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Method for recovering adipose mesenchymal stem cells

A technology of mesenchymal stem cells and fat, applied in the field of resuscitating adipose-derived mesenchymal stem cells, can solve the problems of unfavorable tissue and organ damage repair, low cell viability, etc., and achieve the effect of improving vitality and promoting growth

Active Publication Date: 2016-01-06
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the above resuscitation method is relatively simple and fast, the cell viability after resuscitation by this method is low, which is not conducive to subsequent clinical applications such as tissue and organ damage repair.

Method used

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  • Method for recovering adipose mesenchymal stem cells
  • Method for recovering adipose mesenchymal stem cells
  • Method for recovering adipose mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Culture of adipose-derived mesenchymal stem cells and collection of conditioned medium

[0036] Wash the extracted adult fat with PBS, cut up the adipose tissue, add 0.25 type I collagenase, digest and enzymatically hydrolyze at 37°C, 200rpm for 50 minutes, centrifuge at 1000-2000rpm for 5-10 minutes, and put on Add complete medium (DMEM / F12+10% FBS) to resuspend the cell pellet, then place it at 37°C, 5% CO 2 Cell culture, change the medium every 2-3 days, when the cell fusion degree reaches 80%-90%, digest the cells with 0.25% trypsin, resuspend the pellet with complete medium after centrifugation, (5-10)×10 3 / cm2 cell density to inoculate the culture dish and continue to culture. The P3-P5 generation cells were collected for conditioned medium collection.

[0037] Conditioned medium collection: Adipose-derived mesenchymal stem cells used for conditioned medium, first according to (5-10) × 10 3 / cm 2The cells were inoculated into culture dishes at a de...

Embodiment 2

[0038] Example 2: Recovery of Adipose-derived Mesenchymal Stem Cells

[0039] Take the cryopreserved adipose-derived mesenchymal stem cells out of liquid nitrogen, place them in a water bath at 35-42°C, and incubate with shaking for 60 seconds. After the cells are thawed, immediately add the cryopreservation solution to 5 times the volume of conditioned medium The medium cell suspension was centrifuged at 1000rpm for 5 minutes, the supernatant was removed, and the pellet was resuspended with the conditioned medium; 3 / cm 2 Cells were seeded at a density in Petri dishes and cultured with conditioned medium. Place at 37°C, 5% CO 2 After the incubator was left standing for 24 hours, the conditioned medium was replaced to continue the culture, and the cell expansion was observed every day, and the cells were continued to be cultured until the degree of cell fusion reached 80%-90%.

Embodiment 3

[0040] Embodiment 3: comparison of resuscitated cell viability

[0041] Experimental group: The conditioned medium was prepared according to Example 1, and the frozen adipose-derived mesenchymal stem cells were taken out of liquid nitrogen, placed in a water bath at 35-42°C, and incubated with shaking for 60 seconds. After the cells were thawed, immediately Add the cryopreservation solution to 5 times the volume of conditioned medium, take a small amount of cell suspension, add trypan blue, place it in a cell viability counter for cell viability detection, and centrifuge the rest of the cell suspension at 1000rpm for 5 minutes, remove the supernatant , resuspend the pellet with conditioned medium; gently pipette to mix, and follow the 7×10 3 / cm 2 Cells were seeded in six-well plates and cultured with conditioned medium. Place at 37°C, 5% CO 2 Culture in an incubator for 72 hours, digest the centrifuged cells with trypsin, resuspend the cells with conditioned medium, take a...

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Abstract

The invention relates to the technical field of biology and discloses a method for recovering adipose mesenchymal stem cells. The method comprises the following steps: culturing adipose mesenchymal stem cells which are abdicated to generations P3-P5 by virtue of a complete culture medium, then culturing by virtue of a serum-free DMEM / F12 culture medium, respectively collecting supernatants of the culture medium when the adipose mesenchymal stem cells are cultured for 24h, 48h and 72h, centrifuging each supernatant, and mixing the supernatants, so as to obtain a conditional culture medium; unfreezing the cryopreserved adipose mesenchymal stem cells, adding the adipose mesenchymal stem cells into the conditional culture medium, carrying out centrifugation to remove the supernatant, adding the adipose mesenchymal stem cells into the conditional culture medium again, carrying out re-suspension, and culturing the adipose mesenchymal stem cells until the fusion degree of the cells meets the requirements. According to the method, the supernatant of the culture medium which is used for culturing the adipose mesenchymal stem cells is taken as the culture medium during the recovery and is used for replacing the complete culture medium generally utilized for culturing the adipose mesenchymal stem cells in existing methods, and the growth of the recovered adipose mesenchymal stem cells is promoted by virtue of active substances secreted in the propagation process of multiple cells contained in the supernatant, and therefore, the activities of the recovered adipose mesenchymal stem cells are improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for recovering adipose-derived mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are an important member of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development, and belong to pluripotent stem cells. MSCs were originally found in the bone marrow because of their multi-directional differentiation potential, hematopoietic support and The promotion of stem cell engraftment, immune regulation and self-replication has attracted increasing attention. For example, mesenchymal stem cells can be differentiated into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells under specific induction conditions in vivo or in vitro, after continuous subculture and cryopreservation It still has multi-directional differentiation potential and can be used as an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 王一飞陈海佳葛啸虎冯德龙王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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