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Kit for mesenchymal stem cell culture and application thereof

A cell culture, stem cell technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of long primary culture time and low purity, and achieve fast cell expansion and expansion efficiency. high effect

Inactive Publication Date: 2013-03-13
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

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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  • Kit for mesenchymal stem cell culture and application thereof
  • Kit for mesenchymal stem cell culture and application thereof
  • Kit for mesenchymal stem cell culture and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Preparation of a kit for isolating umbilical cord mesenchymal stem cells

[0037] 1. Preparation of components

[0038] Preparation of cell washing liquid: 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.

[0039] Preparation of cell culture solution A: Dissolve cytokine LIF and cytokine bFGF in sterile Knockout-DMEM medium (liquid), the concentration of cytokine LIF is 100ng / ml, the concentration of cytokine bFGF is 100ng / ml, place in Store in the refrigerator at 4°C for later use.

[0040] Cell culture medium B is sterile fetal bovine serum, which is stored in a -20°C refrigerator for later use.

[0041] Cell digestion solution: dissolve Collagenase II in deionized water so that the concentration of Collagenase II is 0.2% (g / 100ml), and place it in a 4°C refrigerator for later use.

[0042] 2. Packing of components and preparation of kits

[0...

Embodiment 2

[0044] Example 2. Isolation and Rapid Expansion of Umbilical Cord Mesenchymal Stem Cells

[0045] The kit prepared in Example 1 was used for the isolation and rapid expansion of umbilical cord mesenchymal stem cells.

[0046] Preparation of cell culture medium: mix cell culture medium A and cell culture medium B according to the volume ratio of 5:1.

[0047] 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.

[0048] 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 umbilical veins and 1 umbilical artery with a ...

Embodiment 3

[0054] Example 3, Verification of pluripotency of umbilical cord mesenchymal stem cells

[0055] In order to confirm the multilineage differentiation potential of umbilical cord mesenchymal stem cells, this example identifies the potential of the cell samples after passage 15 in Example 2 to differentiate into osteoblasts and adipocytes.

[0056] 1. Osteoblast differentiation

[0057] The cell sample was 3000cells / cm 2 Inoculate, then add osteogenic induction medium (add dexamethasone to 100 nM, ascorbic acid to 50 μM, and β-glycerophosphate to 10 mM to Knockout-DMEM medium), at 37 ° C, 5% CO 2 After culturing in an incubator, the following phenomena can be observed after about 5 to 7 days: cells confluent into a single layer, cell protrusions are connected to each other, and can overlap and grow without contact inhibition; cells that overlap grow gradually form nodules, and then collagen accumulates And calcium salt deposition, dense round opaque mass material appears betwe...

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Abstract

The invention discloses a kit for mesenchymal stem cell culture and its application. The kit provided by the invention includes a cell culture solution A and a cell culture solution B. The cell culture solution A is a Knockout-DMEM culture medium containing 80-120ng / ml cytokine LIF and 80-120ng / ml cytokine bFGF. The cell culture solution B is fetal bovine serum. The kit provided by the invention can separate umbilical cord mesenchymal stem cells from an in-vitro umbilical cord and culture the umbilical cord mesenchymal stem cells, and has the advantages of rapid cell expansion speed and high expansion efficiency. After multiple subculturing, cell totipotency can still be maintained. In the invention, the problems of umbilical cord mesenchymal stem cell separation purity, quantity and primary culture time are solved, and a lot of umbilical cord mesenchymal stem cells can be obtained. Thus, the kit provided in the invention provides abundant sources for making use of umbilical cord mesenchymal stem cells to conduct further experimental research.

Description

technical field [0001] The invention relates to a kit for culturing mesenchymal stem cells and its application. Background technique [0002] Mesenchymal stem cells (MSCs) are important members 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 damage caused by aging and disease. Mesenchyma...

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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