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A method for extracting and culturing human adipose stem cells

A technology of adipose stem cells and human fat, applied in the field of cell engineering, can solve problems such as unclear, small number of stem cells, slow subculture proliferation, etc., to achieve the effect of increasing the number and increasing the number

Active Publication Date: 2021-08-24
朗姿赛尔生物科技(广州)有限公司
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Problems solved by technology

The disadvantage of this method of cultivating stem cells is that the method is complicated, the number of stem cells extracted is small, the purity is not high, and the subculture proliferation is slow.
The existing conventional culture technology uses exogenous serum (such as fetal bovine serum) to promote the proliferation of adipose-derived stem cells. Although this technology can achieve the purpose of proliferation, the primary culture takes a long time, and although most of the components of exogenous serum are known, But there is still a part that is not clear, and the composition and content of serum often vary with the sex, age, physiological conditions and nutritional conditions of the blood-supplying animals

Method used

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  • A method for extracting and culturing human adipose stem cells

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Embodiment 1

[0021] Example 1 Extraction method of human adipose stem cells

[0022] Human fat digestion solution contains: 0.6mg / ml cholic acid, 1mg / ml glycine, 1.5mg / ml α1-antitrypsin, 15μl / ml dimethyl sulfoxide, 205mg / ml 0.9% normal saline, 75mg / ml magnesium chloride, 20mg / mL choline chloride, 5mg / ml Trypsin-EDTA.

[0023] The extraction of human adipose stem cells is achieved by a method comprising the following steps:

[0024] Place the human adipose tissue into a centrifugal separation device, then add physiological saline, fully submerge it, stir well, use a centrifugal device to separate the human adipose tissue, and obtain an upper oil layer, a middle fat layer, and a lower deposition layer; put the middle fat layer in In the centrifugal separation device, add an equal volume of human fat digestion solution and mix evenly. After centrifugation, remove the floating fat and take the upper liquid, then add Dulbecco's phosphate buffer to the upper liquid, then centrifuge, remove the...

Embodiment 2

[0025] Embodiment 2 Culture expansion method of human adipose stem cells

[0026] Adipose stem cell medium contains: DMEM / F12 basal medium, 14μg / ml thyroxine, 8μg / ml insulin, 10mmol / L glutamine, 200ng / ml iron gluconate, 18mg / ml sodium selenate, 12mg / ml vitamin C , 25mg / ml fibroblast growth factor, 9mg / ml L-proline, 2.5mg / ml choline chloride, 1.5mg / ml penicillin.

[0027] The culture and expansion of human adipose stem cells is realized by a method comprising the following steps:

[0028] The 15th passage human adipose stem cells were inoculated in a culture flask containing DMEM / F12 basal medium at a density of 1.5×104 / cm2, cultured at 37°C with 6% CO2 for 13 hours, and replaced with the stem cell medium of the present invention , 37 ° C, 6% CO2 continue to culture for 34 hours.

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Abstract

The invention relates to the technical field of cell engineering, in particular to a method for extracting, culturing and amplifying human adipose stem cells, comprising an extraction step of human adipose stem cells and a step of cultivating and amplifying human adipose stem cells; the extraction of human adipose stem cells is carried out through human fat digestive juice Realization, described human fat digestion liquid comprises: cholic acid, glycine, α1-antitrypsin, dimethyl sulfoxide, 0.9% normal saline, magnesium chloride, choline chloride, Trypsin-EDTA; The growth is realized through the adipose stem cell culture medium, and the adipose stem cell culture medium comprises: DMEM / F12 basal medium, thyroxine, insulin, glutamine, iron gluconate, sodium selenate, vitamin C, fibroblast growth factor, L-proline, choline chloride, penicillin; the method for extracting and culturing human adipose stem cells described in the present invention can significantly improve the quantity, survival rate and activity of the obtained stem cells.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for extracting, culturing and expanding human adipose stem cells. Background technique [0002] Stem cells are primitive cells with self-renewal ability and multi-directional differentiation potential. Under certain conditions, they can also proliferate and differentiate into different functional cells. Therefore, the study of stem cells is of great significance to the renewal and damage repair of various tissues and organs in the living body, and has gradually become the only hope for many incurable diseases, especially those with cell and tissue loss or damage. Adipose-derived stem cells (ADSCs) and ADSC pluripotent cells are a kind of stem cells with multipotential differentiation potential isolated from adipose tissue in recent years. It mainly restores the repair function of tissue cells, promotes cell regeneration, restores a youthful appearance, and at t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2500/12C12N2500/30C12N2500/32C12N2500/38C12N2501/115C12N2501/33C12N2501/395C12N2509/00
Inventor 陈忠平
Owner 朗姿赛尔生物科技(广州)有限公司
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