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Freezing medium, application thereof and adipose tissue freezing method

An adipose tissue and cryopreservation technology, applied in the field of cells, can solve the problem of unstable preservation effect of adipose tissue, and achieve the effect of maintaining good cell activity and good protection.

Inactive Publication Date: 2017-05-31
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no special method for adipose tissue cryopreservation. The classic cryopreservation solution formula is FBS+DMSO, which is mainly used for cell cryopreservation, but the preservation effect of this cryopreservation solution on adipose tissue is not stable.

Method used

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  • Freezing medium, application thereof and adipose tissue freezing method
  • Freezing medium, application thereof and adipose tissue freezing method
  • Freezing medium, application thereof and adipose tissue freezing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～3

[0033] Prepare freezing solution according to the formula in Table 1, and then sterilize:

[0034] Table 1 Example 1-3

[0035] NEAA volume fraction DMSO Glycerol 1,2-propanediol DMEM / F12 Example 1 10mL 200mL 150mL 150mL 490mL Example 2 10mL 100mL 300mL 300mL 290mL Example 3 10mL 200mL 50mL 50mL 690mL

[0036] Collect 30 human adipose tissues, divide them into 3 groups at random, wash them twice with equal volume of normal saline, centrifuge at 500 g for 5 min, discard the upper layer of fat layer and the lower layer of normal saline layer, cut them into pieces with scissors, pass through a 2mm sieve, and then the fat of each group The tissues were mixed with the cryopreservation solutions of Examples 1-3 at a volume ratio of 1:1, and entered into liquid nitrogen. Keep for 3 months.

Embodiment 4

[0043] Adipose tissue without cryopreservation was taken as the control group, and primary separation was performed to obtain P0 adipose stem cells, which were observed under an inverted microscope.

[0044] The adipose tissue after cryopreservation was prepared in Examples 1-3 and Comparative Examples 1-3, and after resuscitation, the primary separation was carried out to obtain P2 generation adipose stem cells, which were observed under an inverted microscope ( figure 1 ).

[0045] The primary separation method is as follows: digest with type I collagenase at a final concentration of 0.2% for 35 minutes, then centrifuge at 1800 RPM-5MIN, keep only the bottom precipitate, wash the precipitate with normal saline, and inoculate and culture to obtain P0 cells.

[0046] The results of statistical separation success rate are shown in Table 3:

[0047] Table 3 separation success rate

[0048] Separation success rate control group 100％ Example 1 100％ E...

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Abstract

The invention relates to the technical field of cells, in particular to a freezing medium, application thereof and an adipose tissue freezing method. The freezing medium comprises a basic culture solution, an NEAA (non-essential amino acid), DMSO, glycerol and 1,2-propanediol, and has a good protection effect on adipose tissue. Cells in adipose tissue which is thawed after being frozen by the freezing medium keep good activity. The success rate of obtaining adipose-derived stem cells by primary isolated culture is 100 percent.

Description

technical field [0001] The invention relates to the field of cell technology, in particular to a cryopreservation solution and its application and a method for freezing adipose tissue. Background technique [0002] Adipose tissue: mainly composed of a large number of clustered fat cells, which are separated into lobules by a thin layer of loose connective tissue. The reticular fibers in adipose tissue are well developed. Yellow (white) adipose tissue is yellow (white in some mammals), which is commonly referred to as adipose tissue. It is formed by the accumulation of a large number of single-vessel fat cells. The fat cells are round or polygonal. There is a large lipid droplet in the center of the cell. The cytoplasm is a thin layer, located at the periphery of the cell, surrounding the lipid droplet. On HE slices, lipid droplets are dissolved into a large vacuole. The nucleus is oblate, pushed to the side of the cell by lipid droplets, and part of the cytoplasm is cresc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 陈海佳葛啸虎王一飞黄幸王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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