WO2019128614A1

WO2019128614A1 - Method for efficiently separating and culturing human primary melanocytes

Info

Publication number: WO2019128614A1
Application number: PCT/CN2018/118382
Authority: WO
Inventors: 吴训伟; 弭军; 徐琳
Original assignee: 山东大学
Priority date: 2017-12-27
Filing date: 2018-11-30
Publication date: 2019-07-04
Also published as: CN108192856B; CN108192856A

Abstract

A method for separating and culturing human primary melanocytes. According to the method, an ROCK inhibitor is added to a culture medium comprising a Ham's F-12 medium, dibutyryl cyclic adenosine monophosphate, 3-isobutyl-1-methylxanthine, sodium orthovanadate, phorbol 12-myristate 13-acetate, fetal bovine serum, and double antibodies.

Description

Method for efficiently separating and culturing human primary melanocytes

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 technique

Melanocytes are a kind of skin cells. They secrete melanin to determine the color of the skin. Melanin is mainly concentrated in the epidermal cells to prevent the skin from being damaged by light radiation. Functional defects in melanocytes, including the synthesis and metabolism of melanin, are a cause of skin pigmentation and are closely related to the development of malignant melanoma. In vitro culture of primary melanocytes is widely used to study melanocyte function, skin pigmentation, and the pathogenesis of melanoma. Moreover, with the development of tissue engineering and regenerative medicine, melanocytes cultured in vitro can be used for clinical treatment of various pigment-deficient diseases, such as vitiligo.

Since melanocytes account for only about 1% of the number of epidermal cells, and their growth is slow and their proliferative capacity is extremely limited, it is very important to efficiently isolate and culture melanocytes. However, the existing separation and culture methods are inefficient, and the growth is slow and the cycle is long, which seriously affects its application.

Chinese Patent Application (CN105112355A) discloses a method of culturing melanocytes. The method comprises pre-culturing for 2-4 h before the melanocyte proliferation culture, wherein the pre-culture medium is: DMEM medium and Ham's F-12 medium are mixed at a volume ratio of 2-4:1; adenine 1.6-2.0 ×10-4M; penicillin 80-100 units/mL; streptomycin 80-100μg/mL; hydrocortisone 0.1-0.5μg/mL; insulin 2-6μg/mL; epidermal growth factor 5-12ng/mL; cholera Toxin 0.8-1.5×10-10M; fetal bovine serum 4-7%. The above culture method promotes adhesion of melanocytes, increases the proportion of melanocytes and cell activity, but there is still a problem that cells grow slowly and the culture period is long.

Summary of the invention

In order to solve the above problems, the present invention provides a method for efficiently separating and culturing human primary melanocytes. By adding the ROCK inhibitor Y-27632 to the isolated medium, the melanocytes can be cloned and the culture period can be shortened by half. It greatly improves the separation efficiency of melanocytes and lays a foundation for future research and clinical transformation of primary melanocytes.

The invention is achieved by the following technical solutions:

A method for efficiently isolating and culturing human primary melanocytes: a ROCK inhibitor is added to a human primary melanocyte culture medium.

Further, the concentration of the ROCK inhibitor is 1-20 μM.

Further, the ROCK inhibitor is Y-27632.

When the concentration of ROCK inhibitor in the medium is in the range of 1-20 μM, the growth of human primary melanocytes can be rapidly promoted, and the culture period is greatly shortened. When the concentration of ROCK inhibitor is less than 1 μM, the effect of promoting melanocyte growth is not obvious; if the concentration is too high, the growth of melanocytes is inhibited.

In the previous study, the present inventors found that only Y27632 of the ROCK inhibitor has the function of promoting the growth of human melanocytes, and the effects of other ROCK inhibitors are not obvious.

Further, the medium comprises the following components: Ham's F-12 medium, dibutyryl cyclic adenosine 1-5×10 ^-4 M, 3-isobutyl-1-methylxanthine 0.2-2× 10 ^-4 M, sodium orthovanadate 0.5-5 μM, phorbol 12-tetradecanoate 13-acetate 10-100 ng/ml, fetal bovine serum 2-10% and double antibody 0.05-0.1%. The medium can promote the growth of human melanocytes, and at the same time effectively inhibit the growth of keratinocytes and fibroblasts. The ROCK inhibitor is added to the medium, and the components can exert a synergistic effect.

Further, the method comprises the steps of:

(1) Remove the subcutaneous tissue of the skin from fresh skin tissue, cut into small pieces, and place in a Petri dish containing a dispersion enzyme solution for overnight culture at 4 ° C;

(2) peeling off the epidermis from the skin tissue cultured in the step (1), and performing melanocyte separation;

(3) The melanocytes isolated in the step (2) were inoculated to a medium containing a ROCK inhibitor, and after 48 hours of culture, the medium containing no ROCK inhibitor was replaced, the culture was continued, and the medium was changed every three days.

Further, the concentration of the dispase solution in the step (1) is 2.5 mg/ml.

Further, the specific step of melanocyte separation in the step (2) is: cutting the exfoliated epidermis into a homogenate with a scalpel, and then digesting with 10 ml trypsin at 37 ° C for 15-30 minutes; adding 10 ml containing 10% serum The DMEM medium was repeatedly blown 20 times or more to obtain a single cell suspension, which was filtered with a 100 μm filter and centrifuged at 1000 rpm for 5 minutes, and the supernatant medium was discarded, and then repeated, and melanocytes were isolated.

Further, the trypsin used was 0.05% trypsin.

Compared with the prior art, the present invention has the following technical advantages:

(1) The present invention adds a ROCK inhibitor to a composition of Ham's F-12 medium, dibutyryl cyclic adenosine, 3-isobutyl-1-methylxanthine, sodium orthovanadate, phorbol 12- In the medium of tetradecanoate 13-acetate, fetal bovine serum and double-antibody, the components interact to make the melanocytes grow in a clone, shorten the culture period by half, and greatly improve the separation efficiency of melanocytes;

(2) The separation and culture method of the present invention is simple, easy to operate, and has a small amount of medium components, and can realize large-scale production of human melanocytes.

DRAWINGS

Figure 1 is a microscopic micrograph of Days 1, 6 and 11 after separation of melanocytes of Example 3 and Comparative Example 1.

Figure 2 Statistics of melanocyte counts on days 6 and 11 after isolation of the melanocytes of Example 3 and Comparative Example 1.

Fig. 3 is a microscopic observation of melanocytes of the cells of Example 3 and Comparative Example 1 for 17 days.

Figure 3 is a graph of melanocyte counts for the Example 3 and Comparative 1 groups on Day 17.

Fig. 5 shows the case where the group 3 of the Example 3 and the group 1 of the comparative example 1 were cultured for two weeks.

Fig. 6 shows the case where the group 3 of the Example 3 and the group 1 of the comparative example 1 were cultured for three weeks.

Fig. 7 shows the case where the passage group P1 of Example 3 and Comparative Example 1 was cultured for four weeks.

Figure 8 is a count of the counts of the group 3 and the control group 1 after passage of the cultured cells P1 for four weeks.

Detailed ways

It should be noted that the following detailed description is illustrative and is intended to provide a further description of the invention. 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, unless otherwise indicated.

It is to be noted that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the exemplary embodiments of the invention. As used herein, the singular " " " " " " There are features, steps, operations, components, and/or combinations thereof.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Ham's F-12 medium, fetal calf serum purchased from Gibco; dibutyryl cyclic adenosine, 3-isobutyl-1-methylxanthine, sodium orthovanadate and phorbol 12-tetradecanoate 13 - Acetate was purchased from Sigma Co.; double antibody was purchased from Invitrogen.

Example 1 A method for efficiently separating and culturing human primary melanocytes

The method comprises the following steps:

(1) Remove the subcutaneous tissue of the skin from fresh skin tissue, cut into small pieces, and place in a Petri dish containing 2.5 mg/ml dispase solution overnight at 4 ° C;

(2) peeling off the epidermis from the skin tissue cultured in the step (1), and performing melanocyte separation. The specific steps are: cutting the peeled epidermis into a homogenate with a scalpel, and then using 10 ml of 0.05% trypsin. Digestion at 37 ° C for 15-30 minutes; add 10 ml of DMEM medium containing 10% serum repeatedly for 20 times or more to obtain a single cell suspension, filter with a 100 μm filter and centrifuge at 1000 rpm for 5 minutes, discard the supernatant medium, then Repeatedly, separating melanocytes;

(3) The melanocytes isolated in the step (2) were inoculated in a medium containing 1 μM ROCK inhibitor Y-27632, and after 48 hours of culture, the medium containing no ROCK inhibitor was replaced, and the culture was continued every three days. Change the fluid once.

The medium contains the following components and their contents: Ham's F-12 medium 60%, dibutyryl cyclic adenosine 1 × 10 ^-4 M, 3-isobutyl-1-methylxanthine 0.2 × 10 ^{- 4} M, sodium orthovanadate 0.5 μM, phorbol 12-tetradecanoate 13-acetate 10 ng/ml, fetal bovine serum 2% and double antibody 0.05%.

Example 2 A method for efficiently separating and culturing human primary melanocytes

The method comprises the following steps:

(3) The melanocytes isolated in the step (2) were inoculated into a medium containing 20 μM ROCK inhibitor Y-27632, and after 48 hours of culture, the medium containing no ROCK inhibitor was replaced, and the culture was continued every three days. Change the fluid once.

The medium contains the following components and their contents: Ham's F-12 medium 80%, dibutyryl cyclic adenosine 5 × 10 ^-4 M, 3-isobutyl-1-methylxanthine 2 × 10 ^{- 4} M, sodium orthovanadate 5 μM, phorbol 12-tetradecanoate 13-acetate 100 ng/ml, fetal bovine serum 10% and double antibody 0.1%.

Example 3 A method for efficiently separating and culturing human primary melanocytes

The method comprises the following steps:

(3) The melanocytes isolated in the step (2) were inoculated into a medium containing 10 μM ROCK inhibitor Y-27632, and after 48 hours of culture, the medium containing no ROCK inhibitor was replaced, and the culture was continued every three days. Change the fluid once.

The medium contains the following components and their contents: Ham's F-12 medium 75%, dibutyryl cyclic adenosine 3.5 × 10 ^-4 M, 3-isobutyl-1-methylxanthine 1.5 × 10 ^{- 4} M, sodium orthovanadate 2.5 μM, phorbol 12-tetradecanoate 13-acetate 40 ng/ml, fetal bovine serum 6% and double antibody 0.07%.

Comparative example 1

The difference from Example 3 is that no ROCK inhibitor is added during the isolation and culture of human melanocytes.

Test Example 1 Human melanocyte culture observation

The human melanocytes were isolated and cultured according to the culture method of Example 3 and Comparative Example 1, respectively, and the melanocytes on the first day, the sixth day, the 11th day and the 17th day of the melanin isolation culture were observed and counted; 4 is shown.

As can be seen from Fig. 1, on the first, sixth and eleventh days after the separation of melanocytes, the number of melanocytes was significantly increased in the group of Example 3 (+Y27632 (10 μM) 48 h) compared with the control group 1 (Control). As can be seen from Fig. 2, the growth rate of the melanocytes in the third group was significantly faster than that in the control group. After 17 days of culture, it can be seen from Fig. 3 that the number of melanocytes in the third group was higher than that in the first group; the melanocytes in the cell culture dishes of the third group and the comparative group 1 were digested with pancreatin and counted, and the results were as follows. As shown in Figure 4, the number of melanocytes in the Example 3 group was about 4-5 times the number of cells in Comparative Example 1.

Test Example 2 ROCK inhibitor Y-27632 shortens melanocyte culture cycle experiment

The primary cells (P0) were cultured for two weeks according to the methods of Example 3 and Comparative Example 1, respectively, and all the cells in each group were digested and then passaged (P1), and the growth of P1 cells in each group is shown in Fig. 5-8. .

Experiments have shown that the primary cells of the first embodiment (P0) have a full time earlier than the comparison group 1 for about 7-10 days.

In summary, the addition of the ROCK inhibitor Y-27632 to the medium used in the present invention can significantly increase the growth rate of human primary melanocytes and shorten the cell culture cycle.

Claims

A method for efficiently separating and culturing human primary melanocytes, which comprises adding a ROCK inhibitor to a human primary melanocyte culture medium.
The method for efficiently separating and culturing human primary melanocytes according to claim 1, wherein the concentration of the ROCK inhibitor is from 1 to 20 μM.
The method for efficiently separating and culturing human primary melanocytes according to claim 1 or 2, wherein the ROCK inhibitor is Y-27632.
The method for efficiently separating and culturing human primary melanocytes according to claim 1 or 2, wherein the medium comprises the following components: Ham's F-12 medium 60% - 80%, dibutyryl adenosine Acid 1-5×10 -4 M, 3-isobutyl-1-methylxanthine 0.2-2×10 -4 M, sodium orthovanadate 0.5-5 μM, phorbol 12-tetradecanoate 13- The acetate is 10-100 ng/ml, the fetal bovine serum is 2-10% and the double antibody is 0.05-0.1%.
A method for efficiently isolating and culturing human primary melanocytes according to claim 1, wherein the method comprises the steps of:

(1) Remove the subcutaneous tissue of the skin from fresh skin tissue, cut into small pieces, and place in a Petri dish containing a dispersion enzyme solution for overnight culture at 4 ° C;

(2) peeling off the epidermis from the skin tissue cultured in the step (1), and performing melanocyte separation;

(3) The melanocytes isolated in the step (2) were inoculated to a medium containing a ROCK inhibitor, and after 48 hours of culture, the medium containing no ROCK inhibitor was replaced, the culture was continued, and the medium was changed every three days.
The method for efficiently separating and culturing melanocytes according to claim 5, wherein the concentration of the dispase solution in the step (1) is 2.5 mg/ml.
The method for efficiently separating and culturing melanocytes according to claim 5, wherein the step of separating melanocytes in the step (2) is: cutting the epidermis under the peeling into a homogenate with a scalpel, and then using 10 ml of the pancreas The enzyme was digested at 37 ° C for 15-30 minutes; 10 ml of DMEM medium containing 10% serum was added and repeatedly beaten 20 times or more to obtain a single cell suspension, which was filtered with a 100 μm filter and centrifuged at 1000 rpm for 5 minutes, and the supernatant culture solution was discarded. Then, it was repeated once and the melanocytes were isolated.
A method for efficiently isolating and culturing melanocytes according to claim 3, wherein the trypsin used is 0.05% trypsin.