CN115786247A

CN115786247A - Serum-free culture medium and application thereof in maintaining hair follicle activity, hair maintenance and transplantation

Info

Publication number: CN115786247A
Application number: CN202211309224.1A
Authority: CN
Inventors: 付廷灵; 滕凌; 石俊芳; 梁庆; 黄继英; 陈美明
Original assignee: Long Peptide Biopharmaceutical Co ltd
Current assignee: Long Peptide Biopharmaceutical Co ltd
Priority date: 2021-12-29
Filing date: 2022-10-25
Publication date: 2023-03-14
Anticipated expiration: 2042-10-25
Also published as: CN114540281A; CN115786247B

Abstract

The invention particularly relates to a serum-free medium and application thereof in maintaining hair follicle activity, hair maintenance and transplantation. The culture medium consists of additive components and a basic culture medium, wherein the additive components comprise at least one of nutritional factors, growth factors, cofactors and apoptosis inhibiting factors. The culture medium provided by the invention has clear components, no animal source and no toxic or side effect on a human body; the culture medium can better maintain the activity of hair follicle cells and skin keratinocytes, the survival rate reaches about 90 percent, and the proliferation capacity is excellent; the culture medium can provide all substances required by the in vitro growth of human hair follicles, can maintain the activity of in vitro hair follicles for more than one week, and can maintain the activity of the hair follicles from stem cell regeneration sources for more than 140 days; the hair follicle cultured by the culture medium can directly grow after being transplanted, and the hair follicle is prevented from entering a resting period.

Description

Serum-free culture medium and application thereof in maintaining hair follicle activity, hair maintenance and transplantation

Technical Field

The invention relates to the technical field of biology, in particular to a serum-free culture medium and application thereof in maintaining hair follicle activity, hair maintenance and transplantation.

Background

At present, under the dual effects of social pressure and bad lifestyle, the alopecia population is continuously strong and shows a trend of youthfulness. Although the physiological health of people is not seriously influenced by alopecia, the hair loss brings great threats to personal image, self-esteem and emotional health, thereby influencing the work and life of people. The hair transplantation operation is a main technical means for solving the problem of alopecia, the hair in the hair transplantation operation is completely consistent with the hair property and the growth rule of a hair supplying part, the hair transplantation operation is safe, has no rejection and no adverse reaction, the density of the hair after the hair transplantation operation is close to the normal density, the appearance is real and natural, and the hair transplantation operation is the best choice for the alopecia patients at present. However, this approach is also ineffective for patients who do not have sufficient provenance in the donor area. Stem cells are the initial source of human body and various tissues, and have the capacity of self-renewal and continuous proliferation and the potential of multidirectional differentiation, so that the problem of alopecia is also solved by culturing the regenerative hair follicles in vitro through the stem cells, and at present, a plurality of researches on the basis of the regenerative hair follicles of the stem cells are also available.

However, both transplant surgery and stem cell regenerative hair follicles share a common problem in that the ex vivo hair follicle activity gradually loses or regresses as the in vitro culture time increases. During the hair transplantation operation, the hair follicle is isolated for about 4-6 hours in vitro, and part of cells in the hair follicle are subjected to apoptosis due to the change of environment and temperature and the lack of nutrient substances in the isolated process, so that the hair follicle can enter a period of resting period after being transplanted. The stem cells induce the hair follicle structure generated in the hair follicle process, and may also cause the degeneration of the hair follicle along with the change of the environment and the lack of nutrition. Therefore, maintaining the activity of hair follicles in culture in vitro for a long time is critical to ensure the survival rate of hair follicle transplantation. At present, the method is a research difficulty, and the invention is just to overcome the difficulty.

Disclosure of Invention

In view of the above, there is a need to provide a serum-free medium and applications thereof in maintaining hair follicle viability, hair maintenance and transplantation, wherein the serum-free medium can solve the problems of low survival rate caused by long-time hair follicle in vitro during hair transplantation, hair follicle degeneration during stem cell hair follicle regeneration, and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a culture medium comprising an additive component and a basal medium, wherein the additive component comprises at least one of a trophic factor, a growth factor, a cofactor and an apoptosis-inhibiting factor.

Further, the basic culture medium is a DMEM/F-12 culture medium or a mixed culture medium of the DMEM/F-12 culture medium and a Neurobasal culture medium.

Preferably, when the basic culture medium is a mixed culture medium of a DMEM/F-12 culture medium and a Neurobasal culture medium, the volume ratio of the DMEM/F-12 culture medium to the Neurobasal culture medium is 1 (0.6-1.4).

Further, the nutritional factor is at least one of a GlutaMAX media supplement, a vitamin A-free B-27 supplement, an N2 supplement, or an insulin.

Preferably, the nutritional factors are GlutaMAX media supplement, vitamin A-free B-27 supplement, N2 supplement, and insulin.

Preferably, the final concentration of the GlutaMAX media supplement in the media is 1 ×, the final concentration of the vitamin a-free B-27 supplement in the media is 0.2 × -1 ×, the final concentration of the N2 supplement in the media is 0.2 × -1 ×, and the final concentration of the insulin in the media is 1-10 μ g/mL.

More preferably, the final concentration of the GlutaMAX media supplement in the media is 1 ×, the final concentration of the vitamin a-free B-27 supplement in the media is 0.5 × -1 ×, the final concentration of the N2 supplement in the media is 0.5 × -1 ×, and the final concentration of the insulin in the media is 1-10 μ g/mL.

Further, the growth factor is at least one of EGF, bFGF, aFGF, VEGFA, TGF-beta, WNT3a, PDGF and KGF, and the final concentration of the growth factor in the culture medium is 2-50ng/mL.

Further, the accessory factor is at least one of hydrocortisone, cholera toxin, triiodothyronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 or inositol.

Preferably, the cofactors are hydrocortisone, cholera toxin, triodothioronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 and inositol.

Preferably, the final concentration of hydrocortisone in the culture medium is 0.2-0.8 mu g/mL; the final concentration of cholera toxin in the culture medium is 0.05-0.15nM; the final concentration of triiodothyronine in the culture medium is 0.1-3ug/mL; the final concentration of the beta mercaptoethanol in the culture medium is 0.05-0.1mM; the final concentration of Normocin in the culture medium is 50-200 mug/mL; the final concentration of Z-VAD-FMK in the culture medium is 30-60 mu M; the final concentration of HA14-1 in the culture medium is 20-50 μ M; the final concentration of inositol in the culture medium is 60-100mg/L.

Further, the apoptosis inhibiting factor is Y-27632, and the final concentration of the apoptosis inhibiting factor in the culture medium is 5-20 mu M.

Furthermore, the additive components of the culture medium also comprise hormone drugs, such as dexamethasone, cortisone, prednisone acetate and betamethasone, and the dosage is 0.1-1ug/mL.

Preferably, the hormone medicine is hydrocortisone, and the dosage of the hydrocortisone is 0.2-0.5ug/mL.

In a second aspect, the present invention provides a method for preparing the above culture medium, wherein the components and contents of the culture medium are uniformly mixed.

In a third aspect, the invention provides the use of the culture medium as described above for the preparation of in vitro hair follicle growth culture products, hair follicle cell culture products and hair follicle nourishing products.

Further, the hair follicle cultured by the in vitro growth of the hair follicle is a human hair follicle or a hair follicle formed by in vitro differentiation of human stem cells.

Preferably, the human hair follicles include, but are not limited to, hair follicles, eyebrow hair follicles, leg hair follicles, and chest hair follicles.

Preferably, the human stem cells include, but are not limited to, adult stem cells of skin origin, induced pluripotent stem cells, or embryonic stem cells.

The invention has the beneficial effects that:

the culture medium provided by the invention has clear components, no animal source and no toxic or side effect on a human body; the culture medium can better maintain the activity of hair follicle cells and skin keratinocytes, the survival rate reaches about 90 percent, and the proliferation capacity is excellent; the culture medium can provide all substances required by the in vitro growth of human hair follicles, can maintain the activity of in vitro hair follicles for more than one week, and can maintain the activity of the hair follicles from stem cell regeneration sources for more than 140 days; the hair follicles cultured by the culture medium can directly grow after being transplanted, so that the hair follicles are prevented from entering a resting period.

Drawings

FIG. 1 is a diagram showing the state of hair follicle stem cells cultured in the medium of example 5 of the present invention;

FIG. 2 is a diagram showing the cell state of the keratinocytes Hacat cultured in the medium and the normal serum medium, respectively, in example 5 of the present invention;

FIG. 3 is a graph showing the viability test of keratinocytes Hacat cultured in the medium of example 5 according to the present invention;

FIG. 4 is a diagram of the culture of human derived hair follicles in the hair follicle organoids cultured in different culture media according to example 5 of the present invention;

FIG. 5 is a graph showing in vivo transplantation growth of mice in which hair follicle organoids derived from hair follicle stem cells or in vitro hair follicles derived from human body were cultured in the medium in example 5 of the present invention.

FIG. 6 is a cell morphology map in a cell proliferation test of example 6, comparative examples 1 to 2 and a control group according to the present invention, wherein FIG. 1) is a cell morphology map corresponding to a culture medium of the control group, FIG. 2) is a cell morphology map corresponding to a culture medium of comparative example 1, FIG. 3) is a cell morphology map corresponding to a culture medium of comparative example 2, and FIG. 4) is a cell morphology map corresponding to a culture medium of example 6.

FIG. 7 shows the results of cell activity assays in cell proliferation assays of example 6, comparative examples 1 to 2 and a control of the present invention, wherein FIG. 1 corresponds to the results of cell activity assays in the first generation of cultures using a medium of the control, FIG. 2 corresponds to the results of cell activity assays in the first generation of cultures using a medium of comparative example 1, FIG. 3 corresponds to the results of cell activity assays in the third generation of cultures using a medium of comparative example 2, and FIG. 4 corresponds to the results of cell activity assays in the third generation of cultures using a medium of example 6. Note: in FIG. 7 the cells were passaged once for 3 days in culture.

FIG. 8 is a proliferation curve in the cell proliferation assay of example 6 of the present invention and comparative examples 1 to 2.

FIG. 9 shows the results of the apoptosis test in example 6 of the present invention, comparative examples 1 to 2 and control group.

FIG. 10 shows the results of cell cycle test of example 6 of the present invention, comparative examples 1 to 2, and control group

FIG. 11 shows the results of human hair follicle viability test for-4 h for example 6 of the present invention, comparative examples 1-2 and control.

FIG. 12 shows the results of-8 h of the human hair follicle activity test for example 6 of the present invention, comparative examples 1 to 2 and a control group.

FIG. 13 shows the results of human hair follicle viability test for-15 h for example 6 of the present invention, comparative examples 1-2 and control.

FIG. 14 shows the results of the human hair follicle activity test for-24 h in example 6 of the present invention, comparative examples 1-2, and a control group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further clearly and completely described below with reference to the embodiments of the present invention. It should be noted that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A serum-free culture medium comprises additive components and a basic culture medium, wherein the additive components are nutritional factors; the basal medium is a mixed medium of a DMEM/F-12 medium and a Neurobasal medium, and the mixing volume ratio of the DMEM/F-12 medium to the Neurobasal medium is 1:1; the nutritional factors are GlutaMAX culture medium additives, vitamin A-free B-27 supplement, N2 supplement and insulin; the final concentration of GlutaMAX media supplement in media was 1X, vitamin A-free B-27 supplement in media was 0.5X, N2 supplement in media was 0.5X, and insulin in media was 5. Mu.g/mL.

Example 2

A serum-free culture medium comprises an additive component and a basic culture medium, wherein the additive component is a growth factor; the basal medium is a mixed medium of a DMEM/F-12 medium and a Neurobasal medium, and the mixed volume ratio of the DMEM/F-12 medium to the Neurobasal medium is 1:1; the growth factor is EGF, and the final concentration of the growth factor in the culture medium is 10ng/mL.

Example 3

A serum-free culture medium comprises an additive component and a basic culture medium, wherein the additive component is an accessory factor; the accessory factors are hydrocortisone, cholera toxin, triiodothyronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 and inositol; hydrocortisone was present at a final concentration of 0.4. Mu.g/mL in the medium, cholera toxin was present at a final concentration of 0.1nM in the medium, triiodothyronine was present at a final concentration of 2nM in the medium, beta mercaptoethanol was present at a final concentration of 0.1mM in the medium, normocin was present at a final concentration of 100. Mu.g/mL in the medium, Z-VAD-FMK was present at a final concentration of 50. Mu.M in the medium, HA14-1 was present at a final concentration of 50. Mu.M in the medium, and inositol was present at a final concentration of 100mg/L in the medium.

Example 4

A serum-free culture medium comprises an additive component and a basic culture medium, wherein the additive component is an apoptosis inhibiting factor; the basal medium is a mixed medium of a DMEM/F-12 medium and a Neurobasal medium, and the mixed volume ratio of the DMEM/F-12 medium to the Neurobasal medium is 1:1; the apoptosis inhibiting factor is Y-27632, and the final concentration of the apoptosis inhibiting factor in the culture medium is 10 mu M.

Example 5

A serum-free culture medium comprises additive components and a basic culture medium, wherein the additive components comprise nutritional factors, growth factors, accessory factors and apoptosis inhibiting factors;

the basal medium is a mixed medium of a DMEM/F-12 medium and a Neurobasal medium, and the mixed volume ratio of the DMEM/F-12 medium to the Neurobasal medium is 1:1;

the nutritional factors are GlutaMAX culture medium additives, vitamin A-free B-27 supplement, N2 supplement and insulin; the final concentration of the GlutaMAX media supplement in the media was 1X, the final concentration of vitamin A-free B-27 supplement in the media was 0.5X, the final concentration of N2 supplement in the media was 0.5X, the final concentration of insulin in the media was 5. Mu.g/mL;

the growth factor is EGF, and the final concentration of the growth factor in the culture medium is 10ng/mL;

the accessory factors are hydrocortisone, cholera toxin, triiodothyronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 and inositol; the final concentration of hydrocortisone in the culture medium is 0.4. Mu.g/mL, cholera toxin in the culture medium is 0.1nM, triodoryronine in the culture medium is 2nM, beta mercaptoethanol in the culture medium is 0.1mM, normocin in the culture medium is 100. Mu.g/mL, Z-VAD-FMK in the culture medium is 50. Mu.M, HA14-1 in the culture medium is 50. Mu.M, and inositol in the culture medium is 100mg/L;

the apoptosis inhibiting factor is Y-27632, and the final concentration of the apoptosis inhibiting factor in the culture medium is 10 mu M.

The normal serum medium used was purchased from gibco.

The medium of example 5 was used for the following experimental tests:

1. hair follicle stem cell culture

The culture medium in example 5 is used for culturing hair follicle stem cells separated from human hair follicles, and the culture method is as follows:

human hair follicle tissue was placed in PBS containing 100U/mL penicillin and 100. Mu.g/mL streptomycin and washed 3 times for 10min each. Subsequently, the hair follicle tissue was transferred to a sterile petri dish containing the medium of example 5, and adherent fat or connective tissue around the lower portion of the hair follicle was trimmed off using sterile scissors and forceps. The hair follicle is adhered to the bottom of the dish through the adhesion of the needle. The follicle is then secured to the plate with a right-handed needle, the needle is rotated 90 degrees clockwise, the beveled edge is left, the pressure on the needle is released, the needle is pulled away from left to right and held in contact with the dish. This process allows the fixation of the follicle on a dish while cutting the follicle, allowing the migration of hair follicle stem cells from the intact follicle structure in a star burst. The adherent follicles were transferred to a cell culture incubator for culture, and the next day, the medium of example 5 was replenished again, and the temperature was continued at 37 ℃ with 5% CO ₂ Culturing in an incubator. After ten days, the cells were checked for growth status, after which the medium was changed every 2-3 days. The growth of hair follicle stem cells is shown in FIG. 1. In fig. 1, a is an isolated human hair follicle, B and C are primary hair follicle cells which climb out of hair follicle tissues after the hair follicle is cultured in an adherent manner under different visual fields, and D are hair follicle cells which are subjected to subculture. As can be seen from FIG. 1, the medium of the present invention can maintain the activity of the hair follicle stem cells as high as 95% or more.

2. Keratinocyte Hacat culture

(1) Keratinocyte Hacat was cultured using the medium of example 5 as follows:

taking out the frozen cells Hacat from the liquid nitrogen tank, quickly thawing in 37 ℃ water bath, and collecting the cellsThe suspension was transferred to a 15mL centrifuge tube containing medium, mixed by gentle blowing, centrifuged at 210g for 5 minutes, and the supernatant was discarded. Adding the medium to resuspend the cells, then transferring to a petri dish, 37 ℃,5% CO ₂ Culturing in an incubator. When the cell confluence rate reaches 70-80%, discarding the old culture solution, washing the cells with PBS, and digesting with pancreatin for 5min. After digestion, the pancreatin was discarded, digestion was stopped by adding fresh medium, the cells were blown into a single cell suspension, the cell suspension was transferred to a 15mL centrifuge tube, centrifuged at 210g for 5 minutes, and the supernatant was discarded. Fresh medium and ordinary serum medium are added to resuspend separately and transferred to new culture dishes for further culture separately. The state diagram of the cells cultured for different days is shown in FIG. 2, panel A. Respectively taking well-grown cells, carrying out trypsinization, centrifuging, removing supernatant, adding a proper amount of fresh culture medium or common serum culture medium, and re-suspending the cells to obtain single cell suspension. Preparing a blood counting cell plate, cleaning and airing, sucking a certain amount of cell suspension, loading the cell suspension on the blood counting cell plate, and counting under a microscope. The cells were cultured again, and 6-well plates were prepared, to each of which 0.4mL of cells (i.e., 1X 10 cells) were added ⁵ Individual cells) and 0.6mL of medium or ordinary serum medium, gently shaking the well plate (shaking in the cross direction) to make the cells uniformly distributed, and performing conventional cell culture (48 h post-culture medium exchange). Sampling is carried out once every 24h, sampled cells are subjected to conventional pancreatin digestion to prepare single cell suspension, and cell counting is carried out under a microscope. The results of counting, with time (d) as the abscissa and the number of cells as the ordinate, were plotted as cell growth curves, as shown in panel B of FIG. 2.

As can be seen from FIG. 2, the culture medium of the invention can promote the proliferation of keratinocyte Hacat, the confluence rate of 2-3 days reaches 80-90%, and the culture medium has good growth capacity, has no significant difference compared with the proliferation capacity of a common serum culture medium, and even has later-stage proliferation capacity superior to that of the common serum culture medium.

(2) The cells cultured in the above medium and having a good growth state were collected, and the old culture medium was aspirated, washed with PBS, and digested with trypsin. After digestion was complete, medium was added in twice the volume of trypsin, the cells were gently pipetted, the pipetted cells were transferred into a centrifuge tube, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. Cells were harvested, gently resuspended in PBS and counted. 5-10 ten thousand cells were centrifuged at 1000g for 5 minutes, the supernatant was discarded, and 195. Mu.L of Annexin V-FITC conjugate was added to gently resuspend the cells. Then 5. Mu.L of Annexin V-FITC was added and mixed gently. Add 10. Mu.L of propidium iodide staining solution and mix gently. Incubate at room temperature (20-25 ℃) for 10-20 minutes in the dark, then place in an ice bath and wrap with aluminum foil in the dark. The cells can be resuspended 2-3 times during incubation to improve staining. Detecting with flow cytometer after 10-20 min. Hacat cell staining As shown in panel A of FIG. 3, annexin V-FITC exhibits green fluorescence and Propidium Iodide (PI) exhibits red fluorescence. A flow cytometer image of Hacat with Annexin-V FITC apoptosis detection is shown in FIG. 3B.

As can be seen from FIG. 3, the culture medium of the invention well maintains the survival rate of the skin keratinocytes cultured for a long time, and the survival rate can reach about 90%.

3. Hair follicle culture

(1) Hair follicle culture from hair follicle stem cells

After day 12 of in vitro culture of the hair follicle organoids, the medium was changed to the medium of example 5, 0.5mL per well, half a change every three days. After 45 days, half of the medium was replaced every other day and complete removal of medium was ensured once a week, increasing the medium volume to 1mL by day 80 of hair follicle organoid differentiation. And similarly, half liquid change every other day and full liquid change in one week are carried out to continue the culture. FIG. 4A is a graph showing the growth of hair follicles in the culture of hair follicles derived from hair follicle stem cells for different days (110, 112, 117, 120 days); fig. 4, panel B, is a graph of cell proliferation of Ki67 staining when hair follicles derived from hair follicle stem cells were cultured for 140 days.

After the hair follicle organoids were cultured in vitro on day 12, the culture medium was changed to normal serum medium, the medium lacking the vitamin A-free B-27 supplement in example 5, and the medium lacking the N2 supplement in example 5, and the culture results are shown in FIGS. 4C, D, and E, respectively. As can be seen from FIG. 4C, after the induced hair follicle organoids were cultured in a normal serum medium, the cells failed to aggregate into balls and then die; as can be seen from Panel D in FIG. 4, when the medium lacks the vitamin A-free supplement B-27, the cells are scattered and not cultured in the form of spheres, and the state is poor; as shown in FIG. 4, in the absence of N2 supplement, although organoids were formed by culturing, the limbal cells gradually fell off and die later in time, which is less than a month.

(2) Human hair follicle culture

The hair follicle stripped in the hair transplantation operation is taken and soaked in normal saline and stored on ice. The hair follicles were first washed with physiological saline to remove surface residual blood, then transferred to D-Hanks buffer containing penicillin (200U/mL) streptomycin (200. Mu.g/mL) for about 3min, and further transferred to D-Hanks buffer containing penicillin (100U/mL) streptomycin (100. Mu.g/mL) for 2 washes, each for 2min, and the whole process was performed on ice. After washing, they were transferred to 24-well plates containing 0.5 mL/well of hair follicle stem cell medium, 1 root per well. The plates were incubated at 37 ℃ and 5% CO ₂ The shape change of the hair follicle was observed daily and the length of the hair follicle was measured. FIG. 4, panel F, shows the growth status of human follicles in the follicles cultured for different days (

days

1,2, 4, 6, 8); FIG. 4 is a microscope photograph of human hair follicles after cultivation; in FIG. 4, the H-plot is the cell proliferation plot of Ki67 staining of human hair follicles.

As can be seen from FIG. 4, the medium of the present invention can maintain the activity of the hair follicle derived from the hair follicle stem cell for 140 days and the in vitro human hair follicle for more than one week.

4. Hair follicle transplantation

The above-mentioned hair follicle organoids derived from hair follicle stem cells or in vitro hair follicles derived from human body were collected, placed on gauze impregnated with the medium of example 5, and stored on ice. Nude mice were prepared for 5-6 weeks. After the nude mouse was anesthetized, a small incision (capable of accommodating a single hair follicle) was made in the skin of the back of the nude mouse using a syringe, and a previously prepared hair follicle derived from hair follicle stem cells or a human body-derived hair follicle was placed in a separate incision site with the hair sphere facing inward, contacted with the muscle layer of the mouse, with the hair shaft facing outward, and exposed to the air. The dressing was placed over the graft area on the back of the mouse and a bandage was wrapped around the body of the nude mouse to tighten the graft area. The bandage was then sutured to hold stability. After surgery, the nude mice were provided with carprofen wet diet to relieve pain. The dressing was removed 7-9 days after surgery. Mice were observed 7 or 14 weeks post-surgery. The effect of the transplantation is shown in fig. 5.

Fig. 5, in which a is a diagram derived from hair follicle organoids cultured from hair follicle stem cells, B is a diagram in fig. 5, in which short hairs appear on the back of a nude mouse after one month of transplantation using the hair follicle organoids, and C is a diagram in fig. 5, in which the length of hair follicles is continuously increased in a nude mouse after two months of transplantation; fig. 5D shows isolated human-derived in vitro hair follicles, fig. 5E shows in vitro hair follicles implanted on the backs of nude mice, and fig. 5F shows hair follicles implanted 2 weeks after transplantation. As can be seen from fig. 5, in the hair follicle transplantation process, no hair follicle shedding and death occur after the hair follicle transplantation process by using the culture medium of the present invention, no matter the hair follicle derived from the hair follicle stem cells or the hair follicle derived from human body, which indicates that the hair follicle has entered the growth phase, thereby avoiding the resting phase and significantly improving the survival rate of the transplanted hair follicle.

Example 6

A serum-free culture medium consists of additive components and a basic culture medium, wherein the additive components comprise nutritional factors, growth factors, accessory factors, apoptosis inhibiting factors and glucocorticoid medicaments;

the basic culture medium is a DMEM/F-12 culture medium;

the nutritional factors are GlutaMAX culture medium additive, vitamin A-free B-27 supplement, N2 supplement and insulin, and the final concentration of the GlutaMAX culture medium additive in the culture medium is 1 x; vitamin a-free B-27 supplement at a final concentration of 0.2 x in the medium; the final concentration of N2 supplement in the medium was 0.2 ×; the final concentration of the insulin in the culture medium is 5 mug/mL;

the growth factor is bFGF, and the final concentration of the growth factor in the culture medium is 10ng/mL;

the accessory factors are Triiodothyronine and inositol, and the final concentration of the Triiodothyronine in the culture medium is 0.4 mu g/mL; the final concentration of inositol in the culture medium is 100mg/L;

the apoptosis inhibiting factor is Y-27632, and the final concentration of the apoptosis inhibiting factor in the culture medium is 5 mu M;

the glucocorticoid drug is hydrocortisone, and the final concentration of the hydrocortisone in the culture medium is 0.4ug/mL.

Comparative example 1

A serum-free culture medium comprises additive components and a basic culture medium, wherein the additive components comprise nutritional factors, growth factors and apoptosis inhibiting factors;

the basic culture medium is a DMEM/F-12 culture medium;

the nutritional factor is a GlutaMAX culture medium additive, and the final concentration of the GlutaMAX culture medium additive in the culture medium is 1 x;

the apoptosis inhibiting factor is Y-27632, and the final concentration of the apoptosis inhibiting factor in the culture medium is 5 mu M.

Comparative example 2

the basic culture medium is a DMEM/F-12 culture medium;

the nutritional factors are GlutaMAX culture medium additive, vitamin A-free B-27 supplement and N2 supplement, and the final concentration of the GlutaMAX culture medium additive in the culture medium is 1 x; vitamin a-free B-27 supplement at a final concentration of 0.2 x in the medium; the final concentration of N2 supplement in the medium was 0.2 ×;

the apoptosis-inhibiting factor is Y-27632, and the final concentration of the apoptosis-inhibiting factor in the culture medium is 5 mu M.

The following tests were carried out on the medium of example 6 (corresponding to the attached figure and formulation 3 hereinafter), of comparative example 1 (corresponding to the attached figure and formulation 1 hereinafter), of comparative example 2 (corresponding to the attached figure and formulation 2 hereinafter) and with physiological saline (control group):

1. cell proliferation assay

The experimental procedure for cell proliferation assay was the same as in example 5, subsection (1) of Hacat culture of keratinocytes.

FIG. 6 shows the morphology of cells cultured in the culture media of example 6, comparative examples 1-2 and control group for 16h, 2 days, 4 days and 6 days, respectively, wherein the cell density and proliferation rate obtained by the culture media of example 6 are significantly better than those of the cells of comparative examples 1-2 and control group.

FIG. 7 shows the results of activity measurements of cells cultured using the culture media of example 6 and comparative examples 1-2, wherein the control medium was used for the first culture, the comparative example 1 was used for the first culture, the comparative example 2 was used for the third culture, and the example 6 was used for the third culture. And cell cultures were propagated every 3 days. Staining by using a kit for detecting Calcein AM cell activity and apoptosis in Byuntian, and taking a picture in confocal manner. Calcein stains as active cells, PI stains as apoptotic cells, and Merge the active cell map stained by Calcein with the apoptotic cell map stained by PI. It was found that the first generation cells, i.e., all apoptotic cells, could not be passaged continuously using saline cultured cells. Formulation 1 (comparative example 1) maintained cell viability with a few apoptotic cells but failed to continue passaging. Both formula 2 (comparative example 2) and formula 3 (example 6) were able to maintain cell viability and promote cell proliferation, with essentially no significant apoptosis, and were passable for at least 3 serial passages.

FIG. 8 shows the proliferation curves in the cell proliferation test obtained for the culture media of example 6, comparative examples 1 to 2 and control, and the cells after 7 days of treatment of

formulation

2,3 showed a normal cell proliferation curve, and formulation 3 was more favorable for cell proliferation than formulation 2, and the cells after 7 days of treatment of formulation 1 were slowly proliferated, and the cells after 7 days of NaCl treatment of control were not proliferated and cell death occurred.

The following conclusions can be drawn from the above cell morphology observation experiment and proliferation experiment:

1) The keratinocytes Hacat in the control group were essentially unable to adhere to the wall when cultured, floated throughout and then died.

2) Formula 1: namely, the cells in the most basic culture medium can be attached to the wall normally, but the proliferation is slow, the proliferation reaches the plateau stage after 4 days, and the cell confluence rate is less than one fourth of that of the culture of the formula II and the formula III after 6 days. Only one generation can be passed.

3) And (2) formula: the cells cultured in this medium had normal cell morphology, cell aggregation and growth, and the proliferation rate was not significantly different from that in the ordinary medium (DMEM +10% FBS). Under the normal culture condition, the continuous passage can be realized after one passage for 3 to 4 days.

4) And (3) formula: the cell morphological proliferation rate of the culture medium is obviously superior to the three culture media and the common culture medium, and the cell number can reach 2 multiplied by 10 after 4 days ⁶ And (4) one cell. Under the normal culture condition, the generation needs to be carried out for 2 days, and the generation can be carried out continuously.

2. Apoptosis assay

The experimental procedure for apoptosis testing was the same as in example 5, subsection (2) of Hacat culture of keratinocytes.

FIG. 9 shows the results of apoptosis tests after 3 days of treatment using example 6, comparative examples 1-2 and control, the degree of apoptosis was not very different after 3 days of treatment with

formulations

1,2,3, with the formulations 1 and 2-cloth 3, and with the control treated with NaCl up to 89.83% of the cells showing late apoptosis and necrosis.

FIG. 10 shows the results of cell cycle measurements 3 days after treatment with example 6, comparative examples 1-2 and control, the cycle 3 days after treatment with

formulations

1,2,3 was normal, the cells 3 days after control treatment were in S phase and G2/M phase more abundant, and the cell cycle was arrested at this time, resulting in abnormal cell proliferation.

3. Human hair follicle activity assay

Human hair follicle activity test experimental method and the third section (2) of hair follicle culture in example 5: the cultured parts of human hair follicles are the same.

FIG. 11 shows the results of the human hair follicle activity test of example 6 and comparative examples 1-2, which shows that there is no significant difference in the proliferation activity of human hair follicles cultured in each of the formulations when cultured in vitro for 4 hours.

FIG. 12 shows the results of-8 h activity test for human hair follicles in example 6 and comparative examples 1-2, wherein the human hair follicle proliferation activity of each formula cultured in the culture medium was not greatly different when cultured in vitro for 8h, and the activity of the control group was slightly lower.

FIG. 13 shows the results of the human hair follicle viability assay of example 6 and comparative examples 1-2, wherein the human hair follicle proliferation activity of each formula culture medium began to decrease after 15h in vitro culture, but there was no significant difference between groups, and the hair follicle viability of the control group was lost.

FIG. 14 shows the results of the human hair follicle viability test-24 h in example 6 and comparative examples 1-2, wherein the human hair follicle proliferation viability cultured in each formula of culture medium was reduced when cultured in vitro for 24h, the hair follicle viability treated by formula three was slightly better than that of formula one and formula two, and the hair follicle viability in the control group was lost.

In conclusion, according to the above results, formula 1 as the most basic culture medium can maintain the morphology and survival rate of cells, but the proliferation is slow, continuous passage cannot be achieved, and the method is not suitable for cell culture. The formula 2 can maintain the normal shape and the normal proliferation rate of cells, can pass more than 3 generations, and can be used for common cell culture. The cells cultured by the formula 3 have excellent proliferation rate, can better maintain the cell viability and the activity of human hair follicles, and can be used for in vitro hair follicle culture.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A serum-free culture medium is characterized by consisting of additive components and a basic culture medium, wherein the additive components comprise at least one of nutritional factors, growth factors, cofactors and apoptosis inhibiting factors.

2. The serum-free medium according to claim 1, wherein the basal medium is DMEM/F-12 medium or a mixed DMEM/F-12 medium and Neurobasal medium.

3. The serum-free medium of claim 1, wherein the nutritional factors are at least one of GlutaMAX medium additives, vitamin A-free B-27 supplements, N2 supplements, or insulins.

4. The serum-free medium of claim 3, wherein the GlutaMAX media supplement is present at a final concentration of 1X in the medium, the vitamin A-free B-27 supplement is present at a final concentration of 0.2X-1X in the medium, the N2 supplement is present at a final concentration of 0.2X-1X in the medium, and the insulin is present at a final concentration of 1-10 μ g/mL in the medium.

5. The serum-free medium according to claim 1, wherein the growth factor is at least one of EGF, bFGF, aFGF, VEGFA, TGF-beta, WNT3a, PDGF, KGF, and the final concentration in the medium is 2-50ng/mL.

6. The serum-free medium according to claim 1, wherein the cofactor is at least one of hydrocortisone, cholera toxin, triiodothyronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 or inositol.

7. The serum-free medium according to claim 6, wherein the hydrocortisone is present in the medium at a final concentration of 0.2 to 0.8 μ g/mL; the final concentration of cholera toxin in the culture medium is 0.05-0.15nM; the final concentration of triiodothyronine in the culture medium is 0.1-3ug/mL; the final concentration of the beta mercaptoethanol in the culture medium is 0.05-0.1mM; the final concentration of Normocin in the culture medium is 50-200 mug/mL; the final concentration of Z-VAD-FMK in the culture medium is 30-60 μ M; the final concentration of HA14-1 in the culture medium is 20-50 μ M; the final concentration of inositol in the culture medium is 60-100mg/L.

8. The serum-free culture medium according to claim 1, wherein the apoptosis-inhibiting factor is Y-27632, and the final concentration of the apoptosis-inhibiting factor in the culture medium is 5-20 μ M.

9. The serum-free medium according to any one of claims 1 to 8, wherein the additive components of the medium further comprise a hormone drug in an amount of 0.1-1ug/mL.

10. The use of the serum-free medium according to any one of claims 1 to 8, wherein the serum-free medium is used for the preparation of hair follicle in vitro growth culture products, hair follicle cell culture products and hair follicle nourishing products.