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Kit and method capable of quickly separating adipose tissue-derived stem cells

A mesenchymal stem cell and kit technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of purity, quantity and primary culture time of adipose-derived mesenchymal stem cells

Inactive Publication Date: 2013-05-08
北京清美联创干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of the purity, quantity and primary culture time of adipose-derived mesenchymal stem cells, the present invention provides a kit for isolating adipose-derived mesenchymal stem cells

Method used

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  • Kit and method capable of quickly separating adipose tissue-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Preparation of a kit for isolating adipose-derived mesenchymal stem cells

[0026] 1. Washing solution: Take 9g NaCl and dilute it to 1000ml with deionized water, autoclave to prepare 0.9% sodium chloride washing solution, and place it in a refrigerator at 4°C for later use.

[0027] 2. Cell culture medium: Knockout-DMEM liquid medium containing 100ng / ml LIF and 100ng / ml bFGF cytokines and sterile fetal calf serum culture medium are mixed according to the volume ratio of 4:1 to prepare the cell culture mixture used in cell culture .

[0028] 3. Cell digestion solution: prepare a collagenase type I enzyme solution with a mass / volume percentage concentration of 0.2%.

Embodiment 2

[0029] Example 2. Packing of the kit for isolating adipose-derived mesenchymal stem cells

[0030] The specification of the kit is 1 time / box, the amount of each component in each box: 1 bottle of washing solution (100ml / bottle), cell culture

[0031] 1 bottle of culture solution A (80ml / bottle), 1 bottle of cell culture solution B (20ml / bottle), 1 bottle of cell digestion solution (100ml / bottle). catch

[0032] Each component in the kit is packaged independently for the above dosage to obtain a kit for isolating adipose-derived mesenchymal stem cells.

Embodiment 3

[0033] Example 3. Isolation and Expansion of Adipose-derived Mesenchymal Stem Cells

[0034] Aseptically collect the fat, immerse it in the cell culture solution (A and B) containing 1% double antibody (penicillin streptomycin), and transport it to the laboratory in a sealed glass container; cut about 1g of fat in a biological safety cabinet, and use physiological The salt water washes the blood from the blood vessels in the fat. Cut the adipose tissue into minced meat, transfer to a centrifuge tube, add 20ml of cell digestion solution, mix well and digest overnight at 37°C for about 6 hours. Centrifuge, resuspend the cells with cell culture medium, inoculate the primary cells in a 0.2% gelatin-coated culture flask, and culture in a 37°C, 5% CO2 incubator.

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Abstract

The invention provides a kit and a method capable of quickly separating adipose tissue-derived stem cells. The kit comprises a washing liquid, a cell culture fluid A, a cell culture medium B and a cell digestive juice, wherein the washing fluid is normal saline (containing 0.9% sodium chloride); the cell culture medium A is a Knockout-DMEM liquid medium containing 100 ng / mL LIF and 100 ng / mL bFGF cytokines; the cell culture medium B is fetal bovine serum; and the cell digestive juice is a collagenase type I enzyme solution with a mass to volume percent concentration of 0.2%. Compared with a conventional separation culture method, the cells obtained by the kit have high purity and fast propagation. Generally, a primary cell culture can be completed in seven days; the primary cells are sub-cultured by using the culture solution, and cell density can reach 90% in 2-3 days. The kit and the method provide a rich source for further experimental study by using the adipose tissue-derived stem cells.

Description

technical field [0001] The invention belongs to the field of tissue engineering and relates to a kit for isolating adipose mesenchymal stem cells, an application of the kit and a method for isolating adipose mesenchymal stem cells. Background technique [0002] Adipose-derived mesenchymal stem cells are a type of stem cells with self-renewal, proliferation and multi-directional differentiation potentials. They have many advantages such as autologous origin, wide range of materials, easy collection, storage and transportation, no allogeneic rejection, and avoiding ethical disputes. Flow cytometry analysis found that adipose-derived mesenchymal stem cells highly expressed mesenchymal cell markers (CD44, CD105), integrin receptors (CD29, CD49b, CD49c, CD51), and did not express hematopoietic lineage markers (CD34, CD45) and endothelial cell markers. Marker CD31. Adipose-derived mesenchymal stem cells can differentiate into bone cells, chondrocytes, liver cells and adipocytes i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 田杰
Owner 北京清美联创干细胞科技有限公司
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