Muscle stem cell in-vitro culture method and application thereof

一种体外培养、干细胞的技术，应用在细胞培养活性剂、非胚胎多能干细胞、组织培养等方向，能够解决肌肉退行性疾病的方法难使用等问题，达到提高数量的效果

Active Publication Date: 2014-12-17
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the existence of this problem, it is difficult to use the method of gene repair in muscle stem cells to treat inherited muscle degenerative diseases in clinical treatment

Method used

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  • Muscle stem cell in-vitro culture method and application thereof
  • Muscle stem cell in-vitro culture method and application thereof
  • Muscle stem cell in-vitro culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Preparation of murine T cell conditioned medium:

[0082] The wild-type C57 / B6 mice were sacrificed, the spleen was taken out, placed in a 70um filter, and 2-3 ml of PBS was added to moisten the spleen, then the spleen was ground with a grinding rod, and an appropriate amount of PBS was added to filter, and the cell suspension was collected. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend the cells with 5ml of erythrocyte lysate, add 5ml of RPMI-1640 culture medium, filter again with a filter to remove cell debris, and collect cells by centrifugation at 1000rpm for 5 minutes. Wash the cells twice with RPMI-1640 medium, adjust the cell density to 1x10 9 cells per liter, inoculated in cell culture flasks, added ConA (final concentration 5mg / L), at 37 degrees CO 2 After culturing in the incubator for 48 hours, add an equal volume of RPMI-1640 culture medium, and after 24 hours, centrifuge at 3000 rpm for 5 minutes, transfer the cell supernatant to ...

Embodiment 2

[0104] Culture and detection of muscle stem cells

[0105] 1) Acquisition of muscle stem cells:

[0106] Mouse muscle stem cells: Several 3-day-old newborn mice were killed, and the limb muscles were placed in DMEM culture medium supplemented with 0.2% type D collagenase, digested at 37 degrees for 1.5 hours, and then washed with PBS three times, each time naturally Sink the muscle for several minutes to collect the muscle.

[0107] Use a Pasteur pipette and an 18G needle to inhale the muscle several times until the muscle mass is broken, filter through a 40um filter to remove non-muscle impurities, centrifuge at 1000rpm for 5 minutes, resuspend in F10 culture medium and inoculate in a 10cm culture After 3 hours, the supernatant was transferred to a 10cm culture dish coated with 0.05% type I collagen, and 5ng / ml FGF cytokines were added at the same time. 2 Incubate overnight in an incubator. The next day, trypsinize the muscle cells, centrifuge at 1500rpm for 5 minutes, was...

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Abstract

Provided is a muscle stem cell in vitro culture method. A muscle stem cell is cultivated in vitro by using a cell culture medium of a cell factor added with a blood cell or a conditioned medium of the blood cell. Also provided are a culture medium used in the muscle stem cell in vitro culture method and an application thereof.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to the fields of cell culture and cell therapy related to muscle stem cells. Background technique [0002] Muscle stem cells are responsible for the growth and regeneration of muscle tissue after birth, and muscle stem cells have no carcinogenic effect in vivo, and there are no ethical issues encountered when using embryonic stem cells, so they are used in various muscle degenerative diseases such as muscle atrophy and myasthenia gravis There are broad application prospects in treatment. An important obstacle to the clinical application of muscle stem cells is the inability to culture muscle stem cells in vitro for a long time. Like most adult stem cells, after muscle stem cells are isolated from the body, they can only divide 2-4 times in the in vitro culture system, and they can hardly be passaged. [0003] The existing methods for culturing muscle stem cells (satellit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074A61K35/34A61P21/00
CPCA61K35/34C12N5/0659C12N2501/2301C12N2501/2302C12N2501/2304C12N2501/2313C12N2501/24C12N2501/25A61P21/00C12N2502/1114C12N2501/20C12N2501/21C12N2501/22C12N2501/2303C12N2501/231C12N2501/2316C12N2501/2317
Inventor 胡苹傅鑫肖俊郭恒江李升刘燕王红艳
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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