Serum-free culture medium for mesenchymal stem cells

A serum-free medium and necessary technology, applied in the biological field, can solve the problems of not supporting MSC primary culture, increasing workload and pollution, and expensive medium, avoiding instability, clear properties, and ensuring consistency Effect

Active Publication Date: 2012-12-19
内蒙古干细胞医学工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these media are expensive, and in the process of culturing MSCs, it is necessary to use auxiliary products to coat the cell culture flasks, and it is possible to introduce animal-derived components during the gelatin coating process, which also increases the workload and pollution. opportunities, and some researchers found that when using these products, they did not support the primary culture of MSC or needed to add 2% serum during the primary culture (Hudson JE, Mills RJ, Frith JE, et al.A defined medium and substrate for expansion of human mesenchymal stromal cell progenitors that enriches for osteo- and chondrogenic precursors. Stem Cells Dev.2011;20:77-87.)
Moreover, these products require high cell seeding density requirements during use, and these media cannot be used when seeding low-density cells (see Table 1)

Method used

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  • Serum-free culture medium for mesenchymal stem cells
  • Serum-free culture medium for mesenchymal stem cells
  • Serum-free culture medium for mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the preparation of culture medium

[0024]The medium SFM composition of the present invention is as follows:

[0025]

[0026]

[0027] More preferably, the medium SFM composition of the present invention is as follows:

[0028]

[0029]

[0030] The adhesive amine in the culture medium of the present invention can be replaced by 1 mg / L recombinant human fibronectin.

[0031] The amino acids mentioned in the table above include L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine , L-Phenylalanine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, L-Alanine, L-Aspartic Acid, L-Asparagine , L-glutamic acid, glycine, L-proline, L-serine;

[0032] The vitamins include calcium pantothenate, choline chloride, folic acid, meso-inositol, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride;

[0033] The lipids comprise arachidonic acid, cholesterol, DL-α-tocopheryl acetate, linoleic acid, linoleni...

Embodiment 2

[0043] Example 2: Isolation and culture of adherent cells in umbilical cord

[0044] Aseptically take 2 cm of the umbilical cord, cut open the blood vessel, rinse with DPBS to remove residual blood, and then digest it with 1 mg / mL type II collagenase DPB S 35 mL at 37 °C for 2 h, centrifuge at 2500 r / min for 10 min (centrifugal radius 22 cm), Remove the sediment from the lower layer and digest it with 0.25% trypsin in PBS for 20min, centrifuge at 2500r / min for 10min (centrifugal radius 22cm), take the precipitate and use PBS to pipette the cells to suspend, filter with a 100μm filter, and centrifuge at 2000r / min for 10min (centrifugal radius 22cm) to obtain single cells , followed by centrifugation with PBS 800r / min for 10min (centrifugal radius 22cm), washing the cells twice, with 1×10 6 Individual / mL density suspended in serum-free medium and serum-free medium of the control group, placed at 37°C, 5% CO 2 Cultivate in an incubator, and then change the medium every 3 to 4 da...

Embodiment 3

[0046] Example 3: Detection of cell phenotype by flow cytometry

[0047] Take the cells passed to the fifth generation, remove the culture medium, wash twice with PBS, digest with 1:1 0.25% trypsin solution, centrifuge at 1000r / min for 5min, wash once with PBS, adjust the cell concentration, and make the concentration for 10 9 L -1 Add 10 μL of antibody to the single cell suspension, incubate at 4°C for 30 min, wash once with PBS, centrifuge at 1000 r / min for 5 min, resuspend the cells in 500 μL of PBS, and then detect on a flow cytometer for comparison. The results are shown in Table 2. The results of comparison of the flow cytometry phenotypes of MSC cultured in the serum-free medium of the present invention and the medium of the control group showed that the MSCs in the serum-free medium provided by the present invention and the medium of the control group positively expressed CD13, CD29, CD44, CD73, CD90, CD105, CD166 and HLA-ABC, negative expression of CD14, CD19, CD45...

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Abstract

The invention relates to the field of biology, and discloses a serum-free culture medium which essentially comprises an IMDM (Iscove Modified Dulbecco Medium), L-glutamine, sodium bicarbonate, Hepes, recombinant human insulin, recombinant human transferrin, recombinant human albumin, 2-mercaptoethanol, protocatechuic acid, lipid, amino acid, vitamins, trace elements, Pluronic F-68, hydrocortisone, vitamin C, bonding amine or recombinant human fibronectin, progesterone, putrescine, heparin, serotonin, epidermal growth factors (EGFs), b-fibroblast growth factors (FGF), platelet derive growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I. The serum-free culture medium is clear in chemical components, free from animal sources and serum and safe and ideal in cell cultivation and avoids the doped animal components and unstable batches, and the results of the cultured mesenchymal stem cells show that the total cellular score, the cell phenotype and the secretory cell factors are normal, so that the serum-free culture medium has good industrial application prospect.

Description

technical field [0001] The invention relates to the biological field, in particular to a serum-free medium for mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells belonging to the mesoderm, which have strong proliferation ability and multi-lineage differentiation potential, and can not only differentiate into osteoblasts, cartilage cells, and Cells, fat cells, and also have the ability to differentiate into muscle cells, liver cells, hematopoietic cells, nerve cells, islet cells and other cells. MSC has a wide range of sources, is easy to isolate, culture, expand and purify, has low immunogenicity, and has no restrictions on moral and ethical issues, so it has great application prospects in tissue engineering, gene therapy and immunotherapy. [0003] The proportion of MSCs in tissues is extremely low, and the number of MSCs obtained through in vitro isolation is very limited, which is far from meeting the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 武晓云康会彦吕岩刘学敏王云虹王黎明高锦
Owner 内蒙古干细胞医学工程技术研究中心
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