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A kind of culture method of Schwann cell and its application

A technology of Schwann cells and culture methods, which is applied in the field of culture of Schwann cells, can solve the problems of slow cell proliferation, limit the clinical application of nerve growth factor, and low content of nerve growth factor, and achieve the effect of great application potential

Active Publication Date: 2020-10-02
重庆市铂而斐细胞生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under certain conditions, Schwann cells cultured in vitro can synthesize and secrete a certain amount of nerve growth factor, but the traditional culture method is slow in cell proliferation and the content of nerve growth factor is not high, which greatly limits the role of nerve growth factor in peripheral nerve injury. Clinical Application in Restoration

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Experimental group 1:

[0028] (1) Isolation, purification and primary culture of Schwann cells:

[0029] 20 SD rats born 3-5 days old were sacrificed and immersed in ethanol with a volume fraction of 75% for 5 minutes. The bilateral sciatic nerves were taken out under sterile conditions. Cut into 0.5mm3 size, add 0.25% trypsin and 0.06% collagenase, mix and digest in 37°C water bath for 30 minutes, shake well and settle for 5 minutes, draw the cell suspension from the list above and transfer it into a container containing 10% fetal bovine serum by volume The digestion was terminated in the DMEM culture medium, the pipette was blown gently to disperse the tissue pieces, and then centrifuged (1000r / min, 5min), and an appropriate amount of DMEM culture medium containing 10% fetal bovine serum was added to form a suspension by blowing. If there is no obvious tissue visible to the naked eye, adjust the cell concentration to 1×10 8 L-1, planted in a 25mL culture bottle pre...

Embodiment 2

[0052] The present invention uses ELISA to detect the level of nerve growth factor secreted by Schwann cells: after culturing for 5 days, take the Schwann cells in logarithmic growth phase of each group, digest them with trypsin-EDTA mixed solution, make cell suspension, and take 5×10 8 The cell concentration of L-1 was inoculated on 24-well culture plates coated with polylysine, 6 wells in each group, and the culture medium was DMEM complete medium containing 10% fetal calf serum, 1 mL per well, placed in 37°C, 5% CO by volume 2 Cultivate in an incubator for 48 hours, centrifuge (1 000r / min) for 5min to take the supernatant, transfer the supernatant to a sterile EP tube, centrifuge again for 5min (1 000r / min), take the supernatant, and refrigerate at -20°C for testing. After the samples were collected, they were detected according to the instructions of the nerve growth factor ELISA test kit. The results are shown in Table 1 below.

[0053] Statistical processing: SPSS16.0 ...

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PUM

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Abstract

The invention discloses a culture method of schwann cells. The culture method comprises the step of applying cycloastragenol and isofluoroether in the culture process. The method comprises the following culture steps: (1) performing steps of separation, purification and primary culture of the schwann cells; (2) subculturing the schwann cells; and (3) performing expression of the nerve growth factors in the schwann cells: when the third-generation cells are about to fully grow at the bottom of a cell bottle, subculturing in a 25mL culture bottle for 5 days, taking a culture solution of cycloastragenol with the mass fraction of 0.5-0.8% as the culture solution, and introducing mixed air containing 3.0% of isofluoroether for 3 hours separately twice.

Description

technical field [0001] The invention relates to a method for cultivating Schwann cells and an application thereof, belonging to the technical field of cell culture. Background technique [0002] Schwann cells, discovered by Theodor Schwann, are formed to insulate and coat the axons protruding from neurons in the peripheral nervous system. They are characterized by phagocytosis and can remove cell debris. Schwann's cells appear in the peripheral nervous system and form myelin to insulate and coat the axons protruding from the neurons of the peripheral nervous system. Myelin does not cover the entire axon. Schwann cells There will be gaps, called Langer's nodes, which speed up the conduction speed of nerve signals in the form of jump conduction. Schwann's cells have phagocytic ability to remove cell debris and provide space for neurons to regenerate. [0003] Nerve growth factor (NGF) is one of the most important bioactive factors in the nervous system, which can promote the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2501/999
Inventor 甘元善颜旭东
Owner 重庆市铂而斐细胞生物技术有限公司