Method for separating keratinocytes derived from human hair follicles in vitro and application of keratinocytes

Abstract

The invention relates to a method for separating keratinocytes from human hair follicles in vitro and application of the keratinocytes. The method comprises the following steps: 1) separating hair follicle tissues from an in-vitro human hair sample; (2) pretreating the hair follicle tissues; and (3) culturing the pretreated hair follicle tissue obtained in the step (2) by adopting a keratinocyte culture medium to obtain the keratinocytes derived from human hair follicles. The invention also provides an application of the keratinocytes derived from human hair follicles in preparation of products for skin inflammation and / or injury recovery and skin regeneration, and provides data support in the aspects of skin physiology, pathology, pharmacology, pharmacotoxicology, tissue engineering, gene therapy and the like. According to the method, the primary separation of the keratinocytes is carried out by taking the hair follicle tissue as a source, the purity of the cells is effectively improved through pretreatment, the operation difficulty of obtaining the pure keratinocytes through separation is reduced, and more feasibility is provided for preparing skin inflammation and / or injury recovery products.

Description

Technical Field

[0001] The present invention relates to the field of medical supplies, and in particular to a method for isolating keratinocytes derived from human hair follicles in vitro, and application of keratinocytes derived from human hair follicles in preparing products for recovery from skin inflammation and / or damage. Background Art

[0002] The skin is mainly composed of two layers, the epidermis and dermis, which are differentiated from the ectoderm and mesoderm during embryonic development. Keratinocytes are derived from the ectoderm and are mainly located in the basal layer of the epidermis. They have strong growth, proliferation and differentiation capabilities. The epidermal cells of the human body are composed of keratinocytes, which produce many large molecular weight molecules (keratin and mucopolysaccharides) to maintain the biological integrity of the skin. The cells that play a leading role in the wound healing process are keratinocytes in the epidermal cells. Therefore, the key to wound healing depends largely on the function of keratinocytes.

[0003] Keratinocytes cultured in vitro have the ability to proliferate and repair, and can be transplanted to burn wounds for repair. Burn patients often face limited donor skin areas when repairing wounds. It is particularly important to cultivate more cells in the smallest donor skin area for wound repair. At present, there have been many reports of successful clinical applications of skin tissue engineering products, which have become a successful example of the clinical application of tissue engineering products. Scholars from various countries have conducted extensive and in-depth research on the isolation, culture and clinical application of keratinocytes. However, in clinical sampling, skin tissue sampling is more precious and difficult.

[0004] Different methods for separating cells from different people are different. In the prior art, most samples of keratinocytes are separated from animal or human skin tissue. When separating human keratinocytes using the method of separating animal keratinocytes, the cells grow slowly and are not easy to adhere to the wall, so they cannot be mass-produced, and some of the reagents used are not suitable for clinical research. At the same time, for the separation of human keratinocytes, the tissues used are all exposed skin tissues, which makes sampling difficult. The risk of skin inflammation or complications after sampling is high, and the recovery period is long. At the same time, human epidermal keratinocytes play a vital role in wound healing, especially the lack of epidermal keratinocytes in patients with severe burns is closely related to the length of the patient's disease course, the occurrence of complications, and the formation of late scars. Therefore, the application of tissue engineering technology to separate, culture and preserve epidermal keratinocytes in vitro and apply them to clinical practice has always been a research hotspot for burn workers.

[0005] Therefore, obtaining keratinocytes from other tissues has become a research direction. Summary of the invention

[0006] Technical issues

[0007] In view of this, the technical problem to be solved by the present invention is how to provide a method for isolating keratinocytes derived from human hair follicles in vitro, and the use of keratinocytes derived from human hair follicles in the preparation of skin inflammation and / or damage recovery products.

[0008] The present invention uses hair follicle tissue as a source to perform primary separation of keratinocytes, effectively improves the purity of cells through pretreatment, reduces the difficulty of separating and obtaining pure keratinocytes, and provides more feasibility for preparing skin inflammation and / or damage recovery products.

[0009] Solution

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a method for isolating keratinocytes derived from human hair follicles in vitro, comprising the following steps:

[0012] 1) Isolate hair follicle tissue from ex vivo human hair samples;

[0013] 2) pre-treating the hair follicle tissue;

[0014] 3) Cultivating the pretreated hair follicle tissue obtained in step 2) using a keratinocyte selection medium to obtain human hair follicle-derived keratinocytes, wherein the keratinocyte selection medium is used for selectively culturing keratinocytes.

[0015] Further, in step 2), the pretreatment method includes: removing the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue, and retaining the middle section as the hair follicle middle section tissue. Optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm.

[0016] Furthermore, in step 2), the pretreatment method includes: removing connective adhesion tissue and / or fat tissue on the hair follicle tissue.

[0017] Furthermore, the method of removing tissue is performed under a stereoscope.

[0018] Furthermore, in step 2), the pretreatment method includes: hydrolyzing the hair follicle tissue with a digestive enzyme, wherein the digestive enzyme is collagenase IV.

[0019] Furthermore, the working concentration of the digestive enzyme is 1-10 mg / mL; optionally, the enzymolysis time is 20-120 min; optionally, the enzymolysis time is 25-35 min; optionally, the enzymolysis time is 30-35 min; optionally, the enzymolysis temperature is 35-38°C; optionally, the enzymolysis temperature is 35-37.5°C.

[0020] Furthermore, in step 1), the hair follicle tissue is a hair follicle unit tissue and / or a single hair follicle tissue;

[0021] Furthermore, in step 2), the hair follicle tissue is a single hair follicle tissue, and optionally, the single hair follicle tissue is separated from a hair follicle unit.

[0022] If the hair follicle tissue in step 1) is a follicular unit tissue, then in the pretreatment of step 2), the follicular unit tissue can be directly pretreated, or separated into single follicular tissues for pretreatment, or the follicular unit tissue can be pretreated and then separated into single follicular tissues.

[0023] In step 2), there are three feasible preprocessing methods, namely:

[0024] Pretreatment method 1: After removing the connective adhesion tissue and fat tissue on the hair follicle tissue, remove the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue, and retain the middle section as the hair follicle middle section tissue. Optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm.

[0025] Pretreatment method 2: After removing the connective adhesion tissue and adipose tissue on the hair follicle tissue, the hair follicle tissue is enzymatically hydrolyzed with collagenase IV.

[0026] Pretreatment method 3: Pretreatment methods include:

[0027] 1) Remove the connective adhesion tissue and fat tissue on the hair follicle tissue;

[0028] 2) removing the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue of step 1), and retaining the middle section as the hair follicle middle section tissue. Optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm;

[0029] 3) the hair follicle midsection tissue of step 2) is enzymatically digested with digestive enzymes;

[0030] Optionally, in step 2), the hair follicle tissue is a single hair follicle tissue. Optionally, the single hair follicle tissue is separated from a hair follicle unit.

[0031] Furthermore, the ex vivo human hair sample is preserved with a storage solution; optionally, the storage solution is lactic acid Ringer's solution; optionally, the storage temperature is 0-15°C; optionally, the storage temperature is 0.5-15°C; optionally, the ex vivo human hair sample is not more than 48 hours after being separated from the body; optionally, the ex vivo human hair sample is stored in a constant temperature transport box.

[0032] Furthermore, before step 1), aseptic treatment and / or aseptic monitoring are also included. Optionally, the aseptic treatment includes: washing with 75% ethanol and / or DPBS / PBS solution containing double antibodies. Optionally, the volume fraction of the double antibodies in the DPBS / PBS solution containing double antibodies is 1-3% (v / v). Optionally, washing with DPBS / PBS solution containing 1-2% (v / v) double antibodies is performed at least 3 times; optionally, aseptic monitoring includes sterility testing before and after sterilization.

[0033] In the second aspect, a method is provided for using keratinocytes derived from human hair follicles in the preparation of skin inflammation and / or damage recovery, or skin regeneration products, and data support is provided in terms of skin physiology, pathology, pharmacology, drug toxicology, tissue engineering and gene therapy.

[0034] Furthermore, the keratinocytes are isolated and cultured from in vitro human hair samples.

[0035] Furthermore, the keratinocytes are obtained by the method described in the first aspect.

[0036] The present invention is very different from the traditional keratinocyte separation method in that the tissue used in the present invention is hair follicle tissue material to separate and obtain keratinocytes, which is more convenient, faster and less damaging than separating skin tissue.

[0037] Studies have found that hair follicle-derived keratinocytes are mainly located in the myelin sheath at the posterior edge of the follicle, but due to the difficulty of experimental operation and separation, they have not received widespread attention. The reason is that hair follicle-derived keratinocytes take a long time to obtain under traditional separation conditions, and are not easy to separate, with a high probability of failure. Hair follicle-derived keratinocytes will only grow slowly after being cultured in vitro for more than 20-30 days. Due to the long time, the application research of hair follicle-derived keratinocytes is limited. In addition, the purity and purity of keratinocytes obtained by traditional separation process are not high, which is also one of the difficulties in separating hair follicle-derived keratinocytes. In addition, experimental operations performed within 12 hours of tissue ex vivo are the basic principles of primary cell separation, which also have great restrictions on cell separation; and improper handling of primary cells, resulting in cell contamination, is also an important limiting factor for primary separation.

[0038] The present invention finds a new method for quickly obtaining keratinocytes derived from hair follicles by comparing and exploring all the methods.

[0039] The human hair follicle-derived keratinocyte separation conditions established by the present invention provide more experimental materials of hair follicle-derived keratinocytes, which is of great significance for conducting skin damage research.

[0040] Beneficial Effects

[0041] (1) The present invention uses hair follicle tissue as a source to separate primary keratinocytes. The present invention uses micromanipulation to perform targeted segmentation, removes the hair bulb tissue and the hair shaft tissue, retains the middle section containing keratinocytes as the hair follicle middle section tissue, and combines enzymatic hydrolysis for separation, thereby overcoming the problems of the prior art in which separation of hair follicle tissue in vitro to obtain hair follicle-derived keratinocytes takes a long time and is difficult to separate. At the same time, this experiment is also the first to operate and culture separate hair follicle / hair bulb / hair papilla tissue, which effectively improves the purity of the cells and reduces the difficulty of isolating and obtaining pure keratinocytes.

[0042] (2) The present invention attempts for the first time to use a constant temperature transport box and storage liquid to transport and store hair follicle tissue. After 24 hours of transportation, keratinocytes can still be separated normally, which prolongs the storage time and solves the problem that the tissue can only be separated immediately or within 12 hours of ex vivo separation, effectively solving the problem of the time limit for tissue ex vivo separation. In addition, aseptic separation and detection itself is also an important experimental environment. Through sufficient washing, processing and detection means, while minimizing damage to the tissue, the aseptic operation of the experiment is quickly guaranteed, and the easy contamination of primary separation is monitored and solved. This research is of great significance for the characteristic cultivation of hair follicle-derived keratinocytes and research in the field of biomedicine.

[0043] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, and to make the above and other purposes, technical features and advantages of the present invention easier to understand, one or more preferred embodiments are listed below and described in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0045] Figure 1 The figure is a real-time monitoring result of temperature control of an embodiment of a constant temperature transport box during transportation of the present invention.

[0046] Figure 2 The figures are the results of the sterility detection experiment before and after the sterile treatment of the present invention, wherein Figure A is the plate control experiment result of liquid A before treatment and the control group; Figure B is the plate experiment comparison result of liquid B after treatment and the control group, and treatment 1 and treatment 2 in Figure B are 2 repetitions of liquid B respectively; the control is a blank SA plate solid culture medium.

[0047] Figure 3 The following are examples of unit hair follicles and single hair follicles, where A is a hair follicle unit and B is a hair follicle tissue.

[0048] Figure 4 This is a schematic diagram of a single hair follicle tissue under a microscope in Example 4.

[0049] Figure 5 This is an example diagram of a single hair follicle tissue under micromanipulation of the hair follicle site in Example 4.

[0050] Figure 6 This is the cell crawling situation of Example 24 (micromanipulation group 4 + digestive enzyme group 4), where A and B are the shooting results of the same sample at different angles.

[0051] Figure 7 This is the identification result of the cell contamination situation of Test Example 1, wherein A is the control group and B is Example 24.

[0052] Figure 8 Examples of cells crawling out of unit hair follicles and single hair follicles, where A represents cells isolated and cultured from the mid-segment tissue of a hair follicle unit, and B represents cells isolated and cultured from the mid-segment tissue of a single hair follicle.

[0053] Fig. 9 This is the immunofluorescence identification result of Test Example 2.

[0054] Fig.10 This is an example diagram of keratinocytes repairing skin damage in Test Example 3. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0056] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0059] In the following example, 1% (V / V) dual-antibody DDPBS / PBS solution was prepared as follows: penicillin-streptomycin (Gibico, 15140122) was added to 1×DPBS / PBS (Gibico, 8121293), and the volume ratio was penicillin-streptomycin:DPBS / PBS=1 mL:99 mL.

[0060] In the following examples, DPBS / PBS is 1×DPBS / PBS buffer (Gibico, 8121293).

[0061] The microscope used in the following examples is a stereo microscope (Leica-SE9).

[0062] The neutralization solution in the following examples is KM culture medium purchased from (ScienCell, 362280).

[0063] In the following examples, the volume of the neutralizing solution added is twice that of the digestive enzyme to neutralize the digestion reaction.

[0064] The ex vivo human hair follicle tissues in the following examples are all from humans (with the subject's knowledge), and are hair follicle tissues from the back of the scalp.

[0065] The digestive enzyme solutions (collagenase I, collagenase II, Dispase, collagenase IV) in the following examples are all digestive enzyme aqueous solutions, which are prepared by dissolving digestive enzymes in water, and the working concentrations are all 1-10 mg / mL.

[0066] The cell culture medium in the following examples is KM medium purchased from (ScienCell, 362280).

[0067] The storage solution in the following examples is lactic acid Ringer's solution, stored at 4° C., purchased from (Shandong Jieshikang, T0447).

[0068] The constant temperature transport box (Hangzhou Duoxie, DX0B821) in the following embodiments is a system that can be used for both temperature maintenance and monitoring.

[0069] In the following examples, the method for collecting human hair samples is as follows: freshly collected ex vivo human hair samples are rinsed with lactic acid Ringer's solution and F12 culture medium (containing 1% (V / V) volume percentage of double-streptomycin), and the skin tissue sample at the back of the head is removed.

[0070] In the following embodiments, the human hair sample transportation method is as follows: the collected and processed human hair sample is immersed in a storage solution (Lactated Ringer's solution) at 4°C, and then transported in a constant temperature transport box (temperature controlled at 0-15°C), and the temperature control of the constant temperature transport box during transportation is monitored in real time. The results are as follows: Figure 1 As shown, the transportation method of the present invention can maintain the temperature of the ex vivo human hair sample at 0-15° C. during transportation, and the transportation time can reach 24 hours.

[0071] In the following examples, aseptic treatment and operation were performed before separating keratinocytes: washing with DPBS / PBS containing double antibodies in different ratios and washing with DPBS / PBS with decreasing concentrations of double antibodies was performed to minimize tissue damage and more quickly achieve the purpose of aseptic treatment and operation. In the following examples, the ex vivo human hair samples (ex vivo time no more than 48 hours) transported by the above method were subjected to the following aseptic treatment and monitoring:

[0072] 1) After taking out the human hair sample from the storage solution, the human hair sample was rinsed three times with a 75% ethanol aqueous solution, and the storage solution after taking out the ex vivo human hair sample was recorded as liquid A (before treatment);

[0073] 2) Washing the ex vivo human hair samples treated in 1) for 3 times in a DPBS / PBS solution containing 2% (V / V) double antibody;

[0074] The DPBS / PBS solution containing 2% (V / V) double-antibody is a solution obtained by mixing double-antibody penicillin-streptomycin with 1×DPBS / PBS, and the volume percentage of double-antibody penicillin-streptomycin is 2% (V / V);

[0075] 3) washing the ex vivo human hair samples treated in 2) for 3 times in a DPBS / PBS solution containing 1% (V / V) double antibody;

[0076] The DPBS / PBS solution containing 1% (V / V) double-antibody is a solution obtained by mixing double-antibody penicillin-streptomycin with 1×DPBS / PBS, and the volume percentage of double-antibody penicillin-streptomycin is 1% (V / V);

[0077] 4) placing the ex vivo human hair sample after treatment in 3) in a clean bench for treatment: washing once with sterile 1×DPBS / PBS, and keeping the residual liquid after washing with sterile 1×DPBS / PBS and marking it as liquid B (after treatment), and recording the hair tissue sample after this treatment as the human hair sample to be separated;

[0078] The liquid A (before treatment) and liquid B (after treatment) collected above were respectively subjected to sterility testing, specifically, the liquids were respectively taken for plate experiments (the culture medium was SA plate solid culture medium purchased from Huankai Microorganisms, CP0201C), and the plate conditions were observed after being placed in a 37°C incubator for 24 hours. The control was sterile 1×DPBS / PBS.

[0079] The results are as follows Figure 2 As shown, the results show that there is a significant difference in the bacterial content in the tissue sample liquid before and after treatment. After treatment, the in vitro human hair sample is sterile and can be carried out to the next step.

[0080] When collecting human hair samples, they are usually collected in follicular units. One follicular unit may contain 1 to 4 hairs with complete follicular tissue structure. Figure 3 As shown in A, a hair follicle tissue Figure 3 As shown in B.

[0081] The human hair sample to be separated obtained by the above aseptic treatment (comprising a number of aseptically treated hair follicle units) is subjected to separation of keratinocytes, some examples of which are as follows:

[0082] Example 1 Micromanipulation Group 1:

[0083] 1) Micromanipulation: An isolated hair follicle unit (human) was washed three times with 1% (V / V) double-antibody DDPBS / PBS solution and then moved to a stereoscope (Leica-SE9). Under a microscope (40X magnification), connective adhesion tissue and fat were removed. Then, the hair follicle unit was washed three times in ordinary 1×DPBS / PBS (Gibico, 8121293) solution to obtain a hair follicle unit tissue with connective adhesion tissue and fat removed.

[0084] 2) Culture: All the hair follicle unit tissues treated in step 1) above were placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0085] Example 2 Micromanipulation 2 Groups:

[0086] 1) Microsurgery: Wash an isolated hair follicle unit (human) three times with 1% (V / V) double-antibody DDPBS / PBS solution and move it under a stereoscope (Leica-SE9). Under a microscope (40X magnification), remove the connective adhesion tissue and fat, and then remove the hair bulb tissue at the front end of the hair follicle and the hair shaft tissue at the rear end ( Figure 3 , Figure 6 , Figure 8), retain the middle tissue containing keratinocytes (about 0.5-2.5mm in length), recorded as the middle tissue of the follicular unit, and then wash the middle tissue of the follicular unit three times in ordinary 1×DPBS / PBS (Gibico, 8121293) solution to obtain the middle tissue of the follicular unit with the front end hair bulb tissue and the rear end hair shaft tissue removed.

[0087] 2) Culture: The mid-section tissues of all hair follicle units treated in step 1) above were placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0088] Example 3 Micromanipulation 3 groups:

[0089] 1) is different from Example 1 in that in step 1), a single hair follicle tissue is separated from a hair follicle unit tissue after the connective adhesion tissue and fat are removed.

[0090] 2) The single hair follicle tissue treated in step 1) was placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0091] Example 4 Micromanipulation 4 groups:

[0092] 1) is different from Example 2 in that, in step 1), a single hair follicle mid-segment tissue is separated from a hair follicle unit tissue by removing connective adhesion tissue, fat, front hair bulb tissue and rear hair shaft tissue, and all the processed single hair follicle mid-segment tissues are placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO2.

[0093] Among them, the single hair follicle tissue under micromanipulation Figure 4 As shown, the micromanipulation of removing the hair bulb tissue and the hair shaft tissue and retaining the tissue in the middle section (including the convex part) is as follows Figure 5 shown.

[0094] Example 5 Digestive enzyme 1 enzymolysis group:

[0095] 1) Enzyme digestion: transfer an isolated hair follicle unit (human) to a 48-well plate and digest it with 50uL of collagenase I solution (Gibico, 2340572, 5mg / mL). After digestion in a 37°C incubator for 30min, add 100uL of neutralizing solution for neutralization reaction for 2min.

[0096] 2) Cultivation: The product after the neutralization reaction in step 1) is placed in KM culture medium (ScienCell, 362280) and cultured in a 37° C., 5% CO 2 incubator.

[0097] Example 6 Digestive Enzyme 2 Enzymatic Group:

[0098] 1) Enzyme digestion: transfer an isolated hair follicle unit (human) to a 48-well plate and digest it with 50uL of collagenase II solution (Gibico, 2240228, 5mg / mL) in a 37°C incubator for 30min, then add 100uL of neutralizing solution for 2min of neutralization reaction.

[0099] 2) Cultivation: The neutralization reaction product of step 1) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0100] Example 7 Digestive enzyme 3 enzymolysis group:

[0101] 1) Enzyme digestion: transfer an isolated hair follicle unit (human) to a 48-well plate and digest it with 50uL of Dispase enzyme solution (Hyclone, J200016, 5mg / mL) in a 37°C incubator for 30min, then add 100uL of neutralizing solution for 2min of neutralization reaction.

[0102] 2) Cultivation: The neutralization reaction product of step 1) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0103] Example 8 Digestive enzyme 4 enzymolysis group:

[0104] 1) Enzyme digestion: transfer an isolated hair follicle unit (human) to a 48-well plate and digest it with 50uL of collagenase IV solution (Gibico, 12604021, 5mg / mL) in a 37°C incubator for 30min, then add 100uL of neutralizing solution for 2min of neutralization reaction.

[0105] 2) Cultivation: The neutralization reaction product of step 1) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0106] Example 9 Micromanipulation 1 group + Digestive enzyme 1 Enzymatic hydrolysis method:

[0107] 1) Micromanipulation: treating an ex vivo hair follicle unit (human) according to the micromanipulation method of step 1) of Example 1 to obtain a hair follicle unit tissue from which connective adhesion tissue and fat are removed;

[0108] 2) Digestion with digestive enzymes: The hair follicle unit tissue separated from the connective adhesion tissue and fat removed in step 1) above was transferred to a 48-well plate and digested with 50uL of collagenase I solution (Gibico, 2340572, 5mg / mL). After digestion in a 37°C incubator for 30min, 100uL of neutralizing solution was added for neutralization reaction for 2min.

[0109] 3) Cultivation: The product after the neutralization reaction in step 2) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0110] Example 10 Micromanipulation 1 group + digestive enzyme 2 enzymatic hydrolysis method:

[0111] 1) Micromanipulation: treating an isolated hair follicle unit (human) according to the micromanipulation method of step 1) of Example 1 to obtain hair follicle tissue from which connective adhesion tissue and fat are removed;

[0112] 2) Digestion with digestive enzymes: The hair follicle unit tissue separated from the connective adhesion tissue and fat removed in step 1) above was transferred to a 48-well plate and digested with 50uL of collagenase II solution (Gibico, 2240228, 5mg / mL) in a 37°C incubator for 30min, and then 100uL of neutralizing solution was added for neutralization reaction for 2min.

[0113] 3) Cultivation: The neutralization reaction product of step 2) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0114] Example 11 Micromanipulation 1 group + digestive enzyme 3 enzymatic hydrolysis method:

[0115] 1) Micromanipulation: treating an isolated hair follicle unit (human) according to the micromanipulation method of step 1) of Example 1 to obtain hair follicle tissue from which connective adhesion tissue and fat are removed;

[0116] 2) Digestion with digestive enzymes: The hair follicle unit tissue separated from the connective adhesion tissue and fat removed in step 1) above was transferred to a 48-well plate and digested with 50uL of Dispase enzyme solution (Hyclone, J200016, 5mg / mL) in a 37°C incubator for 30min, and then 100uL of neutralizing solution was added for neutralization reaction for 2min.

[0117] 3) Cultivation: The neutralization reaction product in step 2) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0118] Example 12 Micromanipulation 1 group + digestive enzyme 4 enzymatic hydrolysis method:

[0119] 1) Micromanipulation: treating an isolated hair follicle unit (human) according to the micromanipulation method of step 1) of Example 1 to obtain hair follicle tissue from which connective adhesion tissue and fat are removed;

[0120] 2) Digestion with digestive enzymes: The hair follicle unit tissue separated from the connective adhesion tissue and fat removed in step 1) above was transferred to a 48-well plate and digested with 50uL of collagenase IV solution (Gibico, 12604021, 5mg / mL) in a 37°C incubator for 30min, and then 100uL of neutralizing solution was added for neutralization reaction for 2min.

[0121] 3) Cultivation: The neutralization reaction product in step 2) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0122] Example 13 Micromanipulation 2 groups + digestive enzyme 1 enzymatic hydrolysis method:

[0123] 1) Micromanipulation: a hair follicle unit (human) isolated from the body is processed according to the micromanipulation method of step 1) of Example 2 to obtain the middle tissue of the hair follicle unit after removing the front hair bulb tissue and the rear hair shaft tissue;

[0124] 2) Digestion with digestive enzymes: Transfer the middle tissue of all hair follicle units from step 1) above, removing the front hair bulb tissue and the rear hair shaft tissue, to a 48-well plate, and digest with 50uL of collagenase I (Gibico, 2340572, 5mg / mL). After digestion in a 37°C incubator for 30 minutes, add 100uL of neutralizing solution for a neutralization reaction for 2 minutes.

[0125] 3) Cultivation: The product after the neutralization reaction in step 2) is placed in KM culture medium (ScienCell, 362280) and cultured in a 37° C., 5% CO 2 incubator.

[0126] Example 14 Micromanipulation 2 groups + digestive enzyme 2 enzymatic hydrolysis method:

[0127] The difference from Example 13 is that the digestive enzyme in step 2) is different, collagenase I is replaced by collagenase II (Gibico, 2240228, 5 mg / mL), and the other operating methods are the same, including the operating methods of steps 1) and 3).

[0128] Example 15 Micromanipulation 2 groups + digestive enzyme 3 enzymatic hydrolysis method:

[0129] The difference from Example 13 is that the digestive enzyme in step 2) is different, collagenase I is replaced by Dispase enzyme (Hyclone, J200016, 5 mg / mL), and the other operation methods are the same.

[0130] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 13.

[0131] Example 16 Micromanipulation 2 groups + Digestive enzyme 4 enzymatic hydrolysis method:

[0132] The difference from Example 13 is that the digestive enzyme in step 2) is different, collagenase I is replaced by collagenase IV (Worthington, 42C22290, 5 mg / mL), and the other operating methods are the same.

[0133] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 13.

[0134] Example 17 Micromanipulation 3 groups + digestive enzyme 1 enzymatic hydrolysis method:

[0135] 1) Micromanipulation: a hair follicle unit (human) isolated from the body is processed according to the micromanipulation method of step 1) of Example 3 to obtain a single hair follicle tissue;

[0136] 2) Digestion with digestive enzymes: The single hair follicle tissue obtained in step 1) above was transferred to a 48-well plate and digested with 50uL of digestive enzyme-collagenase I (Gibico, 2340572, 5mg / mL). After digestion in a 37°C incubator for 30min, 100uL of neutralizing solution was added for neutralization reaction for 2min.

[0137] 3) The neutralization reaction product of step 2) was placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0138] Example 18 Micromanipulation 3 groups + digestive enzyme 2 enzymatic hydrolysis method:

[0139] The difference from Example 17 is that the digestive enzyme in step 2) is different, collagenase I is replaced by collagenase II solution (Gibico, 2240228, 5 mg / mL), and the other operating methods are the same.

[0140] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 17.

[0141] Example 19 Micromanipulation 3 groups + digestive enzyme 3 enzymatic hydrolysis method:

[0142] The difference from Example 17 is that the digestive enzyme in step 2) is different, collagenase I is replaced by Dispase enzyme solution (Hyclone, J200016, 5 mg / mL), and the other operating methods are the same.

[0143] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 17.

[0144] Example 20 Micromanipulation 3 groups + digestive enzyme 4 enzymatic hydrolysis method:

[0145] The difference from Example 17 is that the digestive enzyme in step 2) is different, collagenase I is replaced by collagenase IV (Worthington, 42C22290, 5 mg / mL), and the other operating methods are the same.

[0146] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 17.

[0147] Example 21 Micromanipulation 4 groups + digestive enzyme 1 enzymatic hydrolysis method:

[0148] 1) Micromanipulation: a hair follicle unit (human) isolated from the body is processed according to the micromanipulation method of step 1) of Example 4 to obtain a single hair follicle mid-section tissue after removing connective adhesion tissue, fat, front hair bulb tissue, and rear hair shaft tissue;

[0149] 2) Digestion with digestive enzymes: The single hair follicle midsection tissue from step 1) above was transferred to a 48-well plate and digested with 50uL of digestive enzyme-collagenase I solution (Gibico, 2340572, 5mg / mL). After digestion in a 37°C incubator for 30min, 100uL of neutralizing solution was added for a neutralization reaction for 2min.

[0150] 3) Cultivation: The product after the neutralization reaction in step 2) is placed in KM culture medium (ScienCell, 362280) and cultured in an incubator at 37° C. and 5% CO 2 .

[0151] Example 22 Micromanipulation 4 groups + digestive enzyme 2 enzymatic hydrolysis method:

[0152] The difference from Example 21 is that the digestive enzyme in step 2) is different, collagenase I is replaced by collagenase II solution (Gibico, 2240228, 5 mg / mL), and the other operating methods are the same.

[0153] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 21.

[0154] Example 23 Micromanipulation 4 groups + digestive enzyme 3 enzymatic hydrolysis method:

[0155] The difference from Example 21 is that the digestive enzyme in step 2) is different, collagenase I is replaced by Dispase enzyme solution (Hyclone, J200016, 5 mg / mL), and the other operating methods are the same.

[0156] The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 21.

[0157] Example 24 Micromanipulation 4 groups + Digestive enzyme 4 enzymatic hydrolysis method:

[0158] The difference from Example 21 is that the digestive enzyme in step 2) is different, collagenase I is replaced by collagenase IV solution (Gibico, 12604021, 5 mg / mL), and the other operation methods are the same. The operation methods of steps 1) and 3) are the same as those of steps 1) and 3) of Example 21.

[0159] Test Example 1

[0160] The operation time before culture, the crawling out time after culture, the contamination after culture, the adhesion efficiency after culture, the purity after culture, etc. of Examples 1 to 24 were tested as follows:

[0161] 1. Operation time before cell culture

[0162] The operation time before cultivation in Examples 1 to 24 was recorded, and the operation time refers to:

[0163] Micromanipulation time of step 1) in Examples 1 to 4;

[0164] The digestion time of the digestive enzyme in step 1) of Examples 5 to 8;

[0165] The micromanipulation time of step 1) and the digestion time of digestive enzymes of step 2) in Examples 9 to 24.

[0166] The results of the operation time before cell culture are shown in Table 1 , and the results show that the operation time is less than 1 h.

[0167] 2. Cell crawling out detection

[0168] The culture products of each group of Examples 1 to 24 at different times were observed under a microscope (Nikon, Ts2) to detect whether there were cells scattered and crawled out around the tissue. The time of cell crawling out of different groups was counted and placed in KM medium (ScienCell, 362280) in an incubator at 37°C and 5% CO2. From the first day of culture, it was recorded as Day 1. Whether the cells crawled out and the time of crawling out were judged according to the appearance of cells around the hair follicle tissue as a node, that is, the time when the cells crawled out with correct morphology in the tissue was counted as the time when the cells began to crawl out.

[0169] The specific results are shown in Table 1.

[0170] The results showed that the cells cultured in Examples 2, 4, 8, 16, and 24 crawled out at different times. Among them, Examples 2 and 4 (micromanipulations 2 and 4) used micromanipulation to remove connective adhesion tissue, fat, front hair bulb tissue, and rear hair shaft tissue. The remaining hair follicle unit middle tissue (or single hair follicle middle tissue) crawled out for a long time (>30 days). Example 8, which did not use micromanipulation but only used digestive enzyme-collagenase IV, also had cells crawling out, and the crawling out time was 28 days. Example 16, which used collagenase IV on the basis of micromanipulation 2, had a greatly shortened cell crawling out time (10 days). Example 24, which used collagenase IV digestion on the basis of micromanipulation 4 (stripping off the single hair follicle middle tissue), had an even shorter cell crawling out time (7 days).

[0171] Example 24 The cell crawling out situation on the 7th day of culture is as follows Figure 6 As shown, the results indicate that the cells crawled out well.

[0172] 3. Pollution detection

[0173] 100uL of the culture solution of each group cultured for 24 hours in Examples 1 to 24 was taken and spread on a plate. After culturing in a 37°C incubator for 24 hours, the bacterial growth on the plate was observed. The control group was the sterile culture solution before culturing.

[0174] The criteria for judging contamination is whether there is any contamination or bacterial infection during the culture process, with "yes" or "no" as the final result.

[0175] The results are shown in Table 1. Figure 7 As shown, Figure 7 The results showed that the culture solution of Example 24 was free of contamination.

[0176] The results in Table 1 show that the culture solutions of Examples 2, 4, 8, 16, and 24 were free of contamination.

[0177] 4. Adhesion efficiency test

[0178] The method for judging the adhesion efficiency is to observe the adhesion of the tissue to the culture dish when culturing after isolating the hair follicle tissue. If it adheres to the culture dish within 1-3 days, it is judged to have a high adhesion efficiency. If it adheres to the culture dish within 4-6 days, it is considered to have a medium adhesion efficiency. If it adheres to the culture dish within 7 days or more, it is considered to have a low adhesion efficiency.

[0179] The results are shown in Table 1. The results show that the adhesion efficiency of Examples 2 and 4 is medium, and the adhesion efficiency of Examples 8, 16, and 24 is high.

[0180] 5. Determination of hair follicle purity

[0181] The purity of hair follicles is determined by observing the morphology of the crawling cells and whether there are tissue blocks left behind when cells crawl out: 1) If the crawling cells have various morphologies and there are tissue blocks left behind, the purity is low; 2) If the crawling cells have various morphologies and there are no tissue blocks left behind, the purity is medium; 3) If the crawling cells have a single morphology and there are no tissue blocks left behind, the purity is high.

[0182] The results are shown in Table 1. The results show that the purity of the hair follicles in Examples 2, 8, and 16 is relatively low, while the purity of the hair follicles in Examples 4 and 24 is relatively high.

[0183] 6. Purity evaluation of single hair follicle keratinocytes

[0184] The purity of a single keratinocyte is judged by observing the morphology of the crawling cells, whether there are floating cells, and whether there are tissue blocks left when the cells crawl out. 1) If it is not a single hair follicle, the morphology of the crawling cells is diverse or single, but there are more floating cells and tissue blocks that have not crawled out, then the purity is low; 2) If it is a single hair follicle, the morphology of the crawling cells is messy, then the purity is considered to be medium; 3) If it is a single hair follicle, the morphology of the crawling cells is relatively consistent, then the purity is considered to be high.

[0185] The results are shown in Table 1, which show that the purity of the single hair follicle keratinocytes of Examples 2, 8, and 16 is relatively low, while the purity of the single hair follicle keratinocytes of Examples 4 and 24 is relatively high.

[0186] Table 1 Comparison of methods for isolating hair follicle-derived keratinocytes in Examples 1 to 24

[0187]

[0188]

[0189] As can be seen from Table 1, Examples 1 to 24 found through comparison and improvement of different methods that it is difficult to separate keratinocytes from hair follicles. After continuous attempts, a method that can effectively separate keratinocytes was obtained.

[0190] First, when only micromanipulation was performed, only micromanipulation 2 (Example 2) and micromanipulation 4 (Example 4) finally attached to the wall and separated to obtain keratinocytes, while micromanipulation 1 (Example 1) and micromanipulation 3 (Example 3) were not successful. By comparing Examples 2 and 4 (micromanipulation group 2 and group 4), it was found that the purity of keratinocytes crawling out of Example 4 (micromanipulation group 4) was higher, which depends on whether the single hair follicle tissue in the hair follicle unit tissue is peeled off separately. By comparing the single hair follicle tissue ( Figures 3 to 5 ) can greatly improve the purity of cells crawling out, and the results are as follows Figure 8, Figure 8 The morphology of cells isolated and cultured from the mid-segment of a single hair follicle in B is relatively Figure 8 A comparison of the morphology of cells isolated and cultured from the middle section of the hair follicle unit shows that the purity of the crawling out of a single hair follicle tissue is significantly higher than that of the cells isolated from the hair follicle unit tissue; this indicates that although the hair follicle tissues come from the same individual, the purity of the cells crawling out is significantly reduced because the hair follicle unit tissue contains multiple hair follicle tissues. This may be because there are more tissue residues between adjacent hair follicle tissues, and the treatment is not very thorough compared to a single hair follicle, resulting in more tissue residues in the cell crawling out process. In Examples 13 to 15 and Examples 21 to 23, other digestive enzymes were used for digestion, which may have damaged the cells, resulting in the cells not crawling out.

[0191] Secondly, when only digestive enzymes were used for digestion, only digestive enzyme 4 (collagenase IV) was able to finally separate keratinocytes, while other digestive enzymes were unable to separate keratinocytes. The inventors speculated that the reasons may be: 1) The complete hair follicle structure containing hair bulb tissue mainly adheres to the wall with the front end of the hair bulb tissue being the main part, while other parts are not normally attached to the wall. Due to the selectivity of the KM culture medium, the attached hair bulb tissue does not contain keratinocytes and will not crawl out of the cells, and the non-attached hair follicle tissue also cannot crawl out of the keratinocytes; 2) Only collagenase IV was able to separate keratinocytes through separation by different enzymatic hydrolysis methods, while other digestive enzymes were unable to digest the hair follicle tissue well, and only collagenase IV finally allowed the hair follicle tissue to successfully crawl out of the keratinocytes.

[0192] Among all the experimental groups in which keratinocytes were successfully separated, the methods of Examples 16 and 24 (micromanipulation 2 groups / 4 groups + digestive enzyme 4 enzymatic hydrolysis groups) had shorter cell crawling out time than other groups. After 7-10 days of culture observation, Examples 16 and 24 (micromanipulation 2 groups / 4 groups + digestive enzyme 4 enzymatic hydrolysis groups) - hair follicle-derived keratinocytes were found to crawl out in the hair follicle tissue, which was 10-20 days shorter than the traditional crawling out hair follicle-derived keratinocytes, without contamination, and in good condition, which confirmed the reliability of the separation method. Under normal operation time and no contamination, the time and efficiency of separating hair follicle-derived keratinocytes can be improved more quickly, which confirmed the reliability and effectiveness of the methods of Examples 16 and 24 (micromanipulation 2 groups / 4 groups + digestive enzyme 4 enzymatic hydrolysis groups). Through repeated experiments, the methods of Examples 16 and 24 also have good stability and can make keratinocytes crawl out every time.

[0193] Test Example 2 Identification of keratinocytes from hair follicles by immunofluorescence K19

[0194] The cells crawling out of Examples 2, 4, 8, 16, and 24 detected in Test Example 1 were subcultured, using KM medium, subcultured in an incubator at 37°C and 5% CO2 for 24 hours, and the cultured cells were immunofluorescently stained with K19 antibody to determine whether the cells obtained by separation and culture are keratinocytes from hair follicles: K19 protein is a marker protein specifically expressed by keratinocytes, and immunofluorescence is used to detect whether there is fluorescence expression by incubating K19 antibody with the cells to be tested (fluorescence expression indicates that the cells contain K19 protein that can bind to K19 antibody), and more than 90% of the cells have fluorescence expression, which is confirmed to be keratinocytes. The specific method is as follows:

[0195] The isolated and cultured cells were blocked with blocking solution BSA (Solebo, SW3015) for 30-60 min; primary antibody K19 (LS Bio, B3148) was added and incubated at 4°C overnight; the cells were washed three times with PBS in the dark, and goat anti-rabbit IgG H&L (Alexa 488) pre-adsorbed secondary antibody (abcam, ab150081, hereinafter referred to as "secondary antibody") and incubated in the dark for 1 hour; pour out the secondary antibody (abcam, ab150081) solution, wash the cells 3 times with PBS in the dark; incubate the cells with DAPI (Solabo, C0065) for 1 minute, then rinse the cells with PBS and observe them under a microscope. If the cells show the above expression of more than 90% of K19, it proves that the isolated and cultured cells are keratinocytes derived from hair follicles.

[0196] The cells isolated in Example 24 were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The results of K19 immunofluorescence identification of the cells are as follows: Fig. 9 As shown, it can be seen that K19 expression is above 90% fluorescent expression, confirming that the isolated cells are keratinocytes.

[0197] The identification results of other groups with cell crawling out (Examples 2, 4, 8, and 16) also proved that keratinocytes were separated and obtained.

[0198] Comparative Example 1

[0199] The collected human hair samples were immersed in sterile physiological saline (no storage solution (Lactated Ringer's solution) was used) and transported in the above-mentioned constant temperature transport box. The temperature was maintained at 0-15°C during transportation. After 24 hours of transportation, the operation method of Example 24 (micromanipulation group 4 + digestive enzyme group 4) was referred to. As a result, the cells failed to crawl out normally after being cultured in an incubator at 37°C and 5% CO2 for 25 days.

[0200] Comparative Example 2

[0201] The human hair samples collected and transported were not treated aseptically, and reference was made to Example 24 (micromanipulation group 4 + digestion enzyme group 4), resulting in cell contamination.

[0202] Combining Comparative Examples 1 and 2, it can be seen that the method of the present invention adopts storage in storage liquid and transportation in a constant temperature transport box, so that the transportation time of hair follicle tissue samples is as long as 24 hours. With the help of micromanipulation and enzymatic hydrolysis, keratinocytes can still be crawled out, which can solve the problem of sample collection time limit and overcome the disadvantage of having to collect samples freshly.

[0203] The inventors of the present invention also tried to transport the cells at a temperature below 0°C, but the keratinocytes could not be separated because the temperature was too low.

[0204] The method of the present invention also adopts aseptic treatment and simultaneous monitoring, so that the sample before microscopic separation is already sterile. If there are bacteria, the operation will be stopped to avoid waste of costs.

[0205] Test Example 3 Efficacy of isolated keratinocytes in repairing skin damage

[0206] The keratinocytes are prepared as a gel dressing in the following manner:

[0207] After the KM medium after culture in Example 24 was dried, 1-2 ml of PBS (Invitrogen, C14190500BT) was added to wash 2-3 times, and then the cells were digested with TrypE (Gibco, 12604021) in an incubator at 37°C and 5% CO2 for 10 min. After neutralization with neutralizing solution, the cells were blown away into a single state; the total number of cells was calculated using a Countstar counting plate; and then the cells were counted according to 1×10 7 / mL of cell concentration, mixed with Matrigel (BD, 354234) to form a gel dressing containing keratinocytes, and finally placed in a 37°C, 5% CO2 incubator for culture.

[0208] Four-week-old male nude mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were induced to adapt for one week, anesthetized, and the back skin tissue was disinfected with iodine. Then, the skin was punched with a 1-cm diameter round hole (HYpunch, 010). Fig.10 ); then place the keratinocyte gel dressing prepared above on the punched position and dry it for 30 minutes ( Fig.10 ), recorded as day 0; the condition of the nude mouse back skin was continuously observed, and on the 15th day of growth, it was found that the wounded skin on the nude mouse back showed a phenomenon of gradual healing and recovery ( Fig.10), indicating that the keratinocytes isolated and cultured from hair follicle tissues of the present invention can be used for the recovery of skin damage.

[0209] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. Any simple modifications, equivalent changes and modifications made to the above exemplary embodiments should fall within the scope of protection of the present invention.

Claims

1. A method for isolating keratinocytes derived from human hair follicles in vitro, comprising the following steps: 1) Isolate hair follicle tissue from ex vivo human hair samples; 2) pre-treating the hair follicle tissue; 3) Cultivating the pretreated hair follicle tissue obtained in step 2) using a keratinocyte selection medium to obtain human hair follicle-derived keratinocytes, wherein the keratinocyte selection medium is used for selectively culturing keratinocytes.

2. The method according to claim 1, characterized in that In step 2), the pretreatment method includes: removing the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue, and retaining the middle section as the hair follicle middle section tissue, and optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm; And / or, in step 2), the pretreatment method includes: removing connective adhesion tissue and / or adipose tissue on the hair follicle tissue; Optionally, the method of removing tissue is performed under a stereoscope.

3. The method according to claim 1 or 2, characterized in that: In step 2), the pretreatment method includes: enzymatically digesting the hair follicle tissue with a digestive enzyme; optionally, the digestive enzyme is collagenase IV; Optionally, the working concentration of the digestive enzyme is 1-10 mg / mL; optionally, the enzymolysis time is 20 to 120 min; optionally, the enzymolysis time is 25 to 35 min; optionally, the enzymolysis time is 30 to 35 min; optionally, the enzymolysis temperature is 35 to 38°C; optionally, the enzymolysis temperature is 35 to 37.5°C.

4. The method according to any one of claims 1 to 3, characterized in that: In step 1), the hair follicle tissue is a hair follicle unit tissue and / or a single hair follicle tissue; And / or, in step 2), the hair follicle tissue is a single hair follicle tissue, and optionally, the single hair follicle tissue is separated from a hair follicle unit.

5. The method according to any one of claims 1 to 4, characterized in that: In step 2), the pretreatment method includes: 1) Remove the connective adhesion tissue and fat tissue on the hair follicle tissue; 2) removing the hair bulb tissue and part of the hair shaft tissue in the hair follicle tissue of step 1), and retaining the middle section as the hair follicle middle section tissue. Optionally, the length of the hair follicle middle section tissue is 0.5-2.5 mm; 3) the hair follicle midsection tissue of step 2) is enzymatically digested with digestive enzymes; Optionally, in step 2), the hair follicle tissue is a single hair follicle tissue. Optionally, the single hair follicle tissue is separated from a hair follicle unit.

6. The method according to any one of claims 1 to 5, characterized in that: Before step 1), the ex vivo human hair sample is stored in a storage solution; optionally, the storage solution is lactic acid Ringer's solution; optionally, the storage temperature is 0-15°C; optionally, the storage temperature is 0.5-15°C; optionally, the ex vivo human hair sample is stored for no more than 48 hours after being ex vivo; optionally, the ex vivo human hair sample is stored in a constant temperature transport box; And / or, before step 1), it also includes aseptic treatment and / or aseptic monitoring. Optionally, the aseptic treatment includes: washing with 75% ethanol and / or DPBS / PBS solution containing double antibodies. Optionally, the volume fraction of the double antibodies in the DPBS / PBS solution containing double antibodies is 1-3%. Optionally, washing with DPBS / PBS solution containing 1-2% v / v double antibodies is performed at least 3 times; optionally, aseptic monitoring includes sterility testing before and after sterilization.

7. The method according to any one of claims 1 to 6, characterized in that: In step 3), the keratinocyte selection medium is KM medium.

8. Use of human hair follicle-derived keratinocytes in the preparation of skin inflammation and / or damage recovery, or skin regeneration products.

9. The use according to claim 8, characterized in that: The keratinocytes are separated and cultured from in vitro human hair samples.

10. The use according to claim 8 or 9, characterized in that: The keratinocytes are obtained by the method according to any one of claims 1 to 7.

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