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Differentiation culture medium and preparation method of oligodendrocyte progenitor cells

A technology of precursor cells and culture medium, applied in the field of preparation of culture medium and oligodendrocyte precursor cells, can solve the problems of complicated and lengthy steps, increase the risk of OPs, limit the application of OPs, etc., and achieve the enhancement of cell viability and differentiation process. Controllable and improved antioxidant effect

Active Publication Date: 2018-10-09
HELP STEM CELL INNOVATIONS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current differentiation steps of pluripotent stem cells into oligodendrocyte precursor cells are complicated and lengthy, and the whole culture process takes about 4 months, and even 6 months for the elderly, which severely limits the application of OPs in cell therapy or in the construction of disease models. Application: In the current culture method of neuroepithelial cells differentiated into OPs, fetal bovine serum, non-human albumin or non-human growth factors and other heterologous substances are added to the culture medium, which increases the risk of OPs in clinical application

Method used

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  • Differentiation culture medium and preparation method of oligodendrocyte progenitor cells
  • Differentiation culture medium and preparation method of oligodendrocyte progenitor cells
  • Differentiation culture medium and preparation method of oligodendrocyte progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Pretreatment of bone marrow mesenchymal stem cells (BMSC)

[0050] 1. Material source:

[0051] Sources of bone marrow include, but are not limited to, 30-day-old Sprague-Dawley rats, or adults between 18-40 years old.

[0052] 2. Dilute 1ml Sprague-Dulle rat or adult bone marrow with 9ml CTS Stem Pro Medium, add the diluted bone marrow to a 10cm cell culture dish that has been modified to be suitable for adherent cell culture. The day of adherence culture was defined as the first day (D0) of the differentiation process of oligodendrocyte precursor cells.

[0053] 3. After 48 hours, remove the medium in the cell culture dish, wash off the non-adherent cells with DPBS, add 10ml CTSStem Pro Medium, and then replace the CTS Stem Pro Medium every 72 hours to continue the culture.

[0054] 4. When cultured to D6~D7, bone marrow mesenchymal stem cell (BMSC) clones can be observed in the culture dish; when cultured to D8~D10, aspirate the medium and rinse the non-adherent ce...

Embodiment 2

[0065] Example 2: Differentiation of BMSC into neural stem cells

[0066] 1. Preparation of neural stem cell culture medium

[0067] Configured with a final concentration of 1% CTS GlutaMAX, 2% CTS B27, 40ng / ml GMP bFGF, 40ng / ml GMP EGF, 20ng / ml GMP IGF-1 and 1% P / S Advanced DMEM / F12 medium.

[0068] 2. When BMSC is passed between the 3rd and 10th generation (preferably D15), digest BMSC with CTS Tryple Select, centrifuge and resuspend BMSC with neural stem cell culture medium, and then resuspend BMSC with 1×10 4 Cells / cm 2 The density is inoculated in Ultra Non-adherent culture in 6-well plates.

[0069] 3. Change the neural stem cell culture medium every 48h. As the culture time increases, the neural stem cells form neurospheres and are suspended in the neural stem cell culture medium. On the 5th to 7th day of non-adherent culture, the culture medium is centrifuged at 200g for 5min. Neural stem cells in the form of neurospheres.

Embodiment 3

[0070] Example 3: Identification of neural stem cells

[0071] Using bone marrow mesenchymal stem cells as a control, after dispersing the neurospheres on the 5th day of non-adherent culture described in Example 2, immunofluorescence technology was used to detect the content of cells that positively express Nestin and GFAP in the neurospheres and count them. figure 2 It can be seen that the amount of cells positively expressing Nestin and GFAP in the neurosphere of Example 2 exceeds 85%.

[0072] Experiments have proved that when the number of cells in the neurosphere described in Example 2 that positively express Nestin and GFAP is higher than 75%, the purity and differentiation efficiency of the oligodendrocyte precursor cells further differentiated by the neurosphere are higher.

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Abstract

The invention provides a culture medium for differentiating neural stem cells into oligodendrocyte progenitor cells. The culture medium does not contain exogenous factors, so that pollution of the exogenous factors is avoided, and the oligodendrocyte progenitor cells can be efficiently differentiated. The invention further provides a method for preparing the oligodendrocyte progenitor cells by using the culture medium. By use of the method provided by the invention, the differentiation efficiency of the oligodendrocyte progenitor cells is improved on the premise of increasing the yield of theoligodendrocyte progenitor cells.

Description

Technical field [0001] The invention relates to the technical field of cell differentiation, in particular to a culture medium and a method for preparing oligodendrocyte precursor cells. Background technique [0002] Myelin sheath is an important structure in the nervous system. Demyelinating diseases can cause cognition, memory, motor and other dysfunctions, which seriously affect the quality of life of patients. [0003] Myelin sheath is formed by oligodendrocytes (OLs) wound around the axons of neurons in the vertebrate central nervous system (CNS). In the early stages of neural development, neural precursor cells first produce neurons , Then began to produce oligodendrocyte precursors (oligodendrocyte precursors, OPs) oligodendrocyte precursor cells migrate and proliferate to other parts of the nervous system, and finally differentiate into mature oligodendrocyte precursors (OLs) and form myelin sheath . [0004] At present, both in vitro and in vivo experiments have confirmed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/079
CPCC12N5/0622C12N5/0623C12N2533/52C12N2506/08C12N2502/1358C12N2500/44C12N2501/135C12N2501/11C12N2501/105C12N2501/115C12N2506/1353C12N2501/415C12N2533/32A61P25/28A61K35/30C12N2533/30
Inventor 徐轶冰王嘉显陈应城岑国欣胡立基林楷
Owner HELP STEM CELL INNOVATIONS CO LTD
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