Keratin 15 type hair follicle stem cell culture medium, adherent culture method and application

Abstract

The invention provides a keratin 15 type hair follicle stem cell culture medium, an adherent culture method and application, and belongs to the technical field of stem cell culture. The invention provides a keratin 15 type hair follicle stem cell culture medium, which comprises a basic culture medium, a serum substitute and a nutrition additive, a mixed culture medium containing a DMEM basic culture medium and an F12 basic culture medium is used as the basic culture medium, and an epidermal growth factor, a platelet lysate and a bovine pituitary extract are used as the serum substitute. The keratin 15 type hair follicle stem cells are cultured on the basis of the culture medium, proliferation-terminated autologous mesenchymal cells serve as a feeding layer, the activity of the hair follicle stem cells p63 can be reserved, and the keratin 15 positive hair follicle stem cells are amplified.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a keratin 15 type hair follicle stem cell culture medium and a wall-adherent culture method and application. Background Art

[0002] Around the world, as many as one billion men, women and even children suffer from hair loss. Despite the huge number of people suffering from hair loss, there is currently no effective treatment for hair loss. As the understanding of hair follicle development deepens, scholars have discovered that hair follicles contain many types of stem cells. The cultivation and application of these types of stem cells will help the development of hair loss treatments.

[0003] Hair follicles are composed of epidermal (epithelial) components and dermal (mesenchymal) components. The interaction between the two plays an important role in hair follicle development, regeneration and hair growth. Traditionally, hair follicle stem cells are believed to belong to the epidermis and exist in the hair follicle bulge in the form of cell clusters. These stem cells in the bulge can form sebaceous glands upward and continuously provide stem cells for the basal layer of the epidermis and downward to the root of the hair follicle. In the process of studying hair follicle stem cells using immunohistochemistry and other methods, it was found that CD34, keratin 15 (K15) and keratin 19 (K19) are its specific markers (Lian Xiaohua. New progress in hair follicle stem cell research [J]. Journal of Local Anatomy and Surgery, 2010, 19(003): 157-158.). K15-positive hair follicle stem cells are effective in treating hair loss. Carlos et al. transplanted K15-positive hair follicle stem cells to the backs of nude mice, and obvious hair growth was observed (Chacón-Martínez, Carlos Andrés, Klose M, Niemann C, et al. Hair follicle stem cell cultures reveal self-organizing plasticity of stem cells and their progeny [J]. Embo Journal, 2017, 36 (2): e201694902. DOI: 10.15252 / embj.201694902.). However, Carlos et al. also pointed out that this type of cell is difficult to maintain in culture, especially the common adherent culture method is difficult to maintain, and it is necessary to use three-dimensional organoid culture to maintain it for a few days. These methods are extremely difficult to apply industrially, let alone clinical transformation.

[0004] On the other hand, the dermal structure includes the dermal papilla (DP) and the dermal sheath. The dermal sheath cells of the hair bulb can differentiate into dermal papilla cells (DPCs). DPCs are a typical mesenchymal stem cell that highly expresses mesenchymal cell molecules such as CD90 (Research Progress on Biological Characteristics of DPCs Chinese Journal of Plastic Surgery, 2021, 37(5): 567-570.). Mesenchymal stem cells are easy to culture, especially with mature serum-free culture conditions, but it is still impossible to culture hair follicle stem cells that are positive for K15 expression. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a keratin 15 type hair follicle stem cell culture medium and an adherent culture method and application. The culture medium can be used to complete industrial two-dimensional expansion and expand hair follicle stem cells expressing K15.

[0006] The present invention provides a keratin 15 type hair follicle stem cell culture medium, comprising a basic culture medium, a serum substitute and a nutrient additive;

[0007] The basal culture medium includes a mixed culture medium of DMEM basal culture medium and F12 basal culture medium;

[0008] The serum replacement includes epidermal growth factor, platelet lysate and bovine pituitary extract;

[0009] The nutritional supplements include insulin, hydrocortisone, epinephrine hydrochloride, transferrin, calcium chloride, SB-431542, Y-27632 and L-alanyl-L-glutamine dipeptide.

[0010] In one embodiment of the present invention, the basal culture medium further comprises glucose, and the concentration of glucose is 5-20 mM.

[0011] In one embodiment of the present invention, in the basal culture medium, the volume ratio of DMEM basal culture medium to F12 basal culture medium is 2:1.

[0012] In one embodiment of the present invention, the final concentration of the epidermal growth factor is 25 ng / ml, the final volume percentage concentration of the platelet lysate is 2%, and the final concentration of the bovine pituitary extract is 56 μg / ml;

[0013] The final concentration of insulin was 5 μg / ml, the final concentration of hydrocortisone was 0.4 μg / ml, the final concentration of epinephrine hydrochloride was 0.39 μg / ml, the final concentration of transferrin was 10 μg / ml, the final concentration of calcium chloride was 0.06 mM, the final concentration of SB-431542 was 2 μM, the final concentration of Y-27632 was 2.5 μM, and the final concentration of L-alanyl-L-glutamine dipeptide was 2 mM.

[0014] The present invention also provides a method for culturing keratin 15 type hair follicle stem cells, comprising the following steps: culturing the mesenchymal cells that have stopped proliferation into a continuous sheet and using it as a feeder layer;

[0015] The hair follicle tissue is digested into single cells, which are inoculated onto the feeder layer using the above culture medium to obtain keratin 15 type hair follicle stem cells.

[0016] In one embodiment of the present invention, the preparation of the mesenchymal cells that have stopped proliferation comprises irradiating the mesenchymal cells with an x-ray irradiator equipped with a copper alloy filter to stop the proliferation of the mesenchymal cells.

[0017] In one embodiment of the present invention, the total irradiation dose is 8 to 21 Gy.

[0018] In one embodiment of the present invention, the seeding density of the mesenchymal cells that have stopped proliferation is 133,000 to 200,000 cells / cm 2 .

[0019] The present invention also provides a keratin 15 type hair follicle stem cell line cultured by the above culture method.

[0020] The present invention also provides the use of the keratin 15 type hair follicle stem cell line in preparing hair growth materials.

[0021] Beneficial effects: The present invention provides a keratin 15 type hair follicle stem cell culture medium, comprising a basal culture medium, a serum substitute and a nutrient additive, using a mixed culture medium comprising a DMEM basal culture medium and an F12 basal culture medium as the basal culture medium, and using epidermal growth factor (EGF), platelet lysate and bovine pituitary extract as serum substitutes, thereby avoiding the use of human or animal serum and complying with the relevant provisions of China's "Guidelines for Quality Control and Preclinical Research of Stem Cell Preparations".

[0022] The present invention also cultures K15 hair follicle stem cells based on the culture medium. During the culture, autologous mesenchymal cells that have stopped proliferation are used as a feeder layer to provide a microenvironment for cell survival. The cytokines contained in the culture medium can activate the mitosis of the hair follicle stem cells, and combined with inhibitors, the increase of contaminating cells can be suppressed, thereby meeting the requirements for industrial two-dimensional expansion of hair follicle stem cells. It has been verified that the culture method of the present invention can expand hair follicle stem cells expressing keratin 15 by retaining the activity of hair follicle stem cells p63.

[0023] After culturing by the culturing method of the present invention, a cell group stained brown can be seen, which is confirmed to be a keratin 15 hair follicle stem cell line, namely a KRT15 positive cell group. This group of cells has a significant epithelial cell morphology and presents a typical paving stone shape. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Bar graph showing the results of the BPE ingredient study for epidermal stem cell culture formula screening;

[0025] Figure 2 Bar graph of results for epidermal stem cell culture formula screening - exploration of epidermal growth factor concentration range;

[0026] Figure 3 Result bar graph of epidermal stem cell culture formulation screening-platelet lysate application exploration;

[0027] Figure 4 Figure 1. Results of an irradiation dose optimization experiment for feeder cells.

[0028] Figure 5 The figure is a comparison result of the laying density of mesenchymal stem cell feeder layer after irradiation;

[0029] Figure 6 The figure is a 100x magnified image of hair follicle stem cells cultured using keratin 15. The left figure is the KRT15 staining result after 10 days of hair follicle stem cell culture using feeder layer cells, and the right figure is the hair follicle tissue digested cells cultured without feeder layer conditions;

[0030] Figure 7 The figure shows the mesenchymal cells derived from hair follicles after continuous passage in mesenchymal culture medium. A: The culture field of cells after hair follicle tissue lysis and adherence in mesenchymal cell culture medium for 24 hours (100x); B: The culture field of cells after hair follicle tissue lysis and adherence in mesenchymal cell culture medium for 3 generations (100x);

[0031] Figure 8 This is a tissue section after skin repair with KRT15 hair follicle stem cells (100 times magnification);

[0032] Fig. 9 This is the immunofluorescence staining of nude mouse hair follicle-like tissue (red is human hair follicle tissue bound by anti-human KRT15+ antibody, and blue is cell nuclei stained with DAPI). DETAILED DESCRIPTION

[0033] The present invention provides a keratin 15 type hair follicle stem cell culture medium, comprising a basic culture medium, a serum substitute and a nutrient additive;

[0034] The basal culture medium includes a mixed culture medium of DMEM basal culture medium and F12 basal culture medium;

[0035] The serum replacement includes epidermal growth factor, platelet lysate and bovine pituitary extract;

[0036] The nutritional supplements include insulin, hydrocortisone, epinephrine hydrochloride, transferrin, calcium chloride, SB-431542, Y-27632 and L-alanyl-L-glutamine dipeptide.

[0037] The basal culture medium described in the present invention is a mixed culture medium. In one embodiment, the mixed culture medium includes DMEM basal culture medium and F12 basal culture medium, and in one embodiment, the volume ratio of the DMEM basal culture medium and the F12 basal culture medium is 2:1, referred to as DF12 (2:1) in the embodiment. The present invention can supplement the content of glucose in the mixed culture medium, such as in one embodiment, the glucose content can be supplemented to 5-20mM. When the volume ratio of DMEM basal culture medium and F12 basal culture medium is 2:1, the amino acid types of the culture medium are rich and the ratio is more appropriate. At the same time, the buffering capacity of the culture medium is strong, which can provide sufficient energy metabolism substrates for long-term culture.

[0038] In the culture medium of the present invention, serum substitutes are used to replace human or animal serum, wherein the serum substitutes include epidermal growth factor (EGF), platelet lysate and bovine pituitary extract, wherein the final concentration of the epidermal growth factor is 25ng / ml, the final volume percentage concentration of the platelet lysate is 2%, and the final concentration of the bovine pituitary extract is 56μg / ml. The EGF of the present invention can effectively increase the number of expanded epidermal-derived cells, the platelet lysate can be combined with EGF to expand keratinocytes, which is beneficial to cell expansion, and the bovine pituitary extract can effectively increase the number of expanded epidermal-derived cells.

[0039] The culture medium of the present invention further contains nutrient additives, including insulin, hydrocortisone, epinephrine hydrochloride, transferrin, calcium chloride, SB-431542, Y-27632 and L-alanyl-L-glutamine dipeptide, wherein the final concentration of insulin is 5 μg / ml, the final concentration of hydrocortisone is 0.4 μg / ml, the final concentration of epinephrine hydrochloride is 0.39 μg / ml, the final concentration of transferrin is 10 μg / ml, the final concentration of calcium chloride is 0.06 mM, the final concentration of SB-431542 is 2 μM, the final concentration of Y-27632 is 2.5 μM, and the final concentration of L-alanyl-L-glutamine dipeptide is 2 mM. The insulin of the present invention enhances cell metabolism, hydrocortisone avoids oxidative stress of cells, adrenaline hydrochloride activates related receptors to enhance PKC-mediated signal transduction, transferrin maintains the balance of iron and metal ions in cells, and calcium chloride enhances the activity of keratinocytes. The active ingredient of SB-431542 of the present invention is 4-[4-(1,3-benzodiazole-5-yl)-5-(2-pyridyl)-1H-imidazole-2-yl]-benzamide hydrate, which can inhibit spontaneous differentiation of cells; the active ingredient of Y-27632 is trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide, which can improve the mitotic activity of cells, and L-alanyl-L-glutamine dipeptide enhances the nutritional stability of cell culture medium. In the culture medium of the present invention, components for preventing cell contamination, such as gentamicin, can also be added, and the amount of gentamicin added can be 50 mg / L.

[0040] The present invention does not specifically limit the sources of the components in the culture medium, such as conventional commercial channels. In one embodiment, EGF, bovine pituitary extract, insulin, hydrocortisone, epinephrine hydrochloride, transferrin, SB-431542 and Y-27632 are all purchased from Solebol, and platelet lysate is purchased from PLBioScience.

[0041] The present invention also provides a method for culturing keratin 15 type hair follicle stem cells, comprising the following steps: culturing the mesenchymal cells that have stopped proliferation into a continuous sheet and using it as a feeder layer;

[0042] The hair follicle tissue is digested into single cells, which are inoculated onto the feeder layer using the above culture medium to obtain keratin 15 type hair follicle stem cells.

[0043] The present invention uses mesenchymal cells as a feeder layer for keratin 15 type hair follicle stem cells, wherein the mesenchymal cells are mesenchymal cells whose proliferation is terminated, and the preparation of the mesenchymal cells whose proliferation is terminated includes irradiating the mesenchymal cells by an x-ray irradiator equipped with a copper alloy filter to terminate the proliferation of the mesenchymal cells. The total irradiation dose is 8 to 21 Gy. In one embodiment of the present invention, it is confirmed that even 8 Gy can ensure that the cells fully terminate division. In one embodiment, the irradiation of the present invention is carried out by an x-ray irradiator equipped with a copper alloy filter, and 8 to 21 Gy is used to irradiate the mesenchymal cells to terminate the proliferation of the mesenchymal cells.

[0044] The present invention cultured the mesenchymal cells that stopped proliferation to obtain a feeder layer, specifically comprising culturing the irradiated mesenchymal stem cells at a density greater than 133,000 cells / cm 2 The concentration of cells per unit area was laid in a culture dish. The embodiment confirmed that a continuous feeder layer can be formed when the cell mass per unit area is greater than 133,000 cells / cm. However, the density should not be too high, such as more than 200,000 cells / cm. 2 When the number of cells is too large, there may be local overcrowding and discontinuity in the cell orientation in the feeder layer, which consumes more cells. Therefore, it is necessary to control the number of cells per cm 2 The number of cells is in the interval (133000, 200000).

[0045] In one embodiment of the present invention, the irradiated mesenchymal stem cells are laid in a culture dish at the above concentration for culture, thereby obtaining a feeder layer, wherein before adding other cells to be cultured, a conventional fetal bovine serum culture medium is used to reduce costs. After the irradiated feeder layer cells are revived, they are statically cultured under normal cell culture conditions, generally at 37°C and a carbon dioxide partial pressure of 5% to 7%. The feeder layer obtained by the present invention is a continuous single layer, without gaps but dense, non-proliferative but metabolically active.

[0046] The present invention digests hair follicle tissue into single cells, uses the culture medium to inoculate them onto a feeder layer made of mesenchymal cells, replaces fresh culture medium every 2 days, and obtains cell colonies after about one week. Staining shows that these colonies are keratin 15 (KRT15) positive, that is, keratin 15 (KRT15) positive hair follicle stem cells are obtained.

[0047] The present invention also provides a keratin 15 type hair follicle stem cell line cultured by the above culture method.

[0048] The present invention also provides the use of the keratin 15 type hair follicle stem cell line in preparing hair growth materials.

[0049] The keratin 15 type hair follicle stem cell line of the present invention has a significant epithelial cell morphology and presents a typical paving stone shape.

[0050] To further illustrate the present invention, a keratin 15 type hair follicle stem cell culture medium, adherent culture method and application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] 1. Optimization of the formula of keratin 15 hair follicle stem cell culture medium

[0053] (1) Screening of epidermal stem cell culture formula - Research on ingredients of bovine pituitary extract

[0054] Keratinocytes isolated from adult female skin were used, and 40,000 cells were plated in each well of a 24-well cell culture plate. After the cells adhered to the wall, DF12 (2:1) medium containing 10 μg / mEGF, 5 μg / ml insulin, 0.4 μg / ml hydrocortisone, 0.39 μg / ml epinephrine hydrochloride, 10 μg / ml transferrin, 0.06 mM calcium chloride, 2 μM SB-431542, 2.5 μM Y-27632, and 2 mM L-alanyl-L-glutamine dipeptide was added. In addition, human albumin was added to 0-5 g / L, and BPE was added to 28-84 mg / L and cultured for 72 hours. After 72 hours, all cells were digested and collected, and the number of cells in each well was calculated using a cell counter, and the results were presented in a bar graph.

[0055] The results are as follows Figure 1 As shown, the addition of BPE can effectively increase the number of epidermal-derived cells that have been expanded, especially when human albumin is present, 56 μg / ml of BPE is most conducive to cell expansion. However, when human albumin is present, 84 μg / ml of BPE is no longer conducive to cell expansion.

[0056] (2) Screening of epidermal stem cell culture formula - exploration of epidermal growth factor concentration range

[0057] Skin-derived keratinocytes (isolated from adult female skin) were plated in 24-well cell culture plates at 40,000 cells per well. After the cells adhered to the wall, DF12 (2:1) culture medium containing 5 μg / ml insulin, 0.4 μg / ml hydrocortisone, 0.39 μg / ml epinephrine hydrochloride, 10 μg / ml transferrin, 0.06 mM calcium chloride, 2 μM SB-431542, 2.5 μM Y-27632, and 2 mM L-alanyl-L-glutamine dipeptide was added. The concentration of epidermal growth factor (EGF) was adjusted to 10 ng / ml to 50 ng / ml and human albumin to 0 to 5 g / ml, and the cells were cultured for 72 hours. After 72 hours, all cells were digested and collected, and the number of cells in each well was calculated using a cell counter. The results were presented in a bar graph.

[0058] The results are as follows Figure 2 As shown, the addition of EGF can effectively increase the number of epidermal-derived cells that have been expanded, especially when human albumin is present, 25 ng / ml of EGF is most conducive to cell expansion. However, when human albumin is present, 50 ng / ml of EGF is no longer conducive to cell expansion.

[0059] (3) Screening of epidermal stem cell culture formula-exploration of platelet lysate application

[0060] Skin-derived keratinocytes (isolated from adult female skin) were used and plated in 24-well cell culture plates at 40,000 cells per well. After the cells adhered to the wall, DF12 (2:1) medium containing 5 μg / ml insulin, 0.4 μg / ml hydrocortisone, 0.39 μg / ml epinephrine hydrochloride, 10 μg / ml transferrin, 0.06 mM calcium chloride, 2 μM SB-431542, 2.5 μM Y-27632, and 2 mM L-alanyl-L-glutamine dipeptide was added, and EGF was adjusted to 10 ng / ml to 50 ng / ml, BPE was adjusted to 0 to 56 μg / ml, and 2% platelet lysate or 10% fetal bovine serum was added for comparison. The cells were cultured for 72 hours. After 72 hours, all cells were digested and collected, and the number of cells in each well was calculated using a cell counter, and the results were presented in a bar graph.

[0061] The results are as follows Figure 3As shown in the figure, adding 2% platelet lysate can better combine with EGF to expand keratinocytes than fetal bovine serum, especially when 2% platelet lysate is present, 25ng / ml EGF and 56ug / ml BPE are most conducive to cell expansion. When fetal bovine serum is used to study the same cytokine combination, the difference in cell expansion efficiency is less obvious, and the overall performance is weaker than the combination of 2% platelet lysate, 25ng / ml EGF and 56μg / ml BPE in the serum-free group.

[0062] 2. Optimization of irradiation dose for feeder cell preparation

[0063] (1) 250,000 frozen hair follicle tissue cells were revived and suspended in 15 ml of mesenchymal cell culture medium (DMEM basal medium: F12 basal medium = 2:1 as basal medium, supplemented with glucose to a concentration of 20 mM and 10% fetal bovine serum), and inoculated into a T75 culture flask for culture. Figure 7 As shown in A.

[0064] (2) After about 5 days, fibroblast-like cells can be seen forming colonies ( Figure 7 In the middle (B), a small amount of hair follicle stem cells attached to the wall are scattered in an epidermal-like morphology. After passage, it can be seen that in the culture medium lacking the nutritional conditions related to the growth of epidermal stem cells, mesenchymal stem cells gradually occupy the dominant position. After 3 passages, it can be seen that the cells in the culture bottle of mesenchymal cells are uniform in morphology, all showing a spindle shape, and due to the abundant number of cells, they grow and distribute in a vortex shape.

[0065] (3) The mesenchymal stem cells were harvested by digestion with a cell dissociation solution (Gibco brand TrypLE Select cell dissociation solution) and suspended in a mesenchymal cell culture medium, and the cell density was adjusted to 2000000 / ml. The cell suspension was divided into five identical centrifuge tubes, which were placed in the cabin of an X-ray irradiator (RS2000pro biological X-ray irradiator) and the irradiation distance was adjusted to 50 cm. The filter of the irradiator was a 0.3 mm copper alloy filter. At a voltage of 225 kV, five groups of cells were irradiated for 0 seconds, 399 seconds, 599 seconds, 799 seconds and 999 seconds, respectively, that is, the dose rate was 1.26 Gy / minute, and the total irradiation dose was 0 Gy, 8 Gy, 13 Gy, 18 Gy and 21 Gy.

[0066] (4) The irradiated mesenchymal feeder layer cells were distributed into a 24-well plate in a manner of inoculating 333,000 cells per well. After overnight observation, the irradiated mesenchymal cells formed a continuous feeder layer. Every 24 hours, cells from one well of each group were digested and dissociated, and the changes in the number of cells were counted and plotted as a curve graph.

[0067] The results are as follows Figure 4 As shown in the figure, even the lowest irradiation intensity, 8Gy, can ensure that cells fully terminate division. Compared with the 0Gy group, cells at each irradiation intensity have no obvious proliferation. Considering the priority of safety, the maximum irradiation time of the instrument, 999 seconds, is still used as the irradiation working time for preparing the feeder layer of mesenchymal stem cells.

[0068] 3. Comparison of the laying density of mesenchymal stem cell feeder layer after irradiation

[0069] The irradiated mesenchymal stem cells were plated in culture dishes at different densities to evaluate their effects on the growth of hair follicle stem cells. In the experiment, we set up three different density groups: low density group (133,000 cells / cm 2 ), medium density group (167,000 cells / cm 2 ) and high-density group (200,000 cells / cm 2 ).

[0070] The results are as follows Figure 5 As shown in the figure, the cells in the low-density group cannot form a continuous feeder layer. Under each irradiation intensity, obvious gaps in the feeder layer can be seen, which is not conducive to the growth of hair follicle stem cells. Only cells with medium density or above can form a relatively continuous feeder layer. After high-density cell inoculation, the feeder layer cells are too dense in some areas and the cells are discontinuous, which also consumes more cells. Therefore, only 167,000 cells per square centimeter can be used as the laying density of the mesenchymal stem cell feeder layer after irradiation.

[0071] 4. Identification of KRT15+ hair follicle stem cells by immunohistochemical staining

[0072] The hair follicle stem cells were divided into two groups and inoculated into 24-well culture plates. One group was cultured using the method described in the present invention, and the other group was directly cultured using a cell culture plate without a feeder layer. Ten days after inoculation, conventional immunohistochemical staining was performed using anti-KRT15 rabbit monoclonal antibody (Beyotime, AG2454). The steps are briefly described as follows:

[0073] First, the adherent cells cultured in the 12-well plate were fixed. After washing the cells twice with pre-cooled PBS, 4% paraformaldehyde solution (Biyuntian) was added and fixed at room temperature for 30 minutes.

[0074] Next, the cells were washed three times with PBS for 5 minutes each time, and then permeabilized by adding 0.5% TritonX-100 (Solabol) solution at room temperature for 10 minutes.

[0075] Afterwards, the cells were washed again three times with PBS and incubated with 3% hydrogen peroxide solution at room temperature for 10 min to inactivate endogenous peroxidase.

[0076] Subsequently, the cells were washed with PBS and blocked at room temperature for 30 minutes with the addition of primary antibody diluent (Biyuntian brand P0023A). After blocking, a 1:50 diluted anti-KRT15 rabbit monoclonal antibody solution was added and placed in a wet box for incubation at 37°C for 2 hours. After washing the cells with PBS, a 1:50 diluted secondary antibody (Biyuntian A0208) solution was added and incubated at room temperature for 30 minutes.

[0077] Next, DAB color development was performed, and freshly prepared DAB color development solution (Biosharp, BL732A) was added, and the color development reaction time was controlled until a brown-yellow positive signal appeared. After DAB color development, the cells were washed with PBS, and hematoxylin staining solution (Biosharp, BL700A) was added for counterstaining, and then washed with pure water. The experimental results were observed and photographed using an optical microscope.

[0078] The results are as follows Figure 6 As shown in the figure, the hair follicle stem cell culture using feeder layer cells is compared with the method without feeder layer. A group of cells with dark brown staining can be seen, namely the KRT15 positive cell group. This group of cells has a significant epithelial cell morphology and a typical paving stone shape. The hair follicle tissue digested cells that are not protected by feeder layer technology have lost the typical KRT15 staining after 10 days of culture, and the morphology shows the characteristics of aging epidermis and fibroblasts.

[0079] 5. Animal experiments on KRT15+ hair follicle stem cells

[0080] The hair follicle formation ability of KRT15+ hair follicle stem cells was tested using a nude mouse skin transplantation model (Jensen, KB, Driskell, RR, & Watt, FM (2010). Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nature Protocols, 5 (5), 898-911. doi: 10.1038 / nprot.2010.39). Briefly, 6-week-old Balbc / nu mice were intradermally injected with 1 ml of sterile saline to form a skin mound with a diameter of about 6 mm, and the upper epidermis was removed with surgical scissors to create a skin defect model with a diameter of 6 mm. 500,000 KRT15+ hair follicle stem cells cultured above were taken and mixed with 5,000,000 freshly isolated newborn mouse fibroblasts, and the cell mixture was resuspended in 100 μL of saline. The suspension containing KRT15+ hair follicle stem cells was dripped into the skin defect, and the upper layer was covered with a breathable PET plastic film (0.4 micron pore size) and wrapped with elastic gauze. After 2 weeks, the plastic film and auxiliary materials were removed and the skin was healed by dry method. After 6 weeks, the area with black hair was surgically cut, and the skin healing tissue transplanted with KRT15+ hair follicle stem cells was sliced ​​and observed under a microscope, and detected by immunohistofluorescence with anti-human KRT15+ antibody and counterstained with DAPI dye.

[0081] The results are as follows Figure 8 As shown in the figure, hair follicle stem cells cultured using the feeder layer cells can form hair follicle-like tissue. Immunofluorescence staining experiments show that ( Fig. 9 ), in the hair follicle-like tissue formed on the back of the nude mouse, an area positively stained with anti-human KRT15+ antibody (red fluorescence) can be seen, and the cell nucleus is stained blue with DAPI dye, indicating that the hair follicle contains cells of both human and mouse origin.

[0082] Example 2

[0083] 1. Two-dimensional serum-free culture of keratin 15 hair follicle stem cells using feeder cells

[0084] (1) Digestion of hair follicle tissue

[0085] ① On the disinfected occipital skin, extract about 30 hair follicle units (about 1 mm in diameter and 5 mm in length) from the patient through hair follicle extraction, suspend them in 2 ml of tissue digestion solution (components are F12 culture medium containing 2 mg / ml collagenase IV), and place them in a 37°C constant temperature incubator or constant temperature shaker for tissue digestion. The total digestion time is about 120 minutes. Under a microscope, the hair follicle tissue dissociates into a single cell suspension, which confirms that the tissue digestion is complete. Pass the digested cell suspension through a cell sieve (such as Falcon brand 35235070 micron pore size), and further centrifuge and precipitate to remove the supernatant of the digestion solution. Use phosphate buffer (Cellmax CBS101.05) to further centrifuge and wash the cells three times.

[0086] ② Count the cells using a cell counter (Countstar BioTech) according to the operating procedures, and digest approximately 2,000,000 hair follicle tissue cells. Suspend the digested cells of the hair follicle tissue in a cell freezing solution (the configuration method is: add 1 volume of dimethyl sulfoxide to 9 volumes of keratin 15 hair follicle stem cell culture medium) for freezing.

[0087] (2) Cultivation and irradiation inactivation of hair follicle-derived mesenchymal cells

[0088] ① Take 250,000 frozen hair follicle tissue cells to revive, suspend them in 15 ml of mesenchymal cell culture medium (DMEM basal medium: F12 basal medium = 2:1 as basal medium, supplemented with glucose to a concentration of 20 mM, supplemented with 10% fetal bovine serum), and inoculate them into T75 culture flasks for culture.

[0089] ② After about 5 days, fibroblast-like cells can be seen forming colonies, with a small amount of hair follicle stem cells attached to the wall scattered in an epidermal-like morphology. After passage, it can be seen that in the culture medium lacking the nutritional conditions related to the growth of epidermal stem cells, mesenchymal stem cells gradually occupy a dominant position. After 3 passages, it can be seen that the cells in the culture bottle of mesenchymal cells are uniform in morphology, all showing a spindle shape, and due to the abundant number of cells, they grow and distribute in a vortex shape.

[0090] ③Mesenchymal stem cells were harvested by digestion with cell dissociation solution (Gibco brand, TrypLE Select cell dissociation solution) and suspended in mesenchymal cell culture medium, and the cell density was adjusted to 2000000 / ml. The cell suspension was placed in the cabin of the X-ray irradiator (RS2000pro, biological X-ray irradiator) and the irradiation distance was adjusted to 50cm. The filter of the irradiator was a 0.3mm copper alloy filter, with a voltage of 225kV, irradiation for 999 seconds, that is, a dose rate of 1.26Gy / minute, and a total irradiation dose of 21Gy.

[0091] ④Suspend the irradiated cells in freezing solution (same formula as above), package according to 5,000,000 cells / tube, and freeze the cells in a deep low temperature refrigerator (Haier DW-86L) using a programmed cooling box (Biosharp BS-02-CFC) to obtain feeder layer cells for use.

[0092] (3) Laying of irradiated inactivated mesenchymal cell feeder layer

[0093] Thaw two frozen irradiated mesenchymal cells in a water bath quickly, slowly add them to the mesenchymal cell culture medium, shake them while adding, make up the volume of the mesenchymal cell culture medium to 12 ml and gently blow them evenly, then distribute the irradiated mesenchymal cells to the 6-well plate by adding 2 ml of cell suspension to each well. After overnight observation, the irradiated mesenchymal cells formed a continuous feeder layer.

[0094] (4) Cultivation of KRT15+ hair follicle stem cells using mesenchymal cell feeder layers

[0095] ① After the cell cryopreservation solution obtained by digestion of hair follicle tissue is revived, the supernatant is discarded by centrifugation, and the cells are resuspended in fresh keratin 15 hair follicle stem cell culture medium. The resuspended cells are inoculated into a 6-well plate with a feeder layer at a density of 20,000 cells / cm2. The culture medium is replaced every two days in an incubator at 37°C and 5% CO2.

[0096] ② After about 5 to 10 days, cell colonies are formed, and the expression of keratin 15 (KRT15) is detected by immunofluorescence staining technology to confirm that the obtained cell colonies are KRT15 positive, which are the required hair follicle KRT15+ hair follicle stem cells. It can be seen that the mesenchymal cell feeder layer does not have the staining ability of KRT15 and is pushed to the periphery by the hair follicle cell colonies.

[0097] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A keratin 15 type hair follicle stem cell culture medium, characterized in that: Including basal culture medium, serum replacement and nutrient supplements; The basal culture medium includes a mixed culture medium of DMEM basal culture medium and F12 basal culture medium; The serum replacement includes epidermal growth factor, platelet lysate and bovine pituitary extract; The nutritional supplements include insulin, hydrocortisone, epinephrine hydrochloride, transferrin, calcium chloride, SB-431542, Y-27632 and L-alanyl-L-glutamine dipeptide.

2. The culture medium according to claim 1, characterized in that The basic culture medium also includes glucose, and the concentration of glucose is 5-20 mM.

3. The culture medium according to claim 1 or 2, characterized in that In the basic culture medium, the volume ratio of DMEM basic culture medium to F12 basic culture medium is 2:

1.

4. The culture medium according to claim 1, characterized in that The final concentration of the epidermal growth factor is 25 ng / ml, the final volume percentage concentration of the platelet lysate is 2%, and the final concentration of the bovine pituitary extract is 56 μg / ml; The final concentration of insulin was 5 μg / ml, the final concentration of hydrocortisone was 0.4 μg / ml, the final concentration of epinephrine hydrochloride was 0.39 μg / ml, the final concentration of transferrin was 10 μg / ml, the final concentration of calcium chloride was 0.06 mM, the final concentration of SB-431542 was 2 μM, the final concentration of Y-27632 was 2.5 μM, and the final concentration of L-alanyl-L-glutamine dipeptide was 2 mM.

5. A method for culturing keratin 15 type hair follicle stem cells, characterized in that: The method comprises the following steps: culturing the mesenchymal cells that have stopped proliferation into a continuous sheet layer as a feeder layer; The hair follicle tissue is digested into single cells, and the single cells are inoculated onto the feeder layer using the culture medium according to any one of claims 1 to 4 to obtain keratin 15 type hair follicle stem cells through culture.

6. The culture method according to claim 5, characterized in that: The preparation of the mesenchymal cells that stop proliferation comprises irradiating the mesenchymal cells by an X-ray irradiator equipped with a copper alloy filter to stop the proliferation of the mesenchymal cells.

7. The culture method according to claim 6, characterized in that: The total dose of the irradiation is 8 to 21 Gy.

8. The culture method according to claim 5, characterized in that: The seeding density of the mesenchymal cells that have stopped proliferation is 133,000 to 200,000 cells / cm 2 .

9. A keratin 15 type hair follicle stem cell line cultured using the culture method according to any one of claims 5 to 8.

10. Use of the keratin 15 type hair follicle stem cell line according to claim 9 in preparing hair growth materials.

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