Serum-free medium (SFM) and application thereof in mesenchymal stem cell (MSC) subculture

A medium and stem cell technology, applied in the field of stem cell culture, can solve the problems of insufficient passage and expansion ability, and achieve the effect of improving mesenchymal stem cell passage and expansion ability, maintenance of cell stemness, and good expression

Pending Publication Date: 2021-01-12
BIOISLAND LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of this, the technical problem to be solved by the present invention is to provide a serum-free medium and its application in the subculture of mesenchymal stem cells. The compositio

Method used

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  • Serum-free medium (SFM) and application thereof in mesenchymal stem cell (MSC) subculture
  • Serum-free medium (SFM) and application thereof in mesenchymal stem cell (MSC) subculture
  • Serum-free medium (SFM) and application thereof in mesenchymal stem cell (MSC) subculture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The components of the serum-free medium include:

[0038] Table 1 Serum-free medium components of each group

[0039]

[0040]

[0041] Each component was dissolved according to its respective solubility characteristics, sterilized by filtration through a 0.22m filter membrane, added to the basal medium and mixed evenly, and then penicillin and streptomycin were added to a concentration of 1 vol%.

Embodiment 2

[0043] The serum-free medium of Example 1 was set as the experimental group, the complete medium containing 10% FBS was set as the control group 1, and the MesenCult-XF Medium of BI Company was set as the control group 2, and the following experiments were carried out.

[0044] 1. Morphological observation and activity detection of UC-MSCs serially passaged

[0045] Select the P3 generation UC-MSCs to carry out the experiment, passage every 3-4 days, and the UC-MSCs are all divided into 1×10 5 The amount of cells per well was seeded in a six-well plate, and each group was set up with 3 replicates. placed in 5% CO 2 Culture in an incubator at 37°C. The image of UC-MSCs was collected before each passage, and the cell morphology (100×) was as follows figure 1 shown.

[0046] Table 2 The cell count results of each generation of UC-MSCs activity detection in each group

[0047]

[0048] Note, ** indicates that there is a very significant difference compared with the control...

Embodiment 3

[0053] Example 3 Detection of UC-MSCs surface markers

[0054] UC-MSCs were inoculated in T25 culture flasks at a density of 1×104 / cm2 and cultured at 37°C in a 5% CO2 incubator. After 3 days, 0.25% trypsin solution digested and collected UC-MSCs in each group, according to the MSC surface marker standard (positive markers CD105, CD73, CD90 expression rate ≥ 95%, negative markers CD34, CD45, HLA-DR expression rate ≤ 2%), the surface markers of UC-MSCs of different passages in each group were detected by flow cytometry. The results are shown in Table 3.

[0055] Table 4 Detection results of markers on the surface of UC-MSCs in each group of P5 generation

[0056] Experimental group CD105 CD90 CD73 CD34 CD45 HLA-DR Control group 1 98.98％ 100.00％ 100.00％ 0.03％ 0.00％ 0.02％ Control group 2 97.75％ 99.98％ 100.00％ 0.21％ 0.10％ 0.04％ Experimental group 1 98.35％ 99.99％ 100.00％ 0.03％ 0.03％ 0.02％ Experimental group 2 99.87％ 10...

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Abstract

The invention relates to the technical field of stem cell culture, in particular to a serum-free medium (SFM) and application thereof in mesenchymal stem cell (MSC) subculture. An additive provided bythe invention is suitable for preparing the SFM for multiple passage of MSCs and effectively maintaining stemness, the defect of insufficient passage and amplification capacity of the MSCs can be overcome effectively, and a stable base material for realizing large-scale, standardized and industrialized production of stem cell products in the future is provided. Experiments show that the additivenot only can promote the amplification and passage of the MSCs, but also can well maintain the expression of surface markers of the MSCs, so that the cell stemness is effectively maintained.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a serum-free medium and its application in subculture of mesenchymal stem cells. Background technique [0002] Stem cells and their representative regenerative medicine research have made major technological breakthroughs in the past two decades, which has aroused people's great attention to the industrialization and clinical application of stem cell technology. As of 2019, 16 stem cell products have been launched in the world. Among them, Mesenchymal stem cells (MSCs) have become the type of stem cells with the most products and the fastest development due to their various advantages. Mesenchymal stem cells are derived from the mesoderm in the early stage of development and are a type of non-hematopoietic stem cells that are widely found in bone marrow, subcutaneous fat, periosteum, muscle, synovium, synovial fluid, liver, peripheral tissues, umbilical cord, cord blood...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2500/90C12N2500/32C12N2501/30C12N2500/25C12N2501/115C12N2501/11C12N2501/135C12N2501/15C12N2500/46C12N2500/40C12N2500/44C12N2501/415
Inventor 陈东煌陈海佳王小燕姜交华戚康艺
Owner BIOISLAND LAB
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