Kit for culturing stem cells

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

Inactive Publication Date: 2016-06-08
赵慧慧
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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 PedinophyllolJ 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 the compound PedinophyllolJ 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...

Embodiment 2

[0031] Embodiment 2: the preparation of kit, compare with embodiment 1, only do not add PedinophyllolJ in cell culture medium 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, wash the blood stains with cell washing solution; remove the outer amnion, 2 umbilical ...

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Abstract

The invention discloses a kit for culturing stem cells. The kit comprises cell culture liquid A and cell culture liquid B. The cell culture liquid A is a Knockout-DMEM culture medium containing 80-120ng / ml of cell factor LIF, 80-120ng / ml of cell factor bFGF and 20-30 microgram / ml of Pedinophyllol J. The cell culture liquid B is fetal calf serum. The kit has the advantages that umbilical cord mesenchymal stem cells can still maintain high purity and good totipotency after 15 times of generation passages by the cell culture liquid; good effect can also be achieved when the cell culture liquid is used for culturing mesenchymal stem cells; the results show that the kit can increase the generation passage time of primary mesenchymal stem cells, and the mesenchymal stem cells can still keep good cell totipotency after multiple times of generation passages.

Description

technical field [0001] The invention belongs to the field of stem cell culture, and in particular relates to a 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 and organ d...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N5/0665C12N2501/115C12N2501/235C12N2501/999
Inventor赵慧慧
Owner赵慧慧