CN112375731A - Method for separating and culturing skin fibroblast - Google Patents
Method for separating and culturing skin fibroblast Download PDFInfo
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- CN112375731A CN112375731A CN202011331103.8A CN202011331103A CN112375731A CN 112375731 A CN112375731 A CN 112375731A CN 202011331103 A CN202011331103 A CN 202011331103A CN 112375731 A CN112375731 A CN 112375731A
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Abstract
The invention discloses a method for separating and culturing skin fibroblasts, and belongs to the field of cell culture. The method of the invention carries out trypsin pretreatment on skin tissue, fixes the treated tissue scratch in a culture dish for culture, and separates out skin fibroblast. The method can obtain a large amount of fibroblasts in a short time, improves the efficiency, saves time and raw materials, can ensure the proliferation capacity and passage activity of the obtained fibroblasts, and is favorable for the fibroblasts to fully play a role in subsequent utilization.
Description
Technical Field
The invention relates to a method for separating and culturing skin fibroblasts, belonging to the field of cell culture.
Background
Fibroblasts are the most important cells in the dermal reticular layer of the skin, are mainly present in loose connective tissue, can synthesize and secrete a large amount of collagen, and have an important role in maintaining the elasticity and toughness of the skin. The fibroblasts separated from the tissues have high proliferation potential and are widely applied to the aspects of skin burn repair, cosmetology, regenerative medicine and the like. Also, reprogramming fibroblasts to induce pluripotent stem cells has become an important use of fibroblasts due to the development of stem cell technology. The rapid and efficient isolation of fibroblasts from skin tissue has become an important part of the field of stem cell biotechnology.
The current methods for isolating dermal fibroblasts mainly include two methods, enzyme digestion and tissue mass adherence. The tissue block adherence method enables dermal fibroblasts to climb out of a tiny tissue block by fixing the dermal tissue block, but the operation finds that the method has high demand on the tissue block and is easy to cause tissue waste; moreover, because the tissue is compact, the cells are not easy to obtain nutrition, so that the cell activity and the proliferation capacity are not good; the culture time is long, and the pollution is easy to occur in the culture process.
The enzyme digestion method requires different digestion time from 1 hour to 4 ℃ overnight, has complex operation, large required tissue block and more waste caused by the fact that the tissue can not be cultured for a long time. In the treatment process, the long-time digestion of the enzyme has great damage to the cells, which causes poor cell viability and proliferation capacity, low purity, long cell separation culture period and difficulty in the utilization of subsequent fibroblasts.
Disclosure of Invention
In view of the defects of the prior art, the technical problems to be solved by the invention are as follows: the fibroblast cells separated and cultured by adopting the prior art have the problems of long culture period, slow cell growth and low cell activity and proliferation capacity.
The invention aims to provide a method for separating and culturing skin fibroblasts, which can quickly and efficiently separate the fibroblasts in a short time, can ensure the cell viability and the proliferation capacity, has stable passage and higher cell purity, and provides stable seed cells for subsequent reprogramming and clinical treatment; the method specifically comprises the following steps: the skin tissue is pretreated by trypsin, and then the pretreated tissue is fixed in a culture dish for culture by a scratching method, and fibroblasts are separated.
Preferably, the mass percentage concentration of the trypsin is 0.25% -0.5%.
Preferably, the trypsin pretreatment process of the present invention comprises: the specific process of trypsin pretreatment is as follows: the epidermis was discarded from the skin tissue, the dermal portion was digested with trypsin at 37 ℃ for 1-3 hours, and the supernatant was removed after centrifugation.
Preferably, the culture medium used in the tissue culture process of the pretreatment of the present invention is: DMEM basal medium + 10% fetal bovine serum + 1% NEAA + 1% double antibody.
Preferably, the culture conditions of the pretreated tissue culture process are as follows: culturing at 37 deg.C and 5 vol% CO2The culture time is 4-10 days.
The fibroblasts of the present invention include, but are not limited to, skin fibroblasts of human and mammalian origin.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the invention provides an optimized method for separating and culturing skin fibroblasts, which fully considers the problems of the lack of clinical samples at present and the difficulty in obtaining samples for some special diseases, and has the advantages of saving samples and improving the tissue utilization rate.
(2) The method reduces the tissue compactness through enzyme digestion, improves the resistance of the cells to migrate from the tissue, accelerates the migration time of the cells from the tissue, and improves the separation efficiency.
(3) The method avoids the huge damage to the cells caused by the long-term digestion of the finely-divided tissues by trypsin, improves the activity and the proliferation capacity of the primary fibroblasts, and accelerates the growth speed of the cells.
(4) A large number of research experiments show that the fibroblasts separated and cultured by the method have high purity and good activity maintenance and can be stably passed for many times.
(5) The fibroblast obtained by the invention can be used for supplementing the shortage of fibroblast in skin dermal tissue caused by aging and ultraviolet irradiation, thereby increasing the content of collagen, hyaluronic acid and other substances.
(6) The invention can rapidly obtain the fibroblast, can be applied with biological material substances in a composite way, can heal the wound surface or repair scars, and can shorten the time for repairing the wound.
Drawings
FIG. 1 shows fibroblasts migrated from tissue under condition D in example 1.
FIG. 2 shows fibroblasts isolated from tissue cultured under the conditions of D in example 1.
FIG. 3 is an identification of vimentin from fibroblasts in example 2.
FIG. 4 is an identification of E-cadherin from fibroblasts in example 2.
FIG. 5 is a graph showing the growth and proliferation of fibroblasts in example 3.
FIG. 6 is the area of migration repair of fibroblasts in example 4 after 6 hours of scratching.
FIG. 7 shows the migration repair area of fibroblasts in example 4 after 12 hours from scratching.
FIG. 8 is the area of migration repair of fibroblasts in example 4 after 24 hours of scratching.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited to the examples.
Example 1
A method for separating and culturing skin fibroblasts specifically comprises the following steps:
(1) collecting skin tissue of human waist with skin sampler under aseptic condition, wherein the tissue block size is 1mm × 1mm × 1 mm; on a clean bench, the blood components adhering to the tissue mass were washed clean with PBS containing 2% double antibody.
(2) Using an ophthalmic scissors to trim and remove the adipose tissues below the dermis on the tissue block; the clean tissue blocks are pretreated before the experiment, then the pretreated tissue or cells are inoculated into a culture dish, and are separated and cultured in a culture medium (DMEM basic medium + 10% fetal bovine serum + 1% NEAA + 1% double antibody).
This example uses A, B, C, D four different pretreatment regimes to study the effect of different pretreatment methods on skin tissue:
A. the epidermis was discarded, the dermal part was cut into pieces by ophthalmic scissors and then placed in a 15ml centrifuge tube containing 0.25% trypsin, digested at 37 ℃ for 1 hour, then centrifuged at 300g for 10 minutes, the supernatant was removed, and the pellet containing the tissue cells was resuspended in a culture medium and transferred to a petri dish for isolation and culture.
B. And (3) performing cross scratch on a culture dish by using a sterilized blade, fixing the cleanly processed dermal tissue blocks at the scratch, and adding a culture medium for isolated culture.
C. Putting the cleaned tissue block into a 15ml centrifuge tube, adding 1ml of 0.25% trypsin, digesting the dermal tissue block for 1h at 37 ℃, scratching the dermal tissue block on a culture dish by using a sterilized blade, cleaning the tissue block by using PBS (phosphate buffered saline), cutting the tissue block into blocks, fixing the blocks at the scratched positions, and adding a culture medium for isolated culture.
D. Putting the cleaned tissue block into a 15ml centrifuge tube, adding 1ml of 0.25% trypsin, digesting the dermal tissue block for 2h at 37 ℃, scratching the dermal tissue block on a culture dish by using a sterilized blade, cleaning the tissue block by using PBS (phosphate buffered saline), cutting the tissue block into blocks, fixing the blocks at the scratched positions, and adding a culture medium for isolated culture.
After 4-14 days of culture, the fibroblasts in the tissues subjected to different treatments can be observed to migrate and proliferate from the periphery of the tissue block or the culture dish under a microscope.
The effect of the different treatments on the tissues and cells is summarized in table 1:
TABLE 1 Effect of different treatments on skin tissue
As can be seen from table 1: the tissue under different pretreatment conditions can successfully separate the fibroblasts from the skin tissue, but the separated and cultured cells have large differences: the A group cells have heterogeneous morphology, low purity and slow proliferation speed, and have great influence on the late culture activity of the cells. The migration and proliferation cycle of the cells in the B group and the C group is long, the activity of the cells is low, and the purity is not uniform. The cells in group D can migrate in the shortest time, have uniform shape, higher purity and better activity, and are beneficial to large-scale amplification culture and activity maintenance of fibroblasts (the shapes of primary fibroblasts separated by the treatment method in group D and fibroblasts after passage are shown in figures 1 and 2).
Example 2
The influence of the optimized separation culture method on the purity of the fibroblasts is as follows:
(1) collecting skin tissue of waist with skin sampler under aseptic condition, wherein the tissue block size is 1mm × 1mm × 1 mm; on a clean bench, the blood components adhering to the tissue mass were washed clean with PBS containing 2% double antibody.
(2) Using an ophthalmic scissors to trim and remove the adipose tissues below the dermis on the tissue block; and (3) pretreating the cleaned tissue block before experiment, fixing the pretreated tissue or cell in a culture dish for culture by a scratching method, and performing isolated culture in a culture medium.
The culture medium is as follows: DMEM basal medium + 10% fetal bovine serum + 1% NEAA + 1% double antibody.
The pretreatment method comprises the following steps: 0.25% trypsin, digested at 37 ℃ for 2 hours.
(3) In order to make the cell purity higher and the cell activity more consistent, the cells need to be subcultured, and the subculture of fibroblasts: when the cell fusion degree in the step (1) reaches 70-80%, sucking away the culture medium, washing with 2ml PBS for 3 times, then digesting the cells with 1ml 0.25% trypsin at 37 ℃ for 3-5 minutes, observing under an inverted microscope, adding 2ml culture medium to terminate digestion and collecting the cells into a 15ml centrifuge tube when the cells gradually change from fusiform to round and float, centrifuging for 4 minutes at 200g, removing the supernatant, adding 2ml culture medium to suspend and transferring to a new culture dish for culture.
And (3) detecting the purity of the fibroblasts:
and after the cells are subcultured for 4 days, detecting the purity of the cells when the cell fusion degree reaches 75%.
Firstly, the culture solution is sucked and discarded, the culture solution is washed for 3 times by 0.01M PBS solution, 5min is added each time, and 4 percent paraformaldehyde solution is added for fixation for 30min at room temperature.
② removing paraformaldehyde fixing solution by suction, washing for 3 times with 0.01M PBS solution, 5min each time, adding 0.01M PBS diluted 0.3 percent Triton-X100 membrane for 15 minutes.
③ removing Triton-X100 by suction, washing 3 times with 0.01M PBS solution, 5min each time, adding 10% goat serum, and sealing at room temperature for 30 min.
Fourthly, abandoning the confining liquid, dripping the diluted rabbit anti-mouse vimentin and E-cadherin polyclonal antibody (1:500) on the cell culture, and then incubating overnight in a refrigerator at 4 ℃.
Fifthly, after the primary antibody is incubated overnight, washing is carried out for 3 times by using 0.01M PBS solution, and each washing is carried out for 5 min. FITC fluorescent labeled goat anti-rabbit secondary antibody (1:50) is incubated for 2h at room temperature in the dark.
Sixthly, the secondary antibody is removed by suction, washed for 3 times by 0.01M PBS solution, washed for 5min each time, the DAPI (1:1000 dilution) is dripped to stain the nucleus, and incubated for 10min at room temperature in the dark.
Eighthly, washing the mixture for 3 times with 0.01M PBS solution, and washing for 5min each time; the number of positive cells was observed under a fluorescent inverted microscope, and the purity of the cells was evaluated by the positive cell rate of the cells.
FIG. 3 is an immunofluorescence staining chart of a fibroblast positive marker vimentin, FIG. 4 is an immunofluorescence staining chart of a negative marker E-cadherin, and as can be seen from FIGS. 3 and 4, the purity of cells can reach more than 95%.
Example 3
Effect of optimized isolation culture method on fibroblast proliferation capacity
Separating out fibroblasts by adopting the group D treatment method in the embodiment 1, and culturing the fibroblasts by adopting the method in the step 3 in the embodiment 2; and after the cells are subcultured for 4 days, detecting the proliferation capacity of the cells when the cell fusion degree reaches 75%.
(1) The medium was aspirated off, washed 3 times with 2ml PBS, after which the cells were digested with 1ml 0.25% trypsin at 37 ℃ for 3-5 minutes, observed under an inverted microscope, and when the cells gradually changed from fusiform to round and floated, 2ml of medium stopped the digestion and the cells were collected into a 15ml centrifuge tube, centrifuged at 200g for 4 minutes, the supernatant was removed, and 2ml of medium was added to resuspend and transferred to a new petri dish for culture.
(2) Fibroblast cells were cultured at 2X 104Inoculating a plurality of cells in a 12-hole plate, digesting the cells in one hole every day to prepare a cell suspension, adding trypan blue staining solution into the cell suspension at a ratio of 1:1, sucking 10 mu l of the cell suspension, and slowly dripping the cell suspension from the edge of a counting plate to fill the space between the counting plate and a cover glass; the cells were counted under a microscope, the number of cells per well was counted for 8 days continuously, and the growth curve of the cells was plotted.
As shown in FIG. 5, it can be seen that the growth of fibroblasts is in accordance with the growth curve, the growth of the fibroblasts is slow in 1-3 days after the passage of the cells, the growth of the cells is rapid in the latent period of growth and in the logarithmic growth period in 3-6 days, and then the growth of the cells reaches the plateau period and is slow in growth. This growth curve indicates good cell viability.
Example 4
The influence of the optimized separation culture method on the migration healing capacity of the fibroblasts is as follows:
fibroblasts were isolated by the group D treatment method of example 1 and cultured by the method of step 3 of example 2. And after the cells are subcultured for 4 days, detecting the migration healing capacity of the cells when the cell fusion degree reaches 75%.
(1) The fibroblasts were harvested by digestion with 0.25% trypsin into 15ml centrifuge tubes and cell counting was performed by 1:1 addition of trypan blue stain.
(2) A marker pen is firstly used on the back of a 6-hole plate, a ruler is used for drawing transverse lines uniformly, the transverse lines are drawn approximately every 0.5-1 cm, the transverse lines cross through holes, and each hole at least penetrates through 5 lines.
(3) Cells were plated at 1X 10 per well5The number of the cells is inoculated into a six-well plate for culture, and a cell scratch experiment is carried out when the cell fusion degree reaches 50% after the cells are cultured for 2 to 3 days.
(4) After the cells are stabilized, the gun head is used for comparing with the ruler, scratches are made perpendicular to the transverse line on the back as much as possible, the cells are washed for 3 times by PBS, a culture medium is added and placed into an incubator for culture, and the cells are photographed and sampled according to 0h, 6h, 12h and 24 h. Then calculating the healing capacity of the cells according to the scratch areas at different time points; (12h healing rate ═ 0h scratch area-12 h scratch area)/0 h scratch area).
Fig. 6 shows the scratch area after the fibroblast scratch is scratched for 6 hours, and fig. 7 shows the scratch area after the fibroblast scratch is scratched for 12 hours, it can be seen that the scratch is repaired by the migration of cells after 6 hours, the scratch surface is reduced after 12 hours of cell migration repair, the scratch is almost covered by the migrated cells after 24 hours, and it can be seen that the cells have high migration repair capability.
Claims (5)
1. A method for separating and culturing skin fibroblasts is characterized by comprising the following steps: the skin tissue is pretreated by trypsin, and then the pretreated tissue is fixed in a culture dish for culture by a scratching method, and fibroblasts are separated.
2. The method for isolated culture of dermal fibroblasts according to claim 1, characterized in that: the mass percentage concentration of the trypsin is 0.25-0.5%.
3. The method for isolated culture of dermal fibroblasts according to claim 1 or 2, characterized in that: the specific process of trypsin pretreatment is as follows: the epidermis was discarded from the skin tissue, the dermal portion was digested with trypsin at 37 ℃ for 1-3 hours, and the supernatant was removed after centrifugation.
4. The method for isolated culture of dermal fibroblasts according to claim 1, characterized in that: the culture medium used in the pre-treatment tissue culture process was: DMEM basal medium + 10% fetal bovine serum + 1% NEAA + 1% double antibody.
5. The method for isolated culture of dermal fibroblasts according to claim 1 or 4, characterized in that: the culture conditions of the pretreated tissue culture process are as follows: culturing at 37 deg.C and 5 vol% CO2The culture time is 4-10 days.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115369077A (en) * | 2022-07-29 | 2022-11-22 | 佛山市中科律动生物科技有限公司 | MEFLC cell strain, and construction method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0263659A2 (en) * | 1986-10-06 | 1988-04-13 | Susan C. Winter | Tissue culture media containing L-carnitine |
| US20050164388A1 (en) * | 2001-02-07 | 2005-07-28 | Young-Sook Son | Method of isolating epithelial cells, method of preconditioning cells, and methods of preparing bioartificial skin and dermis with the epithelial cells and preconditioned cells |
| CN101597593A (en) * | 2009-07-27 | 2009-12-09 | 杭州安倍生物科技有限公司 | Method for culturing autologous tissue fibroblast |
| US20120190059A1 (en) * | 2009-07-23 | 2012-07-26 | Beijing Huayuanbochuang Technology Co., Ltd. | Methods for obtaining hepatocytes, hepatic endoderm cells and hepatic progenitor cells by induced differentiation |
| CN202401066U (en) * | 2011-11-23 | 2012-08-29 | 中国水产科学研究院黑龙江水产研究所 | Cell culture dish |
| CN107354129A (en) * | 2017-08-24 | 2017-11-17 | 上海科医联创生物科技有限公司 | A kind of Fibroblast cell-culture method in autologous skin source |
| CN109468271A (en) * | 2018-12-28 | 2019-03-15 | 广州暨大美塑生物科技有限公司 | A kind of skin histology source fibroblast quick separating cultural method |
-
2020
- 2020-11-24 CN CN202011331103.8A patent/CN112375731A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0263659A2 (en) * | 1986-10-06 | 1988-04-13 | Susan C. Winter | Tissue culture media containing L-carnitine |
| US20050164388A1 (en) * | 2001-02-07 | 2005-07-28 | Young-Sook Son | Method of isolating epithelial cells, method of preconditioning cells, and methods of preparing bioartificial skin and dermis with the epithelial cells and preconditioned cells |
| US20120190059A1 (en) * | 2009-07-23 | 2012-07-26 | Beijing Huayuanbochuang Technology Co., Ltd. | Methods for obtaining hepatocytes, hepatic endoderm cells and hepatic progenitor cells by induced differentiation |
| CN101597593A (en) * | 2009-07-27 | 2009-12-09 | 杭州安倍生物科技有限公司 | Method for culturing autologous tissue fibroblast |
| CN202401066U (en) * | 2011-11-23 | 2012-08-29 | 中国水产科学研究院黑龙江水产研究所 | Cell culture dish |
| CN107354129A (en) * | 2017-08-24 | 2017-11-17 | 上海科医联创生物科技有限公司 | A kind of Fibroblast cell-culture method in autologous skin source |
| CN109468271A (en) * | 2018-12-28 | 2019-03-15 | 广州暨大美塑生物科技有限公司 | A kind of skin histology source fibroblast quick separating cultural method |
Non-Patent Citations (2)
| Title |
|---|
| PETER BREDBACKA: "Recent developments in embryo sexing and its field application", 《REPROD NUTR DEV》 * |
| 邵晓琳等: "组织块法结合胰蛋白酶消化法培养分离人口腔黏膜上皮细胞和成纤维细胞", 《中国卫生检验杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115369077A (en) * | 2022-07-29 | 2022-11-22 | 佛山市中科律动生物科技有限公司 | MEFLC cell strain, and construction method and application thereof |
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