In-vitro separation, culture and identification method of human primary sweat gland muscle epithelial cells
Through an in vitro isolation and culture identification method, the problem of difficulty in obtaining high-purity and high-active sweat adenozoic myoepithelial cells in the prior art is solved, and efficient and low-cost cell acquisition is achieved. It is suitable for use in most laboratories and promotes the development of sweat gland research and tissue engineering.
Patent Information
- Application Number
- CN202510438244.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-06-24
AI Technical Summary
The prior art is difficult to efficiently obtain high-purity and high-active sweat adenozoic myoepithelial cells in vitro, and conventional methods have problems such as cell damage, difficulty in controlling digestive enzymes, and expensive flow cell sorting, which hinders the further promotion of sweat gland research.
A method of in vitro isolation, culture and identification of human primary sweat adenomyosis myoepithelial cells is adopted, including obtaining skin specimens, low temperature preservation, cleaning, digestion, irradiation and disinfection, implant attachment, isolation and transplantation, discarding culture medium and observing and taking photos, and high-purity sweat adenomyosis myoepithelial cells are obtained through differential digestion and differential adherence method.
It has achieved efficient acquisition of high-purity and high-active sweat adenozoic myoepithelial cells in vitro, simplified the operation process, reduced costs, and was suitable for use in most laboratories, meeting the needs of basic scientific research and sweat gland tissue engineering regeneration.
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Figure CN120192912A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biology, and specifically refers to a method for in vitro isolation, culture and identification of human primary sweat gland myoepithelial cells. Background Art
[0002] Sweat glands are a type of gland in the human skin, which are divided into two types: apocrine sweat glands (apocrine glands) and eccrine sweat glands (eccrine glands). There are approximately 3 million - 5 million sweat glands in the human body. However, in patients with large - area skin defects such as severe burns, due to the absence of appendages such as sweat glands and sebaceous glands in their skin, the wound surface is then covered with disorderly piled scar tissue. When facing stress or high - temperature weather, such patients often face life - threatening due to the lack of effective heat - dissipation measures. On the contrary, excessive sweating, such as hyperhidrosis, will seriously affect the daily life and social activities of patients and reduce the quality of life of patients. It should be specifically noted that the sweat glands mentioned in this article all refer to eccrine sweat glands.
[0003] The sweat gland is a single tube, composed of a secretory coil and a duct. The secretory coil is a pseudostratified epithelium, composed of myoepithelial cells in the outer basal layer and secretory cells in the lumen layer. The myoepithelial cells in the sweat gland are spindle - shaped. After receiving nerve signals, they can contract the secretory coil to regulate the secretion of sweat, while the secretory cavity cells release sweat into the lumen of the secretory coil. The myoepithelial cells in the secretory duct can self - renew and differentiate into lumen cells, which play a key role in maintaining the sweat gland. In addition, the myoepithelial cells in the sweat gland are embedded in Matrigel matrix gel and can achieve the regeneration of sweat gland structure when cultured in in - vitro and in - vivo environments.
[0004] In order to further understand the growth, development and physiological functions of sweat glands, long - term in - vitro culture of myoepithelial cells is a promising model. However, at present, there are still few studies on in - vitro models of myoepithelial cells in sweat glands. First, people still cannot correctly understand the characteristics of sweat gland myoepithelial cells in vitro and overly focus on the functions of other sweat gland epithelial cells. Second, conventional flow cytometry sorting and immunomagnetic bead adsorption methods require adding various digestive enzymes to digest sweat gland tissues into cell suspensions, and it is necessary to strictly control the types, amounts and digestion times of the added enzymes, which easily causes damage to myoepithelial cells and affects subsequent experiments. In addition, flow cytometry screening is expensive and has low yield, which is even worse for researchers with limited research funds in ordinary laboratories, making it difficult to carry out experiments. These disadvantages have hindered the further popularization of related technologies in the field of sweat gland research.
[0005] Therefore, how to efficiently obtain high - purity and high - activity sweat gland myoepithelial cells in an in - vitro environment and understand their biological characteristics to meet the needs of large - scale basic research, and even the key points of studying the growth and development of sweat glands, is also a driving factor for the development of tissue engineering such as sweat gland regeneration. Summary of the Invention
[0006] The object of the present invention is to solve the problems in the above-mentioned background technology, and provide a method for in vitro isolation, culture and identification of human primary sweat gland myoepithelial cells, which is characterized by including obtaining skin specimens, cryopreservation, washing, digestion, washing, digestion, irradiation disinfection, implanting and adhering, separating and planting, discarding the culture medium and observing and photographing.
[0007] The specific operation mode is as follows:
[0008] (1) Obtaining the skin specimens: Obtain skin specimens through surgery;
[0009] (2) Cryopreservation: Put it into a sterile PBS solution containing 1× penicillin (100 U / ml) - streptomycin (0.1 g / ml) double antibody for cryopreservation;
[0010] (3) Washing: On a super-clean workbench sterilized by ultraviolet irradiation, wash it twice again with PBS containing 1× double antibody to remove residual blood and tissue debris;
[0011] (4) Digestion: Cut the skin tissue into pieces of 0.5 cm3 in size, transfer it to another clean culture dish, with the epidermal side facing down, add 2.5 mg / ml neutral protease solution, and digest it in a 37°C 5% CO2 cell culture incubator for 1 hour;
[0012] (5) Washing: Then take out the culture dish, remove the epidermis with forceps, put the white dermal part into a clean PBS solution and wash it twice, and place it in a 60 mm culture dish for standby;
[0013] (6) Digestion: Use an ophthalmic scissors to cut the dermis into pieces of 1 mm3 in size in the culture dish, add 3 ml of dermal digestion solution containing 20 mg / ml type II collagenase, and digest it in a 37°C cell culture incubator for 2 hours;
[0014] (7) Irradiation disinfection: After 2 hours, under an inverted microscope (40×) sterilized by ultraviolet irradiation, use a 200 um pipette to pick out the sweat gland tissue and place it in a complete sweat gland culture medium for washing 2 - 3 times;
[0015] (8) Implanting and adhering: After the sweat gland explants adhere for 1 - 2 days, myoepithelial cells can grow out, and then 200 ul of complete culture medium can be supplemented, and the culture medium is changed every 2 - 3 days;
[0016] (9)Separation and seeding: One week after the sweat gland tissue grows cells, use 0.25% trypsin digestion solution to digest and isolate primary sweat gland myoepithelial cells, and add complete medium to terminate the digestion. Centrifuge the cell suspension at 1000 r / s for 5 min, resuspend it with complete medium, seed it in a 6-well plate, and culture it in a cell incubator at 37°C for 2 hours. Then discard the old medium and replenish it with fresh pre-warmed complete medium. After that, change the medium by half every 2 - 3 days, and record it as the first-generation cells. Passage can be carried out when the cell growth confluence reaches 80 - 90%.
[0017] (10)Observation and photography: Use forceps to pick out the coverslip and invert it on a clean glass slide, then observe and photograph it under an inverted fluorescence microscope (Olympus).
[0018] Preferably, discard the medium, wash it twice with PBS, fix it with 4% paraformaldehyde for 15 min, then wash it three times with PBS, add 0.3% Triton X-100 solution to permeabilize for 5 min, wash it three times with PBS again, then block it with 5% BSA at room temperature for 1 h. After discarding the blocking solution, add the prepared primary antibody solution (dilution ratio 1:100), incubate it in a 4°C refrigerator for 12 h. Discard the primary antibody solution, then wash it three times with PBS, add the prepared secondary antibody solution (dilution ratio 1:1000), incubate it at room temperature in the dark for 1 h. Discard the secondary antibody solution, then wash it three times with PBS, add 10 μg / ml DAPI solution, incubate it in the dark for 5 min. After discarding the DAPI solution, wash it three times with PBS.
[0019] The beneficial effects of the present invention are as follows: The present invention uses the adherent method of sweat gland tissue blocks to culture sweat gland cells. Among them, at the time point of one week after the cells grow, trypsin digestion solution is used to obtain and isolate sweat gland myoepithelial cells. In addition, through the difference in cell adhesion ability, highly pure sweat gland myoepithelial cells are extracted, which is inconsistent with the method reported in the current literature of digesting sweat gland tissue into cell suspension with collagenase, Accutase enzyme or hyaluronidase and obtaining sweat gland myoepithelial cells by flow cytometry screening using myoepithelial cell marker antibodies. This method has the advantages of simple operation, low technical threshold, high cell viability, and low cost, and is suitable for the use of most laboratories. Through long-term in vitro amplification culture, a large number of sweat gland myoepithelial cells with high cell viability and high purity are obtained, meeting the needs of basic scientific research and sweat gland tissue engineering regeneration.
[0020] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.
[0021] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings
[0022] Figure 1 It is a schematic flowchart of the method for isolating primary sweat gland myoepithelial cells provided by an embodiment of the present invention;
[0023] Figure 2 It is a morphological photograph of the isolated sweat gland secretory coil observed under an inverted optical microscope;
[0024] Figure 3 It is an optical microscope scan of the sweat gland myoepithelial cells grown after 3 days of the sweat gland tissue adhering to the wall;
[0025] Figure 4 It is an optical microscope scan of the sweat gland myoepithelial cells and other sweat gland epithelial cells grown after 7 days of the sweat gland tissue adhering to the wall;
[0026] Figure 5 It is an optical microscope scan of the isolated 3rd generation of human primary sweat gland myoepithelial cells;
[0027] Figure 6 It is an optical microscope scan of the isolated 12th generation of human primary sweat gland myoepithelial cells;
[0028] Figure 7 It is a schematic diagram of the immunofluorescence staining results of epithelial marker CK7 (green), mesenchymal marker α-SMA (red), and nuclear staining DAPI (blue) in the 3rd generation of human primary sweat gland myoepithelial cells. Detailed Description of the Invention
[0029] The following uses specific embodiments to illustrate the present invention, which is not a limitation to the present invention.
[0030] As Figures 1-7 shown, a method for in vitro isolation, culture and identification of human primary sweat gland myoepithelial cells
[0031] The skin specimens obtained surgically were placed in a sterile PBS solution containing 1× penicillin (100 U / ml) - streptomycin (0.1 g / ml) double antibiotics and stored at low temperature; on a super clean bench sterilized by ultraviolet irradiation, they were washed twice again with PBS containing 1× double antibiotics to remove residual blood and tissue debris; the skin tissue was cut into pieces of 0.5 cm3 in size, transferred to another clean petri dish with the epidermal surface facing down, and 2.5 mg / ml neutral protease solution was added, and digested in a 37°C 5% CO2 cell culture incubator for 1 hour; then the petri dish was taken out, the epidermis was removed with forceps, and the white dermal part was washed twice in a clean PBS solution and placed in a 60 mm petri dish for standby; in the petri dish, the dermis was cut into pieces of 1 mm3 in size with ophthalmic scissors, 3 ml of dermal digestive solution containing 20 mg / ml type II collagenase was added, and digested in a 37°C cell culture incubator for 2 hours; after 2 hours, under an inverted microscope (40×) sterilized by ultraviolet irradiation, the sweat gland tissue was picked out with a 200 um pipette and placed in the complete medium for sweat glands and washed 2 - 3 times; then the sweat gland tissue was picked out again and placed in a 24-well plate, and 3 - 4 sweat gland tissues were planted in each well; an appropriate amount of complete medium was added, just covering the sweat gland tissue without suspending it; the complete medium for sweat glands was based on DMEM / F12 supplemented with 10% FBS, 1× penicillin-streptomycin double antibiotics, 2 ng / ml triiodothyronine, 10 ug / ml transferrin, 10 ng / ml rhEGF, 1 ug / ml insulin, 2 mmol / L L-glutamine, 0.4 ug / ml hydrocortisone; after the sweat gland explants adhered for 1 - 2 days, myoepithelial cells could grow out, and then 200 ul of complete medium could be supplemented and the medium was changed every 2 - 3 days; after 1 week when cells grew out from the sweat gland tissue, 0.25% trypsin digestive solution was used to digest and isolate primary sweat gland myoepithelial cells, and complete medium was added to terminate digestion. The cell suspension was centrifuged at 1000 r / s for 5 min, resuspended with complete medium, and planted in a 6-well plate. After culturing in a 37°C cell culture incubator for 2 hours, the old medium was discarded and freshly preheated complete medium was replenished, and then the medium was changed by half every 2 - 3 days, which was recorded as the first generation of cells; when the cell growth confluence reached 80 - 90%, subculture could be carried out;
[0032] Human sweat gland myoepithelial cells were seeded onto 24-well plates containing cell culture slides. When the cell confluence reached 60 - 80% and the growth state was good, the following experiments could be carried out; discard the culture medium, wash twice with PBS, fix with 4% paraformaldehyde for 15 min; wash three times with PBS again, add 0.3% Triton X-100 solution for permeabilization for 5 min; wash three times with PBS again, then block with 5% BSA at room temperature for 1 h; discard the blocking solution and add the prepared primary antibody solution (dilution ratio 1:100), incubate in a 4°C refrigerator for 12 h; discard the primary antibody solution, then wash three times with PBS, add the prepared secondary antibody solution (dilution ratio 1:1000), incubate at room temperature in the dark for 1 h; discard the secondary antibody solution, then wash three times with PBS, add 10 μg / ml DAPI solution, incubate in the dark for 5 min; discard the DAPI solution, wash three times with PBS; use forceps to pick out the slide and invert it onto a clean glass slide, and then observe and take pictures under an inverted fluorescence microscope (Olympus).
[0033] After the sweat glands ( Figure 2 ) adhered for about 1 - 2 days, it could be seen that cells began to crawl out of the sweat gland explants ( Figure 3 ). After 3 - 4 days, the number of migrating cells increased. These cells were spindle-shaped "long fusiform", with small cell bodies and long protrusions, and had a fast migration and proliferation ability. They were the myoepithelial cells of the sweat glands ( Figure 3 ). As the myoepithelial cells migrated out, after about 7 - 10 days, other epithelial cells (secretory cells and duct cells) in the sweat glands also gradually migrated out, showing a "paving stone-like" polygon, and covered the adjacent myoepithelial cells, forming a multi-layer structure ( Figure 4 ).
[0034] We obtained sweat gland myoepithelial cells by differential digestion and differential attachment methods. Under an inverted optical microscope, the cells were fibroblast-like, with slender protrusions, and were difficult to distinguish from dermal fibroblasts with the naked eye ( Figure 5 ). However, when the sweat gland myoepithelial cells grew confluent, they did not form the "spiral-like" structure of fibroblasts. As the number of passages increased, the myoepithelial cells gradually aged, manifested as slower cell proliferation, nuclear pyknosis, longer protrusions, and the cells grew in a disorderly and stacked manner ( Figure 6 ). The sweat gland myoepithelial cells had the epithelial cell marker CK7 and also expressed the mesenchymal cell marker α-SMA, and could be determined as the myoepithelial cells of the sweat glands ( Figure 7 ).
[0035] It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.
[0036] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made under the inventive concept of the present invention by using the content of the specification and drawings of the present invention, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.
[0037] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for isolating, culturing and identifying primary human sweat gland myoepithelial cells in vitro, characterized in that: Including obtaining skin specimens, low temperature preservation, cleaning, digestion, cleaning, digestion, irradiation and disinfection, implant attachment, separation and planting, discarding culture medium and observation and photography, The specific operation modes are as follows: (1) Obtaining a skin specimen: Obtaining a skin specimen by surgery; (2) The cryopreservation: placing in a sterile PBS solution containing 1× penicillin (100 U / ml)-streptomycin (0.1 g / ml) for cryopreservation; (3) Cleaning: On a clean bench sterilized by ultraviolet irradiation, wash twice with PBS containing 1× double antibody to remove residual blood and tissue debris; (4) Digestion: Cut the skin tissue into 0.5 cm3 pieces, transfer to another clean culture dish, with the epidermis facing downward, add 2.5 mg / ml neutral protease solution, and digest in a 37°C 5% CO2 cell culture incubator for 1 hour; (5) Cleaning: After that, take out the culture dish, remove the epidermis with tweezers, wash the white dermis twice in a clean PBS solution and place it in a 60 mm culture dish for later use; (6) Digestion: Cut the dermis into pieces of 1 mm3 in size using ophthalmic scissors in a culture dish, add 3 ml of dermal digestion solution containing 20 mg / ml type II collagenase, and digest in a cell culture incubator at 37°C for 2 hours; (7) Irradiation disinfection: After 2 hours, under an inverted microscope (40×) that has been disinfected by ultraviolet irradiation, the sweat gland tissue is picked out using a 200 μm pipette and placed in a complete sweat gland culture medium and washed 2-3 times; (8) The explant adheres to the wall: After the sweat gland explant adheres to the wall for 1-2 days, myoepithelial cells can grow out, and then 200ul of complete culture medium can be added, and the culture medium is replaced every 2-3 days; (9) Isolation and planting: After the sweat gland tissue has grown cells for 1 week, the primary sweat gland myoepithelial cells are isolated by digestion with 0.25% trypsin digestion solution, and complete culture medium is added to terminate the digestion. The cell suspension is centrifuged at 1000 r / s for 5 min, resuspended with complete culture medium, and planted in a 6-well plate. After culturing in a 37°C cell culture incubator for 2 hours, the old culture medium is discarded and fresh preheated complete culture medium is added again. After that, half of the medium is replaced every 2-3 days. The cells are recorded as the first generation of cells. When the cell confluence reaches 80-90%, they can be passaged. (10) Observation and photography: Use tweezers to pick up the slide and place it upside down on a clean glass slide, and then observe and photograph it under an inverted fluorescence microscope (Olympus).
2. The in vitro isolation, culture and identification method of primary human sweat gland myoepithelial cells according to claim 1, characterized in that: The culture medium was discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, permeabilized with 0.3% Triton X-100 solution for 5 min, washed three times with PBS, blocked with 5% BSA at room temperature for 1 h, the blocking solution was discarded, the prepared primary antibody solution (dilution ratio 1:100) was added, incubated in a 4°C refrigerator for 12 h, the primary antibody solution was discarded, the cells were washed three times with PBS, the prepared secondary antibody solution (dilution ratio 1:1000) was added, incubated in the dark at room temperature for 1 h, the secondary antibody solution was discarded, the cells were washed three times with PBS, 10 ug / ml DAPI solution was added, incubated in the dark for 5 min, the DAPI solution was discarded, and the cells were washed three times with PBS.