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A method for isolating and culturing skin keratinocytes

The technology of a keratinocyte and separation method is applied in the field of separation and acquisition of skin keratinocytes, which can solve the problems of rejection reaction and low efficiency, and achieve the effect of avoiding pollution and high vitality.

Active Publication Date: 2019-12-06
NOVAPRINT THERAPEUTICS SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the current problems of keratinocyte isolation and culture such as low efficiency and rejection, the present invention provides a method for keratinocyte isolation and culture

Method used

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  • A method for isolating and culturing skin keratinocytes
  • A method for isolating and culturing skin keratinocytes
  • A method for isolating and culturing skin keratinocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Successful isolation and expansion of human epidermal cells from adult normal chest skin

[0062] (1) Materials and methods

[0063] Sample: adult normal chest skin (including epidermis and dermis) discarded during hospital surgery, about 3cm 2 .

[0064] Cell culture medium: KGM-Gold.

[0065] (2) Cell isolation and culture

[0066] a) The parts where the skin is taken, the parts with hair should be removed first, and the cuticle should be removed in advance if the cuticle is too thick, and then sterilized with 75% alcohol cotton balls, and the tissue slices were collected.

[0067] b) Fresh skin samples are placed in HBSS / DPBS / DMEM, stored at 4°C and transported to the laboratory, and the area is measured.

[0068] c) Disinfect the sample in iodine tincture and 75% alcohol respectively, and then wash it with DPBS.

[0069] d) removing the yellow fat layer and most of the white dermis in the tissue sheet, retaining the epidermis and the 0.2-2 mm thick d...

Embodiment 2

[0078] Example 2: Successful isolation and expansion of human epidermal cells from adult normal chest skin

[0079] (1) Materials and methods

[0080] Sample: adult normal chest skin (including epidermis and dermis) discarded during hospital surgery, about 3cm 2 .

[0081] Cell culture medium: DKSFM.

[0082] (2) Cell isolation and culture

[0083] a) The parts where the skin is taken, the parts with hair should be removed first, and the cuticle should be removed in advance if the cuticle is too thick, and then sterilized with 75% alcohol cotton balls, and the tissue slices were collected.

[0084] b) Fresh skin samples are placed in HBSS / DPBS / DMEM, stored at 4°C and transported to the laboratory, and the area is measured.

[0085] c) Disinfect the sample in iodine tincture and 75% alcohol respectively, and then wash it with DPBS.

[0086] d) removing the yellow fat layer and most of the white dermis in the tissue sheet, retaining the epidermis and the 0.2-2 mm thick derm...

example 2

[0094] Example 2: Preparation of epidermal cells from healthy male foreskin

[0095] (1) Materials and methods

[0096] Sample: circumcised skin from a healthy male

[0097] (2) Cell separation and cultivation: the method is the same as in Example 1.

[0098] (3) Results: The isolated cells are easy to adhere to the wall, have more clones, and can quickly connect into sheets in the shape of "paving stones". Figure 4 shown. The cells are relatively pure, and can still maintain a good state and proliferation ability after passage and cryopreservation.

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Abstract

In order to solve the problems of an existing keratinocyte isolation and culture method which is low in efficiency, causes a rejection reaction and the like, the invention provides a keratinocyte isolation and culture method; digestion is carried out in two steps by virtue of Trypsin / EDTA digestion liquid and I-shaped collagenase, and keratinocytes which are relatively strong in proliferative capacity are mainly extracted from a basal layer and extracted around hair follicle; relatively simplex keratinocytes are obtained; and the keratinocytes are high in activity, easy in adhesion to wall and easy in proliferation.

Description

technical field [0001] The invention relates to a method for separating and obtaining skin keratinocytes, in particular to a method for separating and culturing skin keratinocytes for culturing and obtaining tissue-engineered epidermis. Background technique [0002] Skin keratinocyte cultures are widely used in scientific research, beauty products and medical fields. The keratinocytes with proliferative ability extracted from the epidermis and part of the dermis can be expanded and cultured to establish an epidermal cell bank with high vitality. [0003] In the medical field, when treating skin defects such as vitiligo, burns, melanoma, scars, and stretch marks, traditional drug therapy or photochemotherapy will have unstable outcomes and require continuous treatment. However, if the skin is transplanted from other parts of the body, it will cause skin defects at the site where the skin is taken, or there will be a shortage of skin sources. Therefore, keratinocyte cultures...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12Q1/02A61L27/38A61L27/60
Inventor 刘洪李琳杨熙
Owner NOVAPRINT THERAPEUTICS SUZHOU CO LTD
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