1. Subculture and Cell lines
Chapter 13

2. Propagation of Subculture
Subculture – important transition from culture
Need to SC – primary culture has occupied all substrate
Biological significance – culture can be propagated, characterized and stored
Homogeneity inc. and hetero dec.

3. Cell line and Strain Primary culture is subcultured – Cell line
Cell line transforms in vitro – Continuous Cell line
Specific properties – Cell Strain
Selected or cloned and characterized - Continuous Cell Strain – Table 13.3
Finite cell lines - die

4. SC Passage number – number of times the culture has been subcultured
Generation number – number of doublings the culture has undergone
No account of cell loss through necrosis, apoptosis, premature aging and withdrawal from cycle
Cell loss occurs btwn sc

5. Culture Age
Cell lines with limited culture life spans – finite cell lines
Reproduce - limited cell generations
Cell lines escaped from senescence - Continuous Cell line
Generation no is less imp., and no of passages is imp.,
Split ratios, cell conc., at SC is imp.,
One cell line grown under same conditions – should show same doublings. CCl show inc. cell proliferation and satura. Density

6. Cell line designations
Given code or designation – accession number by cell bank
NHB – Normal Human Brain
NHB1, NHB2 – cell strain and cell line number
If cloned – NHB2-1, NHB2-2
Split ratios – NHB2/2
Cell bank – should follow exactly the no

7. Choosing a cell line
Finite vs. continuous cell line - prefer continuous lines
Normal or transformed – prefer non-tumorgenic
Species – nonhuman cell line
Growth characteristics
Availability – stocks or prepare own line
continuous lines are easy to maintain, grow faster, more clones, can adapt to serum – free media. With non-human cl there are less biohazard restrictions and more tissue is accessible

8. Choosing a cell line Validation – characterized or not
Phenotypic expression
Control cell line
Stability – cloned or not, length of cloning and can you make frozen stocks?

9. Routine maintenance Periodic medium change or feeding
Nonproliferating cultures - medium change is needed – exhausted
Proliferating cultures – trypsinization + medium change are needed
Intervals vary – cell lines

10. Significance of cell morphology
Check for contamination – granularity around nucleus, cytoplasmic vacuolation and rounding of detached cells from substrate
Indicates inadequate toxic medium or serum, microbial contamination or senescence of cell line
Indicates medium change or SC

11. Replacement of medium Four factors indicate replacement
A drop in pH – Cells stop growing – pH 7.0 to 6.5, lose viability btwn 6.5 and 6.0
Medium changes from red through orange to yellow
Change 0.1 pH units/day - can be left longer
Change 0.4 pH units/day – change hrs

12. Replacement of medium
Cell concentration – High cell conc. Exhaust faster – indicated by pH change
Cell Type – Normal cells deteriorate less than transformed cells
Morphological deterioration – allowed longer can lead to apoptosis

13. Replacement of medium
Holding medium – used when mitosis is undesirable at high cell densities
Will hold finite life span lines without using limited cell generations
Serum conc. Is less or absent
Not suitable to transformed cell lines

14. Replacement of medium Volume, depth and surface area
Medium volume to surface area = ml/cm2
Cells with high o2 do better in shallow medium – 2mm
Cells with low o2 do better in deep medium – 5mm
> 5mm – gaseous diffusion limitation

15. When to Subculture? Seeding - Lag period
Log/expo. Phase – cell density or no more growth
Removal of medium + dissociation of cells with trypsin + diluting in new bottles
Sensitivity of cells to proteolysis
Medium must be changed or cells divided

17. Criteria for subculture
Density of Culture – Normal and transformed cells – confluency – reseeded
Exhaustion of medium – fall in pH
Time since last SC – routine SC
Less density – more seeding and viceversa
Requirements – No SC in lag period
- Taken btwn middle of log phase and time before plateau phase of previous SC
Contamination or exhaustion

18. Propagation in suspension
Nonadhesive or mechanical suspension
No trypsin treatment and media replacement
Culture diluted and expanded or diluted and excess discarded
2-5 mm medium for gaseous exchange + agitation by pendulum

19. Subculture of suspension cells
Cell concentration – should not exceed 1 x 10 6 cells/ml
pH – declines as cell conc. Goes up
Time since last SC – regular schedule
Contamination increases with any buildup of minor spillage on neck of flask during dilution
Standardize amount and type of media with serum conc.

20. SC
Check the use of antibiotics
Maintain provenance

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