MSC (mesenchymal stem cell) culture kit

A quality stem cell and cell culture technology, applied in the field of stem cell culture, can solve the problems of low purity and long primary culture time, and achieve the effect of improving the passage time

Inactive Publication Date: 2016-06-29
赵顺英
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no unified standard for the isolation, culture and expansion of umbilical cord mesenchymal stem cells, and there are disadvantages such as low purity and long primary culture time.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the preparation of kit

[0024] 1. Preparation of components

[0025] Preparation of cell washing solution: Dissolve 9 g of NaCl in deionized water and adjust the volume to 1000 ml with deionized water, autoclave it and place it in a refrigerator at 4°C for later use.

[0026] Preparation of cell culture medium A: Dissolve cytokine LIF, cytokine bFGF and compound Granulodiene A in sterile Knockout-DMEM medium (liquid), the concentration of cytokine LIF is 100ng / ml, and the concentration of cytokine bFGF is 100ng / ml , the concentration of compound GranulodieneA was 25 μg / ml. Store in a 4°C refrigerator for later use.

[0027] Cell culture medium B is sterile fetal bovine serum, which is stored in a -20°C refrigerator for later use.

[0028] Cell digestion solution: dissolve Collagenase II in deionized water so that the concentration of Collagenase II is 0.2g / 100ml, and store in a 4°C refrigerator for later use.

[0029] 2. Packing of components and prep...

Embodiment 2

[0031] Example 2: Preparation of the kit, compared with Example 1, only the cell culture solution A does not add Granulodiene A

[0032] 1. Preparation of components

[0033] Preparation of cell washing solution: Dissolve 9 g of NaCl in deionized water and adjust the volume to 1000 ml with deionized water, autoclave it and place it in a refrigerator at 4°C for later use.

[0034] Preparation of cell culture solution A: cytokine LIF and cytokine bFGF were dissolved in sterile Knockout-DMEM medium (liquid), the concentration of cytokine LIF was 100 ng / ml, and the concentration of cytokine bFGF was 100 ng / ml. Store in a 4°C refrigerator for later use.

[0035] Cell culture medium B is sterile fetal bovine serum, which is stored in a -20°C refrigerator for later use.

[0036] Cell digestion solution: dissolve Collagenase II in deionized water so that the concentration of Collagenase II is 0.2g / 100ml, and store in a 4°C refrigerator for later use.

[0037] 2. Packing of componen...

Embodiment 3

[0039] Example 3: Isolation and rapid expansion of umbilical cord mesenchymal stem cells

[0040] The kits prepared in Example 1 (experimental group) and Example 2 (control group) were used for the isolation and rapid expansion of umbilical cord mesenchymal stem cells.

[0041] Preparation of cell culture medium: Mix cell culture medium A and cell culture medium B at a volume ratio of 5:1.

[0042] 1. Aseptically collect the isolated umbilical cord (the source is human), immerse in the cell culture medium containing double antibody (the cell culture medium containing double antibody is composed of cell culture medium, penicillin and streptomycin, the concentration of penicillin is 1g / 100ml, The concentration of streptomycin is 1g / 100ml), and the glass container is sealed and transported to the laboratory.

[0043] 2. Cut the umbilical cord about 10 cm long in the biological safety cabinet, and clean the blood stains with cell washing solution; remove the outer amnion, 2 umbil...

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Abstract

The invention discloses a MSC (mesenchymal stem cell) culture kit. The MSC culture kit comprises a cell culture solution A and a cell culture solution B, wherein the cell culture solution A is a Knockout-DMEM (dulbecco's modified eagle medium) containing 80-120 ng / ml of cell factor LIF, 80-120 ng / ml of cell factor bFGF and 20-30 mu g / ml of Granulodiene A; the cell culture solution B is fetal calf serum. After the umbilical cord MSCs are subjected to passage for 15 times by the aid of the cell culture solutions, the cells still maintain high purity and good totipotency; when the cell culture solutions are adopted to culture bone marrow MSCs, the same good effect is realized; the results indicate that the primary cell passage time of the MSCs can be prolonged by means of the culture kit, so that the cell totipotency is still maintained after the MSCs are subjected to passage for multiple times.

Description

technical field [0001] The invention belongs to the field of stem cell culture, and in particular relates to a mesenchymal stem cell culture kit. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSC) is an important member of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. Originally found in bone marrow, MSCs have attracted increasing attention because of their multi-lineage differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. 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 ideal seed cell for the repair of tissue...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N5/0668C12N2500/84C12N2501/115C12N2501/2306
Inventor赵顺英
Owner赵顺英