CN108192856B - Method for efficiently separating and culturing human primary melanocytes - Google Patents
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a method for efficiently separating and culturing human primary melanocytes. The ROCK inhibitor is added into a culture medium which contains Ham' sF-12 culture medium, dibutyryl cyclic adenosine monophosphate, 3-isobutyl-1-methylxanthine, sodium orthovanadate, phorbol 12-myristate 13-acetate, fetal calf serum and diabase as components, all the components interact with each other, so that melanocytes grow in a clone mode, the culture period is shortened by half, and the separation efficiency of the melanocytes is greatly improved; the separation and culture method is simple, easy to operate and low in culture medium component, and can realize large-scale production of the humanized melanocytes.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a method for efficiently separating and culturing human primary melanocytes.
Background
Melanocytes, which are one of the constituents of skin cells, secrete melanin, which determines the color of the skin, and melanin is mainly accumulated in epidermal cells to protect the skin from damage by light radiation. Functional defects of melanocytes, including the problems of melanin synthesis and metabolism, are responsible for skin pigment diseases and are closely related to the occurrence of malignant melanoma. In vitro culture of primary melanocytes is widely used for studying melanocyte function, skin pigment diseases and pathogenesis of melanoma. With the development of tissue engineering and regenerative medicine, the melanocytes cultured in vitro can be applied to the clinical treatment of various pigment-deficient diseases, such as vitiligo.
Since melanocytes account for only about 1% of the number of epidermal cells, and because melanocytes grow slowly and have very limited proliferation capacity, efficient isolation and culture of melanocytes is very important. However, the existing separation culture method has low efficiency, slow growth and long period, and seriously influences the application of the separation culture method.
Chinese patent application (CN105112355A) discloses a method for culturing melanocytes. The method comprises the following steps of pre-culturing for 2-4h before melanocyte proliferation culture, wherein the pre-culture medium comprises: mixing a DMEM culture medium and a Ham' sF-12 culture medium according to a volume ratio of 2-4: 1; adenine 1.6-2.0X 10-4M; 80-100 units/mL of penicillin; streptomycin 80-100 mug/mL; hydrocortisone 0.1-0.5 μ g/mL; 2-6 mug/mL of insulin; 5-12ng/mL of epidermal growth factor; cholera toxin 0.8-1.5X 10-10M; 4-7% of fetal bovine serum. The culture method promotes the attachment of the melanocytes and improves the proportion and the activity of the melanocytes, but the problems of slow cell growth and long culture period still exist.
Disclosure of Invention
In order to solve the problems, the invention provides a method for efficiently separating and culturing human primary melanocytes, which can lead the melanocytes to grow in a cloning manner by adding ROCK inhibitor Y-27632 in a separation culture medium, shortens the culture period by a half, greatly improves the separation efficiency of the melanocytes, and lays a foundation for future scientific research and application of clinical transformation of the primary melanocytes.
The invention is realized by the following technical scheme:
a method for efficiently separating and culturing human primary melanocytes comprises the following steps: adding ROCK inhibitor into human primary melanocyte culture medium.
Further, the concentration of the ROCK inhibitor is 1-20 mu M.
Further, the ROCK inhibitor is Y-27632.
When the concentration of ROCK inhibitor in the culture medium is in the range of 1-20 mu M, the growth of human primary melanocytes can be rapidly promoted, and the culture period is greatly shortened. When the concentration of the ROCK inhibitor is lower than 1 mu M, the effect of promoting the growth of the melanocyte is not obvious; if the concentration is too high, the growth of melanocytes is inhibited.
In the previous research, the technical personnel of the invention find that only Y27632 in ROCK inhibitors has the function of promoting the growth of humanized melanocytes, and the action effects of other ROCK inhibitors are not obvious.
Further, the medium comprises the following components: ham's F-12 Medium, dibutyryladenosine 1-5X 10-4M, 3-isobutyl-1-methylxanthine 0.2-2X 10-4M, sodium orthovanadate 0.5-5 mu M, phorbol 12-myristate 13-acetate 10-100ng/ml, fetal calf serum 2-10% and double antibody 0.05-0.1%. The culture medium can promote the growth of human-derived melanocytes, and can effectively inhibit the growth of keratinocytes and fibroblasts. ROCK inhibitors are added to the medium and the components act synergistically.
Further, the method comprises the steps of:
(1) removing subcutaneous tissue of skin from fresh skin tissue, cutting into small pieces, and culturing at 4 deg.C overnight in culture dish containing dispase solution;
(2) peeling off the epidermis from the skin tissue cultured in the step (1) and separating melanocytes;
(3) inoculating the melanocyte separated in the step (2) into a culture medium containing a ROCK inhibitor for culture, after 48 hours of culture, replacing the culture medium without the ROCK inhibitor, continuing culture, and replacing the culture solution once every three days.
Further, the concentration of the dispersed enzyme solution in the step (1) was 2.5 mg/ml.
Further, the specific steps of the melanocyte separation in the step (2) are as follows: cutting the peeled epidermis into homogenate sample by a scalpel, and then digesting the homogenate sample for 15 to 30 minutes by 10ml of pancreatin at 37 ℃; adding 10ml DMEM medium containing 10% serum, repeatedly blowing and beating for more than 20 times to obtain single cell suspension, filtering with 100 micron filter screen, centrifuging for 5 min at 1000 rpm, discarding the upper layer culture solution, repeating the above steps once, and separating to obtain melanocyte.
Further, pancreatin used was 0.05% of pancreatin.
Compared with the prior art, the invention has the following technical advantages:
(1) the ROCK inhibitor is added into a culture medium which contains Ham' sF-12 culture medium, dibutyryl cyclic adenosine monophosphate, 3-isobutyl-1-methylxanthine, sodium orthovanadate, phorbol 12-myristate 13-acetate, fetal calf serum and diabase as components, all the components interact with each other, so that melanocytes grow in a clone mode, the culture period is shortened by half, and the separation efficiency of the melanocytes is greatly improved;
(2) the separation and culture method is simple, easy to operate and low in culture medium component, and can realize large-scale production of the humanized melanocytes.
Drawings
FIG. 1 is a microscopic image of melanocytes isolated at 1 st, 6 th and 11 th days after example 3 group and comparative example 1 group.
FIG. 2 statistics of melanocyte counts in days 6 and 11 after isolation of melanocytes in example 3 group and comparative example 1 group.
FIG. 3 is a microscopic observation of melanocytes in 17 days of cell culture in example 3 group and comparative example 1 group.
Fig. 4 statistics of melanocyte counts for example 3 and comparative example 1 groups on day 17.
FIG. 5 cases of two weeks in culture of passaged cells P1 of the example 3 group and the comparative example 1 group.
FIG. 6 shows the case where the passaged cells P1 of the example 3 group and the comparative example 1 group were cultured for three weeks.
FIG. 7 cases in which the passaged cells P1 of the example 3 group and the comparative example 1 group were cultured for four weeks.
FIG. 8 statistics of counts after four weeks of culture of passaged cells P1 in example 3 and comparative example 1.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, 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 invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, elements, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Ham' sF-12 medium, fetal bovine serum were purchased from gibco; dibutyryl cyclic adenosine, 3-isobutyl-1-methylxanthine, sodium orthovanadate and phorbol 12-myristate 13-acetate from sigma; double antibody was purchased from Invitrogen.
Example 1A method for efficiently isolating and culturing human Primary melanocytes
The method comprises the following steps:
(1) removing subcutaneous tissue of fresh skin tissue, cutting into small pieces, and culturing at 4 deg.C overnight in culture dish containing 2.5mg/ml dispase solution;
(2) peeling off the epidermis from the skin tissue cultured in the step (1) and separating melanocytes, wherein the method comprises the following specific steps: cutting the peeled epidermis into homogenate with a scalpel, and digesting with 10ml of 0.05% pancreatin at 37 ℃ for 15-30 minutes; adding 10ml DMEM medium containing 10% serum, repeatedly blowing and beating for more than 20 times to obtain single cell suspension, filtering with 100 micron filter screen, centrifuging for 5 minutes at 1000 rpm, discarding the upper layer culture solution, repeating the steps for one time, and separating to obtain melanocytes;
(3) inoculating the melanocyte separated in the step (2) into a culture medium containing 1 mu M of ROCK inhibitor Y-27632 to culture, after culturing for 48 hours, replacing the culture medium without the ROCK inhibitor, continuing to culture, and replacing the liquid every three days.
The culture medium comprises the following components and contents: ham' sF-12 Medium 60%, dibutyryladenosine 1X 10-4M, 3-isobutyl-1-methylxanthine 0.2X 10-4M, sodium orthovanadate 0.5 mu M, phorbol 12-myristate 13-acetate 10ng/ml, fetal bovine serum 2% and diabodies 0.05%.
Example 2A method for efficiently isolating and culturing human Primary melanocytes
The method comprises the following steps:
(1) removing subcutaneous tissue of fresh skin tissue, cutting into small pieces, and culturing at 4 deg.C overnight in culture dish containing 2.5mg/ml dispase solution;
(2) peeling off the epidermis from the skin tissue cultured in the step (1) and separating melanocytes, wherein the method comprises the following specific steps: cutting the peeled epidermis into homogenate with a scalpel, and digesting with 10ml of 0.05% pancreatin at 37 ℃ for 15-30 minutes; adding 10ml DMEM medium containing 10% serum, repeatedly blowing and beating for more than 20 times to obtain single cell suspension, filtering with 100 micron filter screen, centrifuging for 5 minutes at 1000 rpm, discarding the upper layer culture solution, repeating the steps for one time, and separating to obtain melanocytes;
(3) inoculating the melanocyte separated in the step (2) into a culture medium containing 20 mu M of ROCK inhibitor Y-27632 to culture, after culturing for 48 hours, replacing the culture medium without the ROCK inhibitor, continuing to culture, and replacing the liquid every three days.
The culture medium comprises the following components and contents: ham' sF-12 Medium 80%, dibutyryladenosine 5X 10-4M, 3-isobutyl-1-methylxanthine 2X 10-4M, 5 mu M sodium orthovanadate, 100ng/ml phorbol 12-myristate 13-acetate, 10% fetal bovine serum and 0.1% diabody.
Example 3A method for efficiently isolating and culturing human Primary melanocytes
The method comprises the following steps:
(1) removing subcutaneous tissue of fresh skin tissue, cutting into small pieces, and culturing at 4 deg.C overnight in culture dish containing 2.5mg/ml dispase solution;
(2) peeling off the epidermis from the skin tissue cultured in the step (1) and separating melanocytes, wherein the method comprises the following specific steps: cutting the peeled epidermis into homogenate with a scalpel, and digesting with 10ml of 0.05% pancreatin at 37 ℃ for 15-30 minutes; adding 10ml DMEM medium containing 10% serum, repeatedly blowing and beating for more than 20 times to obtain single cell suspension, filtering with 100 micron filter screen, centrifuging for 5 minutes at 1000 rpm, discarding the upper layer culture solution, repeating the steps for one time, and separating to obtain melanocytes;
(3) inoculating the melanocyte separated in the step (2) into a culture medium containing 10 mu M of ROCK inhibitor Y-27632 to culture, after culturing for 48 hours, replacing the culture medium without the ROCK inhibitor, continuing to culture, and replacing the liquid every three days.
The culture medium comprises the following components and contents: ham' sF-12 Medium 75%, dibutyryladenosine 3.5X 10-4M, 3-isobutyl-1-methylxanthine 1.5X 10-4M, sodium orthovanadate 2.5 mu M, phorbol 12-myristate 13-acetate 40ng/ml, fetal bovine serum 6% and diabodies 0.07%.
Comparative example 1
The difference from example 3 is that no ROCK inhibitor is added during the isolation and culture of the human melanocytes.
Test example 1 culture Observation of human-derived melanocytes
Isolating and culturing human melanocytes according to the culture methods of example 3 and comparative example 1, observing melanin pigment cells on the 1 st, 6 th, 11 th and 17 th days of the isolation culture of melanin, and counting; the results of the experiment are shown in FIGS. 1 to 4.
As is clear from FIG. 1, on days 1, 6 and 11 after the isolation of melanocytes, the number of melanocytes was significantly increased in the group of example 3 (+ Y27632 (10. mu.M) 48h) compared to the group of comparative example 1 (Control). As can be seen from FIG. 2, the rate of increase of melanocytes was significantly higher in the example 3 group than in the control group. After 17 days of culture, as can be seen from FIG. 3, the number of melanocytes was greater in the example 3 group than in the comparative example 1 group; the melanocytes in each of the cell culture dishes of the group of example 3 and the group of comparative example 1 were digested with pancreatin and counted, and as a result, as shown in FIG. 4, the number of melanocytes in the group of example 3 was about 4 to 5 times that of the cells in the group of comparative example 1.
Test example 2 experiment for shortening melanocyte culture cycle Using ROCK inhibitor Y-27632
After culturing primary cells (P0) for two weeks according to the method of example 3 and comparative example 1, respectively, all cells in each group were digested and then subcultured (P1), and the growth of P1 cells in each group is shown in FIGS. 5-8.
Experiments have shown that the primary cells (P0) of example 3 group grew well about 7-10 days earlier than the group of comparative example 1.
In conclusion, when the ROCK inhibitor Y-27632 is added into the culture medium, the growth speed of the human primary melanocytes can be obviously improved, and the cell culture period is shortened.
Claims (5)
1. A method for efficiently separating and culturing human primary melanocytes is characterized in that a ROCK inhibitor is added into a culture medium for culturing the human primary melanocytes;
the ROCK inhibitor is Y-27632, and the concentration is 1-20 mu M;
the culture medium comprises the following components: ham' sF-12 culture medium 60% -80%, dibutyryladenosine 1-5X 10%-4M, 3-isobutyl-1-methylxanthine 0.2-2X 10-4M, sodium orthovanadate 0.5-5 mu M, phorbol 12-myristate 13-acetate 10-100ng/ml, fetal calf serum 2-10% and double antibody 0.05-0.1%.
2. The method for efficiently isolating and culturing human primary melanocytes according to claim 1, characterized in that it comprises the following steps:
(1) removing subcutaneous tissue of skin from fresh skin tissue, cutting into small pieces, and culturing at 4 deg.C overnight in culture dish containing dispase solution;
(2) peeling off the epidermis from the skin tissue cultured in the step (1) and separating melanocytes;
(3) inoculating the melanocyte separated in the step (2) into a culture medium containing a ROCK inhibitor for culture, after 48 hours of culture, replacing the culture medium without the ROCK inhibitor, continuing culture, and replacing the culture solution once every three days.
3. The method for efficiently isolating and culturing human primary melanocytes according to claim 2, wherein the concentration of the solution of dispase in step (1) is 2.5 mg/ml.
4. The method for efficiently isolating and culturing human primary melanocytes according to claim 2, wherein the isolation of melanocytes in step (2) comprises the following steps: cutting the peeled epidermis into homogenate sample by a scalpel, and then digesting the homogenate sample for 15 to 30 minutes by 10ml of pancreatin at 37 ℃; adding 10ml DMEM medium containing 10% serum, repeatedly blowing and beating for more than 20 times to obtain single cell suspension, filtering with 100 micron filter screen, centrifuging for 5 min at 1000 rpm, discarding the upper layer culture solution, repeating the above steps once, and separating to obtain melanocyte.
5. The method for efficiently isolating and culturing human primary melanocytes according to claim 4, wherein pancreatin used is 0.05% pancreatin.
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| PCT/CN2018/118382 WO2019128614A1 (en) | 2017-12-27 | 2018-11-30 | Method for efficiently separating and culturing human primary melanocytes |
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| CN108192856B (en) * | 2017-12-27 | 2020-05-12 | 山东大学 | Method for efficiently separating and culturing human primary melanocytes |
| CN109735486B (en) * | 2019-01-31 | 2022-02-22 | 中国疾病预防控制中心辐射防护与核安全医学所 | Primary melanocyte culture method for researching premature senility caused by UVB irradiation |
| CN110951675B (en) * | 2019-12-23 | 2021-03-23 | 中国水产科学研究院珠江水产研究所 | Method for separating and culturing melanocytes of orange-colored double-crowned fish |
| CN111100838B (en) * | 2019-12-27 | 2023-05-09 | 广东博溪生物科技有限公司 | Low-temperature preservation and transportation culture medium |
| CN114058565A (en) * | 2020-07-31 | 2022-02-18 | 上海尚瑞生物医药科技有限公司 | Serum-free melanoblast culture solution and culture method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532950A (en) * | 2008-12-31 | 2009-09-16 | 程树军 | Method for analyzing toxicity and effect of skin whitening agent through human being skin melanocyte |
| CN103785064A (en) * | 2013-08-26 | 2014-05-14 | 济南磐升生物技术有限公司 | Method for regenerating human intact skin tissue by use of in vitro cultured cells |
| CN105112355A (en) * | 2015-09-07 | 2015-12-02 | 北京艾美特夫生物科技有限公司 | Melanocyte culture method |
| CN105132358A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Culture method for acquisition of tissue-engineered epidermis and application of culture method |
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| ES2688019T3 (en) * | 2012-06-05 | 2018-10-30 | The Regents Of The University Of California | Methods and compositions for accelerated production of retinal pigmented epithelial cells from pluripotent cells |
| CN104099289A (en) * | 2013-04-08 | 2014-10-15 | 瑞田投资有限公司 | Epidermal tissue culturing method, and prepared epidermal tissue culture and application thereof |
| CN108192856B (en) * | 2017-12-27 | 2020-05-12 | 山东大学 | Method for efficiently separating and culturing human primary melanocytes |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532950A (en) * | 2008-12-31 | 2009-09-16 | 程树军 | Method for analyzing toxicity and effect of skin whitening agent through human being skin melanocyte |
| CN103785064A (en) * | 2013-08-26 | 2014-05-14 | 济南磐升生物技术有限公司 | Method for regenerating human intact skin tissue by use of in vitro cultured cells |
| CN105132358A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Culture method for acquisition of tissue-engineered epidermis and application of culture method |
| CN105112355A (en) * | 2015-09-07 | 2015-12-02 | 北京艾美特夫生物科技有限公司 | Melanocyte culture method |
Non-Patent Citations (4)
| Title |
|---|
| A ROCK inhibitor promotes keratinocyte survival and paracrine secretion, enhancing establishment of primary human melanocytes and melanocyte–keratinocyte co‐cultures;Jun Mi,Yang Feng,Jie Wen,Yiqun Su,Lin Xu,Tingjian Zu;《PIGMENT CELL & MELANOMA》;20190806;1-15页 * |
| Efficient procurement of epithelial stem cells from human tissue specimens using a rho-associated protein kinase inhibitor Y-27632;Terunuma A, Limgala RP, Park CJ, Choudhary I, Vogel JC;《Tissue engineering, Part A》;20101231;全文 * |
| Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells;Wen Jie, Zu Tingjian, Zhou Qian, Leng Xue, Wu Xunwei;《Journal of tissue engineering and regenerative medicine》;20170727;1251-1255页 * |
| 人类皮肤再生效率的提高及应用的研究;温洁;《中国博士学位论文全文数据库医药卫生科技辑》;20170815;摘要、正文第20、29、36、38页 * |
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