Subculture of cells from primary culture

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BACKGROUND

Methods of extracting cells from an animal and subsequent growth in an artificially controlled environment termed as primary cell culture. Subculture is a new culture originated from the primary cell culture.

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REQUIREMENTS

Cells:              Cultured cells in a confluent stage

Apparatus:      Petri dishes – 60 mm

Pipettes
Centrifuge tubes – 15 ml
Centrifuge machine

Chemicals:      1X HBSS

Trypsin solution – 0.5%

Cell culture media (DMEM)

Serum

Antibiotics/Antimycotics

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PROCEDURE

Cell culture media composition

After confluence, the cells from primary culture plates are subcultured into new plates by discarding the media from the cultured plates and then wash the cells with HBSS (5 ml/60 mm petri dish). The cells are then added with trypsin (0.25%) in the cell plate (5 ml/60 mm petri dish) for few minutes (1-2 minutes) and verified the detachment of cells in light microscope. After complete detachment of cells, add cell culture media containing 10% serum and then centrifuge it for 5 min at 5000 rpm at 4⁰C. Discard the supernatant (optional washing with HBSS), add cell culture media with serum, and antibiotics/antimycotics mix the cells and transfer the solution in the plates after cell counting incubate in 37⁰C with 5% CO₂.¹

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CONCLUSION

The cells are thus subcultured from the primary culture in to the secondary culture.

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REFERENCES

1. Kumar A, Mishra HK, Dwivedi P, Subramaniam. Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway. Annals of neuroscience. 2015;22(2):97-106.