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Culture medium, kit for cell culture, and cell culture method

A kit and culture medium technology, applied in the field of culture medium, cell culture kit and cell culture, to achieve the effect of prolonging the number of culture generations

Active Publication Date: 2017-11-07
深圳涌泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, what is currently stored in biobanks only includes frozen tissues, fixed tissues, DNA, RNA, proteins, blood, plasma, serum and urine, etc. These extremely precious resources are gone immediately and cannot be regenerated

Method used

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  • Culture medium, kit for cell culture, and cell culture method
  • Culture medium, kit for cell culture, and cell culture method
  • Culture medium, kit for cell culture, and cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] The culture passage of embodiment 1Swiss 3T3 cell

[0130] Swiss 3T3 cells were obtained from the laboratory of Professor Howard Green at Harvard University.

[0131] Reagents required:

[0132] Complete DMEM medium: DMEM (GIBCO#11965-092), 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin

[0133] Trypsin Digest Solution: 0.05% Trypsin / EDTA (GIBCO #25300-062)

[0134] Calcium Magnesium Phosphate Buffer: Ca 2+ , Mg 2+ free PBS

[0135] Subculture operation steps:

[0136] Inoculate 1×10 5 Put Swiss 3T3 cells in a T75 cell culture dish, add 10ml of the above-mentioned complete DMEM medium, and place in CO 2 Incubator at 5% CO 2 , cultured at 37°C for 2-5 days. When the cell monolayer abundance is 80-90%, remove the culture medium in the culture dish, wash the cell monolayer twice with 10ml of calcium-magnesium-free phosphate buffer, add 2ml of trypsin digestion solution, and incubate at 37°C for 1 minute . Tap the culture dish to ful...

Embodiment 2

[0137] Embodiment 2HL culture medium

[0138] HL medium 1 is a mixed medium of DMEM (GIBCO#11965-092) and Ham's F-12NUTRIENTMIX (GIBCO#11765-054), the volume ratio is 3:1, and 5% fetal bovine serum and 0.4μg / ml cortisol (hydrocortisone), 5μg / ml insulin (insulin), 8.4ng / ml cholera toxin (cholera toxin), 10ng / ml epidermal growth factor (epithelial growth factor (EGF)), 24μg / ml adenine (adenine) , 100U / ml penicillin (penicillin) and 100μg / ml streptomycin (streptomycin), 0.25μg / ml amphotericin B (Fungizone,) 30μM Fasudil (Fasudil) (or 0.5μM H-1152 or 5μM Y- 27632, 30μM HA1100, 10uM GSK429286), the above culture medium needs to be filtered through a 0.22μ pore filter membrane.

[0139] HL medium 2 is DMEM (Low Glucose, No Glutamine, GIBCO #11054-020) supplemented with 10% fetal bovine serum, and 0.4 μg / ml cortisol (hydrocortisone), 5 μg / ml insulin (insulin), 8.4ng / ml cholera toxin, 10ng / ml epithelial growth factor (EGF), 24μg / ml adenine, 100U / ml penicillin and 100μg / ml streptom...

Embodiment 3

[0144] Example 3 Preparation of feeder cells (Swiss 3T3 cells that have lost the ability to divide) (mitomycin C treated swiss3T3 cells)

[0145] 1) The swiss 3T3 cells used as feeder cells should be the cells in the early stage of subculture. When swiss 3T3 cells grow to fullness, add mitomycin C (dissolved in water, stock solution concentration: 0.5 mg / mL) with a final concentration of 10 μg / mL to the culture medium, and treat at 37°C for 2 hours;

[0146] 2) Then add warm-bathed 1xPBS or serum-free medium (DMEM) to wash 3 times, and discard the washing solution;

[0147] 3) Add 0.05% trypsin / EDTA to pre-digest the cells for 30-40 seconds, discard them, add 0.05% trypsin / EDTA to digest again for 30 seconds, tap the culture dish to disperse the cells, and then add complete medium (containing 10% fetal DMEM) neutralization reaction of bovine serum;

[0148] 4) Low-speed centrifugation (1000rpm) to remove the supernatant and obtain cell pellets;

[0149] 5) The precipitated ...

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PUM

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Abstract

The present invention relates to a culture medium, a kit for cell culture, and a cell culture method, and in particular to a culture medium for epithelial cell culture, a kit for cell culture, and a cell culture method. The medium contains serum, calcium components and ROCK inhibitors.

Description

[0001] This application is a divisional application of the Chinese invention patent application (application date is May 17, 2011) whose application number is 201110127667.4 and the invention name is medium, a kit for cell culture and a method for cell culture. technical field [0002] The present invention relates to a culture medium, a kit for cell culture, and a cell culture method, and in particular to a culture medium for epithelial cell culture, a kit for cell culture, and a cell culture method. Background technique [0003] Vital organs such as lung, kidney, liver, pancreas and skin are composed of organ-specifically differentiated epithelial cells. These specifically differentiated epithelial cells are directly related to the specific functions of different organs, such as the gas exchange function of the lungs, the filtering function of the kidneys, the detoxification and neutralization functions of the liver, the production of insulin by pancreatic cells, and the pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07
Inventor 李晖
Owner 深圳涌泰生物科技有限公司
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