SFM (serum-free medium) for culturing MSCs (mesenchymal stem cells)

A serum-free medium and quality stem cell technology, applied in the field of serum-free medium, can solve the problems of increasing the chance of cell culture workload contamination, high difficulty in quality control, and difficulty in standardizing cells, so as to increase the chance of contamination and the complexity of operation. The effect of ensuring the stability of batches and avoiding heterologous contamination

Active Publication Date: 2014-02-05
BEIJING DONGFANG HUAHUI BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (3) High batch-to-batch variability makes it difficult to standardize the cell production process;
[0008] (4) Serum may also contain growth inhibitory factors and cytotoxic substances;
[0009] (5) It is very difficult to carry out quality control to reduce various pollution risks;
[0010] (6) The effects of other unknown factors, hormones and other substances are yet to be studied
Serum-free medium has the characteristics of clear

Method used

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  • SFM (serum-free medium) for culturing MSCs (mesenchymal stem cells)
  • SFM (serum-free medium) for culturing MSCs (mesenchymal stem cells)
  • SFM (serum-free medium) for culturing MSCs (mesenchymal stem cells)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] This example provides a BPS-SFM medium, the specific components of which are shown in Table 2:

[0042] Table 2

[0043] Component

Content

α-MEM

10.2g / L

Sodium bicarbonate

2.4g / L

[0044] L-glutamine

5mM

Poloxamer 188

100mg / L

Recombinant Human Albumin

8g / L

Recombinant human transferrin

20mg / L

Recombinant human insulin

10mg / L

Hepes

5mM

β-mercaptoethanol

50nM

Cholesterol

0.5mM

Arachidonic acid

50nM

Palmitic acid

0.26mg / L

Palmitoleic acid

0.25mg / L

Stearic acid

0.28mg / L

Oleic acid

0.28mg / L

Linoleic acid

0.28mg / L

Linolenic acid

0.28mg / L

Vitamin PP

50mg / L

Vitamin C

20mg / L

Cu

5nM

Se

30nM

Zn

1mM

Ga

0.3mM

Cr

5μM

Mg

0.3mM

Mn

5nM

Glutathione

1mg / L

P-aminobenzoic acid

1mg / L

Hydrocortisone

50ng / mL

Compound of formula I

10μM

Compound represented by formula II

20μM

Progesterone

15ng / mL

Putrescine

10mg / L

Heparin

10IU / mL

EGF

10ng / mL

b-FGF

10ng / mL

HGF

10ng / mL

VEGF

10ng / mL

...

Embodiment 3

[0059] Example 3 Detection of cell proliferation ability

[0060] In this example, the cell proliferation ability of the BPS-SFM medium provided in Example 1 and the control group were compared and tested, specifically according to the following steps:

[0061] Treat the P4 generation umbilical cord mesenchymal stem cells in two different serum-free media of the BPS-SFM medium and the control group, respectively, with 4×10 4 / mL and 6×10 4 Inoculate a 96-well plate at a density of 100 μL per well, and inoculate 5 parallel wells for each group of cells; add CCK-8 reagent at a ratio of 1:10 after the cells adhere to the wall for 24 hours at 37℃, 5% CO 2 Incubate for 2h in the incubator, and measure the OD value.

[0062] See the cell proliferation results Figure 2a with Figure 2b Shown, where, Figure 2a Is 4×10 4 The cell proliferation results of the two when inoculated at a density of / mL, Figure 2b Is 6×10 4 The cell proliferation results of both when inoculated at a density of / m...

Embodiment 4

[0063] Example 4 Detection of cell phenotype by flow cytometry

[0064] In this example, the cell phenotype of the P4 generation cells in the BPS-SFM medium provided in Example 1 and the control group MSCM-SF was performed according to the following steps:

[0065] TrypLE was used to digest and collect the P4 generation cells in the experimental group BPS-SFM medium and the control group MSCM-SF. They were incubated with fluorescently-labeled CD29, CD90, CD105, CD34 and CD45 and other surface antibodies, and were incubated with FITC and PE-labeled small cells. Mouse IgG isotype antibody was used as a control; incubate at 4°C for 45min and centrifuge to collect the cells; after washing 3 times with PBS buffer, resuspend the cells in 400μL PBS buffer and perform flow cytometry analysis on the machine. The specific results are as follows Table 3 shows.

[0066] table 3

[0067]

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Abstract

The invention relates to an SFM (serum-free medium) for culturing MSCs (mesenchymal stem cells). Based on volume, the SFM comprises the following components: 10.2 grams per liter of alpha-MEM (alpha-minimum essential medium), 2.4 grams per liter of sodium bicarbonate, 1 to 5 millimoles of L-glutamine, 50 to 300 milligrams per liter of poloxamer 188, 2 to 8 grams per liter of recombinant human albumin, 10 to 20 milligrams per liter of recombinant human transferrin, 2 to 10 milligrams per liter of recombinant human insulin, 1 to 5 millimoles per liter of Hepes, 50 nanomoles of beta-mercaptoethanol, 0.1 to 1 milligram per liter of lipid, 1 to 5 milligrams per liter of trace element, 0.1 to 5 milligrams per liter of glutathione, 0.5 to 5 milligrams per liter of para-aminobenzoic acid, 1 to 50 nanograms per milliliter of hydrocortisone, 20 to 50 milligrams per liter of vitamin PP, 5 to 50 milligrams per liter of vitamin C, 2 to 10mu M of compound shown in a formula I, 5 to 20mu M of compound shown in a formula II, 10 to 20 nanograms per milliliter of progestin, 1 to 10 milligrams per liter of putrescine, 1 to 10 international units per liter of heparin, 1 to 10 nanograms per milliliter of EGF (epidermal growth factor), 1 to 10 nanograms per milliliter of b-FGF (b-fibroblast growth factor), 1 to 10 nanograms per milliliter of HGF (hepatocyte growth factor) and 1 to 10 nanograms per milliliter of VEGF (vascular endothelial growth factor). The SFM for culturing the MSCs is a BPS-SFM which has determinate chemical components and is free of animal-derived substances.

Description

Technical field [0001] The invention relates to a serum-free medium for culturing mesenchymal stem cells, belonging to the technical field of cell engineering and biomedicine. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells that are widely present in human tissues and organs. Because of their high self-renewal ability and multi-directional differentiation potential, they are used in clinical hematopoietic support, promotion of stem cell implantation, and immune regulation. It has potential application prospects. At present, large-scale preclinical and clinical studies have confirmed that MSCs are directly infused as cell therapy products or combined with scaffold materials to construct tissue-engineered tissues and organs (skin, bone or cartilage, etc.) for transplantation. It has good safety and effectiveness in the treatment of burns, bone defects, soft tissue filling, end-stage liver disease, myocardial infarction, diabetes and other trauma or...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 赵侃王春有赵宇刘湘连李莉莉
Owner BEIJING DONGFANG HUAHUI BIOMEDICAL TECH
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