A method and use for extracting and culturing mouse primary keratinocytes and fibroblasts

Abstract

The application discloses a method and application for extracting and culturing mouse primary keratinocytes and fibroblasts, and belongs to the technical field of cell culture. The method comprises the following steps: obtaining mouse skin tissue, washing and removing subcutaneous fat; using a type I collagenase solution to perform low-temperature digestion on the obtained skin tissue, and then mechanically separating an epidermis layer and a dermis layer; collecting the epidermis layer and the dermis layer respectively; cutting the epidermis layer, adding trypsin for digestion; cutting the dermis layer, adding a type I collagenase solution for digestion; respectively terminating the digestion of the epidermis layer and the dermis layer, removing tissue fragments through filtration, and centrifuging to collect cell precipitates; respectively resuspending the collected epidermis layer and dermis layer cell precipitates in corresponding complete culture media, inoculating and culturing, and obtaining primary keratinocytes and fibroblasts. The application does not need ultrasonic treatment and low-temperature recovery in the whole process, is simple to operate, has a short digestion time, effectively protects cell activity, and has high cell purity.

Description

Technical Field

[0001] This invention belongs to the field of tissue cell biology and cell culture technology, specifically relating to a method and application for extracting and culturing mouse primary keratinocytes and fibroblasts. Background Technology

[0002] The skin, as the largest organ of the body, consists of the epidermis and the dermis. The main cells of the epidermis are keratinocytes, which participate in the formation of the skin barrier and are widely used in research on the pathological mechanisms of scar formation and psoriasis. The most important cells in the dermis are fibroblasts, which are responsible for secreting extracellular matrix proteins such as collagen and play a key regulatory role in skin repair and regeneration. Simultaneously obtaining highly active primary keratinocytes and fibroblasts in vitro is of great value for research in skin toxicology, burn treatment, cosmetic medicine, and the pathogenesis of skin diseases.

[0003] In existing technologies, enzymatic digestion is commonly used to separate and culture these two cell types, but this method has significant shortcomings. For example, Chinese invention patent application CN116004518A discloses a method for extracting human primary keratinocytes and fibroblasts. This method involves treating skin tissue with dispase II at 37°C for 30-60 minutes, followed by low-intensity ultrasound treatment at 4°C, then mechanically separating the epidermis and dermis, followed by low-temperature awakening and trypsin digestion. In this method, the ultrasound step easily causes mechanical damage to cells and reduces cell viability, while the low-temperature awakening procedure is cumbersome, increasing operational complexity and the risk of contamination. Some methods, to effectively separate the epidermis and dermis, require overnight digestion at 4°C (e.g., CN106047793A), with digestion times exceeding 12 hours. This not only prolongs the experimental cycle but also further affects the survival rate and proliferation capacity of primary cells. Furthermore, prolonged action of digestive enzymes can easily damage fibroblasts, resulting in low cell yield and poor cell condition.

[0004] In keratinocyte culture, the epidermis inevitably contains a small number of fibroblasts, which proliferate rapidly and easily contaminate keratinocytes, leading to decreased purity. Traditional methods to eliminate this contamination rely on increasing the number of passages to gradually eliminate fibroblasts. However, repeated passages are time-consuming and accelerate the aging and differentiation of keratinocytes, causing them to lose their stem cell characteristics. Although serum-free culture media (such as CN1560235A) exist for keratinocyte culture without the need for feeder cells, it remains difficult to simultaneously solve the problems of cell adhesion difficulties and fibroblast contamination.

[0005] Therefore, there is an urgent need for a culture method that is simple to operate, has a short processing cycle, can effectively protect cell viability, and can simultaneously obtain high-purity primary keratinocytes and fibroblasts. Summary of the Invention

[0006] This invention provides a method for extracting and culturing mouse primary keratinocytes and fibroblasts, which can effectively solve the problems of cumbersome separation steps and complex operations such as reliance on ultrasonic treatment or low-temperature awakening in the prior art; at the same time, it solves the problems of decreased cell activity and increased risk of contamination due to excessive digestion time; and it can also effectively solve the problem of repeated passages required during keratinocyte culture due to fibroblast contamination, which accelerates cell aging and differentiation and makes it difficult to stably obtain high-purity primary keratinocytes.

[0007] The first inventive point of this invention is: a method for extracting and culturing mouse primary keratinocytes and fibroblasts, comprising the following steps: (1) Obtain mouse skin tissue, clean and remove subcutaneous fat; (2) The skin tissue obtained in step (1) was digested at low temperature using a type I collagenase solution, followed by mechanical separation of the epidermis and dermis; (3) Collect the epidermis and dermis from step (2) respectively: cut the epidermis into pieces and add trypsin for digestion; cut the dermis into pieces and add type I collagenase solution for digestion; (4) The epidermis and dermis in step (3) are digested separately, tissue fragments are removed by filtration, and cell pellets are collected by centrifugation; (5) The epidermal and dermal cell pellets collected in step (4) are resuspended in the corresponding complete culture medium, inoculated and cultured to obtain primary keratinocytes and fibroblasts.

[0008] Preferably, in step (2), the concentration of the type I collagenase solution is 1-5 mg / mL, and the amount added is 3-5 times the volume of the skin tissue sample; the low-temperature digestion is digestion at a temperature of 1-8℃ for 2-6 hours; the mechanical separation is performed entirely on an ice box to maximize the maintenance of the high activity of the tissue and the separated cells.

[0009] Preferably, in step (3), the epidermis is digested using 0.05%-0.5% trypsin in a water bath at 35-38°C for 15-45 minutes, and the amount of trypsin added is 3-5 times the volume of the skin tissue sample; the dermis is digested using a type I collagenase solution of 1-5 mg / mL at 1-8°C for 1.5-4 hours, and the amount of type I collagenase solution added is 3-5 times the volume of the tissue sample.

[0010] Preferably, in step (4), a 20-100 μm cell sieve is used for filtration; the centrifugation speed is 700-1300 rpm and the time is 4-7 minutes.

[0011] Preferably, the complete culture medium used for the keratinocytes includes EpiLife™ medium, EpiLife™ growth supplement, antibiotics and fetal bovine serum, in a volume ratio of 100:(0.5-2):(0.5-2):(0.1-1).

[0012] Preferably, the complete culture medium used for the fibroblasts includes DMEM high glucose medium, fetal bovine serum and antibiotics, in a volume concentration ratio of 100:(10-20):(0.5-2).

[0013] Preferably, the cell resuspension is inoculated onto a culture plate pre-coated with rat tail collagen for culture.

[0014] Preferably, the keratin 14 (K14) positivity rate of the keratinocytes is ≥93% and the vimentin positivity rate of the fibroblasts is ≥98% as determined by immunofluorescence.

[0015] The second inventive point of this invention is: the application of the above-described method in the preparation of a skin research model.

[0016] The third inventive point of this invention is: the application of the above-described method in the preparation of a skin drug screening model.

[0017] Compared with the prior art, the advantages of the present invention are: This invention provides a method for extracting and culturing primary mouse keratinocytes and fibroblasts, which has significant advantages compared to existing technologies. Firstly, this invention uses type I collagenase to briefly digest skin tissue at 4°C (preferably 4 hours), after which the epidermis and dermis can be mechanically separated by forceps. The entire process requires no ultrasonic treatment or low-temperature rewarming step, simplifying the operation and significantly reducing operational complexity and the risk of mechanical damage to cells. Simultaneously, the digestion time is shortened from the commonly used "overnight digestion" (over 12 hours) to 4 hours, greatly improving experimental efficiency and keeping the total operation time from tissue acquisition to obtaining cell pellet within 8 hours. This also effectively avoids damage to cell viability caused by prolonged enzymatic action and reduces the chance of bacterial or other microbial contamination. The entire process is performed on an ice box or at 4°C, synergistically maintaining a high cell viability during separation and ensuring the quality of the starting cells for subsequent culture.

[0018] Secondly, this invention designs a differentiated and gentle stepwise digestion scheme tailored to the extracellular matrix characteristics of the epidermis and dermis: the epidermis is digested with trypsin for a short time, while the dermis is digested with type I collagenase at low temperature. This avoids cell damage caused by excessive action of a single enzyme, further ensuring the viability and yield of fibroblasts. In the culture stage, a complete culture medium specifically for keratinocytes containing a low concentration of fetal bovine serum is used. This effectively promotes keratinocyte proliferation while significantly inhibiting the growth of mixed fibroblasts, eliminating the need for cell purification through increased passage times and reducing the risk of cell aging and differentiation associated with passage operations. Simultaneously, the use of rat tail collagen pre-coated culture plates enhances the adhesion efficiency of keratinocytes. The primary keratinocytes obtained using this method have a K14 positivity rate ≥93%, and the primary fibroblasts have a Vimentin positivity rate ≥98%, stably obtaining high-purity primary cell lines of both types, providing an ideal cell source for research on skin-related diseases and the construction of drug screening models. Attached Figure Description

[0019] Figure 1 Experimental flowchart; Figure 2 Diagram of keratinocyte morphology; Figure 3 Fibroblast morphology diagram; Figure 4 Immunofluorescence identification image of keratinocytes; Figure 5 Immunofluorescence identification image of fibroblasts; Figure 6 Statistical chart of K14 and Vimentin positivity rates in primary cells.

[0020] Specific implementation methods To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0022] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0023] Example 1 The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0024] This embodiment provides a method for extracting and culturing mouse primary keratinocytes and fibroblasts, including the following steps: (1) Obtain mouse skin tissue, clean and remove subcutaneous fat; (2) The skin tissue obtained in step (1) was digested at low temperature using a type I collagenase solution, followed by mechanical separation of the epidermis and dermis; (3) Collect the epidermis and dermis from step (2) respectively: cut the epidermis into pieces and add trypsin for digestion; cut the dermis into pieces and add type I collagenase solution for digestion; (4) The epidermis and dermis in step (3) are digested separately, tissue fragments are removed by filtration, and cell pellets are collected by centrifugation; (5) The epidermal and dermal cell pellets collected in step (4) are resuspended in the corresponding complete culture medium, inoculated and cultured to obtain primary keratinocytes and fibroblasts.

[0025] This invention employs a process of "low-temperature digestion with type I collagenase → mechanical separation of epidermis and dermis → epidermal trypsin digestion and dermal type I collagenase digestion → separate culturing". In step (2), type I collagenase specifically degrades the collagen components of the dermis under low-temperature conditions, loosening the basement membrane connection between the dermis and epidermis, thus allowing the subsequent mechanical separation operation to be completed without ultrasonic treatment, avoiding mechanical damage to cells caused by ultrasound. In step (3), a differentiated enzymatic digestion strategy is adopted based on the different extracellular matrix compositions of the epidermis and dermis. Trypsin mainly dissociates the intercellular connections of keratinocytes in the epidermis, while type I collagenase continues to digest the residual collagen in the dermis, releasing fibroblasts. This "stepwise differentiated digestion" design ensures that both types of cells can be released in a highly active state. In steps (4) and (5), the separated cells are cultured under suitable conditions, ultimately obtaining two types of primary cells simultaneously. Therefore, these five steps, from "tissue separation" to "cell dissociation" and then to "culture acquisition", constitute a complete and inseparable technical loop, embodying the core invention of "one method to obtain two types of cells simultaneously".

[0026] As a further preferred embodiment, in step (2), the concentration of the type I collagenase solution is 1-5 mg / mL, and the amount added is 3-5 times the volume of the skin tissue sample; the low-temperature digestion is digestion at a temperature of 1-8℃ for 2-6 hours; the mechanical separation is performed entirely on an ice box to maximize the maintenance of the high activity of the tissue and the separated cells.

[0027] When the concentration of type I collagenase solution is too low (below 1 mg / mL), the collagenase activity is insufficient to fully degrade dermal collagen, making it difficult to separate the epidermis from the dermis, requiring additional mechanical force and increasing the risk of cell damage. Conversely, a concentration that is too high (above 5 mg / mL) may cause non-specific degradation of cell membrane proteins, reducing cell viability. The range of "low-temperature digestion at 1-8℃ for 2-6 hours" is optimally defined at 4℃. The low-temperature environment has two benefits: firstly, it reduces the enzyme reaction rate, making the digestion process gentle and controllable, avoiding excessive damage to cells from high temperatures; secondly, the low temperature itself inhibits cell metabolism, reduces apoptosis, and maintains cells in a quiescent state, which is beneficial for preserving their viability. "Operating entirely on an icebox" further extends the low-temperature protection to the entire separation process, ensuring that cells are under low-temperature protection from tissue excision to completion of separation.

[0028] As a further preferred embodiment, in step (3), the epidermis is digested using 0.05%-0.5% trypsin in a water bath at 35-38°C for 15-45 minutes, and the amount of trypsin added is 3-5 times the volume of the skin tissue sample; the dermis is digested using a type I collagenase solution of 1-5 mg / mL at 1-8°C for 1.5-4 hours, and the amount of type I collagenase solution added is 3-5 times the volume of the tissue sample.

[0029] Epidermal layer digestion uses 0.05%-0.5% trypsin at 35-38℃ for 15-45 minutes. The principle behind this technique is that trypsin is a serine protease that primarily cleaves intercellular adhesion proteins and cell-matrix junctions; 37℃ is its optimal temperature. The choice of concentration and time is a balance. If the trypsin concentration is too low or the time is too short, keratinocytes cannot be fully dissociated from the epidermal tissue; if the concentration is too high or the time is too long, excessive degradation of cell membrane surface proteins will occur, damaging cells and reducing their adhesion ability. A 0.25% concentration digestion for 25 minutes is preferred, as it can fully dissociate cells while maximizing cell viability.

[0030] The dermis is digested separately using 1-5 mg / mL type I collagenase at 1-8°C for 1.5-4 hours, demonstrating a targeted design based on the characteristics of the dermis: the dermis is rich in type I collagen, and type I collagenase has substrate-specific degradation capabilities for it. Compared to existing techniques that treat the dermis and epidermis together with trypsin, this method uses collagenase to treat the dermis separately, allowing fibroblasts to be gently released from the collagen matrix, rather than being "torn" off by strong trypsin.

[0031] As a further preferred embodiment, a 20-100 μm cell sieve is used for filtration; the centrifugation speed is 700-1300 rpm and the time is 4-7 minutes.

[0032] Cell filtration is a crucial step in removing incompletely digested tissue fragments and ensuring the quality of single-cell suspensions. Pore sizes that are too small (below 20 μm) may trap larger fibroblasts or clumps of cells, reducing cell yield; pore sizes that are too large (above 100 μm) cannot effectively remove tissue debris, resulting in excessive impurities in the culture system and affecting cell adhesion and growth. The preferred pore size in this invention is 40 μm.

[0033] As a further preferred embodiment, in step (5), the complete culture medium used for the keratinocytes includes EpiLife™ medium, EpiLife™ growth supplement, double antibiotics and fetal bovine serum, which are mixed in a volume concentration ratio of 100:(0.5-2):(0.5-2):(0.1-1).

[0034] This formulation creates a culture environment with a "selective growth window" for both cell types. Keratinocyte proliferation in vitro requires exogenous growth factors, while the introduced fibroblasts also rely on serum mitogens for rapid division. The core discovery of this invention is that when the serum concentration is precisely controlled within the low range of 0.1%-1%, it achieves the dual effect of "meeting the requirements for keratinocyte proliferation but falling below the serum concentration threshold required for rapid fibroblast expansion." Keratinocytes, due to the sufficient specific signals such as epidermal growth factor provided by EDGS, have a relatively low dependence on serum and can still adhere and divide normally under low serum conditions. Fibroblasts, as mesenchymal-derived cells, have proliferation signaling pathways that are highly sensitive to serum concentration; a serum level of 0.1%-1% is insufficient to activate them into the logarithmic growth phase, thus inhibiting their growth. It is this "selective inhibition" mechanism, based on the fundamental difference in serum dependence between the two cell types, that allows keratinocytes to naturally gain a growth advantage without the need for repeated passages to eliminate fibroblasts, ultimately achieving high-purity culture.

[0035] As a further preferred embodiment, in step (5), the complete culture medium used for the fibroblasts includes DMEM high glucose medium, fetal bovine serum and double antibiotics, which are mixed in a volume concentration ratio of 100:(10-20):(0.5-2).

[0036] This formula provides fibroblasts with an optimal nutritional environment that is highly matched to their tissue origin and metabolic characteristics. Dermal fibroblasts are already in a collagen-rich matrix environment in vivo, and their natural metabolism is vigorous. Their demand for glucose and serum growth factors is much higher than that of epidermal keratinocytes. The glucose concentration provided by the DMEM high-glucose medium can meet their active energy metabolism needs, while the higher concentration of serum (10%-20%) provides sufficient mitogen signals such as fibroblast growth factor and platelet-derived growth factor, promoting their rapid adhesion and massive proliferation.

[0037] As a further preferred embodiment, in step (5), the cell resuspension is inoculated onto a culture plate pre-coated with rat tail collagen for culture.

[0038] Keratinocytes are epithelial cells and have poor adhesion to ordinary plastic culture plates. Rat tail collagen, a type I collagen, can mimic the extracellular matrix environment of the in vivo basement membrane, providing natural adhesion sites for keratinocytes and significantly improving their adhesion efficiency. Simultaneously, the collagen coating layer promotes keratinocyte spreading, maintains their undifferentiated epithelial cell morphology, and prevents cells from prematurely entering the differentiation process and losing their proliferative capacity.

[0039] As a further preferred embodiment, the keratin 14 (K14) positivity rate of the keratinocytes was ≥93% and the vimentin positivity rate of the fibroblasts was ≥98% as determined by immunofluorescence.

[0040] Cell purity was determined using immunofluorescence. K14 (cytokeratin 14) is a positive marker for basal keratinocytes; microscopic examination revealed positive K14 immunofluorescence staining in cultured cells, confirming that the cultured cells were basal keratinocytes. Vimentin is a positive marker for fibroblasts; microscopic examination revealed positive Vimentin immunofluorescence staining in cultured cells, confirming that the cultured cells were fibroblasts.

[0041] K14 (cytokeratin 14) is a specific intermediate filament protein of keratinocytes in the basal layer of the epidermis. Its positive rate directly reflects the purity of keratinocytes and the degree of preservation of stem cell characteristics. Vimentin is an intermediate filament protein marker of mesenchymal-derived cells (such as fibroblasts). A high positive rate indicates high purity of the fibroblast population. Immunofluorescence, as an identification method, has the advantages of direct observation of cell morphology and accurate marker localization, providing a reliable technical guarantee for defining product characteristics.

[0042] Example 2 Application of a method for extracting and culturing mouse primary keratinocytes and fibroblasts in the preparation of skin research models.

[0043] Application of a method for extracting and culturing mouse primary keratinocytes and fibroblasts in the preparation of a skin drug screening model.

[0044] Example 3 Experimental materials: DMEM high-glucose medium (Guangzhou Angfei Biotechnology Co., Ltd.), 0.25% Trypsin-EDTA (1X), Phenol Red (Beijing Bailongxingda Biotechnology Co., Ltd.), double antibiotics (Guangzhou Zhichuang Biotechnology Co., Ltd.), type I collagenase (Guangzhou Shikenai Life Technology Co., Ltd.), rat tail collagen type I (Shenzhen Yide Biotechnology Co., Ltd.), EpiLife TM Culture medium (Shenzhen Jiuhong Technology Co., Ltd.), Cytokeratin 14 Recombinant antibody (Beijing Dongge Boye Biotechnology Co., Ltd.), Anti-Vimentin antibody (Guangzhou Squirrel Biotechnology Co., Ltd.), SA00013-3 CoraLite594-conjugated Goat Anti-Mouse IgG (H+L) (Shenzhen Baitai Technology Co., Ltd.), EdU-594 cell proliferation detection kit (Beijing Huaxia Yuanyang Technology Co., Ltd.), Ginsenoside Rg1 (Hangzhou Xincheng Biotechnology Co., Ltd.) Experimental procedure: This experimental example provides a method for extracting and culturing mouse primary keratinocytes and fibroblasts. The experimental procedure is as follows: Figure 1 As shown, the specific steps are as follows: (1) Take 1-3 day old Balb / c mice, euthanize them on ice, and immediately disinfect them in 75% alcohol for 5 minutes. Remove them, cut off their limbs and tails, and cut open the skin from one side to obtain the skin of the back and abdomen. Remove the internal fat layer with forceps, wash the skin three times in pre-cooled PBS containing 2% penicillin and antibiotics, and then wash it twice with PBS to remove blood. Put the washed skin tissue into a 2 mg / mL type I collagenase solution, with the amount of type I collagenase added being 4 times the sample volume, and digest it at 4°C for 4 hours.

[0045] (2) Take out the digested tissue and place it in a 10 cm culture dish. The entire process is carried out on an ice box. Use tweezers to mechanically separate the epidermis and dermis.

[0046] (3) The epidermis was cut into pieces to obtain tissue homogenate, which was placed in a 15 mL centrifuge tube and 0.25% trypsin was added at 4 times the sample volume. The homogenate was then digested in a 37°C water bath for 25 minutes. The dermis was cut into pieces to obtain tissue homogenate, which was placed in a 15 mL centrifuge tube and 2 mg / mL type I collagenase solution was added at 4°C for 2.5 hours.

[0047] (4) Add culture medium containing 10% serum to the digestion solution of the epidermis and dermis respectively to terminate digestion. Filter the solution using a 40μm cell sieve to remove tissue debris, centrifuge the filtrate at 1000 rpm for 5 minutes, discard the supernatant, and obtain the cell pellet.

[0048] (5) Resuspend the epidermal cell pellet in keratinocyte complete culture medium and seed it onto a rat tail collagen pre-coated culture plate. The keratinocyte complete culture medium formula is: EpiLife™ medium + 1% EDGS + 1% penicillin-dip antibiotics + 0.3% fetal bovine serum. Resuspend the dermal cell pellet in fibroblast complete culture medium and seed it for culture. The fibroblast complete culture medium formula is: DMEM high glucose medium + 15% fetal bovine serum + 1% penicillin-dip antibiotics.

[0049] (6) Place the culture plate in a 37℃, 5% CO2 incubator. Observe the cell morphology under a microscope after culture. Figure 2 The image shows keratinocytes obtained through culture, exhibiting a typical cobblestone-like morphology; Figure 3 The image shows fibroblasts obtained through culture, exhibiting a typical long spindle-shaped morphology.

[0050] (7) Immunofluorescence assay for cell purity. Keratin 14 (K14) antibody was used for immunofluorescence staining of keratinocytes, and vimentin antibody was used for immunofluorescence staining of fibroblasts. Results are as follows: Figure 4 As shown, keratinocytes showed positive K14 immunofluorescence staining; Figure 5 As shown, fibroblasts showed positive results for Vimentin immunofluorescence staining. Figure 6 As shown, statistical analysis revealed that the positive rate of K14 in keratinocytes was ≥93%, and the positive rate of Vimentin in fibroblasts was ≥98%, indicating that the method in this embodiment can obtain high-purity primary keratinocytes and fibroblasts.

[0051] in conclusion: In summary, the method for simultaneous extraction and culture of mouse primary keratinocytes and fibroblasts provided by this invention utilizes low-temperature, short-time collagenase digestion combined with mechanical separation, eliminating cumbersome steps such as ultrasound and low-temperature reawakening, and controlling the total operation time to within 8 hours, significantly reducing the risk of cell damage and contamination. Differential enzymatic digestion protocols are designed for the epidermis and dermis, combined with selective culture media and mouse tail collagen coating, effectively inhibiting fibroblast contamination. High-purity primary cells with K14 positivity ≥93% and Vimentin positivity ≥98% can be obtained without repeated passages. This method is simple to operate, has a short cycle, and produces high cell viability, providing a reliable cell source for basic skin research, disease model construction, and drug screening, and has good prospects for widespread application.

[0052] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for extracting and culturing mouse primary keratinocytes and fibroblasts, characterized in that, Includes the following steps: (1) Obtain mouse skin tissue, clean and remove subcutaneous fat; (2) The skin tissue obtained in step (1) was digested at low temperature using a type I collagenase solution, followed by mechanical separation of the epidermis and dermis; (3) Collect the epidermis and dermis from step (2) separately: cut the epidermis into small pieces and add trypsin for digestion; The dermis is cut into small pieces and digested with type I collagenase solution; (4) The epidermis and dermis in step (3) are digested separately, tissue fragments are removed by filtration, and cell pellets are collected by centrifugation; (5) The epidermal and dermal cell pellets collected in step (4) are resuspended in the corresponding complete culture medium, inoculated and cultured to obtain primary keratinocytes and fibroblasts.

2. The method according to claim 1, characterized in that, In step (2), the concentration of the type I collagenase solution is 1-5 mg / mL, and the amount added is 3-5 times the volume of the skin tissue sample; the low-temperature digestion is digestion at a temperature of 1-8℃ for 2-6 hours.

3. The method according to claim 1, characterized in that, In step (3), the epidermis is digested using 0.05%-0.5% trypsin in a water bath at 35-38°C for 15-45 minutes, and the amount of trypsin added is 3-5 times the volume of the skin tissue sample; the dermis is digested using a type I collagenase solution of 1-5 mg / mL at 1-8°C for 1.5-4 hours, and the amount of type I collagenase solution added is 3-5 times the volume of the tissue sample.

4. The method according to claim 1, characterized in that, In step (4), a 20-100 μm cell sieve is used for filtration; the centrifugation speed is 700-1300 rpm and the time is 4-7 minutes.

5. The method according to claim 1, characterized in that, In step (5), the complete culture medium used for the keratinocytes includes EpiLife™ medium, EpiLife™ growth supplement, antibiotics and fetal bovine serum, in a volume concentration ratio of 100:(0.5-2):(0.5-2):(0.1-1).

6. The method according to claim 1, characterized in that, In step (5), the complete culture medium used for the fibroblasts includes DMEM high glucose medium, fetal bovine serum and antibiotics, which are mixed in a volume concentration ratio of 100:(10-20):(0.5-2).

7. The method according to claim 1, characterized in that, In step (5), the cell resuspension is seeded onto a culture plate pre-coated with rat tail collagen for culture.

8. A mouse primary keratinocyte and fibroblast obtained by the method according to any one of claims 1-7, characterized in that, Immunofluorescence assays showed that the keratin 14 (K14) positivity rate of the keratinocytes was ≥93%, and the vimentin positivity rate of the fibroblasts was ≥98%.

9. The application of the method according to any one of claims 1-7 in the preparation of skin research models.

10. The application of the method according to any one of claims 1-7 in the preparation of a skin drug screening model.

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