Preparation method of autologous skin fibroblasts

A fibroblast and skin technology, applied in the field of preparation of autologous skin fibroblasts, can solve the problems of long digestion time, pollution, complicated operation steps, etc., and achieve the effect of saving filtration steps

Inactive Publication Date: 2021-06-18
纳美细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above patents have a long digestion time or need to be digested twice or more, or need to be filtered after digestion, the operation steps are cumbersome, and it is easy to cause pollution due to human operation.

Method used

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  • Preparation method of autologous skin fibroblasts
  • Preparation method of autologous skin fibroblasts
  • Preparation method of autologous skin fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation method of autologous skin fibroblast proposed by the present invention comprises the following steps:

[0028] S1: Cut a piece of human autologous skin tissue from the cheek or behind the ear, completely immerse the tissue in a tissue treatment solution mixed with 1% double-antibody and 0.9% saline for 20 minutes in a sterile environment, and remove the adipose tissue and connective tissue from the soaked tissue Tissue, the rest of the tissue was placed in a sterile petri dish and shredded, the size of the shredded skin tissue was about 0.1mm 3 ;

[0029] S2: Mix 0.5ml dispase, 1.0ml DNase, 0.5ml collagenase II, 0.5ml collagenase IV, and 1.0ml hyaluronidase, add Hank's balanced salt solution to prepare a mixed enzyme solution, and heat it at 37°C warm up;

[0030] S3: Add the shredded tissue to 7ml of mixed enzyme digestion solution containing Hank’s balanced salt solution, and digest on a shaking table at 37°C and 80rpm for 30min. After the digestion ...

Embodiment 2

[0035] The preparation method of autologous skin fibroblast of the present invention comprises the following steps:

[0036] S1: Cut a piece of human autologous skin tissue from the cheek or behind the ear, completely immerse the tissue in a tissue treatment solution mixed with 1% double-antibody and 0.9% saline for 20 minutes in a sterile environment, and remove the adipose tissue and connective tissue from the soaked tissue Tissue, the remaining tissue is placed in a sterile petri dish and chopped, the size of the skin tissue obtained by chopping is about 0.2mm 3 ;

[0037] S2: Mix 1.0ml dispase, 0.5ml DNase, 0.7ml collagenase II, 1.5ml collagenase IV, and 0.3ml hyaluronidase, add Hank's balanced salt solution to prepare a mixed enzyme solution, and heat it at 37°C warm up;

[0038] S3: Add the shredded tissue to 10ml of mixed enzyme digestion solution containing Hank’s balanced salt solution, shake and digest on a shaking table at 37°C and 80rpm for 30min, and add stop di...

Embodiment 3

[0043] The preparation method of autologous skin fibroblast of the present invention comprises the following steps:

[0044] S1: Cut a piece of human autologous skin tissue from the cheek or behind the ear, completely immerse the tissue in a tissue treatment solution mixed with 1% double-antibody and 0.9% saline for 20 minutes in a sterile environment, and remove the adipose tissue and connective tissue from the soaked tissue Tissue, the rest of the tissue was placed in a sterile petri dish and shredded, and the size of the shredded skin tissue was about 0.3mm 3 ;

[0045] S2: Mix 0.2ml dispase, 0.8ml DNase, 1.5ml collagenase II, 1.0ml collagenase IV, and 0.5ml hyaluronidase, add Hank's balanced salt solution to prepare a mixed enzyme solution, and heat it at 37°C warm up;

[0046] S3: Add the shredded tissue to 14ml of mixed enzyme digestion solution containing Hank’s balanced salt solution, shake and digest on a shaking table at 37°C and 80rpm for 30min, and add stop diges...

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Abstract

The invention discloses a preparation method of autologous skin fibroblasts. The preparation method comprises the following steps: S1, cutting a piece of human autologous cheek or retroauricular skin tissue, completely soaking the tissue in a tissue treatment solution for 10-20 min in a sterile environment, removing adipose tissue and connective tissue from the soaked tissue, and cutting the residual tissue into pieces in a sterile culture dish; S2, adding the cut tissue into a mixed enzyme digestive juice preheated at 37 DEG C for digestion, and adding a digestion termination juice after digestion is ended to terminate digestion; S3, merging the obtained tissue fluid into a centrifugal tube, centrifuging, removing supernatant, and adding a cell culture fluid into the cell precipitate to obtain P0 fibroblasts; and S4, inoculating the P0-generation cells into a culture bottle, collecting the cells after culture is finished to obtain P1-generation fibroblasts, sampling, and counting the number and motility rate of the cells. The mixed digestive juice of multiple enzymes is adopted, digestion can be directly carried out, the operation process is simple, and consumed time is short.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for preparing autologous skin fibroblasts. Background technique [0002] The skin is the tissue that wraps around the muscles and covers the whole body. It is the largest organ of the human body and can be divided into three layers: the epidermis, the dermis and the subcutaneous tissue. Skin fibroblast (Fbs) is the main structural component of the skin dermis, which can synthesize and secrete collagen fibers, elastic fibers, reticular fibers, hyaluronic acid and other extracellular matrices, which are important for maintaining skin strength and elasticity, repairing damage and Beauty and body care play an important role. Its normal proliferation and differentiation is to maintain the normal structure and physiological functions of the skin. [0003] As people grow older, the number of skin fibroblasts decreases and their activity decreases. Correspondingly, the su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2509/00C12N2509/10C12N2501/998C12N2501/115C12N2501/11
Inventor 李怡林
Owner 纳美细胞科技有限公司
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