Method for culturing middle turbinate derived olfactory ensheathing cells

A technology of olfactory ensheathing cells and turbinates, applied in the field of in vitro cell culture, can solve the problems of difficulty in obtaining olfactory ensheathing cells, and achieve the effects of increasing the ratio of olfactory ensheathing cells, high rejection risk, and low safety

Active Publication Date: 2018-08-24
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem of difficulty in obtaining OECs, the present invention provides a method for isolating and culturing OECs from the autologous middle turbinate, the method has a high ratio of double-positive OECs

Method used

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  • Method for culturing middle turbinate derived olfactory ensheathing cells
  • Method for culturing middle turbinate derived olfactory ensheathing cells
  • Method for culturing middle turbinate derived olfactory ensheathing cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Prepare cleaning solution: 10 mL of DPBS balanced salt solution containing Antibiotic-Antimycotic mixture (1:100) per liter;

[0032] Prepare the primary culture medium: 500 mL of DMEM / F-12 medium, 50 mL of fetal bovine serum, 2 mM L-glutamine, and 5 mL of Antibiotic-Antimycotic mixture (1:100);

[0033] Prepare passage medium: each 250 mL contains 50 mL fetal bovine serum, 50% endothelial progenitor cell conditioned medium (EPC-CM) 250 mL, BPE 20μg / mL, Forskolin 2uM, L-glutamine 2 mM, Antibiotic-Antimycoticmixture ( 1:100) 5 mL of DMEM / F-12 medium.

[0034] (1) Collect healthy middle turbinate mucosa, put the sample in a storage and transportation bottle and label it in a 4-25℃ transportation box for storage and transportation; the sample will be processed within 2 hours after collection: perform 75% on the outer surface of the storage and transportation bottle. Wipe and disinfect with% alcohol, open the cap of the storage and transportation bottle in the biological safety c...

Embodiment 2

[0039] Example 2 Indirect immunofluorescence identification of olfactory ensheathing cells

[0040] After the pressure screening in step (4) of Example 1, the cells in the culture dish grew to 80% confluence, the cell slides in the dish were taken out and placed in a six-well plate, rinsed once with PBS balanced salt solution; After fixation in formaldehyde solution at room temperature for 20 minutes, aspirate the fixative solution; then add 0.1% TritonX-100 solution, incubate at room temperature for 10 minutes, and then aspirate all the liquid. Rinse the slides with PBS balanced salt solution 5 times for 5 minutes each time; Donkey serum was diluted with the primary antibody Anti-human S100β / Anti-human GFAP to a final concentration of 1:400; the diluted primary antibody was added to the cell slide and incubated overnight at 4°C; after incubation, the slide was rinsed with PBS balanced salt solution 3 times, 5min each time; add a secondary antibody labeled with a fluorophore at a...

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Abstract

The invention provides a method for culturing middle turbinate derived olfactory ensheathing cells. The method comprises the following steps: firstly, carrying out two times of digestion on middle turbinate mucosa to remove blood cells and an epithelial layer; then carrying out primary culture on digested tissue blocks; carrying out first-time passage after the cells grow to a certain fusion degree; carrying out pressurized screening on first-generation cells and then removing a screening reagent; identifying after the cells grow to the certain fusion degree. The method for culturing the olfactory ensheathing cells has the advantages of no ethical problems, easiness for collecting tissue sources, high safety and the like. Finally, the obtained cells are of a 10<7> order of magnitude and the purity reaches the international same level; the method is suitable for scientific research experiments and pre-clinical researches. All reagents used by the method are common reagents and the costof the unit cell amount is relatively low.

Description

Technical field [0001] The invention relates to a method for separating and culturing olfactory ensheathing cells from the middle turbinate, and belongs to the technical field of in vitro cell culture. Background technique [0002] In 1992, Devon and Doucete formally proved for the first time that olfactory ensheathing cells (OECs) can be cultured to form myelin sheath in vitro. In 2000, Barnet reported for the first time that intraoperatively removed olfactory bulbs were successfully cultured and expanded human OECs in vitro, and proved that these cells have good physiological functions. Implantation into the demyelinated area of ​​rodents can make myelinated axons without tumorigenicity. . [0003] Domestic Professor Huang Hongyun first reported on OECs. He removed the olfactory bulbs of fetuses who were more than 4 months old, carried out a series of processing, culture and purification, and transplanted them into a group of 23 patients with advanced spinal cord injury (SCI). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0622C12N2500/32C12N2500/40C12N2509/00
Inventor 陈恒宋振涛刘小盾曲廷瑜
Owner SHANDONG QILU STEM CELL ENG
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