Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof

A technology of mesenchymal stem cells and stem cells, which is applied in the field of preparation of adipose-derived mesenchymal stem cells and their preparations, can solve the problems of limitations and low purity, and achieve the effects of accelerating regeneration and replacement, high purity, and promoting proliferation

Inactive Publication Date: 2015-07-08
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there are the following disadvantages: 1. The induced hematopoietic stem cells are mainly hematopoietic stem cells, which are functionally limited to the blood system; 2. The purity of the hematopoietic stem cells obtained by culture is 5-40%, which is too low

Method used

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  • Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof
  • Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof
  • Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Adipose-derived mesenchymal stem cell extraction

[0052] 1. Obtain the fat solution and transfer it to a laboratory ultra-clean bench at 4°C;

[0053] 2. Divide adipose tissue evenly into 50mL centrifuge tubes, add 0.5% type I collagenase to each tube, seal and shake back and forth horizontally to mix well. The volume ratio of the type I collagenase to the fat is 0.5-1:1.

[0054] 3. Transfer to a constant temperature air shaker, digest at 37°C and 100R for 50 minutes; add 1mL FBS with a 1mL pipette tip to stop the digestion, blow evenly with a Pasteur pipette, balance the centrifuge tube, put it in a centrifuge, and centrifuge at 1500rpm / min for 5min .

[0055] 4. Aspirate the supernatant with a Pasteur pipette, add 40mL PBS to each tube to resuspend the cells, take 20uL of the cell suspension and count it with a cell counter, balance the centrifuge tubes, place them in a centrifuge, and centrifuge at 1500rpm / min for 5min.

[0056] 5. Use a Pasteur pipett...

Embodiment 2

[0058] Example 2 Flow cytometric identification of adipose-derived mesenchymal stem cells

[0059] Such as figure 2 As shown, from the results of flow cytometry, the surface markers of adipose-derived mesenchymal stem cells

[0060] CD59+CD90+CD45-HLA-DR- up to 99.7%.

Embodiment 3

[0061] Example 3 Adipose-derived mesenchymal stem cell culture

[0062] After 1.48 hours, use a Pasteur pipette to suck off the medium in the dish prepared in Example 1, add 10 mL of PBS to each dish for washing, and repeat twice.

[0063] 2. Inoculate 10mL of cell suspension into a 10cm culture dish, and add 100uL of 1ug / mL EGF to each dish. Shake the culture dish 5 times to make the cell suspension evenly distributed in the culture dish, transfer to 5% CO 2 , 37°C, and a cell incubator with a saturated humidity of 95% to continue culturing, observe the state of the cells and take pictures every day.

[0064] 3. When the cells grow to 80% confluence, discard the culture medium, add 10ml of PBS to wash once, add 4ml of 0.25w / v% EDTA-trypsin solution to digest for 1-2min. The concentration of EDTA-trypsin in the EDTA-trypsin solution is 0.25w / v%, and the ratio of the adherent area of ​​the adipose-derived mesenchymal stem cells to the EDTA-trypsin solution is: every 10cm 2 A...

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Abstract

The invention relates to the field of biotechnology, and in particular relates to a preparation method and an application of adipose-derived mesenchymal stem cells and a preparation thereof. The preparation method comprises the following steps: fat is obtained; pretreatment, enzymolysis and centrifugation are carried out; precipitate is collected, such that the adipose-derived mesenchymal stem cells are obtained; the adipose-derived mesenchymal stem cells are cultured; when the adipose-derived mesenchymal stem cells grow to 80% fusion, the adipose-derived mesenchymal stem cells are mixed with EDTA-pancreatin for digestion; digestion is terminated, and centrifugation is carried out; and subculturing is carried out. The method provided by the invention has the following advantages: 1, the purity of cultured stem cells is high; 2, various factors secreted during an adipose-derived mesenchymal stem cell culturing process are recovered, and epidermal cell proliferation can be substantially promoted, which means that epidermal cell renewing and replacement can be accelerated, and a significant anti-aging effect can be achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of adipose-derived mesenchymal stem cells and preparations thereof. Background technique [0002] Adipose-derived stem cells (ADSCs) are a kind of stem cells isolated from adipose tissue in recent years with multipotential differentiation potential. Studies have found that ADSCs cells can proliferate stably in vitro and have a low rate of death. At the same time, it has the advantages of easy extraction, a large number of stem cells can be obtained from a small amount of tissue, suitable for large-scale culture, and less damage to the body. Moreover, it has a wide range of sources and a large reserve in the body. Suitability for autologous transplantation has gradually become one of the new research hotspots in recent years. At present, adipose stem cells are mainly used in cosmetic industries such as breast enhancement and wrinkle removal in adi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P39/06A61K8/98A61Q19/08A23L1/29A23L33/00
Inventor 王一飞陈海佳葛啸虎应杰王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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