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Culture medium, cell culture kit and cell culture method

The technology of a medium and a kit is applied in the field of medium for epithelial cell culture to achieve the effect of prolonging the culture generation.

Active Publication Date: 2012-11-21
深圳涌泰生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effect that this patented technology allows for effective culturing of epothilone-producing cell lines while maintaining their original genes has been achieved through use of an improved method called Culture Media Kit (CMT).

Problems solved by technology

This patents discusses two technical methods: 1) traditional methodologies like immortality and chemotherapics, 2) molecularly imprintable virus technology called XFISH, developed through induced transformation of soma labeled retrovirus vectors carrying replication origins. Current bio bank systems require extensive amounts of sample material and donory costs associated therewith. Additionally, existing bioengineering tools limit usability because they affect both the quality and efficiency of studying cancer growth and developing novel medicines.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1 Culture and passage of Swiss 3T3 cells

[0129] Swiss 3T3 cells were obtained from the laboratory of Professor Howard Green at Harvard University.

[0130] Required reagents:

[0131] Complete DMEM medium: DMEM (GIBCO#11965-092), 10% fetal bovine serum (FBS), 100U / ml penicillin (penicillin) and 100μg / ml streptomycin (streptomycin)

[0132] Trypsin digestion solution: 0.05% pancreatin / EDTA (GIBCO#25300-062)

[0133] Calcium-free magnesium phosphate buffer: Ca 2+ , Mg 2+ free PBS

[0134] Subculture operation steps:

[0135] Vaccination 1x10 5 Swiss 3T3 cells are placed in a T75 cell culture dish, add 10ml of the above complete DMEM medium, and place in CO 2 Incubator in 5% CO 2 Incubate at 37°C for 2-5 days. When the cell monolayer abundance is 80-90%, remove the culture medium from the culture dish, wash the cell monolayer twice with 10ml calcium-magnesium-free phosphate buffer, add 2ml trypsin digestion solution, and incubate at 37℃ for 1 minute . Tap the petri dish t...

Embodiment 2

[0136] Example 2HL medium

[0137] HL medium 1 is a mixed medium of DMEM (GIBCO#11965-092) and Ham's F-12NUTRIENTMIX (GIBCO#11765-054), with a volume ratio of 3:1, while adding 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 (EGF), 24μg / ml adenine (adenine) , 100U / ml penicillin (penicillin) and 100μg / ml streptomycin (streptomycin), 0.25μg / ml amphotericin B (Fungizone,) 30μM Fasudil (or 0.5μM H-1152 or 5μM Y- 27632, 30μM HA1100, 10uM GSK429286), the above medium needs to be filtered through a 0.22μ pore filter.

[0138] HL medium 2 is DMEM (Low Glucose, No Glutamine, GIBCO#11054-020) supplemented with 10% fetal calf serum, 0.4μg / ml cortisol (hydrocortisone), 5μg / ml insulin (insulin), 8.4ng / ml cholera toxin, 10ng / ml epidermal growth factor (EGF), 24μg / ml adenine, 100U / ml penicillin and 100μg / ml streptomycin , 0.25μg / ml Amphotericin B (Fungizone,) 30μM Fasudil (or 0.5μ...

Embodiment 3

[0143] Example 3 Preparation of feeder cells (Swiss 3T3 cells that have lost the ability to divide) (mitogen C treatment of swiss 3T3 cells)

[0144] 1) As a feeder cell, swiss 3T3 cells are suitable for early subculture. When swiss 3T3 cells grow to full abundance, add mitogen C (dissolved in water, stock solution concentration of 0.5mg / mL) at a final concentration of 10μg / mL to the medium, and treat for 2 hours at 37°C;

[0145] 2) Add warm bath 1xPBS or serum-free medium (DMEM) and wash 3 times, discard the washing solution;

[0146] 3) Add 0.05% trypsin / EDTA to pre-digest the cells for 30-40 seconds and then discard them, add again 0.05% trypsin / EDTA to digest for 30 seconds, tap the petri dish to disperse the cells, and then add complete medium (containing 10% fetus). DMEM) neutralization reaction of bovine serum;

[0147] 4) Centrifuge at low speed (1000 rpm) to remove the supernatant to obtain cell pellet;

[0148] 5) The precipitated cells can be used directly as feeder cells ...

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Abstract

The invention relates to a culture medium, a cell culture kit and a cell culture method, and especially relates to a culture medium, a cell culture kit and a cell culture method which are used for the epithelial cell culture. The culture medium contains a serum, a calcium component, and an ROCK inhibitor.

Description

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Claims

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Application Information

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Owner 深圳涌泰生物科技有限公司
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