Method for culturing adult hair follicle convex cells

Abstract

The invention relates to a method for culturing adult hair follicle protuberance cells, which belongs to the technical field of biology and comprises the following steps: S1, cleaning hair follicle tissues to obtain hair follicle tissues containing protuberance cells; s2, the hair follicle tissue containing the protuberant cells is soaked in an Advanced DMEM / F12 culture medium mixed solution containing collagenase, dispersing enzyme, EGF, bFGF and 1 * green-streptomycin, and digestion is carried out; s3, chopping hair follicle tissues containing the protuberant cells, centrifuging after stopping digestion, adding a cell culture dissociation agent into precipitated tissues, digesting twice, collecting supernatant after stopping digestion, centrifuging, collecting precipitates, re-suspending, and filtering; and S4, inoculating the cells into a cell six-well plate for 3D suspension culture, and changing the solution and blowing and beating the cells every other day. According to the method, a two-step method is adopted to digest the tissue part of the hair follicle convex cells, so that a larger number of hair follicle convex cells are obtained, and the characteristics and dryness of the hair follicle convex cells are improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for culturing adult hair follicle bulge cells. Background Art

[0002] Hair follicles are an easily accessible and abundant source of human stem cells. The hair follicle bulge cells are a well-characterized niche for adult stem cells. During hair follicle development, hair follicle stem cells reside as a single layer in a niche called the "bulge", providing the driving force for the periodic growth of hair. The growth cycle of hair follicles goes through the growth phase, the regression phase, and the resting phase, a process known as the hair cycle. Hair follicle stem cells can produce and regulate daughter cells that can affect their own activities, stimulate proliferation in the early stage of the hair cycle, and maintain long-term silence in the subsequent regeneration cycle. As an important component of hair follicle stem cells, hair follicle bulge cells are one of the cell sources for hair follicle regeneration, and can differentiate into various cell types of hair follicles, participating in the reconstruction of hair follicle structure.

[0003] Hair follicle stem cells are usually cultured in specially treated culture dishes or plates to promote cell adhesion and growth. Commonly used basal culture media such as DMEM or DMEM / F12 are supplemented with serum and growth factors such as EGF and bFGF. However, cells cultured on the wall usually have reduced cell characteristics. At the same time, a culture system containing serum will lead to reduced cell differentiation and stemness. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing adult hair follicle bulge cells in order to overcome the problems of reducing cell characteristics, reducing cell differentiation, and stemness in the prior art of culturing hair follicle stem cells.

[0005] The purpose of the present invention can be achieved by the following technical solutions: The technical solution of the present invention is to provide a method for culturing adult hair follicle bulge cells, comprising the following steps: S1, cleaning and processing hair follicle tissue to obtain hair follicle tissue containing bulge cells; S2, immersing the hair follicle tissue containing bulge cells obtained in step S1 in a mixed solution of Advanced DMEM / F12 medium containing collagenase, dispase, EGF, bFGF, and 1× penicillin-streptomycin for digestion; S3, mincing the hair follicle tissue containing bulge cells after the treatment in step S2, centrifuging and removing the supernatant after terminating the digestion, adding a cell culture dissociation agent to the precipitated tissue and continuing digestion twice, collecting the supernatant after terminating the digestion and centrifuging, collecting the precipitate, resuspending it, and filtering it; S4. Inoculate the cells filtered in step S3 into a six-well cell plate for 3D suspension culture, change the medium and blow the cells every other day.

[0006] In some specific embodiments, in step S1, the hair follicle tissue is cleaned by: The hair follicle tissue was washed several times by soaking in physiological saline, and the fat and dermal tissue were removed. Then, the tissue was washed several times by soaking in PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, and finally the dermal hair papilla was removed.

[0007] In some specific embodiments, in step S2, the final concentration of collagenase in the mixed solution is 200-300 U / mL, the final concentration of dispase is 5-10 U / mL, the final concentration of EGF is 50 ng / mL, the final concentration of bFGF is 200 μg / mL, the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL.

[0008] In some embodiments, the collagenase is collagenase IV.

[0009] In some embodiments, the dispase is dispase.

[0010] In some specific embodiments, in step S2, the digestion temperature is 37° C. and the digestion time is 12 to 18 h.

[0011] In some specific embodiments, in step S3, the cell culture dissociation agent is TrypLE cell culture dissociation agent.

[0012] In some specific embodiments, in step S3, the digestion temperature is 37° C. and the digestion time is 20 to 30 min.

[0013] In some specific embodiments, in step S3, the filtration is specifically performed by filtering with a 200-mesh screen.

[0014] In some embodiments, in step S4, the culture conditions are: 37°C, 5% CO 2 The cells were cultured in an incubator at 400 μg / mL for 7 days.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts a two-step method to digest the hair follicle bulge cell tissue site, uses collagenase and dispase to gently decompose the tissue cells, and uses a cell culture dissociation agent for digestion. The cell viability after one digestion is 70%-85%, and the cell viability after two digestions is 65%-80%. Taking both the cell yield and viability into consideration, the present invention performs two digestions to obtain a larger number of hair follicle bulge cells, and combines the serum-free culture system to perform 3D suspension culture of the hair follicle bulge cells, thereby improving the characteristics and stemness of the hair follicle bulge cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The morphology of the hair follicle bulge cell cluster.

[0017] Figure 2 Flow cytometry identification of hair follicle bulge cells, co-labeled with K15, CD200, and ITGA6.

[0018] Figure 3 QPCR identification of K19, LGR5, SOX9, and LHX2 in hair follicle bulge cells.

[0019] Figure 4 The expression of cell markers (CD200, K15, LHX2, TCF3, NFATC1, SOX9) of hair follicle bulge cells in 2D adherent culture and 3D suspension culture.

[0020] Figure 5 The CD200+ cell ratio of hair follicle bulge cells in 2D adherent culture and 3D suspension culture is compared. DETAILED DESCRIPTION

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0022] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0023] Example 1

[0024] (1) 30 units of hair follicle tissue were obtained from human scalp by FUE, soaked and washed several times with saline, and excess fat and dermal tissue were removed with a sterile surgical blade. The hair follicle tissue was stored in PBS buffer and transported back to the laboratory. (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time; (3) Under a stereomicroscope, the dermal papilla was removed and the remaining tissue was placed in a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL collagenase IV, 5 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) and digested at 37°C for 16 h. (4) Mince the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (5) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 10 min to allow the tissue to precipitate. Collect the supernatant into another clean centrifuge tube. (6) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 10 min. Add Advanced DMEM / F12 containing EGF 50 ng / mL, bFGF 200 μg / mL, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to terminate the digestion. Mix the supernatant collected in the previous step, centrifuge at 1800 rpm for 5 min, and discard the supernatant. (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellets, filter through a 200-mesh filter, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (8) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells, count them, and inoculate them into low-adhesion six-well plates for 3D suspension culture; (9) Change the medium and blow the cells every other day. Collect the cells for identification after one week.

[0025] Example 2

[0026] (1) 50 units of hair follicle tissue were obtained from human scalp by FUE, soaked and washed several times with saline, and excess fat and dermal tissue were removed with a sterile surgical blade. The hair follicles were stored in PBS buffer and transported back to the laboratory. (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time; (3) Under a stereomicroscope, the dermal papilla was removed and the remaining tissue was placed in an Advanced DMEM / F12 mixed solution containing 200 U / mL collagenase IV, 10 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) and digested at 37°C for 18 h. (4) Mince the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (5) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 15 min to allow the tissue to precipitate. Collect the supernatant into another clean centrifuge tube. (6) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 15 min. Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to terminate the digestion. Mix the supernatant collected in the previous step, centrifuge at 1800 rpm for 5 min, and discard the supernatant. (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellets, filter through a 200-mesh filter, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (8) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells, count them, and inoculate them into low-adhesion well plates for 3D suspension culture; (9) Change the medium and blow the cells every other day. Collect the cells for identification after one week.

[0027] Example 3

[0028] (1) 60 units of hair follicle tissue were obtained from human scalp by FUE, soaked and washed several times with saline, and excess fat and dermal tissue were removed with a sterile surgical blade. The hair follicles were stored in PBS buffer and transported back to the laboratory. (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time; (3) Under a stereomicroscope, the dermal papilla was removed and the remaining tissue was placed in an Advanced DMEM / F12 mixed solution containing 200 U / mL collagenase IV, 10 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) and digested at 37°C for 18 h. (4) Mince the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (5) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 15 min to allow the tissue to precipitate. Collect the supernatant into another clean centrifuge tube. (6) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 15 min. Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to terminate the digestion. Mix the supernatant collected in the previous step, centrifuge at 1800 rpm for 5 min, and discard the supernatant. (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellets, filter through a 200-mesh filter, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (8) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells, count them, and inoculate them into low-adhesion well plates for 3D suspension culture; (9) Change the medium and blow the cells every other day. Collect the cells for identification after one week.

[0029] Comparative Example 1 Compared with Example 1, the only difference is that the TrypLE cell dissociation agent is used to digest the tissue only once. The specific steps are as follows: (1) 30 units of hair follicle tissue were obtained from human scalp by FUE, soaked and washed several times with saline, and excess fat and dermal tissue were removed with a sterile surgical blade. The hair follicle tissue was stored in PBS buffer and transported back to the laboratory. (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time; (3) Under a stereomicroscope, the dermal papilla was removed and the remaining tissue was placed in a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL collagenase IV, 5 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) and digested at 37°C for 16 h. (4) Mince the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (5) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 10 min. Add Advanced DMEM / F12 containing EGF, bFGF, and 1× penicillin-streptomycin to terminate the digestion. Centrifuge at 1800 rpm for 5 min and discard the supernatant. (6) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellets, filter through a 200-mesh filter, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells and count them.

[0030] Comparative Example 2 Compared with Example 2, most of the steps are the same, except that the tissue is digested only once using TrypLE cell dissociation agent. The specific steps are as follows: (1) 50 units of hair follicle tissue were obtained from human scalp by FUE, soaked and washed several times with saline, and excess fat and dermal tissue were removed with a sterile surgical blade. The hair follicles were stored in PBS buffer and transported back to the laboratory. (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time; (3) Under a stereomicroscope, the dermal papilla was removed and the remaining tissue was placed in a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL collagenase IV, 10 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) and digested at 37°C for 18 h. (4) The hair follicle tissue was minced with a sterile surgical blade, and digested with Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL). The cells were centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. (5) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 15 min. Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to terminate the digestion. Centrifuge at 1800 rpm for 5 min and discard the supernatant. (6) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellets, filter through a 200-mesh filter, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells and count them.

[0031] Comparative Example 3 Compared with Example 3, most of the steps are the same, except that the tissue is digested only once using TrypLE cell dissociation agent. The specific steps are as follows: (1) 60 units of hair follicle tissue were obtained from human scalp by FUE, soaked and washed several times with saline, and excess fat and dermal tissue were removed with a sterile surgical blade. The hair follicles were stored in PBS buffer and transported back to the laboratory. (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time; (3) Under a stereomicroscope, the dermal papilla was removed and the remaining tissue was placed in a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL collagenase IV, 10 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) and digested at 37°C for 18 h. (4) Mince the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (5) Add TrypLE cell dissociation agent to continue digesting the tissue at 37°C for 15 min. Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to terminate the digestion. Centrifuge at 1800 rpm for 5 min and discard the supernatant. (6) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellets, filter through a 200-mesh filter, centrifuge at 1800 rpm for 5 min, and discard the supernatant; (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells and count them.

[0032] The content and steps of the identification are as follows: (1) The hair follicle bulge cells cultured for 7 days in Example 1 were Figure 1 The bulge cell clusters shown under the microscope were digested with 0.25% trypsin-EDTA for 20 min. After the digestion was stopped with DMEM medium containing 10% FBS, the cells were resuspended into a single cell suspension for flow cytometry identification.

[0033] (2) Flow cytometry identification of CD200 and ITGA6: The single cell suspension was centrifuged and the supernatant was discarded; the cell density was 1×10 6 / mL. Add 0.5 μg CD200 antibody and 0.5 μg ITGA6 antibody, incubate at 4°C in the dark for 60 min, and set up a control; wash twice with flow cytometry blocking buffer, resuspend with flow cytometry blocking buffer, and detect on the machine.

[0034] (3) Flow cytometry identification of K15: K15 is a cytoplasmic protein, so the cells need to be permeabilized before flow cytometry. After centrifugation of the digested single cell suspension and discarding the supernatant, fix with 4% PFA at room temperature in the dark for 10-15 min. Centrifuge, discard the supernatant, and wash twice with PBS. Add membrane permeabilization blocking solution (0.1% TX100 + flow staining buffer) and resuspend, incubate at room temperature in the dark for 10 min. Centrifuge and discard the supernatant, resuspend with flow staining buffer, and the cell density is 1×10 6 / mL, add 0.5 μg of K15 antibody, incubate at 4 degrees in the dark for 60 min; and set up a control; wash twice with flow cytometry buffer, resuspend in flow cytometry buffer, and detect on the machine.

[0035] like Figure 2 As shown, K15 ( Figure 2 A) and CD200, ITGA6 co-labeled ( Figure 2 B) The cell ratio was basically maintained, indicating that the characteristics of hair follicle bulge cells can be maintained under this culture condition.

[0036] like Figure 3 As shown, the expression of genes K19, LGR5, SOX9, and LHX2, which are closely related to the stemness of cells, was detected by qPCR. The stemness of cells refers to the ability of cells to maintain their differentiation potential.

[0037] The expression of the K19 gene is related to the self-renewal and differentiation potential of specific stem cells; the LGR5 gene is related to stem cell maintenance and regeneration; the SOX9 gene plays a key regulatory role in the differentiation of stem cells into specific cell lineages; and the LHX2 gene plays a regulatory role in maintaining the state of stem cells and their ability to differentiate into specific cell types.

[0038] Among them, it can be seen that taking culture day 0 (D0) as the control, the expression of K19, LGR5, SOX9, and LHX2 in the cells is significantly increased, indicating that under the culture conditions of the present invention, the stemness of hair follicle bulge cells can be improved.

[0039] Table 1 shows the number of hair follicle bulge cells obtained by two dissociation agent digestions and one dissociation agent digestion in Examples 1 to 3 and Comparative Examples 1 to 3, respectively: Table 1: Number of hair follicle bulge cells in different digestion times

[0040] Note: *The average number of cells obtained from digestion of 10 hair follicles.

[0041] Comparative Example 4 Compared with Example 1, most of the steps are the same, except that in step (8), "3D suspension culture" is replaced by "2D adherent culture".

[0042] like Figure 4 As shown, taking 2D adherent culture as the control, the expression levels of CD200, K15, LHX2, TCF3, NFATC1, and SOX9 markers in P0 cells cultured in 3D suspension were significantly higher than those in P0 cells cultured in 2D adherent culture.

[0043] like Figure 5 As shown, taking 2D adherent culture as the control, the proportion of CD200+ cells in P0 cells cultured in 3D suspension was significantly higher than that in P0 cells cultured in 2D adherent culture.

[0044] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for culturing adult hair follicle bulge cells, characterized in that: The steps include: S1, cleaning and processing hair follicle tissue to obtain hair follicle tissue containing bulge cells; S2, immersing the hair follicle tissue containing bulge cells obtained in step S1 in a mixed solution of Advanced DMEM / F12 medium containing collagenase, dispase, EGF, bFGF, and 1× penicillin-streptomycin for digestion; S3, mincing the hair follicle tissue containing bulge cells digested in step S2, centrifuging to remove the supernatant after terminating the digestion, adding a cell culture dissociation agent to the precipitated tissue and continuing digestion twice, collecting the supernatant after terminating the digestion, centrifuging, collecting the precipitate, resuspending it, and filtering it; S4. Inoculate the cells filtered in step S3 into a six-well cell plate for 3D suspension culture, change the medium and blow the cells every other day.

2. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S1, the method of cleaning the hair follicle tissue is: The hair follicle tissue was washed several times by soaking in physiological saline, and the fat and dermal tissue were removed. Then, the tissue was washed several times by soaking in PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, and finally the dermal hair papilla was removed.

3. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S2, the final concentration of collagenase in the mixed solution is 200-300 U / mL, the final concentration of dispase is 5-10 U / mL, the final concentration of EGF is 50 ng / mL, the final concentration of bFGF is 200 μg / mL, the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL.

4. The method for culturing adult hair follicle bulge cells according to claim 3, characterized in that: The collagenase is collagenase IV.

5. The method for culturing adult hair follicle bulge cells according to claim 3, characterized in that: The dispase is dispase.

6. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S2, the digestion temperature is 37°C and the digestion time is 12 to 18 h.

7. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S3, the cell culture dissociation agent is TrypLE cell dissociation agent.

8. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S3, the digestion temperature is 37°C and the digestion time is 20-30 min.

9. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S3, the filtering is specifically filtering using a 200-mesh screen.

10. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S4, the culture conditions are: culture in an incubator at 37° C. and 5% CO 2 concentration for 7 days.

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