Preparation of cell line III_animal cell culture.pptx

Preparation of cell line III_animal cell culture

1. Preparation and Maintenance of Cell Line (III) Routine Maintenance

2. Routine Maintenance Once a culture is initiated, whether it is a primary culture or a subculture of a cell line, it will need- Feeding Subculture Intervals between medium changes and between subcultures vary from one cell line to another, depending on the rate of growth and metabolism. Rapidly growing transformed cell lines: HeLa Feeding- 4 days interval Subculture- 7 days interval Slow growing, non-transformed cell lines: WI 38 Feeding- 7 days interval Subculture- 3-4 weeks interval

3. Feeding Feeding: Periodic medium change that allows replenishing with new nutrients and equilibrating the pH Factors dictate the necessity of feeding: A drop in pH Cell concentration Cell type Morphological deterioration

4. Subculture Subculture- dividing a culture Subculture involves- Removal of the spent medium Dissociation of the cells in the monolayer Harvesting cells Appropriate dilution in fresh medium in new culture vessel

5. Criteria for Subculture Density of culture- cell confluence Exhaustion of the medium Time since last subculture Requirements for other procedures

6. Types of Subculture Methodology Subculturing may vary based on Monolayer culture Suspension culture

7. Subculture of Monolayer Remove the medium and rinse the monolayer Expose the cells briefly to trypsin ; remove the trypsin Incubate the cells then disperse in medium Count, dilute, and reseed the subculture

8. Subculture of Monolayer Cells

9. Propagation in Suspension Non-adhesive cells (e.g. leukemia cells) and transformed cells mechanically kept in suspension show growth characteristics like microbial suspension culture Suspension cultures have a number of advantages- No need for trypsinization Feeding is not required, only subculture for routine maintenance Regular culture flask can be used for growing cells Faster Less traumatic for the cells Scale-up is easier

10. Subculture of Suspension Cells Withdraw a sample of the cell suspension Count the cells Seed an appropriate volume of the cell suspension into fresh medium in a new flask (restoring the cell concentration to the initial seeding level)

11. Subculture of Suspension Cells

12. Comparison between monolayer and suspension culture Criteria Monolayer Suspension Culture requirement Cyclic maintenance Steady -state maintenance Trypsin passage and dilution Dilution Limited by surface area Limited by volume (gas exchange) Growth properties Contact inhibition Homogenous suspension Cell interaction Diffusion boundary Useful for Cytology Bulk production Mitotic shake-off Batch harvesting In situ extraction Continuous product harvesting Applicable to Most cell types, including primary Only blood derived and transformed cells

13. Comparison of Finite and Continuous Cell Lines

14. Choosing a Cell Line Finite or continuous Normal or transformed Species Growth characteristics Availability Validation Phenotypic expression Control cell line Stability

15. Features of Commonly Used Cell Lines

16. Granularity around the nucleous Cytoplasmic vacuolation Cell rounding and detachment

17. Cellular confluence