CN117660306A - Method for culturing keratinocytes - Google Patents
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- CN117660306A CN117660306A CN202311710007.8A CN202311710007A CN117660306A CN 117660306 A CN117660306 A CN 117660306A CN 202311710007 A CN202311710007 A CN 202311710007A CN 117660306 A CN117660306 A CN 117660306A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for culturing keratinocytes, which comprises the following steps: isolation of primary keratinocytes: taking human skin tissue, cleaning, shearing, and sequentially adding digestive juice, pancreatin and DNase for digestion; filtering with 100 μm cell filter, centrifuging to obtain cell precipitate, re-suspending in inoculating culture medium, culturing, and separating fibroblast and keratinocyte by pancreatin digestion time difference; (II) three-dimensional culture of keratinocytes: resuspending keratinocytes in the seeding medium and culturing for 3 days; the liquid is changed into keratinocyte culture liquid, and the keratinocyte culture liquid is cultured for 7 days. The method does not need to separate two layers of tissues, and avoids long-time digestion and complex experimental operation; can promote cell proliferation, promote cell adhesion and growth, and improve keratinocyte proliferation and mobility. The keratinocyte culture method of the present invention is of great importance for the culture of keratinocytes.
Description
Technical Field
The invention relates to a method for culturing keratinocytes, and belongs to the technical field of cell culture.
Background
The skin is the largest organ of the human body and is the first protective barrier to protect tissue integrity from external stimuli. The skin structure is divided into two layers: skin layers and dermis layers. Wherein the most predominant cells in the epidermis are keratinocytes, accounting for more than 95% of the epidermis cells. Keratinocytes can differentiate at a certain stage from basal layer to spiny, to granular, and finally to stratum corneum with protective effect, thus forming complete whole skin, thereby achieving barrier effect and playing an important role in innate immune response. The most predominant cells in the dermis are fibroblasts, whose main function is to make extracellular matrix such as collagen, etc., to move the skin repair process towards regeneration or scarring, while fibroblasts secrete cytokines to regulate immune cells.
Three-dimensional (3D) cell culture is a powerful in vitro technique for studying keratinocyte stratification and differentiation. Three-dimensional (3D) culture has been shown to provide cells with more appropriate physiological conditions than conventional planar 2D culture systems, and is more suitable for proliferation, migration or protein expression of these functional cells.
Clinically refractory skin ulcers, including diabetic complications, venous ischemia, pyoderma gangrenosum ulcers, and some other trauma or burns and scalds, have been headache-prone, and conventional treatments often fail to achieve satisfactory results, requiring functional, permanent skin substitutes, providing barrier function to the patient's skin.
In the prior art, when keratinocytes are cultured, skin tissues need to be digested overnight to separate epidermis from dermis, and the digestion time is long, so that the activity of primary cells is influenced. The Chinese patent application CN 116004518A discloses a method for extracting and culturing human primary keratinocytes and fibroblasts, which comprises the steps of carrying out low-intensity ultrasound on tissues at 4 ℃ and then mechanically separating to obtain epidermis layers and dermis layers, respectively carrying out low-temperature reviving on the tissues, carrying out complex operation and easy bacterial contamination, and carrying out culture by using a product culture medium Epilife in the primary stage, wherein the proliferation speed is low, the dryness of the epidermis cells is weak, and the cells are easy to age.
Disclosure of Invention
In view of the above prior art, the present invention provides a method for culturing keratinocytes.
The invention is realized by the following technical scheme:
a method of culturing keratinocytes comprising the steps of:
isolation of (one) Primary keratinocytes
(1) Taking human skin tissue, cleaning, shearing to obtain tissue homogenate;
(2) Adding the digestion solution, and digesting for 25-35 minutes, preferably 30 minutes;
(3) Adding pancreatin, and digesting for 8-12 min, preferably 10 min;
(4) Adding DNase, and digesting for 4-6 minutes, preferably 5 minutes;
(5) Stopping digestion, and blowing and uniformly mixing for 15-25 times, preferably 20 times, so as to fully release cells;
(6) Filtering with 100 μm cell filter to remove tissue fragments, centrifuging supernatant to obtain cell precipitate;
(7) Resuspending the cell pellet in seeding medium, placing in T75 cell culture flask, 37 ℃ and 5% CO 2 Culturing under saturated humidity condition;
(8) When primary cells are cultured to have a fusion rate of 85% -90%, removing the culture medium, adding pancreatin, digesting for 30 seconds, obtaining the cells obtained by digestion, namely fibroblasts, and centrifuging (300 g,10 min) to obtain the fibroblasts; adding pancreatin into the supernatant again, digesting for 5 min, obtaining keratinocytes as cells obtained by the second digestion, and centrifuging (300 g,10 min) to obtain keratinocytes; the fibroblasts and keratinocytes were cultured separately until passaging. In this step, fibroblasts and keratinocytes are separated by a differential pancreatin digestion time.
Three-dimensional culture of keratinocytes
(9) Will be put onThe obtained keratinocytes (primary or passage) were resuspended in inoculation medium, plated uniformly on pre-coated plates by pipetting gun, 37 ℃,5% co 2 Culturing for 3 days under saturated humidity condition;
(10) Changing the inoculating culture medium into keratinocyte culture medium, 37 deg.C, 5% CO 2 Continuously culturing for 7 days under the saturated humidity condition;
the keratinocyte culture solution comprises the following components in percentage by volume: penicillin, 80-120U/ml; streptomycin, 0.08-0.12 mg/ml; FGF-2 (fibroblast growth factor 2), 75-85 ng/mL; EGF (epidermal growth factor) 45-55 ng/mL; b27 has no serum additive and is 1.8 to 2.2 percent; hydrocortisone, 0.8-1.2. Mu.M; 15 to 25 portions of progesterone, nM; transferrin, 8-12 μg/μl; adenine, 35-45. Mu.M; umbilical cord mesenchymal stem cell conditioned medium, 0-8%; the balance being cell culture medium. Preferably, the keratinocyte culture fluid comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 80 ng/mL; EGF,50 ng/mL; b27 no serum additive, 2%; hydrocortisone, 1 μm; progesterone, 20 nM; transferrin, 10 μg/μl; adenine, 40. Mu.M; umbilical cord mesenchymal stem cell conditioned medium, 5%; the balance being DMEM/F12 medium.
Further, in the step (1), the specific mode of cleaning is as follows: washing 1 time with PBS buffer solution, washing 1 time with 70% (volume percent) ethanol solution, and incubating human skin tissue with the washing solution for 2 times for 5 minutes each time; the washing liquid comprises the following components: penicillin, 1.5-2.5U/ml; streptomycin 1.5-2.5 mg/l; the balance being PBS buffer. Preferably, it is: penicillin, 2U/ml; streptomycin, 2 mg/l; the balance being PBS buffer.
Further, in the step (2), the digestive juice comprises the following components: neutral proteinase, 4-6 mg/ml; collagenase type I, 4-6 mg/ml; the balance being cell culture medium. Preferably, it is: neutral protease, 5 mg/ml; collagenase type I, 5 mg/ml; the balance being F12 medium.
Further, in the steps (3) and (8), pancreatin is added in the form of 0.25% pancreatin cell digest.
Further, in the step (5), a neutralization medium is added to stop digestion; the neutralization culture medium comprises the following components: penicillin, 80-120U/ml; streptomycin, 0.08-0.12 mg/ml; fetal bovine serum, 8% -12% (volume ratio); the balance being cell culture medium. Preferably, it is: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; fetal bovine serum, 10%; the balance being DMEM/F12 medium.
Further, in the step (7), the inoculating culture medium comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 70-90 ng/mL; EGF, 45-55 ng/mL; the balance being cell culture medium. Preferably, it is: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 80 ng/mL; EGF,50 ng/mL; the balance being DMEM/F12 medium.
Further, in the step (9), the pre-coated culture plate is obtained by: taking a porous high polymer medical polyvinyl chloride sheet (PVC sheet), cutting a circular sheet with the diameter of 8.0-9.0 mm at the center to form a donut structure; immersing the PVC sheet in a cell culture medium containing hyaluronic acid and chitosan, and pre-coating for 1.5-2.5 hours to form a porous culture environment and medium between gel and liquid, so as to increase the adhesiveness, and have the effects of promoting cell adhesion and proliferation, shortening the culture time and preventing keratinocyte aging; taking out, and cleaning with physiological saline; spreading the PVC sheet on a cell culture plate to obtain the final product.
Compared with the traditional separation method (which requires overnight digestion, separation of dermis and epidermis by physical methods such as instruments and the like, and digestion respectively) in the prior art, the culture method of keratinocytes does not need to separate two layers of tissues, only needs to treat the tissues into homogenates, separates two types of skin cells through pancreatin hydrolysis time difference after cell adhesion, avoids long-time digestion and complex experimental operation, and only needs 3 hours in the whole extraction process; meanwhile, incomplete epidermis separation is avoided, and the probability of pollution of the mixed cells is reduced. The culture medium prepared by combining the high polymer material 3D culture and the donut structure adopted during culture can promote the proliferation speed of cells, central cells can also contact with culture solution, the adhesion and growth are rapid, the skin dryness is uniform, and the expression of the microtubule actin factor 1 is obviously higher than that of the traditional culture mode. The cell culture solution can make the proliferation and mobility of keratinocyte faster than the traditional method and age slowly, and can fully utilize the condition culture medium of umbilical cord mesenchymal stem cells abandoned by amplifying umbilical cord mesenchymal stem cells in daily work, thereby reducing the culture cost. The culture method of the invention has important significance for the culture of keratinocytes.
Drawings
Fig. 1: scanning electron microscope pictures of PVC sheets.
Fig. 2: ELISA detection results are schematically shown.
Fig. 3: the result of the cell scratch experiment is schematically shown, wherein the left side is a photograph of a control group, and the right side is a photograph of an experimental group.
Fig. 4: western blot results are schematically shown.
Fig. 5: CCK8 shows the result of detecting cell proliferation, wherein the configured culture medium represents an experimental group and EPIlife represents a control group.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.
EXAMPLE 1 culture of keratinocytes
Main experimental reagent: PBS buffer (burning), green streptomycin mix (100X) (penicillin content 10 kU/ml, streptomycin content 10 mg/ml) (Gibco), neutral protease (sigma), type I collagenase (Soy treasure), F12 medium (Gibco), 0.25% pancreatin cell digest (Gibco), DNase (Soy treasure), fetal bovine serum (cell-box), DMEM/F12 medium (Gibco), FGF-2 (MCE), EGF (MCE), B27 serum free additive (Gibco), hydrocortisone (Soy treasure), progesterone (Biyun), transferrin (Soy treasure), adenine (Soy treasure), nutriStem MSCXF Basal medium medium (BI), chitosan (sigma 448869), epile medium (Gibco).
The method comprises the following steps:
isolation of (one) Primary keratinocytes
(1) Human skin tissue 1 g was taken, washed 1 time with 10 mL of PBS buffer and then with 70% ethanol for 1 minute. Human skin tissue was then incubated with 10 mL washes for 2 times, 5 minutes each, with fresh 100 mm cells each time to culture the sterile petri dishes.
Human skin tissue was transferred to another 100 mm cell culture sterile petri dish, and the tissue was thoroughly minced with tissue scissors to obtain a tissue homogenate, 200 μl of PBS buffer was added every 5 minutes, and this was done within 10 minutes to ensure tissue wettability.
The components of the washing liquid are as follows: penicillin, 2U/ml; streptomycin, 2 mg/l; the balance being PBS buffer.
(2) Adding 10 ml digestive juice into the tissue homogenate, and performing water bath digestion for 30 minutes at 37 ℃ to obtain primary digestive juice; the digestive juice comprises the following components: neutral protease, 5 mg/ml; collagenase type I, 5 mg/ml; the balance being F12 medium.
(3) Adding 0.25% pancreatin cell digestion solution with total volume of 1/10 into the preliminary digestion solution, and performing water bath digestion at 37 ℃ for 10 minutes to obtain pancreatin digestion solution.
(4) Adding DNase with final concentration of 10 mg/ml into the pancreatin digestion solution, and standing at room temperature for 5 minutes to obtain DNase treatment solution.
(5) An equal volume of neutralization medium was added to the above cell digestate to stop digestion, and the mixture was blown and mixed 20 times to release the cells thoroughly. The components of the neutralization medium are as follows: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; fetal bovine serum, 10%; the balance being DMEM/F12 medium.
(6) The resulting solution was filtered through a 100 μm cell filter to remove tissue debris, and the supernatant was centrifuged at 300 g for 10 minutes to obtain a cell pellet.
(7) Resuspending the cell pellet in seeding medium, placing in T75 cell culture flask, 37 ℃ and 5% CO 2 Culturing under saturated humidity condition.
The inoculating culture medium comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 80 ng/mL; EGF,50 ng/mL; the balance being DMEM/F12 medium.
(8) When the primary cells are cultured to 90% of fusion rate, the culture medium is discarded, 0.25% pancreatin cell digestive juice of 1 ml is added, and the cells obtained by digestion are fibroblasts, and the fibroblasts are obtained by centrifugation (300 g,10 min); adding 0.25% pancreatin cell digestive juice of 1 ml into the supernatant again, digesting for 5 min, obtaining keratinocytes as cells obtained by the second digestion, centrifuging (300 g,10 min), obtaining keratinocytes; the fibroblasts and keratinocytes were cultured separately until passaging. In this step, fibroblasts and keratinocytes are separated by a differential pancreatin digestion time.
(II) culture of keratinocytes
(9) Resuspending the keratinocytes obtained by centrifugation in an inoculation medium, uniformly spreading on a pre-coated culture plate by a pipetting gun, and at 37 ℃ 5% CO 2 Culturing for 3 days under saturated humidity condition.
The pre-coated plates were obtained by: cutting a porous high polymer medical polyvinyl chloride sheet (PVC sheet) to 6 pore plate sizes (127 mm multiplied by 85 mm), cutting a circular sheet (equivalent to the pore diameter of a 24 pore plate) with the diameter of about 8.5 mm at the center, and forming a donut structure; adding 2 ml sodium hyaluronate solution (the concentration of the sodium hyaluronate solution is 5 mg/L) and 1 g chitosan into 50 DMEM/F12 culture medium of ml, immersing the cut PVC sheet into the culture medium, and pre-coating the PVC sheet in a 37 ℃ incubator for 2 hours to form a porous culture environment and medium between gel and liquid, so as to increase adhesiveness, and have the effects of promoting cell adhesion and proliferation, shortening culture time and preventing keratinocyte aging; taking out, and cleaning with physiological saline; and (5) tiling the pretreated PVC sheet on the bottom of the 6-pore plate.
(10) Changing the inoculating culture medium into keratinocyte culture medium, 37 deg.C, 5% CO 2 Culturing under saturated humidity for 7 days.
The keratinocyte culture solution comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 80 ng/mL; EGF,50 ng/mL; b27 no serum additive, 2%; hydrocortisone, 1 μm; progesterone, 20 nM; transferrin, 10 μg/μl; adenine, 40. Mu.M; umbilical cord mesenchymal stem cell conditioned medium, 5%; the balance being DMEM/F12 medium.
The umbilical cord mesenchymal stem cell conditioned medium is a commercial medium: nutriStem MSCXF Basal medium medium.
Example 2 Performance test
(1) Electron microscope experiment: the pretreated PVC sheet of step (9) of example 1 was fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1M sodium carbonate buffer (pH 7.3); after washing with PBS buffer, fixation in 1% osmium tetroxide aqueous solution for 1 hour, then transfer from ethanol to acetic acid amide, and then critical point drying with liquid carbon dioxide; the dried sample was sputter coated with platinum palladium (Hitachi, E1030, japan) and observed with Hitachi S4700, as shown in FIG. 1, and as can be seen from FIG. 1, the porous and adherent coated hyaluronic acid component is a suitable cell scaffold for cell adhesion and proliferation.
(2) ELISA detection FGF, TGF-. Beta.1 assay: the content of FGF and TGF-beta 1 in a sample is determined by a human FGF and TGF-beta 1 quantitative factor ELISA kit, and the amount of the specific binding antigen is detected by using a corresponding specific antibody (a species specific antibody coupled to horseradish peroxidase) provided in the matched kit. The color development was stopped and the intensity of the color was measured at 450 nm.
The samples of the experimental group are: the culture broth obtained in step (10) of example 1 was cultured for 7 days, and centrifuged (300 g,10 min) to obtain a supernatant.
The control group samples were: example 1 in step (10), the inoculation medium was changed to Epilife medium, 37℃and 5% CO 2 Culturing for 7 days under saturated humidity condition, centrifuging the culture solution (300 g,10 min) to obtain supernatant.
As shown in FIG. 2, the absolute quantitative values of FGF and TGF-beta 1 are obtained by ELISA, and as can be seen from FIG. 2, the content of FGF and TGF-beta 1 in the experimental group is far greater than that in the control group, which shows that the effect of the keratinocyte culture solution of the present invention is superior to that of the Epilife culture medium.
(3) Cell scratch assay
Experimental group: taking culture solution 1 ml obtained by culturing in step (10) of example 1 for 7 days, adding 0.25% pancreatin cell digestive juice of 1 ml, and digesting for 3 minutes; centrifuging (300 g,10 min), re-suspending the obtained cell sediment in 1 ml keratinocyte culture solution, inoculating into a 6-well plate, and culturing for 24 hours; at 95% fusion level, 10 μg/mL mitomycin C was added to prevent cell proliferation. The medium was removed, streaked onto cells with 200 μl microtube tip, the resulting wound lines were rinsed with PBS buffer, and 1 ml keratinocyte culture broth was added for culture, and recorded by photographing at 0 and 24 hours.
Control group: example 1 in step (10), the inoculation medium was changed to Epilife medium, 37℃and 5% CO 2 Culturing under saturated humidity for 7 days. Taking culture solution 1 ml, adding 0.25% pancreatin cell digestive juice of 1 ml, and digesting for 3 minutes; centrifuging (300 g,10 min), and re-suspending the obtained cell sediment in an Epilife culture medium of 1 ml, inoculating the cell sediment into a 6-well plate for culturing for 24 hours; at 95% fusion level, 10 μg/mL mitomycin C was added to prevent cell proliferation. The medium was removed, streaked onto cells with 200. Mu.l microtube tip, the resulting wound line was rinsed with PBS buffer, and 1 ml of Epilife medium was added for culture, and the recordings were taken at 0 and 24 hours.
The results of the cell scarification experiment are shown in FIG. 3. As can be seen from fig. 3, after 24 hours, the experimental group cultured keratinocytes were greater in both healing rate and area than the control group.
(4) Western blot experiment: total proteins in cells of the experimental group sample and the control group sample were extracted by RIPA (4 parallel experiments were performed, numbered 1, 2, 3, 4), and the extract was centrifuged by a pre-cooling centrifuge 10000 g at 4℃for 30 min. The supernatant was collected and the protein concentration in the supernatant was determined using BCA protein assay kit (MCE). The supernatant was subjected to SDS-PAGE and transferred to PVDF membrane. Anti-microtubule actin crosslinking factor 1 (1:1000 dilution, santa), anti-tumor associated dockerin (N-RAP) (1:1000 dilution, santa) was used for detection and secondary antibody was incubated with HRP conjugated goat anti-rabbit IgG.
The samples of the experimental group are: example 1 cells after 7 days of culture in step (10) were subjected to RIPA extraction to give total protein.
The control group samples were: example 1 in step (10), the inoculation medium was changed to Epilife medium, 37℃and 5% CO 2 And (3) continuously culturing the cells for 7 days under the saturated humidity condition, and extracting total protein from the cells by RIPA.
The result of the western blot experiment is shown in fig. 4, and the loading sequence is (corresponding to the bands in fig. 4, from left to right): experimental group 1, experimental group 2, control group 1, control group 2, experimental group 3, experimental group 4, control group 3, control group 4. As can be seen from FIG. 4, the expression of the microtubule actin crosslinking factor 1 in the experimental group is higher than that in the control group, and the expression of the anti-tumor related anchoring protein is also higher than that in the control group, which indicates that the cells cultured by the method of the invention have stronger biological functions, higher cell activity and better dryness, and are convenient for subsequent differentiation to form full-thickness skin for clinical treatment.
(5) CCK8 assay for cell proliferation
Experimental group: the culture broth in the flask after 7 days of the culture in step (10) of example 1 was discarded, 0.25% pancreatin cell digest of 1 ml was added, digested for 3 minutes, centrifuged (300 g,10 min), and the resulting cell pellet was resuspended in 1 ml keratinocyte culture broth to obtain a cell suspension. 100 μl of cell suspension was added to each well of the 96-well plate, three wells per group. This procedure was performed in 1 96-well plate, 7 96-well plates were used in total, and the culture was performed for 7 days. 10 μl of cck8 solution was added to each well of the plates at the same time every day, and the plates were incubated in the incubator for 2 hours; the absorbance at 450 nm was measured with a microplate reader and continuously measured for 7 days.
Control group: example 1 in step (10), the inoculation medium was changed to Epilife medium, 37℃and 5% CO 2 Culturing under saturated humidity for 7 days. The flask was discarded, 1 ml of 0.25% pancreatin cell digest was added, digested for 3 minutes, centrifuged (300 g,10 min) and the resulting cell pellet resuspended in 1 ml of epiif medium to give a cell suspension. 100 μl of cell suspension was added to each well of the 96-well plate, three wells per group. This procedure was performed in 1 96-well plate, 7 96-well plates were used in total, and the culture was performed for 7 days. 10 μl of cck8 solution was added to each well of one of the plates at the same time every day, and the plates were incubated in the incubator for 2 hours; the absorbance at 450 nm was measured with a microplate reader and continuously measured for 7 days.
The results of CCK8 detection are shown in FIG. 5. As can be seen from fig. 5, the proliferation rate of keratinocytes in the experimental group is significantly higher than that in the control group, and it is also proved that the PVC sheet pretreated by the present invention is an excellent cell scaffold without toxicity to cells.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (10)
1. A method of culturing keratinocytes, comprising the steps of:
isolation of (one) Primary keratinocytes
(1) Taking human skin tissue, cleaning, shearing to obtain tissue homogenate;
(2) Adding digestive juice, and digesting for 25-35 minutes;
(3) Adding pancreatin and digesting for 8-12 minutes;
(4) Adding DNase, and digesting for 4-6 minutes;
(5) Stopping digestion, and blowing and mixing uniformly to fully release cells;
(6) Filtering with 100 μm cell filter to remove tissue fragments, centrifuging supernatant to obtain cell precipitate;
(7) Resuspending the cell sediment in an inoculation culture medium for culture;
(8) When the primary cells are cultured to have the fusion rate of 85-90%, the culture medium is discarded, pancreatin is added, the primary cells are digested for 30 seconds, and the primary cells are centrifuged to obtain fibroblasts; adding pancreatin into the supernatant again, digesting for 5 minutes, centrifuging to obtain keratinocytes;
three-dimensional culture of keratinocytes
(9) Resuspending the keratinocytes obtained above in an inoculation medium, spreading on a pre-coated culture plate, at 37deg.C, 5% CO 2 Culturing for 3 days under saturated humidity condition;
(10) Changing the inoculating culture medium into keratinocyte culture medium, 37 deg.C, 5% CO 2 Continuously culturing for 7 days under the saturated humidity condition;
the keratinocyte culture solution comprises the following components: penicillin, 80-120U/ml; streptomycin, 0.08-0.12 mg/ml; FGF-2, 75-85 ng/mL; EGF, 45-55 ng/mL; b27 has no serum additive and is 1.8 to 2.2 percent; hydrocortisone, 0.8-1.2. Mu.M; 15 to 25 portions of progesterone, nM; transferrin, 8-12 μg/μl; adenine, 35-45. Mu.M; umbilical cord mesenchymal stem cell conditioned medium, 0-8%; the balance being cell culture medium.
2. The method of claim 1, wherein in the step (1), the washing is performed by: washing with PBS buffer solution for 1 time, washing with 70% ethanol solution for 1 time, and incubating human skin tissue with the washing solution for 2 times for 5 minutes each time; the washing liquid comprises the following components: penicillin, 1.5-2.5U/ml; streptomycin 1.5-2.5 mg/l; the balance being PBS buffer.
3. The method of culturing keratinocytes according to claim 1, wherein in the step (2), the composition of the digestive juice is as follows: neutral proteinase, 4-6 mg/ml; collagenase type I, 4-6 mg/ml; the balance being cell culture medium.
4. The method for culturing keratinocytes according to claim 1, wherein: in the steps (3) and (8), pancreatin is added in the form of 0.25% pancreatin cell digest.
5. The method for culturing keratinocytes according to claim 1, wherein: in the step (5), a neutralization medium is added to stop digestion; the neutralization culture medium comprises the following components: penicillin, 80-120U/ml; streptomycin, 0.08-0.12 mg/ml; fetal bovine serum, 8% -12%; the balance being cell culture medium.
6. The method for culturing keratinocytes according to claim 1, wherein: in the step (7), the inoculating culture medium comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 70-90 ng/mL; EGF, 45-55 ng/mL; the balance being cell culture medium.
7. The method for culturing keratinocytes according to claim 2, 3, 5 or 6, wherein: the washing liquid comprises the following components: penicillin, 2U/ml; streptomycin, 2 mg/l; the balance of PBS buffer solution;
the digestive juice comprises the following components: neutral protease, 5 mg/ml; collagenase type I, 5 mg/ml; the balance being F12 culture medium;
the neutralization culture medium comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; fetal bovine serum, 10%; the balance of DMEM/F12 culture medium;
the inoculating culture medium comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 80 ng/mL; EGF,50 ng/mL; the balance being DMEM/F12 medium.
8. The method of claim 1, wherein in step (9), the pre-coated culture plate is obtained by: cutting a PVC sheet at the center, and cutting a circular sheet with the diameter of 8.0-9.0 mm; immersing the PVC sheet into a cell culture medium containing hyaluronic acid and chitosan, and pre-coating for 1.5-2.5 hours; taking out, and cleaning with physiological saline; spreading the PVC sheet on a cell culture plate to obtain the final product.
9. The method of claim 8, wherein the pre-coated culture plate is obtained by: cutting a PVC sheet to the size of a 6-pore plate, and cutting a circular sheet with the diameter of 8.0-9.0 mm at the center to form a donut structure; adding 2 ml sodium hyaluronate solution with the concentration of 5 mg/L and 1 g chitosan into 50 DMEM/F12 culture medium of ml, immersing the cut PVC sheet into the solution, and pre-coating the PVC sheet in a culture box at 37 ℃ for 2 hours; taking out, and cleaning with physiological saline; and (5) spreading the PVC sheet on the bottom of the 6-pore plate.
10. The method of claim 1, wherein in step (10), the keratinocyte culture fluid comprises the following components: penicillin, 100U/ml; streptomycin, 0.1 mg/ml; FGF-2, 80 ng/mL; EGF,50 ng/mL; b27 no serum additive, 2%; hydrocortisone, 1 μm; progesterone, 20 nM; transferrin, 10 μg/μl; adenine, 40. Mu.M; umbilical cord mesenchymal stem cell conditioned medium, 5%; the balance being DMEM/F12 medium.
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