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Method for efficient separation and culture of human primary melanin cells

A high-efficiency technology for melanocytes, applied in the field of high-efficiency separation and culture of human primary melanocytes, can solve the problems of slow cell growth and long culture period, achieve simple separation and culture methods, shorten the culture period, and realize large-scale production Effect

Active Publication Date: 2018-06-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above culture method promotes the adhesion of melanocytes, improves the proportion of melanocytes and cell activity, but there are still problems of slow cell growth and long culture period.

Method used

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  • Method for efficient separation and culture of human primary melanin cells
  • Method for efficient separation and culture of human primary melanin cells
  • Method for efficient separation and culture of human primary melanin cells

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 A method for efficiently isolating and culturing human primary melanocytes

[0036] Described method comprises the following steps:

[0037] (1) Remove the subcutaneous tissue of the fresh skin tissue, cut it into small pieces and put it into a petri dish containing 2.5mg / ml dispase solution for overnight cultivation at 4°C;

[0038] (2) The epidermis is peeled off from the skin tissue after step (1) culture, and the melanocytes are separated. The specific steps are: cut the peeled epidermis into a homogenate sample with a scalpel, and then use 10ml of 0.05% trypsin Digest at 37°C for 15-30 minutes; add 10ml of DMEM medium containing 10% serum and pipette repeatedly more than 20 times to obtain a single-cell suspension, filter it with a 100-micron filter and centrifuge at 1000 rpm for 5 minutes, discard the upper culture medium, and then Repeat the process once to obtain melanocytes;

[0039] (3) Inoculate the melanocytes obtained in step (2) into a culture ...

Embodiment 2

[0041] Example 2 A method for efficiently isolating and culturing human primary melanocytes

[0042] Described method comprises the following steps:

[0043] (1) Remove the subcutaneous tissue of the fresh skin tissue, cut it into small pieces and put it into a petri dish containing 2.5mg / ml dispase solution for overnight cultivation at 4°C;

[0044](2) The epidermis is peeled off from the skin tissue after step (1) culture, and the melanocytes are separated. The specific steps are: cut the peeled epidermis into a homogenate sample with a scalpel, and then use 10ml of 0.05% trypsin Digest at 37°C for 15-30 minutes; add 10ml of DMEM medium containing 10% serum and pipette repeatedly more than 20 times to obtain a single-cell suspension, filter it with a 100-micron filter and centrifuge at 1000 rpm for 5 minutes, discard the upper culture medium, and then Repeat the process once to obtain melanocytes;

[0045] (3) Inoculate the melanocytes isolated in step (2) in a medium cont...

Embodiment 3

[0047] Example 3 A method for efficiently isolating and culturing human primary melanocytes

[0048] Described method comprises the following steps:

[0049] (1) Remove the subcutaneous tissue of the fresh skin tissue, cut it into small pieces and put it into a petri dish containing 2.5mg / ml dispase solution for overnight cultivation at 4°C;

[0050] (2) The epidermis is peeled off from the skin tissue after step (1) culture, and the melanocytes are separated. The specific steps are: cut the peeled epidermis into a homogenate sample with a scalpel, and then use 10ml of 0.05% trypsin Digest at 37°C for 15-30 minutes; add 10ml of DMEM medium containing 10% serum and pipette repeatedly more than 20 times to obtain a single-cell suspension, filter it with a 100-micron filter and centrifuge at 1000 rpm for 5 minutes, discard the upper culture medium, and then Repeat the process once to obtain melanocytes;

[0051] (3) Inoculate the melanocytes obtained in step (2) in a medium con...

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Abstract

The invention belongs to the technical field of cell culture, and in particular relates to a method for efficient separation and culture of human primary melanin cells. According to the method provided by the invention, an ROCK inhibitor is added to a culture medium which consists of the following ingredients: a Ham's F-12 medium, dibutiryl cyclic adenosine monophosphate, 3-isobutyl-1-methylxanthine, sodium orthovanadate, phorbol 12-myristinate 13-acetate, fetal calf serum and double-antibody; on the basis of mutual actions of the various ingredients, the melanin cells can achieve cloning growth, so that a culture cycle can be shortened by half and separation efficiency of the melanin cells can be greatly improved; the separation and culture method provided by the invention is simple and easy to operate and is low in amount of medium ingredients; and large-scale production of the human primary melanin cells is achieved.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for efficiently separating and culturing human primary melanocytes. Background technique [0002] Melanocytes are a type of skin cells that secrete melanin to determine the color of the skin. Melanin is mainly gathered in epidermal cells to prevent skin from being damaged by light radiation. The functional defects of melanocytes, including the synthesis and metabolism of melanin, are closely related to the occurrence of skin pigmentation diseases and malignant melanoma. Primary melanocytes cultured in vitro are widely used to study the function of melanocytes, skin pigment diseases and the pathogenesis of melanoma. Moreover, with the development of tissue engineering and regenerative medicine, melanocytes cultured in vitro can be used in the clinical treatment of various pigment-deficient diseases, such as vitiligo. [0003] Since melanocytes only acco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0626C12N2501/70C12N2509/00
Inventor 吴训伟弭军徐琳
Owner SHANDONG UNIV
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