Mixed cultivation method of keratinocyte and melanocyte and application

A technology of keratinocytes and melanocytes, applied in the field of mixed culture of keratinocytes and melanocytes, which can solve the problems of inefficiency, shortage of skin sources for patients, poor uniformity, etc., and achieve the effect of avoiding pollution

Inactive Publication Date: 2015-12-02
赫柏慧康生物科技无锡有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current treatment methods include topical corticosteroids, NB-UVB / PUVA, excimer laser, etc., but the efficacy is low and accompanied by certain side effects
[0004] Other methods for treating the above-mentioned diseases include: 1) Surgical transplantation techniques, such as negative pressure blistering, edge-thickness skin grafts, and micro-skin grafts, but there is a shortage of skin sources for patients with large areas; Melanocyte transplantation method, such as the melanocyte transplantation coating carrier disclosed in CN102266586B, the melanocyte culture method disclosed in CN101892286B, and the melanocyte suspension preparation disclosed in CN100354001C, can be used for melanocyte transplantation, but there is melanocyte The risk of tumor complications and poor uniformity, and the biggest technical difficulty lies in the poor growth of melanocytes, which cannot be expanded in a large amount in a short period of time
[0005] On the other hand, there is no specific method for isolating melanocytes other than culture medium, and the number of melanocytes directly isolated from the living body is extremely small and has limited uniformity and proliferation ability, so it is not efficient at all

Method used

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  • Mixed cultivation method of keratinocyte and melanocyte and application
  • Mixed cultivation method of keratinocyte and melanocyte and application
  • Mixed cultivation method of keratinocyte and melanocyte and application

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0052] Example 1: Successful isolation and expansion of human epidermal cells from adult normal breast skin

[0053] (1) Materials and methods

[0054] Sample: adult normal chest skin (including epidermis and dermis) discarded during hospital surgery, about 3cm 2 .

[0055] Skin Media: KGM-Gold vs. KGM

[0056] (2) Cell isolation and skin slice culture

[0057] a) The skin samples were transported to the laboratory in refrigerated DMEM medium, and the area was measured.

[0058] b) Wash the sample with PBS; sterilize with iodine and alcohol and then wash the sample again, remove the yellow fat layer and most of the white dermis in the tissue slice, keep the epidermis and 0.2-3mm thick dermis, and decompose the skin slice into tiny particles .

[0059] c) 0.05% trypsin cold digestion sample for 12-18 hours; add trypsin stop solution; filter to collect epidermal tissue.

[0060] d) Add type I collagenase and magnetically stir for 2-6 hours to heat digest, filter, centr...

example 2

[0068] Example 2: The foreskin skin of a healthy male after circumcision was successfully isolated and expanded to culture human epidermal cells

[0069] (1) Materials and methods

[0070] Sample: Foreskin skin (including epidermis and dermis) of healthy males after circumcision, about 4cm 2 .

[0071] Skin Media: KGM-Gold vs. KGM

[0072] (2) Cell isolation and skin slice culture

[0073] a) The skin samples were transported to the laboratory in refrigerated DMEM medium, and the area was measured.

[0074] b) Wash the sample with PBS; sterilize with iodine and alcohol and then wash the sample again, remove the yellow fat layer and most of the white dermis in the tissue slice, keep the epidermis and 1-3mm thick dermis, and decompose the skin slice into tiny particles .

[0075] c) 0.05% trypsin cold digestion sample for 15 hours; add trypsin stop solution; filter to collect epidermal tissue.

[0076] d) adding type I collagenase and magnetic stirring for 5 hours of t...

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Abstract

The invention provides a mixed cultivation method of keratinocyte and melanocyte comprising: breaking the skin sample reserving epidermal layer and at least part of corium layer, cold digesting by pancreatin; collecting deposition organization; heat digesting by I type collagenase, collecting cells; primary inoculating, culturing in KGM-Gold medium; subcultring to the cells merge, culturing in KGM-Gold medium to the cells differentiate and stratify; collecting the formed skin sheet. The mixed cultivation method of keratinocyte and melanocyte uses the medium only for skin keratinocyte and melanocyte with which can avoid pollution of the other cells, and can get skin sheet including functional melanocyte and distributing uniformly. The mixed cultivation method of keratinocyte and melanocyte can get large area of skin sheet from quite small area of autologous sample, solve the problem of shortage of the skin source at skin grafting for the patient with large-area skin diseases, and apply in skin diseases of pigment deficiency such as leucoderma and so on.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a method for mixed culture of keratinocytes and melanocytes (melanocytes) and its application. Background technique [0002] The difference in skin color mainly depends on the amount, size, type and distribution of melanin in the skin. Melanin is a protein derivative produced by melanocytes in the epidermis. [0003] White spots appear on various parts of the body of some patients with skin diseases because the skin melanocytes are missing, damaged or non-functional, which in turn affects the skin pigment manufacturing system. For example, vitiligo is a common acquired localized or generalized skin depigmentation disease, which is caused by the disappearance of melanocytes in the skin. Other similar diseases include moles, chloasma, freckles, nevus of Ota, etc. Pigmentary disorders. The disease can cause severe physical and psychological damage to the sufferer. The current treatment m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61L27/38A61L27/60
Inventor 刘洪李琳杨熙
Owner 赫柏慧康生物科技无锡有限公司
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