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Culture medium for mesenchymal stem cells and large-scale culture method thereof

A large-scale culture of stem cell technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of high price, cell shape change, cell aging, etc., achieve good proliferation and differentiation ability, delay Aging phenomenon, effect of maintaining characteristics

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

AI Technical Summary

Problems solved by technology

In the routine culture of mesenchymal stem cells, the most common culture system is to use DMEM / F12 medium plus 10% fetal bovine serum (FBS) for in vitro culture and expansion of mesenchymal stem cells, but occasionally After the 5th generation of mesenchymal stem cells, the cell morphology has changed, and some cells have aged
Moreover, although FBS can provide some necessary nutrients for mesenchymal stem cells, FBS is an animal-derived substance with complex components, which has potential risks in clinical application and increases the chance of cell contamination
Moreover, FBS is a relatively expensive biological product. In the large-scale cultivation of mesenchymal stem cells, it will undoubtedly greatly increase the cost of production.

Method used

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  • Culture medium for mesenchymal stem cells and large-scale culture method thereof
  • Culture medium for mesenchymal stem cells and large-scale culture method thereof
  • Culture medium for mesenchymal stem cells and large-scale culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Primary isolation and culture of umbilical cord mesenchymal stem cells

[0039] Obtain the bovine umbilical cord tissue block, cut it into pieces, add complete medium to the tissue block, pipette evenly and transfer to the cell culture dish; shake the culture dish to make the tissue block evenly distributed in the culture dish, transfer to 5% CO 2 , Cultivate in a cell incubator at 37°C, and observe the growth of the cells regularly; when the cells climb out, discard the old medium and tissue pieces, wash the culture dish with PBS, add complete medium and continue to culture in the cell incubator ; When the cell confluency reaches 80% to 90%, discard the old medium, wash the culture dish with PBS, add the digestion solution containing 0.25v / v% trypsin and 0.04v / v% EDTA to digest for 1 to 2min , after adding complete medium to terminate the digestion, the cells were pipetted into a single cell suspension to obtain umbilical cord MSCs, which were recorded as P1...

Embodiment 2

[0040] Embodiment 2, large-scale culture and cell identification of umbilical cord MSC

[0041] After the P1 generation cells obtained in Example 1 were collected by centrifugation, the Count Star cell counter was used for cell counting, and the complete medium was adjusted to 5 × 10 cells according to the counting results. 4 / mL of the optimal seeding density, and inoculated into a 15cm large Petri dish, placed in 5% CO 2 , 37 ℃ incubator to continue culturing. When the cell fusion degree reaches 80%-90%, it can be subcultured again, and it will be recorded as P2 generation cells. Repeated subculture by analogy for large-scale expansion culture, and when it is passed to P10 generation cells, a large number of cells that meet clinical needs can be obtained. dose of MSC. The complete medium is the medium of the mesenchymal stem cells, including serum-free medium and epidermal growth factor and platelet-derived factor, wherein the concentration of epidermal growth factor is 5 ...

Embodiment 3

[0042] Embodiment 3, the synergistic effect of EGF and PDGF in the culture process of umbilical cord MSC

[0043] Take the P10 generation cells cultivated in Example 2, digest them with trypsin containing 0.25v / v% trypsin and 0.04v / v% EDTA, and press 1×10 with Ultra CULTURE serum-free medium 4 / mL was inoculated onto a six-well plate with 2 mL of the system per well. PDGF and EGF were added according to different addition methods, and the addition schemes are shown in Table 1. Regularly observe the growth state and morphological changes of the cells, change the medium every 3 days, and take pictures of the cells on the 7th day, the results are as follows: figure 1 . The figure below is a comparison picture of cell morphology, in which figure 1 Among them, A, B, C, D, E and F correspond to the addition schemes of groups (1), (2), (3), (4) and (5) in Table 1, respectively. Cells were collected by trypsinization and counted by a cell counter. The results of the cell counts a...

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Abstract

The invention relates to the field of cells, and discloses a culture medium for mesenchymal stem cells and a large-scale culture method thereof. The culture medium for mesenchymal stem cells disclosed by the invention comprises a serum-free culture medium, EGF and PDGF, wherein the concentration of the EGF is 5-10ng / mL, and the concentration of the PDGF is 5-10ng / mL. The large-scale culture method for mesenchymal stem cells disclosed by the invention comprises the following steps: taking P1-generation mesenchymal stem cells which are primarily isolated and cultured, adjusting an inoculum density to be 2*10<4> / mL to 3*10<5> / mL by the culture medium for mesenchymal stem cells, culturing to a cell fusion degree of 80-90% and then recording as P2-generation, and repeatedly passing to P10-generation cells. The culture medium disclosed by the invention reduces a cell pollution probability, promotes MSC amplification, and delays the ageing speed of MSC in in-vitro culture. The large-scale culture method disclosed by the invention is capable of obtaining lots of high-purity mesenchymal stem cells, and keeping the good stem cell characteristics of the mesenchymal stem cells.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a medium for mesenchymal stem cells and a large-scale culture method thereof. Background technique [0002] Stem cells are a kind of pluripotent cells with self-renewing ability. Under certain conditions, it can differentiate into a variety of functional cells. Mesenchymal stem cells (MSCs) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. Mesenchymal stem cells have the common characteristics of stem cells, namely, the ability of self-renewal, multidirectional differentiation and homing . In a specific in vitro differentiation environment, it can be induced to differentiate into various tissue cells such as nerve, heart, bone, cartilage, fat, epithelium, etc. It is considered to be one of the most promising source cells for cell therapy technology. research hotspot. As an important regenerative medical resource...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 王一飞陈海佳葛啸虎王小燕万桦罗二梅
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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