CN115786247B

CN115786247B - Serum-free culture medium and application thereof in aspects of hair follicle activity maintenance and hair transplantation

Info

Publication number: CN115786247B
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: 2023-10-20
Anticipated expiration: 2042-10-25
Also published as: CN114540281A; CN115786247A

Abstract

The invention particularly relates to a serum-free culture medium and application thereof in the aspects of hair follicle activity maintenance, hair maintenance and transplantation. The culture medium consists of additive components and basic culture medium, wherein the additive components comprise at least one of nutritional factors, growth factors, auxiliary factors and apoptosis inhibiting factors. All components of the culture medium provided by the invention are definite, no animal source exists, and no toxic or side effect is caused to human bodies; the culture medium can well maintain the activities 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 the isolated hair follicles for more than one week, and can maintain the activity of hair follicles from stem cell regeneration sources for more than 140 days; after the hair follicle cultured by the culture medium provided by the invention is transplanted, the hair follicle can directly grow, and the hair follicle is prevented from entering the resting stage.

Description

Serum-free culture medium and application thereof in aspects of hair follicle activity maintenance and hair transplantation

Technical Field

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

Background

At present, under the dual actions of social pressure and bad life style, the alopecia group is continuously strong and the trend of younger is presented. Although alopecia does not seriously affect the physiological health of people, the alopecia brings great threat to personal image, self-esteem and emotional health, thereby affecting the work and life of people. The hair implantation operation is a main technical means for solving the problem of alopecia, the hair in the hair implantation operation is completely consistent with the hair property and growth rule of the hair supply part, the hair implantation operation is safe and has no rejection and adverse reaction, the hair density after the implantation is close to the normal density, the appearance is real and natural, and the hair implantation operation is the best choice for patients suffering from alopecia at present. But this approach is also ineffective for patients whose donor area is not sufficiently sourced. 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 multi-directional differentiation, so that the regeneration of hair follicles by in vitro culture of stem cells is also a potential direction for solving the problem of alopecia, and many researches based on stem cell regeneration are also carried out at present.

However, there is a common problem in both hair-setting surgery and stem cell regenerating hair follicles, in that the activity of the hair follicle in vitro is gradually lost or degenerated with the increase of the in vitro culture time. During the hair implantation operation, the hair follicle is isolated for about 4-6 hours, and part of cells in the hair follicle are apoptotic 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 resting period after being transplanted. The stem cells induce the hair follicle structure generated during the hair follicle process, and the degeneration of the hair follicle can be caused along with the change of environment and the lack of nutrition. Thus, maintaining the activity of long-term in vitro culture of hair follicles is critical in ensuring survival of hair follicle transplantation. This is a difficulty of research at present, and the present invention is to overcome this difficulty.

Disclosure of Invention

In view of the above, it is necessary to provide a serum-free medium and its application in maintaining hair follicle activity, hair maintenance and transplantation, which can solve the problems of low survival rate caused by long-time hair follicle ex-vivo in hair transplantation process, hair follicle degeneration in stem cell regeneration process, etc.

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

in a first aspect, the 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 basal medium is DMEM/F-12 medium or a mixed medium of DMEM/F-12 medium and Neurobasal medium.

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

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

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

Preferably, the final concentration of the Glutamax medium additive in the medium is 1×, the final concentration of the vitamin A free B-27 supplement in the medium is 0.2× -1×, the final concentration of the N2 supplement in the medium is 0.2× -1×, and the final concentration of the insulin in the medium is 1-10 μg/mL.

More preferably, the final concentration of the Glutamax medium additive in the medium is 1×, the final concentration of the vitamin A free B-27 supplement in the medium is 0.5× -1×, the final concentration of the N2 supplement in the medium is 0.5× -1×, and the final concentration of insulin in the medium is 1-10 μg/mL.

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

Further, the cofactor is at least one of hydrocortisine, cholera toxin, triodothitronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 or inositol.

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

Preferably, the final concentration of hydrocortisone in the medium is 0.2-0.8 μg/mL; the final concentration of cholera toxin in the culture medium is 0.05-0.15nM; the final concentration of triedothiron in the culture medium is 0.1-3ug/mL; the final concentration of beta mercaptoethanol in the culture medium is 0.05-0.1mM; normocin is present in the medium at a final concentration of 50-200. Mu.g/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. Mu.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.

Further, the culture medium also comprises hormone medicines 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 is 0.2-0.5ug/mL.

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

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

Furthermore, the hair follicle in vitro growth culture 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 follicles, leg follicles, and chest 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 beneficial effects of the invention are as follows:

all components of the culture medium provided by the invention are definite, no animal source exists, and no toxic or side effect is caused to human bodies; the culture medium can well maintain the activities 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 the isolated hair follicles for more than one week, and can maintain the activity of hair follicles from stem cell regeneration sources for more than 140 days; after the hair follicle cultured by the culture medium provided by the invention is transplanted, the hair follicle can directly grow, and the hair follicle is prevented from entering the resting stage.

Drawings

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

FIG. 2 is a cell state diagram of the culture medium and the common serum culture medium of example 5 of the present invention for culturing keratinocytes Hacat, respectively;

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

FIG. 4 is a diagram showing the culture of human-derived hair follicles by culturing hair follicle organoids derived from hair follicle stem cells in different culture media according to example 5 of the present invention;

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

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

FIG. 7 shows the results of cell activity measurements in the cell proliferation assays of examples 6, comparative examples 1-2 and control groups according to the present invention, wherein FIG. 1 corresponds to the result of cell activity measurement in the first generation of culture in the medium of the control group, FIG. 2 corresponds to the result of cell activity measurement in the first generation of culture in the medium of comparative example 1, FIG. 3 corresponds to the result of cell activity measurement in the third generation of culture in the medium of comparative example 2, and FIG. 4 corresponds to the result of cell activity measurement in the third generation of culture in the medium of example 6. And (3) injection: in FIG. 7, the cells were cultured for 3 days for one passage.

FIG. 8 is a graph showing the proliferation of cells in the test of example 6 and comparative examples 1 to 2 according to the present invention.

FIG. 9 shows apoptosis test results of inventive example 6, comparative examples 1 to 2 and control group.

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

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

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

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

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

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further clearly and completely described in the following in conjunction with the embodiments of the present invention. It should be noted that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

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

Example 2

A serum-free medium, which consists of additive components and a basic medium, wherein the additive components are growth factors; the basic culture medium is a mixed culture medium of a DMEM/F-12 culture medium and a Neurobasal culture medium, and the mixed volume ratio of the DMEM/F-12 culture medium to the Neurobasal culture medium is 1:1, a step of; the growth factor is EGF, and the final concentration of the EGF in the culture medium is 10ng/mL.

Example 3

A serum-free culture medium consists of additive components and a basic culture medium, wherein the additive components are auxiliary factors; the cofactors are hydrocortisine, cholera toxin, triodothionine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 and inositol; the final concentration of hydrocortisine in the medium was 0.4. Mu.g/mL, of cholera toxin in the medium was 0.1nM, of triodothioronine in the medium was 2nM, of beta mercaptoethanol was 0.1mM, of Normocin was 100. Mu.g/mL, of Z-VAD-FMK was 50. Mu.M, of HA14-1 was 50. Mu.M, and of inositol was 100mg/L.

Example 4

A serum-free culture medium consists of additive components and a basic culture medium, wherein the additive components are apoptosis inhibiting factors; the basic culture medium is a mixed culture medium of a DMEM/F-12 culture medium and a Neurobasal culture medium, and the mixed volume ratio of the DMEM/F-12 culture medium to the Neurobasal culture medium is 1:1, a step of; 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 medium, which consists of additive components and a basic medium, wherein the additive components comprise nutritional factors, growth factors, auxiliary factors and apoptosis-inhibiting factors;

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

the nutritional factors are Glutamax medium additive, vitamin A-free B-27 supplement, N2 supplement and insulin; the final concentration of the GlutaMAX medium additive in the medium is 1X, the final concentration of the vitamin A-free B-27 supplement in the medium is 0.5X, the final concentration of the N2 supplement in the medium is 0.5X, and the final concentration of the insulin in the medium is 5 mug/mL;

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

the cofactors are hydrocortisine, cholera toxin, triodothionine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 and inositol; the final concentration of hydrocortisine in the medium was 0.4. Mu.g/mL, of cholera toxin in the medium was 0.1nM, of triodothioronine in the medium was 2nM, of beta mercaptoethanol was 0.1mM, of Normocin was 100. Mu.g/mL, of Z-VAD-FMK was 50. Mu.M, of HA14-1 was 50. Mu.M, of inositol was 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 common 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 was used to culture the isolated hair follicle stem cells from human hair follicles as follows:

human hair follicle tissue was placed in PBS containing 100U/mL penicillin and 100 μg/mL streptomycin and washed 3 times for 10min each. The follicle tissue was then transferred to a sterile petri dish containing the medium of example 5, and the adherent fat or connective tissue surrounding the lower portion of the follicle was trimmed off using sterile scissors and forceps. Hair follicles were attached to the bottom of the dish by needle attachment. Then use the right hand needle to removeThe hair follicle is secured to the plate and the needle is rotated 90 degrees clockwise with the beveled edge facing left, releasing pressure on the needle, pulling the needle away from left to right and into contact with the dish. This procedure can fix the hair follicle on a dish while cutting the hair follicle so that the hair follicle stem cells migrate out of the intact hair follicle structure in a starburst format. The attached hair follicle was transferred to a cell incubator for culturing, and the culture medium of example 5 was replenished the next day, and the culture medium was continued at 37℃with 5% CO ₂ Culturing in an incubator. Ten days later, the cell growth status was checked, after which the medium was changed every 2-3 days. The growth of hair follicle stem cells is shown in figure 1. In fig. 1, a is an isolated human hair follicle, B, C is primary hair follicle cells which climb out of hair follicle tissue after the hair follicle is subjected to adherent culture in different fields of view, and D is the hair follicle cells subjected to subculture. As can be seen from FIG. 1, the culture medium of the present invention can maintain the activity of hair follicle stem cells up to 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 in a liquid nitrogen tank, quickly thawing in a water bath at 37 ℃, transferring the cell suspension into a 15mL centrifuge tube containing a culture medium, gently blowing, mixing uniformly, centrifuging at 210g for 5 minutes, and removing the supernatant. The cells were resuspended by addition of medium and then transferred to a petri dish at 37℃with 5% CO ₂ Culturing in an incubator. When the cell confluency rate reaches 70-80%, discarding old culture solution, washing cells with PBS, and digesting with pancreatin for 5min. After digestion is completed, pancreatin is discarded, fresh medium is added to terminate digestion, cells are blown into single cell suspension, the cell suspension is transferred into a 15mL centrifuge tube, 210g is centrifuged for 5 minutes, and the supernatant is discarded. Fresh medium and normal serum medium were added separately for resuspension and transferred separately to new dishes for further culture. Cell status charts of the different days of culture are shown in FIG. 2A. Taking well-grown cells, performing pancreatin digestion, centrifuging, removing supernatant, and adding a proper amount of fresh culture medium or common serum culture medium to resuspend the cells so as to form single cell suspension. Preparing a blood cell counting plate, cleaning, airing, sucking a certain amount of cell suspension, and adding the sample toOn a hemocytometer, the count is under a microscope. Cell re-culture, preparation of 6-well plates, 0.4mL of cells per well (i.e., 1X 10) ⁵ Individual cells) and 0.6mL of a medium or a common serum medium, gently shaking the well plate (shaking in a cross direction) to make the cells uniformly distributed, and performing conventional cell culture (48 h later liquid change culture). Once every 24h, cells were sampled for routine pancreatin digestion, single cell suspensions were prepared, and cell counts were performed under a microscope. As a result of the counting, a cell growth curve was drawn with time (d) as an abscissa and the number of cells as an ordinate, as shown in FIG. 2B.

As shown in FIG. 2, the culture medium of the invention can promote the proliferation of keratinocytes Hacat, the confluence rate reaches 80-90% in 2-3 days, and the culture medium has good growth capacity, and compared with the proliferation capacity of a common serum culture medium, the culture medium has no significant difference, and even has better later proliferation capacity than the common serum culture medium.

(2) And (3) taking cells cultured by the culture medium and having good growth state, sucking out old culture solution, washing by PBS, and digesting by trypsin. After digestion was completed, twice the volume of medium was added to trypsin, the cells were gently blown, the blown cells were transferred to a centrifuge tube, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. Cells were collected, resuspended gently with PBS and counted. 5-10 ten thousand cells were taken, centrifuged at 1000g for 5min, the supernatant was discarded, and 195. Mu.L of Annexin V-FITC conjugate was added to gently resuspend the cells. Subsequently, 5. Mu.L of Annexin V-FITC was added and gently mixed. Add 10. Mu.L propidium iodide staining solution and mix gently. Incubation at room temperature (20-25deg.C) for 10-20min in the dark, and then placing in ice bath, and wrapping with aluminum foil in the dark. Cells may be resuspended 2-3 times during incubation to improve staining. And detecting by a flow cytometer after 10-20 min. Hacat cells stained as shown in FIG. 3, panel A, annexin V-FITC for green fluorescence and Propidium Iodide (PI) for red fluorescence. The flow cytometer image of Hacat through Annexin-V FITC apoptosis detection is shown in FIG. 3, panel B.

As can be seen from FIG. 3, the culture medium of the invention better maintains the long-term culture survival rate of the skin keratinocytes, and the survival rate can reach about 90%.

3. Hair follicle culture

(1) Hair follicle culture derived from hair follicle stem cells

After day 12 of in vitro culture of the follicular organoids, the medium was changed to that of example 5, 0.5mL per well, and half changed every three days. After 45 days, half of the medium was replaced every other day with medium, and complete removal of medium was ensured once a week, increasing the medium volume to 1mL by day 80 of follicular organodifferentiation. The culture is continued by half liquid exchange every other day and full liquid exchange every week. FIG. 4A shows the growth of hair follicles (110, 112, 117, 120 days) of hair follicle stem cells from different days of culture; panel B in FIG. 4 is a graph of cell proliferation of Ki67 staining when hair follicles derived from hair follicle stem cells were cultured for 140 days.

After day 12 of in vitro culture of the follicular organoids, the culture medium was replaced with a normal serum medium, a medium lacking the vitamin A-free B-27 supplement of example 5, and a medium lacking the N2 supplement of example 5, respectively, and the culture results were shown in FIG. 4, C, D, E, respectively. As can be seen from fig. 4C, after the induction of follicular organoids by culture with normal serum medium, cells cannot aggregate into spheres, and then die; as can be seen from the graph D in FIG. 4, when the B-27 supplement containing no vitamin A is absent in the culture medium, the cells scatter, cannot be cultured in a balling manner, and the condition is poor; as can be seen from the graph E in FIG. 4, when the N2 supplement is absent from the medium, the organoids are formed by culturing, but the limbal cells gradually fall off, and the culture time is less than one month after the later period until death.

(2) Culture of human hair follicle

The hair follicle peeled in the hair implantation operation is taken, soaked in normal saline and preserved on ice. The hair follicle was first washed with physiological saline to remove residual blood on the surface, then transferred to D-Hanks buffer containing penicillin (200U/mL) streptomycin (200. Mu.g/mL) for about 3min, and then transferred to D-Hanks buffer containing penicillin (100U/mL) streptomycin (100. Mu.g/mL) for 2 times, each for 2min, with the whole procedure being performed on ice. After washing clean, it was transferred to a 24-well plate containing 0.5 mL/Kong Maonang stem cell medium, 1 per well. The plates were placed at 37℃with 5% CO ₂ Is cultured in an incubator of (2), the morphological changes of the hair follicle are observed daily and the hair follicle length is measured.FIG. 4F is a graph showing the growth state of human hair follicles after culturing for different days (days 1,2, 4, 6, 8); FIG. 4 is a microscopic image of the cultured human hair follicle; FIG. 4, panel H, shows the cell proliferation of Ki67 staining of human hair follicles.

As shown in FIG. 4, the culture medium of the invention can be used for culturing hair follicles from hair follicle stem cells, and can maintain the activity of the hair follicles for 140 days and more than one week.

4. Hair follicle transplantation

The above follicular organoids derived from hair follicle stem cells or in vitro hair follicles of human origin 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 anesthetizing a nude mouse, a small incision (capable of accommodating a single hair follicle) is made in the skin of the back of the nude mouse by using a syringe, and the prepared hair follicle derived from the hair follicle stem cells or the hair follicle derived from the human body is placed in a separate incision site, mao Qiumian is faced inward, contacted with the muscle layer of the mouse, and the hair shaft is faced outward and exposed to the air. The dressing was applied over the transplanted area on the back of the mice and wound around the body of the nude mice using a bandage to tighten the transplanted area. The bandages were then sewn to remain stable. After surgery, bare 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 after surgery. The effect of the transplantation is shown in fig. 5.

FIG. 5A shows a follicular organoid derived from a culture of hair follicle stem cells, FIG. 5B shows a nude mouse after one month after transplantation of the follicular organoid, and shorter hair appears on the back of the nude mouse, and FIG. 5C shows a nude mouse after two months of transplantation, with the hair follicle length increasing; fig. 5D shows isolated human-derived in vitro hair follicles, fig. 5E shows in vitro hair follicles implanted in the back of nude mice, and fig. 5F shows hair follicles after 2 weeks of transplantation. As can be seen from fig. 5, in the hair follicle transplantation process, the culture medium of the present invention is used to prevent hair follicle from falling off and dying after transplantation, i.e. the hair follicle has entered the anagen phase, avoid the telogen phase, and significantly improve the survival rate of the transplanted hair follicle.

Example 6

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

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

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

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

the cofactors are triodothiuron and inositol, and the final concentration of the triodothiuron in the culture medium is 0.4 mug/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 nutrient factors are Glutamax culture medium additives, and the final concentration of the Glutamax culture medium additives in the culture medium is 1X;

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 1X; the final concentration of vitamin a-free B-27 supplement in the medium was 0.2×; the final concentration of N2 supplement in the medium was 0.2×;

The following tests were performed on example 6 (corresponding to the drawing and formulation 3 below), comparative example 1 (corresponding to the drawing and formulation 1 below), comparative example 2 (corresponding to the drawing and formulation 2 below), and medium with physiological saline (control group):

1. cell proliferation assay

The experimental procedure for cell proliferation assay was the same as that of section (1) of example 5 in which two keratinocytes Hacat were cultured.

FIG. 6 shows cell morphology patterns of 16h, 2 days, 4 days, and 6 days of culture using the culture media of example 6, comparative examples 1 to 2, and the control group, respectively, wherein the cell density and proliferation rate obtained using the culture media of example 6 are significantly superior to those of the cells of comparative examples 1 to 2 and the control group.

FIG. 7 shows the results of cell viability assays using the media of example 6 and comparative examples 1-2, wherein the control media was used to culture the first generation, the comparative example 1 media was used to culture the first generation, the comparative example 2 media was used to culture the third generation, and the example 6 media was used to culture the third generation. And cell culture transmitted the first generation every 3 days. And (3) staining by using a Biyundian Calcein AM cell activity and apoptosis detection kit, and confocal photographing. Calcein staining was active cells, PI staining was apoptotic cells, and Merge represents the fusion of the Calcein-stained active cell pattern with the PI-stained apoptotic cell pattern. Cells cultured with physiological saline were found to be unable to be serially passaged for the first generation of cells, i.e., all apoptosis. Formulation 1 (comparative example 1) maintained cell viability with little apoptosis but no serial passage. Both formulation 2 (comparative example 2) and formulation 3 (example 6) maintained cell viability and promoted cell proliferation, substantially without apparent apoptosis, and were passaged continuously for at least 3 passages.

FIG. 8 shows the proliferation curves in the cell proliferation test obtained for the culture media of example 6, comparative examples 1-2 and control, the cells after 7 days of treatment of formulation 2,3 showed normal cell proliferation curves, and formulation 3 was more favorable for cell proliferation than formulation 2, the cells after 7 days of treatment of formulation 1 proliferated slowly, the cells after 7 days of treatment of control NaCl did not proliferate, and cell death occurred.

From the above cell morphology observation experiments and proliferation experiments, the following conclusions were drawn:

1) Keratinocytes Hacat in the control group were not substantially adherent when cultured, were all floating, and died.

2) Formula 1: that is, the cells in the basic culture medium can be adhered normally, but the proliferation is slow, the proliferation reaches the plateau after 4 days, and the cell confluency rate is less than one fourth of that in the culture of the formula II and the formula III after 6 days. Only one generation can be transmitted.

3) Formula 2: the cells cultured by the culture medium have normal cell morphology, the cell aggregation growth and the proliferation rate are not obviously different from those of the common culture medium (DMEM+10% FBS). Under normal culture conditions, the culture medium can be continuously passaged after 3-4 days.

4) Formula 3: the cell form proliferation rate of the culture medium is obviously superior to that of the three culture mediums and the common culture medium, and the cell number can reach 2 multiplied by 10 after 4 days ⁶ Individual cells. Under normal culture conditions, the culture medium needs to be passaged for 2 days, and can be continuously passaged.

2. Apoptosis test

The apoptosis test experiment method is the same as that of section (2) in which keratinocyte Hacat is cultured in example 5.

FIG. 9 shows the results of apoptosis tests of example 6, comparative examples 1-2 and control groups for 3 days, with less difference in the degree of apoptosis of formulas 1,2,3 after 3 days, formula 1<2<3, and advanced apoptosis and necrosis of up to 89.83% of cells after 3 days of NaCl treatment.

FIG. 10 shows the results of cell cycle tests after 3 days of treatment with example 6, comparative examples 1-2 and control, the cycle after 3 days of treatment with formulations 1,2,3 showed normal, and cells after 3 days of control were in more S phase than G2/M phase, and cell cycle arrest was in this phase, resulting in abnormal cell proliferation.

3. Human hair follicle activity test

Human hair follicle activity test experimental method and three, hair follicle cultures in example 5 (2) section: the culture parts of human hair follicles are identical.

FIG. 11 shows the results of human hair follicle activity test-4 h for example 6 and comparative examples 1-2, without significant differences in human hair follicle proliferative activity in each of the formulation media when cultured in vitro for 4 h.

FIG. 12 shows the results of the human hair follicle activity test-8 h in example 6 and comparative examples 1-2, wherein the human hair follicle proliferation activity in each of the culture media 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 activity test-15 h for example 6 and comparative examples 1-2, in which the human hair follicle proliferation activity in each of the formulation media began to decrease when cultured in vitro for 15h, but there was no significant difference from group to group, and the hair follicle activity in the control group was lost.

FIG. 14 shows the results of the human hair follicle activity test-24 hours for example 6 and comparative examples 1-2, wherein the human hair follicle proliferation activity in each of the formulation culture media was reduced when cultured in vitro for 24 hours, and the formulation III treated hair follicle activity was slightly superior to the formulation one and formulation two, and the hair follicle activity of the control group was lost.

In summary, according to the above results, formula 1 was used as the most basic medium to maintain the morphology and viability of cells, but the proliferation was slow, and continuous passage was not possible, and it was not suitable for cell culture. The formula 2 can maintain the normal form and the normal proliferation rate of cells, can transmit more than 3 generations, and can be used for common cell culture. The cells cultured in 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 foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A serum-free culture medium, which is characterized by comprising additive components and a basal culture medium, wherein the additive components comprise nutritional factors, growth factors, auxiliary factors and apoptosis-inhibiting factors; the basic culture medium is a mixed culture medium of a DMEM/F-12 culture medium and a Neurobasal culture medium; the nutritional factors are Glutamax medium additive, vitamin A-free B-27 supplement, N2 supplement and insulin; the growth factor is EGF; the cofactors are hydrocortisone, cholera toxin, triiodothyronine, beta mercaptoethanol, normocin, Z-VAD-FMK, HA14-1 and inositol; the apoptosis inhibiting factor is Y-27632; the final concentration of the GlutaMAX culture medium additive in the culture medium is 1×, the final concentration of the vitamin A-free B-27 supplement in the culture medium is 0.5× -1×, the final concentration of the N2 supplement in the culture medium is 0.5× -1×, and the final concentration of the insulin in the culture medium is 1-10 μg/mL; the final concentration of the growth factors in the culture medium is 10ng/mL; the final concentration of hydrocortisone in a serum-free medium is 0.4 mug/mL, the final concentration of cholera toxin in the serum-free medium is 0.05-0.15nM, the final concentration of triiodothyronine in the serum-free medium is 2nM, the final concentration of beta mercaptoethanol in the serum-free medium is 0.05-0.1mM, the final concentration of Normocin in the serum-free medium is 50-200 mug/mL, the final concentration of Z-VAD-FMK in the serum-free medium is 30-60 mu M, the final concentration of HA14-1 in the serum-free medium is 20-50 mu M, and the final concentration of inositol in the serum-free medium is 60-100 mg/L; the final concentration of the apoptosis inhibiting factors in the culture medium is 5-20 mu M.

2. The serum-free medium according to claim 1, wherein the final concentration of the GlutaMAX medium additive in the medium is 1×, the final concentration of the vitamin a-free B-27 supplement in the medium is 0.5×, the final concentration of the N2 supplement in the medium is 0.5×, and the final concentration of insulin in the medium is 5 μg/mL.

3. Use of a serum-free medium according to claim 1 or 2, characterized in that 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.