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Melanocyte culture method

A melanocyte and culture method technology, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of low proportion and total number of melanocytes, low proliferation and differentiation activities, and achieve good adherence performance. , good activity, reducing the effect of cell damage

Active Publication Date: 2015-12-02
AMT BIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of low proportion and total number of melanocytes and low proliferation and differentiation activity in melanocyte culture in the prior art, a culture method that can produce melanocytes with high proportion and high differentiation activity is provided

Method used

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  • Melanocyte culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Add 20 mL of DMEM medium, penicillin, and streptomycin (penicillin 100 units / mL; streptomycin 100 μg / mL) into a 50 mL centrifuge tube as a skin bleb preservation solution.

[0054] Add 1mL, 10g / L type I collagenase into a Φ60mm Petri dish and set aside. Put the isolated epidermis (acquired by sucking blisters, 8-15 sucking blisters) into the above-mentioned petri dish, and use ophthalmic scissors to cut the epidermis into pieces of 2mm×2mm.

[0055] After shredding, add 1 mL of 2.5 g / L trypsin·0.38 g / LEDTA solution to the petri dish, and put it in a 37°C incubator for 1 hour.

[0056] Every half hour, take out the petri dish and observe the status of digestion under the microscope. After 1 hour, the petri dish was taken out and the skin fragments were blown and observed under a microscope. If it was found that the cells on the epidermis were basically completely digested, the pre-medium was added to terminate the digestion.

[0057] Recover the digested skin pieces an...

Embodiment 2

[0070] Add 20 mL of DMEM medium, penicillin, and streptomycin (penicillin 100 units / mL; streptomycin 100 μg / mL) into a 50 mL centrifuge tube as a skin bleb preservation solution.

[0071] Add 1mL, 10g / L type I collagenase into a Φ60mm Petri dish and set aside. Put the isolated epidermis (acquired by sucking blisters, 8-15 sucking blisters) into the above-mentioned petri dish, and use ophthalmic scissors to cut the epidermis into pieces of 2mm×2mm.

[0072] After shredding, add 1 mL of 2.5 g / L trypsin·0.38 g / LEDTA solution to the petri dish, and put it in a 37°C incubator for 1 hour.

[0073] Every half hour, take out the petri dish and observe the status of digestion under the microscope. After 1 hour, the petri dish was taken out and the skin fragments were blown and observed under a microscope. If it was found that the cells on the epidermis were basically completely digested, the digestion was terminated.

[0074] The digested skin pieces and suspension were recovered int...

Embodiment 3

[0078] Add 20 mL of DMEM medium, penicillin, and streptomycin (penicillin 100 units / mL; streptomycin 100 μg / mL) into a 50 mL centrifuge tube as a skin bleb preservation solution.

[0079] Add 1mL, 10g / L type I collagenase into a Φ60mm Petri dish and set aside. Put the isolated epidermis (acquired by sucking blisters, 8-15 sucking blisters) into the above-mentioned petri dish, and use ophthalmic scissors to cut the epidermis into pieces of 2mm×2mm.

[0080] After shredding, add 1 mL of 2.5 g / L trypsin·0.38 g / LEDTA solution to the petri dish, and put it in a 37°C incubator for 0.5 hours.

[0081] Every 15 minutes, remove the Petri dish and observe the digestion status under the microscope. After 0.5 h, take out the petri dish and blow the skin fragments, observe under a microscope, if it is found that the cells on the epidermis are basically completely digested, then add the pre-culture solution to stop the digestion.

[0082] The digested skin pieces and suspension were recov...

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Abstract

The invention discloses a melanocyte culture method. The method comprises the following step: preculturing for 2-4 hours before melanocyte enrichment culture, wherein the preculture medium is prepared by mixing a DMEM (dulbecco's modified eagle medium) culture medium and a Ham's F-12 culture medium in a volume ratio of (2-4):1, and is composed of 1.6-2.0*10<-4>M adenine, 80-100 unit / mL penicillin, 80-100 mu g / mL streptomycin, 0.1-0.5 mu g / mL hydrocortisone, 2-6 mu g / mL insulin, 5-12 ng / mL epidermal growth factor, 0.8-1.5*10<-10>M choleratoxin and 4-7% of fetal calf serum. The preculture in the method promotes wall adherence of melanocytes, and enhances the proportion and cell activity of the melanocytes. After the subculture, the melanocyte content is enhanced from 1% to 90% at the maximum, and the melanocytes have stable shape, functions and hereditary characters.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for culturing melanocytes. Background technique [0002] Vitiligo is a skin and mucous membrane disease characterized by localized or generalized depigmentation caused by the dysfunction and loss of the tyrosine system in melanocytes (MC) of the skin and hair follicles. Melanocytes are located in the basal layer of the epidermis and together with epidermal cells form the epidermal melanin unit, whose main function is to produce melanosomes to protect the skin from damage. [0003] The change process of melanocytes in skin lesions of patients with vitiligo is from early functional inactivation to late loss. Based on this pathological change, the principle of effective treatment is to transplant their own melanocytes from healthy skin to the leukoplakia without melanocytes, and in the leukoplakia without melanocytes. Survival after transplantation produce...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 罗卫李明刁云珍徐印祥
Owner AMT BIO
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