CN116875534A - Method for improving expression level of Sox2 in DP (DP) cells - Google Patents

Abstract

The application belongs to the technical field of cell culture, and particularly relates to a method for improving the expression level of Sox2 in DP cells. The present application employs a method for isolating DP cells comprising the steps of: cleaning a hair follicle sample; after removing at least part of fat and connective tissue, segmenting the rest hair follicle sample, and collecting a part containing DP cells to obtain DP cell segment tissue; adding enzymolysis liquid into the collected DP cell segment tissue for enzymolysis, so that DP cells are exposed; stopping enzymolysis, repeatedly blowing to drop DP cells from the DP cell segment tissue, and centrifuging; and (3) carrying out solid-liquid separation to obtain the DP cells, and cleaning the obtained DP cells. The passage cells obtained by carrying out the adherence culture on the primary DP cells separated by the method have higher Sox2 expression level compared with the passage cells of the same generation obtained by carrying out the passage on the DP cells separated by a microdissection method.

Description

Method for improving expression level of Sox2 in DP (DP) cells

Technical Field

The application belongs to the technical field of cell culture, and particularly relates to a method for improving the expression level of Sox2 in DP cells.

Background

Hair is an accessory organ of skin, and plays an important role in controlling body temperature, protecting skin and the like. Both disease and medication can lead to hair loss, which, although not directly threatening human life, can cause some confusion to the patient's mind and quality of life. At present, the most common cause of alopecia is androgenic alopecia, and hair follicle stem cells of such patients are not damaged in early stages, whereas the signal transduction of Dermal Papilla (DP) cells is disturbed, so that hair growth cannot be maintained. Thus, DP cells are a key target point for the treatment of hair loss.

At present, two methods of drug treatment and autologous hair follicle transplantation are mainly adopted for treating alopecia. Although the medicines for treating alopecia obtained in the market have certain effects, the medicines can not promote the formation of new hair follicles, so the symptoms are cured and the root causes are not cured. Autologous hair follicle transplantation often requires a large number of DP cells with hair follicle regenerating function. On the one hand, the autologous human scalp papilla cells available for transplantation in some patients with severe alopecia are very limited in source, and on the other hand, these transplanted cells cannot be expanded, so that the feasibility of obtaining a sufficient amount of DP cells from patients for surgical transplantation is not great clinically, and a method for rapidly obtaining a large amount of DP cells with hair follicle regeneration function is needed. Hair follicle formation results from the interaction of the epidermis and dermis, with DP cells having a decisive role in inducing hair follicle regeneration and hair cycle. The ability of DP cells to induce hair regrowth is rapidly lost when cultured in vitro, so how to obtain a large number of DP cells during in vitro culture while maintaining their ability to induce hair regrowth has become a hotspot and difficulty in current research and development.

DP cells are "signal centers" in the process of hair follicle growth and development, control the differentiation process from the hair follicle stem cells in the carina area to the hair matrix cells, and participate in the regulation of the hair follicle cycle. The number of DP cells may be involved in determining the differential developmental process of the primary/secondary hair follicle, and thus the size of the hair follicle. Meanwhile, various cytokines and growth factors secreted by the DP cells can induce the hair follicle reconstruction process under the in-vitro condition by a xenograft method, and are beneficial to analyzing the functions of the hair follicle in the development biological process. DP cells are one of the most representative key cells for the regulation of the follicular cycle, and their identification and isolation work is imperative. In recent years, scholars at home and abroad succeed in separating and culturing various hair follicle cells, and main separation methods include microdissection and enzyme digestion. The microdissection method has the advantages of complicated operation, large labor capacity, large sample quantity required by the enzyme digestion method, low purity of the separated cells and waste of the samples.

The DP tissue is obtained by adopting the integrated microscope microdissection method, which is time-consuming and labor-consuming, the hair induction characteristic of the DP tissue is reduced or even lost due to overlong operation time at room temperature, in addition, the DP tissue obtained by adopting the microdissection method is scratched and attached, the DP cell climbing-out period is longer, and the cell climbing-out rate is low; when other DP cells are obtained by adopting an enzyme digestion method, the cells in all the hair root sheath areas are directly digested, and the lack of a step of separating the DP cell mass from other cells can lead to the mixed collection of various cells of hair follicles, so that the DP cells are not easy to obtain independently, and the cell purity is lower.

Accordingly, there is a need to provide an improved solution to the above-mentioned deficiencies of the prior art.

Disclosure of Invention

The application aims to provide a method for improving the expression level of Sox2 in DP cells, which at least solves or improves the problem that the hair follicle induction characteristics of the DP cells isolated in the prior art are required to be improved.

In order to achieve the above object, the present application provides the following technical solutions: a method for increasing Sox2 expression levels in DP cells, the DP cells being isolated by a method comprising the steps of: s01: cleaning a hair follicle sample; s02: after removing at least part of fat and connective tissue, segmenting the rest hair follicle sample, and collecting a part containing DP cells to obtain DP cell segment tissue; s03: adding enzymolysis liquid into the collected DP cell segment tissue for enzymolysis, so that DP cells are exposed; s04: stopping enzymolysis, repeatedly blowing to drop DP cells from the DP cell segment tissue, and centrifuging; s05: solid-liquid separation is carried out to obtain the DP cells, and the obtained DP cells are cleaned; in step S03, the components of the enzymatic hydrolysate include collagenase a, dispase II and calcium salt.

Preferably, in the enzymolysis solution, the concentration of collagenase A is 1-5mg/mL, the concentration of dispese II is 2-4mg/mL, and the concentration of calcium salt is 2-6mM.

Preferably, in the enzymolysis solution, the concentration of the collagenase A is 2-4mg/mL.

Preferably, the calcium salt is calcium chloride, and the component of the enzymolysis liquid further comprises HBSS.

Preferably, in step S03, the enzymolysis is performed on a constant temperature shaker; the enzymolysis temperature is 37 ℃ and the enzymolysis time is 20-120min; the rotating speed of the constant temperature shaking table is 100-200rpm.

Preferably, in step S02, the DP cell segment is organized as the lower 1/3-1/2 portion of the hair follicle sample that remains after removal of at least a portion of fat and connective tissue.

Preferably, in step S04, the centrifugal force of the centrifugation is 180-250g, and the centrifugation time is 2-5min.

Preferably, the washing is performed by adding the culture medium again to the obtained hair papilla cells after discarding the supernatant, repeatedly blowing, and centrifuging to remove the supernatant.

Preferably, in step S01, the hair follicle sample is washed with a hair follicle preservation solution; the hair follicle washout solution was DMEM/f12+10% fbs with 1% penicillin/streptomycin.

The beneficial effects are that:

the method for improving the expression level of the Sox2 of the DP cells is beneficial to improving the separation efficiency of the primary DP cells of the hair follicle and shortening the separation time; the method has the advantages that the generation cells obtained by the adherent culture of the primary DP cells obtained by the separation method are higher in Sox2 expression level compared with the generation cells obtained by the generation of the DP cells obtained by the separation of microdissection method; the method of the application for increasing the Sox2 expression level of DP cells helps to avoid or reduce the reduction or even loss of hair-inducing properties of DP tissue.

Compared with the DP cells separated by adopting a microdissection method, the DP cells separated by the method for improving the expression level of the Sox2 of the DP cells are beneficial to shortening the cell climbing-out period and improving the cell climbing-out rate and the harvesting quantity.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. Wherein:

FIG. 1 is a chart showing the generation of cells after the enzymatic separation and attachment of primary hair follicle papilla tissue provided in example 1 of the present application; wherein, (a) a hair follicle sample graph is extracted for a hair follicle extractor, and the whole hair follicle structure graph is cut in three sections; (b) The liquid drops are in a state when the hair follicle sample tissue is stripped under a split microscope; (c) DP tissue harvested after 1/3 of the lower end of hair follicle tissue is subjected to enzymolysis under an integral microscope; (d) A cell morphology diagram of 5 th day is obtained by inverting a microscope after the purified hair papilla grows in an adherence manner after enzymolysis; (e) A cell morphology diagram of the hair papilla cells under an inverted microscope before P0 generation of harvest;

FIG. 2 is a graph of the generation of cells after cutting and separating hair follicle primary papilla tissue and attaching wall provided in comparative example 1 of the present application; wherein, (a) is a schematic diagram of fixing and attaching the purified hair papilla in a culture dish after cutting and stripping under a split microscope; (b) Cutting under a split microscope, peeling off the purified hair papilla, growing the hair papilla by attaching to the wall, and then inverting the cell morphology map of the cells on the 5 th day under the microscope;

FIG. 3 is a graph showing the comparison of the loosening and falling of the outer root sheath wrapping DP after the 1/3 structure at the lower end of the hair follicle provided in example 2 is subjected to enzymolysis with collagenase of different concentrations for different times, and the exposure degree of the DP tissue structure; wherein the concentration of collagenase A is 1mg/mL, 3mg/mL and 5mg/mL respectively, and the enzymolysis time is 20min, 40min and 60min respectively;

FIG. 4 is an immunofluorescence comparison chart of P2 generation secondary cells obtained after the enzymolysis separation and passage of the hair follicle primary papilla tissue provided by the experimental example 1 of the application and P2 generation cells obtained after the DP passage of the fixed adherence culture after the microdissection under the integrated microscope of the comparative example 1; among the markers detected are Sox2, LEF1, α -SMA, ALP.

Detailed Description

The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the application, fall within the scope of protection of the application.

The present application will be described in detail with reference to examples. It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other.

Aiming at the problem of low hair follicle induction characteristic of DP cells separated by adopting a microdissection method at present, the application provides a method for improving the expression level of Sox2 of the DP cells, which comprises the following steps of: s01: cleaning a hair follicle sample; s02: after removing at least part of fat and connective tissue, segmenting the rest hair follicle sample, and collecting the part containing DP cells to obtain DP cell segment tissue; s03: adding enzymolysis liquid into the collected DP cell segment tissue for enzymolysis to expose DP cells; s04: stopping enzymolysis, repeatedly blowing to drop DP cells from DP cell segment tissue (DP cells are suspended in the upper suspension, and collecting the upper suspension), and centrifuging (centrifuging the obtained upper suspension); s05: performing solid-liquid separation to obtain DP cells, and cleaning the obtained DP cells; in step S03, the components of the enzymolysis liquid comprise collagenase A, dispase II and calcium salt.

In step S03, the end point of enzymolysis is: under a stereoscopic microscope, the dermis sheath is basically completely hydrolyzed, but the hair papilla is not hydrolyzed. In the enzymolysis process, the bottom of the tube is flicked every 20min, and the mixture is gently mixed.

In step S04, after the separation to obtain the upper suspension, a step of adding a medium (e.g., DMEM/F12 medium containing 10% fbs) to the lower residue to wash and blow the lower residue, and collecting the upper suspension after the washing and blowing are completed. Preferably, the step of cleaning the blow is 5 times.

By isolating the DP cells according to the method comprising the above steps, the expression level of Sox2 of the DP cells can be increased, and the DP cells can have better hair follicle inducing properties. In addition, the method for improving the expression level of the DP cell Sox2 is beneficial to improving the separation efficiency of primary DP cells of hair follicles, shortening the separation time, shortening the climbing cycle of the DP cells and improving the climbing rate and the cell harvesting rate of the cells.

In a preferred embodiment of the application, the concentration of collagenase A in the enzymatic hydrolysate is 1-5mg/mL (e.g., 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL or 5 mg/mL), the concentration of Dispase II is 2-4mg/mL (e.g., 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL or 4 mg/mL), and the concentration of calcium salt is 2-6mM (e.g., 2mM, 3mM, 4mM, 5mM or 6 mM). Wherein, the addition of calcium salt is helpful to improve the enzymolysis efficiency of the tissue of the hair papilla section, shorten the enzymolysis time, and also has the functions of maintaining the cell integrity and controlling the permeability of the membrane. According to the application, collagenase A is applied to enzymolysis of the papilla section tissues, so that the cell harvest amount is increased along with the prolongation of the enzymolysis time under a lower concentration.

In a preferred embodiment of the application, the concentration of collagenase A in the enzymatic hydrolysate is 2-4mg/mL (e.g., 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL or 4 mg/mL).

In the preferred embodiment of the application, the concentration of collagenase A is 3mg/mL, and the enzymolysis time is 40min.

In a preferred embodiment of the application, the calcium salt is calcium chloride, and the enzymatic hydrolysate further comprises HBSS (i.e., hank's balanced salt solution).

In the preferred embodiment of the present application, in step S03, the enzymolysis is performed on a constant temperature shaking table; the enzymolysis temperature is 37deg.C, and the enzymolysis time is 20-120min (e.g. 20min, 50min, 80min, 100min or 120 min); the rotation speed of the thermostatic shaker is 100-200rpm (e.g., 100rpm, 130rpm, 150rpm, 180rpm or 200 rpm).

In a preferred embodiment of the application, in step S02, the DP cell segment tissue is the lower 1/3-1/2 (e.g., 1/3, 2/5, 3/7, 3/8 or 1/2) portion of the hair follicle sample that remains after removal of at least a portion of the fat and connective tissue. If the segment is less than 1/3, damage may be caused to the DP portion of the hair follicle by manipulation of the dissection tool. If the fragment is larger than 1/2, there may be a part of stem cells remaining in the bulk region, and the remaining cells express Sox2 protein, thereby affecting the determination of DP cells by Sox2 expression.

In a preferred embodiment of the application, in step S04, the centrifugal force is 180-250g (e.g. 180g, 200g, 220g or 250 g) and the centrifugation time is 2-5min (e.g. 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5 min), which step is low, only the DP cell pellet is centrifuged while other digested single cells are prevented from being centrifuged.

In the preferred embodiment of the application, the cleaning is to add culture medium again to the obtained DP cells after discarding the supernatant, repeatedly blow, and then centrifuge to remove the supernatant;

preferably, the number of washes is 3 to aid in removal of digested foreign cells.

In a preferred embodiment of the present application, in step S01, a hair follicle preserving fluid is used to wash a hair follicle sample; the hair follicle washings were DMEM/F12 medium with 1% penicillin/streptomycin and 10% fbs.

In a preferred embodiment of the present application, the hair follicle sample is a hair follicle sample containing a hair follicle skin sample or extracted by a hair follicle extractor.

In a preferred embodiment of the present application, the method further comprises the steps of: s06: inoculating DP cells obtained by the treatment of step S05, and inoculating DP cells with 5% CO at 37deg.C ₂ Culturing in an incubator, changing the liquid when culturing to the 5 th day, and changing the culture medium every 3 days; s07: after the primary tissue of the DP cells is attached, the cells are harvested when the confluence degree reaches 80% -95%, the supernatant is discarded, and the cells are digested and cleaned to obtain P1 generation cells; s09: and (3) subculturing the P1 generation cells, and in the subculture process, carrying out passage when the cell growth confluence reaches 80% -95%, until the DP cells of required generation times are obtained.

The method for increasing Sox2 expression level in DP cells according to the present application will be described in detail by way of specific examples.

EXAMPLE 1 isolation of human hair follicle papilla tissue structures

The method for increasing the expression level of Sox2 in DP cells of this example adopts a method comprising the steps of:

(1) Hair follicle tissue sample tissue obtained from a hospital comprises intact hair follicle tissue and surrounding connective tissue: the skin tissue is required to be intact and structurally distinct (see fig. 1 (a)).

(2) Collecting, preserving and transporting hair follicle tissues: hair follicle tissue collected by a hair-planting medical institution is filled into a 15mL centrifuge tube containing 13mL of hair follicle preservation solution (DMEM/F12+10% FBS of 1% penicillin/streptomycin) and is placed in a low-temperature preservation box (2-8 ℃) for transportation;

(3) Tissue cleaning: the hair follicle tissue sample in the sterile stock solution is poured into a 70 μm sieve, and the tissue is washed 3-5 times with a washing solution.

(4) Extraction of intact hair follicles: hair follicle tissue was transferred to a new 10cm dish cover drop (DMEM/f12+10% fbs medium with 1% penicillin/streptomycin) and connective tissue surrounding the hair follicle tissue was carefully isolated under a stereoscopic microscope using microscopic forceps (see fig. 1 (b)). The excised intact hair follicle tissue was placed in 10cm dishes containing 10mL of 1% penicillin/streptomycin DMEM/F12+10% FBS medium.

(5) Enzymolysis of the 1/3 part structure of the lower end of the hair follicle: the removed hair follicle tissue was transferred to a new 10cm dish cap drop (DMEM/F12+10% FBS medium containing 1% penicillin/streptomycin), the lower third of the hair follicle (containing hair papilla) was cut off and retained under a stereoscopic microscope using a pair of micropeels and Venus shears, the lower third of the hair follicle tissue was carefully placed at the bottom of a 1.5mL EP tube, the medium carried by the hair follicle tissue was aspirated as much as possible using a pipette gun, and 1.5mL of enzymatic hydrolysate (the components of the enzymatic hydrolysate were: collagenase A1mg/mL, dispase II 1mg/mL, caCl) was added to the 1.5mL EP tube ₂ 5mM and HBSS mixed solution; the reagent volumes were formulated as the case may be). A shaking table is maintained at 37deg.C and 200rpm/min for 40min (under the microscope, the dermis sheath is substantially completely hydrolyzed while the hair papilla is not yet hydrolyzed). Carefully flick the bottom of the tube every 20min during digestion, mix gently.

(6) After enzymolysis, the dermis sheath is basically completely hydrolyzed under the microscope, and the hair papilla is not hydrolyzed yet, after digestion of the culture medium (DMEM/F12+10% FBS culture medium) is stopped, the hair papilla which is not hydrolyzed is gently blown by a pipette, and the hair papilla and the hair shaft which are not hydrolyzed are completely separated, and the upper suspension is collected. This step was repeated 5 times (washing and blowing; specifically, washing and blowing means that fresh medium was added to the lower sample after the upper suspension was collected, gentle blowing was performed with a pipetting chamber to promote complete separation of the hair papilla from the hair shaft, and the upper suspension was collected after the blowing was completed), and all the upper suspensions were collected into the same 15mL centrifuge tube. Centrifuging at 180r/min for 5min at low speed, discarding supernatant, and collecting precipitate.

This washing step was repeated 5 times, and the precipitate was a purified hair papilla (see fig. 1 (c)).

(7) Inoculating hair papilla at proper density at 37deg.C with 5% CO ₂ Culturing under the environment, standing for 5 days, observing photographing and changing liquid (see fig. 1 (e)), changing liquid 2 times per week, and subculturing under the condition of incomplete fusion.

(8) After culturing for 7-10 days (see FIG. 1 (d)), the culture medium was discarded after observing that the cell density in 6well could reach 80% or more, PBS was washed 1 time, 0.5mL of TrypLE (GIBCO, cat# 12604013; 1X concentration) was added to the bottom of the six-well plate, and then the mixture was allowed to digest for 3 minutes at room temperature, 1mL of the culture medium (DMEM/F12+10% FBS medium) was added to the dish using a 1mL pipette to terminate the digestion, the supernatant was taken out and placed into a 15mL centrifuge tube, and then the bottom of the bottle was washed 1 time with 1mL of the culture medium (DMEM/F12+10% FBS medium), and the washing liquid was added to the centrifuge tube. The supernatant was centrifuged at 400g in a centrifuge for 5min, the supernatant was discarded, 1mL of medium (DMEM/F12+10% FBS medium) was added for resuspension, and the counted inoculations were transferred to P1 generation cells.

And (3) performing enzymolysis test on the complete structure of the hair follicle by adopting an enzymolysis liquid before the step (5):

the complete structure of hair follicle obtained in step (4) of example 1 was subjected to enzymatic hydrolysis, and collagenase required for enzymatic hydrolysis was: the final concentration of collagenase A in the enzymolysis liquid is respectively prepared to be 1mg/mL (the enzymolysis liquid also comprises 1mg/mL of Dispase II and CaCl) ₂ 5mM and HBSS mixed solution), and respectively adding into a 1.5mL EP tube with the same number of hair follicles in complete structure, carrying out enzymolysis at 37 ℃ by a constant-temperature shaking table at 100rpm for 2h and 3h, and measuring the cell activity and the number of living cells of the enzymolysis supernatant after the enzymolysis is finished.

Meanwhile, collagenase A in the enzymolysis liquid is replaced by collagenase I, collagenase II and collagenase IV with equal concentration (other components in the enzymolysis liquid are unchanged), and the enzymolysis effect of the collagenase A is verified by comparison.

The complete hair follicle structure obtained by shearing in the step (4) in the example 1 is respectively placed into 8 EP pipes of 1.5mL according to the same quantity, four groups are respectively placed into the EP pipes of each group, the concentrations of the prepared collagenase I, collagenase II, collagenase IV and collagenase A are respectively 1mg/mL, each pipe of the prepared collagenase I, collagenase II, collagenase IV and collagenase A is 1.5mL, the pipes are placed into a constant temperature shaking table at 37 ℃ for enzymolysis at 100rpm for 2h and 3h, and the cell viability and the living cell number of the enzymolysis supernatant are measured after the enzymolysis is finished, so that the measurement results are shown in the following table 1.

TABLE 1 harvesting of primary cells after complete structure of hair follicle by collagenase enzyme of different types

As shown in Table 1, the complete structure of hair follicle is hydrolyzed by a shaking table at a constant temperature of 37 ℃ and 100rpm, the enzymolysis effect of collagenase A is best in the enzymolysis process, and the number of the obtained living cells is gradually increased along with the lengthening of the enzymolysis time, so that the enzymolysis process is a progressive enzymolysis process.

Example 2

The optimal selection of collagenase A contained in the enzymatic hydrolysate in the enzymatic hydrolysis of the hair follicle in example 1 is that of collagenase A, so that the enzymatic hydrolysis liquid in the structure of the lower end 1/3 of the hair follicle obtained in step (5) in example 1 is also selected, the concentration of collagenase A is increased, the enzymatic hydrolysis time is shortened, namely, the concentration of collagenase A is 1mg/mL-5mg/mL, and the enzymatic hydrolysis time is 20min-60min.

The lower 1/3 structure of hair follicle obtained in experimental example 1 is put into three 1.5mL EP pipes according to the same quantity, collagenase A with the concentration of 1mg/mL, 3mg/mL and 5mg/mL is respectively added into the 1.5mL EP pipes, enzymolysis is carried out by a constant temperature shaking table with the volume of 1.5mL and the temperature of 37 ℃ and the speed of 100rpm, the enzymolysis time is 20min, 30min and 60min, and tissues are placed under a split microscope to be observed and photographed every time when one enzymolysis time point is reached, and the loosening and falling of the outer hair root sheath wrapping DP and the exposure degree of the DP tissue structure are observed.

As shown in the result of FIG. 3, the concentration of collagen A in the enzymolysis liquid is 3mg/mL when the structure of the lower end of the hair follicle is subjected to enzymolysis, and the enzymolysis time is 40min, so that the best effect is achieved, wherein the outer hair root sheath wrapping DP basically falls off after repeated blowing and sucking by the gun head, and DP tissues are exposed and left in enzymolysis supernatant.

Example 3

The DP tissue structure obtained after enzymolysis and centrifugation in the step (6) of the experimental example 1 is subjected to adherence culture, and the method comprises the following steps:

s01: the human hair papilla DP tissue collected in example 1 was resuspended in medium, inoculated at a DP number of about 80 per T25 flask, supplemented with appropriate medium, and placed at 37℃with 5% CO ₂ In the incubator, liquid change and photographing were performed by the time of culturing to the 5 th day, after which the medium was changed every three days.

S02: cells were harvested when the confluence of cell growth reached 80-95% after attachment of DP primary tissue, the supernatant from the flask was discarded, the flask was washed once with 5mL PBS, 1mL TrypLE digestive enzyme was added to the flask for 3 minutes at room temperature, 3mL of TrypLE digestive enzyme was added after digestion was completed, the digestion was stopped by adding 3mL of LPBS, the supernatant after digestion was collected in a 15mL centrifuge tube, the flask was washed once with 3mL PBS and the supernatant was collected in the same 15mL centrifuge tube, centrifuged at 1500rpm for 5 minutes, and counted (see Table 2). Passage to generation P1. The P1 generation cell growth confluence reaches 80% -95% and can be passed.

S03: and collecting cells when the cell growth confluence reaches 80% -95%, and repeating the step S02 until the required generation number is obtained through subculture.

Amplification data were collected for DP primary cell growth (see table 2 below)

TABLE 2 harvesting of primary cells after enzymatic hydrolysis of papilla

Comparative example 1

The comparative example provides a method for conventional isolation and culture of hair follicle papilla tissue, comprising the following steps: (A) the same as in (1) in Experimental example 1.

(B) Hair follicle papilla separation: the hair follicle tissue is washed 3 times by PBS containing gentamycin, the hair follicle tissue structure in the hair follicle sample is clear and complete, the hair follicle tissue structure comprises sebaceous glands, connective tissues, external root sheaths, hair shafts and the like, 5 drops of DMEM/F12 culture medium (about 150 mu L per drop) are added into a 100mm culture dish, 2-3 hair follicles are placed in each drop, the hair follicle bulb is cut off by a 1mL syringe, the dermal sheath outside the hair follicle bulb is fixed by a syringe needle in the left hand, the hair follicle bulb is turned over by a syringe needle in the middle of the hair follicle bulb, the hair papilla part is cut off along a depression, the hair papilla is fixed on the 100mm culture dish by a needle (see fig. 2 (a)), and the DP hair papilla obtained after the hair follicle sample tissue is peeled off under a microscope is shown in the figure; black arrow indicates hair papilla, takes the structure of flesh color heart-like), cover the petri dish lid after finishing fixing, place in the superclean bench for 3-5min, until hair papilla and orifice plate are laminated more. 2mL of medium (DMEM/F12 medium, preventing the hair papilla from blowing up) was slowly replenished along the well plate wall. The cell names and dates were labeled, and the cells were cultured in a 5% CO2 cell incubator at 37℃for 24 hours, followed by addition of 3mL of the corresponding medium (DMEM/F12 medium). And then, the culture is carried out for four days without liquid supplementing or moving. The presence or absence of cell climbing out around the hair papilla was observed, and if there was cell climbing out (see fig. 2 (b), after the hair papilla was attached, cultured cells climbed out on day 8), the liquid was changed every 3 days.

(C) Primary harvesting of hair follicle papilla cells: amplification data of cell growth after DP primary adherence was collected as in step (8) of example 1.

(D) Subculturing to obtain P2 generation cells.

Amplification data were collected for DP primary cell growth (see table 3 below)

TABLE 3 harvesting of Primary cells after adhesion of papilla by microdissection

Sample numbering	Total number of hair follicles	Primary culture specification	Days of culture	Harvesting cell mass
					20210800101	10	6well×1	14	1.18E+05
20210900202	16	6well×1	23	7.85E+04
					20210900303	18	6well×1	21	5.67E+04
20211000303	20	6well×1	24	8.14E+04
					20211000404	15	6well×1	27	8.34E+04
20211100505	15	6well×1	22	6.32E+04
					20211100707	25	6well×1	16	9.73E+04
20211200909	55	6well×2	18	2.00E+05
					20211201010	55	6well×3	21	2.55E+05

From a combination of the experimental data in tables 2 and 3, it can be seen that: the amount of primary cells obtained from hair papilla by the enzymolysis method of the present application (example 3) was higher than that obtained after microdissection (comparative example 1) of the adherent primary cells.

Experimental example

1. Immunofluorescence assay hair papilla cell marker obtained by two different methods of example 1 and comparative example 1 and induction of hair gene expression.

The marker expression of P2 generation cells obtained by subculture of example 1 (DP purified after enzymatic digestion) and P2 generation human hair papilla cells obtained by subculture of comparative example 1 (DP cultured by fixed adherence after microdissection under a split microscope) were examined by immunofluorescence technique.

Immunofluorescence detection method: after the P2 generation secondary cells which are conventionally cultured in 2D are directly abandoned from culture supernatant, PBS is used for cleaning once, 4% paraformaldehyde is added for fixing at room temperature for 10min, and the dyeing step is carried out after 3 times of cleaning.

Cells were first blocked with blocking solution containing 5% BSA and 0.3% triton at room temperature for 1h, after dilution of the antibody at 1:200, primary antibody was incubated overnight at 4℃and washed 3 times with PBS, after 1:2000 dilution of secondary antibody was added and incubated at room temperature for 1h, washed 3 times with PBS, and then nuclei were stained with appropriate amount of DAPI for 5min, and photographed under observation under a fluorescent microscope.

The detection results are shown in FIG. 4.

The results show that: the Sox2 expression of P2 generation cells obtained by subculture in example 1 of the present application (DP purified after enzymatic digestion, corresponding to "digested+adherent" sample in fig. 4) is significantly higher than that of P2 generation cells obtained by subculture in comparative example 1 (DP cultured after fixed adherent after microdissection under an integrated microscope, corresponding to "microdissection+adherent" sample in fig. 4); there was no significant difference in expression of LEF1, α -SMA, ALP.

It was demonstrated that the hair follicle induction characteristics of the P2 generation sub-human hair papilla cells obtained by subculture of example 1 (DP purified after enzymatic digestion) were higher than those of the P2 generation cells obtained by subculture of comparative example 1 (DP cultured by fixed adherence after microdissection under a split microscope).

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (9)

1. A method for increasing Sox2 expression levels in DP cells, comprising isolating DP cells by a method comprising the steps of:

s01: cleaning a hair follicle sample;

s02: after removing at least part of fat and connective tissue, segmenting the rest hair follicle sample, and collecting a part containing DP cells to obtain DP cell segment tissue;

s03: adding enzymolysis liquid into the collected DP cell segment tissue for enzymolysis, so that DP cells are exposed;

s04: stopping enzymolysis, repeatedly blowing to drop DP cells from the DP cell segment tissue, and centrifuging;

s05: solid-liquid separation is carried out to obtain the DP cells, and the obtained DP cells are cleaned;

in step S03, the components of the enzymatic hydrolysate include collagenase A, dispaseII and calcium salt.

2. The method for increasing Sox2 expression level of DP cells according to claim 1, wherein the concentration of collagenase a in the enzymatic hydrolysate is 1-5mg/mL, the concentration of DispaseII is 2-4mg/mL, and the concentration of calcium salt is 2-6mM.

3. The method for increasing Sox2 expression level of DP cells according to claim 2, wherein the concentration of collagenase a in the enzymatic hydrolysate is 2-4mg/mL.

4. The method for increasing Sox2 expression level of DP cells according to claim 2, wherein said calcium salt is calcium chloride and said enzymatic hydrolysate further comprises HBSS.

5. The method for increasing Sox2 expression level of DP cells according to claim 1, wherein in step S03, the enzymatic hydrolysis is performed on a constant temperature shaker;

the enzymolysis temperature is 37 ℃ and the enzymolysis time is 20-120min;

the rotating speed of the constant temperature shaking table is 100-200rpm.

6. The method of claim 1, wherein in step S02, the DP cell stage tissue is the lower 1/3-1/2 portion of the hair follicle sample remaining after removal of at least a portion of fat and connective tissue.

7. The method for increasing the Sox2 expression level of DP cells according to claim 1, wherein in step S04, the centrifugal force is 180-250g and the centrifugal time is 2-5min.

8. The method for increasing Sox2 expression level of DP cells according to claim 1, wherein said washing is to add the culture medium again to the obtained papilla cells after discarding the supernatant, repeatedly blow, and then centrifuge to remove the supernatant.

9. The method for increasing expression level of Sox2 in DP cells according to claim 1, wherein in step S01, the hair follicle sample is washed with a hair follicle preservation solution;

the hair follicle washout solution was DMEM/f12+10% fbs with 1% penicillin/streptomycin.