Lecture 7

Question 1

list errors that cause cross-contaminations

Answer 1

1. poor pipetting technique
2. sharing media/pipettes among cell lines
3. generation of aerosols when leaving flasks open
4. mislabeling and seeding the wrong flask
5. poor inventory control in freezer

Question 2

explain how homogeneity changes from primary cultures to cell strains

Answer 2

the primary culture is very heterogenous from tissue. with subculturing it gets more homogenous as certain lines die off while others thrive. Once the hay flick limit is reached, all finite cells die and only a few lines remain (the transformed immortal cells). Now the culture is very homogeneous and can be used to get more homogenous by making specialized cell strains.

Question 3

describe the proper cell culture terminology

Answer 3

primary culture: 1st culture derived from tissue
Cell line: includes secondary culture, territory culture, and so on. it’s the splitting of the primary. can be finite or continuous
cell strains: subline with defined special properties. derived from continuous cell lines

Question 4

describe the relationship between passage number and generation number

Answer 4

passage number: # of times a culture was subcultured (split)
generation number: # of population doublings (PD)
split ratio is how much you dilute with each passage. you can use this number to compare passage number with generation number
split ratio of 1:2 means the passage number = generation number (1 passage = 1 PD)
split ratio of 1:8 means that 1 passage = 3 PD
etc.

Question 5

describe common properties of finite and continuous cell lines

Answer 5

finite: cells have limited culture life spans (20-80 PDs). general properties include: anchorage dependence, contact inhibition, density limit of proliferation, high serum requirement, low cloning efficiency, retain special functions, slow growth rate, normal ploidy (euploid or diploid)
continuous: cells are immortal transformed. general properties include: no anchorage dependence, no contact inhibition, reduced density limitation, can be suspension, low serum requirement, high cloning efficiency, lost special functions, rapid growth rate, and abnormal chromosome ploidy (aneuploid, heteroploid)

Question 6

explain a typical cell growth curve

Answer 6

Lag phase: just after seeding the cells are slow to grow
exponential “log” phase: cells grow rapidly and lots of feeding necessary
plateau phase: cells have reached confluence, contact inhibition slows growth.
subculturing should occur just before the plateau phase!

Question 7

describe criteria for subculture

Answer 7

1. Density of culture: for normal cells, subculture asap when approaching confluence. for transformed cells subculture on reaching confluence
2. exhaustion of medium: if pH is low needs a medium change. drop in pH indicates increased cell density
3. time since last subculture: should follow schedule for reproducibility and consistent/correct seeding density determined by growth curve. deviation from regular pattern indicates problems, slow growth = environment wrong, fast growth = contamination of other fast cells

Question 8

explain consequences of splitting ratio errors

Answer 8

too high: seeding too often so each split seeds fewer and fewer cells until there are just not enough to keep going.
too low: seeding too infrequently so each split is at a higher concentration that will reach plateau phase sooner. Cells start dying due to plateau signaling

Question 9

list the individual steps involved in subculturing suspension cells

Answer 9

no trypsin needed, typically not fed. Options are:
1. diluted and expanded
2. diluted and excess discarded
3. withdraw bulk of cell suspension and dilute residue back to appropriate seeding density
can continue in same vessel, but contamination risk increases. keep concentration < 1x10^6
keep culture conditions standard including media, serum, and flasks/dishes (including batch/supplier consistency!)

Question 10

factors indicating a need for feeding maintenance

Answer 10

1. pH drop (cells lose viability pH 6.0-6.5)
2. cell concentration too dense
3. cell type (?)
4. morphological deterioration (?)
   Add 0.2-0.5 ml/cm3 of medium. consider O2 requirements, the deeper the medium the lower the O2 access.

Question 11

factors indicating a need for feeding maintenance

Answer 11

1. pH drop (cells lose viability pH 6.0-6.5)
2. cell concentration too dense
3. cell type (?)
4. morphological deterioration (?)
   Add 0.2-0.5 ml/cm3 of medium. consider O2 requirements, the deeper the medium the lower the O2 access.

Question 12

list the individual steps involved in subculturing adherent cells

Answer 12

must use trypsin to detach and reseed with new “food” media. can dilute and expand, discard excess, and bring back to appropriate seeding density.