture plate at 300 μL / well, and allowed to stand in an incubator at 37°C for more than 30 minutes. Thereafter, the diluent was removed and used.

[0079] (2) Culture of mesenchymal stem cells The hair of the subject (human) was plucked and immediately immersed in the antibiotic mixture for a while. Next, the hair whose hair bulb was covered by the dermal root sheath tissue was selected, and the hair including the hair bulb part and the part where the outer root sheath was attached was removed. The obtained hair was seeded one by one on a 24-well plate coated with iMatrix containing 150μL DS-MSC culture medium.

[0080] 150 μL DS-MSC medium was supplemented 1 and 5 days after seeding, and 300 μL DS-MSC medium was further supplemented 7 days after seeding. Thereafter, the DS-MSC medium was continuously supplemented and replaced according to the cell proliferation level.

[0081] Once cells proliferate to 50-80% confluence, they are passaged using the following procedure. That is, 0.2 mL of TrypLE Select was added, and the cells were allowed to stand in a 37°C incubator, and then the cells were detached by pipetting. After that, the cells were collected in a 1.5 mL centrifuge tube, centrifuged, the supernatant was removed, the cells were resuspended in 1 mL of DS-MSC medium, and the cells were counted. Then, the cells were seeded in a 6-well plate coated with iMatrix at a density of 50,000 / well using DS-MSC medium.

[0082] After seeding the cells, they were cultured in an incubator at 37°C. Starting from the second day after seeding, supplementation and full replacement of StemFit for mesenchymal stem cells (Ajinomoto, hereinafter also referred to as StemFit for MSC) were performed to proliferate mesenchymal stem cells (including hair-derived mesenchymal stem cells of the dermal root sheath (DS); DS-MSC).

[0083] (4) Results The figure shows the state of cells extending from the vicinity of the hair bulb 7 days after seeding into a 24-well plate. Figure 2 As shown, a large number of mesenchymal stem cell-like cells proliferated near the hair bulb. also, Figure 3 The state of cells extending from the vicinity of the outer root sheath attached to the hair 6 days after seeding into a 24-well plate is shown. Figure 3 As shown, cells extending from the vicinity of the outer root sheath attached to the hair do not exhibit a mesenchymal stem cell-like morphology, but rather a keratinocyte-like morphology.

[0084] Furthermore, when cells proliferating near the hair bulb and cells proliferating near the outer root sheath attached to the hair are mixed and passaged in the above-mentioned (2), the keratinocyte-like cells proliferating near the outer root sheath die, and only the mesenchymal stem cell-like cells proliferate.

[0085] As can be seen from the above, according to the culture method of the present invention in Example 1, it is clear that mesenchymal stem cells are cultured from hair including a hair bulb covered by a dermal hair root sheath tissue. Then, although there are cells similar to keratinocytes proliferating near the outer hair root sheath attached to the hair, only mesenchymal stem cells proliferate as the passage continues. Therefore, it can be considered that according to the culture method of the present invention, even if the region including the outer hair root sheath is not removed, mesenchymal stem cells can be isolated and cultured.

[0086] <Example 2> Induced differentiation into osteoblasts, adipocytes, and chondrocytes The hair-derived mesenchymal stem cells (DS-MSCs) cultured in Example 1 were induced to differentiate into osteoblasts, adipocytes, and chondrocytes according to the following procedures from the second day after the third passage.

[0087] (1) Differentiation induction of osteoblasts and adipocytes After confirming that the cells in the 24-well plate have reached 80-90% confluence, the medium is replaced with a medium specifically for inducing differentiation of osteoblasts and adipocytes, respectively. Specifically, MSC osteogenic differentiation medium (Promo Cell) is used for induction of osteoblasts, and MSC adipogenic differentiation medium (Promo Cell) is used for induction of adipocytes.

[0088] After replacing the culture medium with differentiation induction medium or undifferentiated maintenance medium (StemFit for MSC) for 2 weeks, osteoblasts were stained with Alizarin Red and adipocytes were stained with Oil Red. Alizarin Red staining was performed using Alizarin Red S staining kit (Bio Mirai Kobo) according to the existing protocol. Oil Red staining was performed using Oil Red O staining kit (Bio Mirai Kobo) with OR staining solution diluted to 60% with purified water.

[0089] In addition, as controls, commercially available adipose tissue-derived mesenchymal stem cells (AD-MSC, Takara Bio, C-12977, batch number: 458Z017.2), bone marrow-derived mesenchymal stem cells (BM-MSC, Takara Bio, C-12974, batch number: 479Z025), and umbilical cord-derived mesenchymal stem cells (UC-MSC, Takara Bio, C-12971, batch number: 458Z025.1) were cultured in the same manner as DS-MSC using differentiation induction medium or StemFit for MSC, and cell staining was performed. Next, use an optical microscope to observe the state of the stained cells. Figure 4 The results of Oil Red staining are shown in Figure 5 shown.

[0090] (2) Chondrocyte differentiation induction After confirming that the cells in the 24-well plate have reached 80-90% confluence, the medium is replaced with a special medium for chondrocyte differentiation induction (MSC chondrogenic differentiation medium (Promo Cell)). After 3 weeks of replacing the medium with the differentiation induction medium, total RNA is extracted from the cells and reverse transcribed to synthesize cDNA. Using the obtained cDNA, the expression levels of chondrocyte marker genes (SOX9, ACAN) and the housekeeping gene GAPDH are measured, and the relative expression levels of each chondrocyte marker gene relative to the expression level of GAPDH are analyzed by quantitative PCR. In addition, as a positive control, marker gene expression analysis was also performed on BM-MSC. The chart showing the expression level of each gene is as follows Figure 6 and Figure 7 shown.

[0091] (3) Results like Figure 4 As shown, DS-MSC, AD-MSC, BM-MSC, and UC-MSC cultured with differentiation induction medium all showed Alizarin Red staining. From this result, it can be seen that mesenchymal stem cells differentiated into osteoblasts. In addition, there were differences in the percentage of stained cells depending on the source of MSC.

[0092] In addition, if Figure 5 As shown, Oil Red staining was observed in all DS-MSCs, AD-MSCs, BM-MSCs, and UC-MSCs cultured using differentiation induction medium. From this result, it can be seen that mesenchymal stem cells differentiate into adipocytes. In addition, there are differences in the percentage of stained cells depending on the source of MSCs.

[0093] like Figure 6 and Figure 7 As shown in Figure 2, the expression levels of SOX9 and ACAN in BM-MSCs cultured with differentiation induction medium increased compared with those in BM-MSCs cultured with undifferentiated maintenance medium. In addition, the expression levels of SOX9 and ACAN in DS-MSCs cultured with differentiation induction medium also increased compared with those in DS-MSCs cultured with undifferentiated maintenance medium. Therefore, it was confirmed that DS-MSCs according to the present invention differentiated into chondrocytes.

[0094] It can be seen from the above that the DS-MSCs of the present invention have the ability to differentiate into osteoblasts, adipocytes and chondrocytes.

[0095] <Example 3> Confirmation of Mesenchymal Stem Cell Markers The hair-derived mesenchymal stem cells (DS-MSCs) cultured in Example 1 were subjected to fluorescence activated cell sorting (FACS) using the following procedure. DS-MSCs that were passaged three times were cultured and proliferated in T75 flasks, and the cells were collected by centrifugation. The obtained cell pellet was resuspended in DPBS and the cells were dispensed into 1.5 mL centrifuge tubes to a cell concentration of 1.0 × 10 6 The cell pellet was centrifuged to obtain a cell precipitate, 100 μL of 2% FBS / DPBS was added to the obtained cell precipitate for resuspending, and the following antibodies were added.

[0096] [Antibody] (A) Isotype control CD73 (PE mouse IgG1 isotype control (FC) (Sony)): 1 μL + isotype control CD105 (AlexaFluor 488 mouse IgG1 isotype control (Sony)): 1 μL (B) Isotype control CD90 (Brilliant Violet 605 mouse IgG1 isotype control (Sony)): 2 μL (C) CD73 (PE mouse anti-human CD73 (BD ​​Biosciences)): 5 μL + CD105 (Alexa Fluor 488 anti-human CD105 (Sony)): 0.5 μL (D) CD90 (Brilliant Violet 605 anti-human CD90 (Thy1) (Sony)): 2 μL

[0097] The antibody and cells were mixed and placed on ice in the dark for 30 minutes. The cells were collected by centrifugation and resuspended in 500 μL of 2% FBS / PBS to dissolve the cell pellet. The obtained samples were subjected to FACS detection using CELL SORTER SH800S (Sony), and the fluorescence of the isotype control and the target sample were compared.

[0098] like Figure 8 As shown, CD105, CD73, and CD90 were confirmed to have stronger fluorescence than the isotype control in the target sample. On the other hand, when the expression of negative markers (CD45, CD34, CD14, CD11b, CD79a, CD19, and HLA DR) was confirmed using the same procedure, no fluorescence was observed. It can be seen from the above that the cells obtained in Example 1 express mesenchymal stem cell markers positively and do not express negative markers.

[0099] From the above results, it can be seen that mesenchymal stem cells can be efficiently cultured from plucked hair according to the present invention of Example 1. In addition, the hair-derived mesenchymal stem cells including the dermal hair root sheath obtained by the present invention have been confirmed to have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes that mesenchymal stem cells can usually differentiate into. Industrial Applicability

[0100] The present invention can be applied to the development of pharmaceutical preparations using mesenchymal stem cells, etc. Explanation of symbols

[0101] S1 Acquisition Process S2 disinfection process S3 cultivation process S31: static culture step.

Claims

1. A method for culturing mesenchymal stem cells, the method comprising a culturing step of culturing hair in a culture medium, wherein the hair is obtained by plucking and contains a hair bulb covered by dermal root sheath tissue.

2. The culture method according to claim 1, wherein The mesenchymal stem cells are derived from the hair bulb.

3. The culture method according to claim 1, wherein The culturing step includes a static culturing step of statically culturing the hair while replenishing a fresh culture medium without replacing the entire amount of the culture medium.

4. The culture method according to claim 3, wherein: The culturing time in the static culturing step is 6 days or more from the start of the static culturing.

5. The culture method according to claim 1, wherein Prior to the culturing step, the culturing method includes a step of contacting the obtained hair with a disinfectant for less than 1 minute before culturing.

6. The culture method according to claim 1, wherein Prior to the culturing step, the culturing method includes a step of sowing one hair per well in a culture vessel.

7. The culture method according to claim 1, wherein The culture method does not include a degradation step of degrading components of the extracellular matrix in the hair before the culture step.

8. The culture method according to claim 7, wherein: The degrading step is a step of treating the hair with collagenase.

9. The culture method according to claim 1, wherein: In the culturing step, the mesenchymal stem cells are subjected to adhesion culture.

10. The culture method according to claim 9, wherein: The adhesion culture is performed on a culture surface coated with laminin or a fragment thereof.

11. The culture method according to claim 1, wherein The culture solution contains L-ascorbic acid, selenium, transferrin and insulin.

12. The culture method according to claim 11, wherein The culture medium contains basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF). 13 . A method for producing mesenchymal stem cells, comprising the culturing method according to any one of claims 1 to 12. 14 . A mesenchymal stem cell obtained by the culture method according to claim 1 .

15. The mesenchymal stem cells according to claim 14, wherein The mesenchymal stem cells have the ability to differentiate into one or more cells selected from osteoblasts, adipocytes and chondrocytes.

16. A method for producing differentiated cells, the method comprising: A step of obtaining a cell population containing mesenchymal stem cells by the culture method according to any one of claims 1 to 12; A step of culturing a cell population containing mesenchymal stem cells in a differentiation induction medium to obtain a cell population containing one or more differentiated cells selected from osteoblasts, adipocytes, and chondrocytes. 17 . A differentiated cell produced by the production method according to claim 16 .

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