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Preserving fluid for adipose tissue-derived stromal cells

A technology of mesenchymal stem cells and preservation solution, which is applied in the field of stem cell preservation, can solve problems such as differences between serum batches, human adipose-derived mesenchymal stem cell damage, and decreased proliferation ability, so as to avoid damage, improve preservation survival rate, and reduce damage Effect

Inactive Publication Date: 2019-03-29
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DMSO is the most commonly used refrigerant in cell preservation, but it also has certain toxicity to cells. It can denature intracellular proteins at room temperature, thereby damaging frozen human adipose-derived mesenchymal stem cells, and even affecting human adipose-derived mesenchymal stem cells. The survival rate of stem cells is unfavorable, resulting in poor cryopreservation effect
[0005] FBS is the most commonly used protective agent in cell preservation. However, animal-derived serum has the risk of infection with pathogens such as viruses and bacteria, and mesenchymal stem cells will endocytose serum substances when exposed to fetal bovine serum. When using fetal bovine serum to preserve After the mesenchymal stem cells are reinfused into the human body, some proteins in them may cause immune rejection caused by heterologous proteins, which reduces the success rate of mesenchymal stem cell transplantation and at the same time leads to adverse reactions, which may cause allergies in severe cases Sexual shock
In addition, serum has problems such as batch-to-batch variation and instability
[0006] Therefore, the existing storage solution of adipose-derived mesenchymal stem cells has the defects of poor recovery rate and decreased proliferation ability for long-term storage.

Method used

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  • Preserving fluid for adipose tissue-derived stromal cells
  • Preserving fluid for adipose tissue-derived stromal cells
  • Preserving fluid for adipose tissue-derived stromal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1) Aseptically absorb the fat extract from the liposuction operation, wash it twice with normal saline to remove the drugs and blood cells used in the liposuction operation; digest with 0.5% type I collagenase at 37°C for 0.5 hour, centrifuge at 400g / min for 5min, After absorbing the upper layer of fat, mix well and filter with a 200-mesh sieve; centrifuge the obtained cell suspension at 500g / min for 8min, wash the cell pellet with normal saline, and adjust the cell concentration to 7×10 4 cells / mL, suspended in DMEM / F12 medium containing 10% serum by volume, inoculated in a 10cm culture dish, placed at 37°C, 5% CO by volume 2 Cultivate in an incubator; after 48 hours, suck out the suspended cell liquid and replace with a new medium; change the medium once every 3 days, and subculture when the cells reach more than 80% confluence after about 10 days;

[0037]2) Digest the cells with 0.25% trypsin, discard the culture medium in the culture dish or carefully suck off the ...

Embodiment 2

[0044] 1) Aseptically absorb the fat extract from the liposuction operation, wash it twice with normal saline to remove the drugs and blood cells used in the liposuction operation; digest with 0.5% type I collagenase at 37°C for 0.5 hour, centrifuge at 400g / min for 5min, After absorbing the upper layer of fat, mix well and filter with a 200-mesh sieve; centrifuge the obtained cell suspension at 500g / min for 8min, wash the cell pellet with normal saline, and adjust the cell concentration to 7×10 4 cells / mL, suspended in DMEM / F12 medium containing 10% serum by volume, inoculated in a 10cm culture dish, placed at 37°C, 5% CO by volume 2 Cultivate in an incubator; after 48 hours, suck out the suspended cell liquid and replace with a new medium; change the medium once every 3 days, and subculture when the cells reach more than 80% confluence after about 10 days;

[0045] 2) Digest the cells with 0.25% trypsin, discard the culture medium in the culture dish or carefully suck off the...

Embodiment 3

[0052] 1) Aseptically absorb the fat extract from the liposuction operation, wash it twice with normal saline to remove the drugs and blood cells used in the liposuction operation; digest with 0.5% type I collagenase at 37°C for 0.5 hour, centrifuge at 400g / min for 5min, After absorbing the upper layer of fat, mix and filter with a 200-mesh sieve; centrifuge the obtained cell suspension at 1200r / min for 8min, wash the cell pellet with normal saline, and adjust the cell concentration to 7×10 4 cells / mL, suspended in DMEM / F12 medium containing 10% serum by volume, inoculated in a 10cm culture dish, placed at 37°C, 5% CO by volume 2 Cultivate in an incubator; after 48 hours, suck out the suspended cell liquid and replace with a new medium; change the medium once every 3 days, and subculture when the cells reach more than 80% confluence after about 10 days;

[0053] 2) Digest the cells with 0.25% trypsin, discard the culture medium in the culture dish or carefully suck off the cul...

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Abstract

The invention provides a preserving fluid for adipose tissue-derived stromal cells. The preserving fluid contains glycerol, trehalose and human serum albumin. The preserving fluid for adipose tissue-derived stromal cells provided by the invention is an animal serum-free and dimethyl sulfoxide-free freezing medium for adipose tissue-derived stromal cells. Cells cryopreserved with the freezing medium have the characteristics of higher recovery rate after long-term preservation, no influence on cell reproductive capacity and capability of keeping diversity of stem cells after the recovery of cells.

Description

technical field [0001] The invention relates to the technical field of stem cell preservation, in particular to a preservation solution of adipose-derived mesenchymal stem cells. Background technique [0002] Stem cells are a class of primitive and unspecialized pluripotent cells capable of self-replication. Under certain conditions, it can differentiate into a variety of functional cells. Adipose-derived mesenchymal stem cells are an important type of stem cells. Adipose-derived mesenchymal stem cells are a kind of stem cells with multi-directional differentiation potential isolated from adipose tissue in recent years. specialty. Human adipose-derived mesenchymal stem cells are a type of adult stem cells widely used in the fields of tissue engineering and regenerative medicine. They have the same multidirectional differentiation potential as bone marrow mesenchymal stem cells. Adipose-derived mesenchymal stem cells grew like fibroblasts, with abundant cytoplasm and nucle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 陈海佳葛啸虎刘帅王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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