Lecture 14 Flashcards
explain advantages, disadvantages and limitations of cell culture cytotoxicity assays.
it’s difficult to monitor systemic and physiological effects in vitro, so most assays stay at cellular level. cheap, easily quantified, and reproducible.
limitations- in vitro measurements should be interpreted in terms of in vivo or at least differences should be clearly understood
pharmacokinetics: complex drug exposure is difficult to recreate in vitro
metabolism: do toxins reach the cells in vitro in the same form as they would in vivo? no, diff metabolism
tissue and systemic responses: in vivo response might rely on systemic responses not in individual cells (inflammation, fibrosis, kidney failure)
explain the difference between cytotoxic and cytostatic effect, and how to interpret cell culture cytotoxicity assays accordingly
cytotoxicity = being toxic to cells (broad)
cytocidal effects = cells are killed
cytostatic effect = cells growth is inhibited
cytotoxicity measures either growth or survival
list and describe the different classes of cytotoxicity assays
viability: an immediate or short term response directly after traumatic procedure (cell count)
survival: long term retention of self-renewal capability (plate colonies)
metabolic response: measure during or shortly after exposure
genotoxicity/transformation: growth control, malignant transformation
irritancy: cell analogue to inflammation, cytokine release in vitro (hard to model in vitro)
describe and interpret cell survival curves
how to make:
- calculate plating efficiency at each drug concentration
- calculate the relative plating efficiency (compared to control)
- plot the surviving fraction on a log scale against concentration
- determine the IC50 or IC90 (concentration of compound causing 50% or 90% inhibition of colony formation
- compare curves for differences in sensitivity. see slide 15! flatter curves indicate resistance
describe and explain variable factors in cytotoxicity (survival) assays (8)
- concentration of agent. start wide and narrow down
- invariant and variable agents/concentrations. serum should be varied (can mask low toxicity)
- various time parameters. duration of exposure, time between exposure and assay, total assay time. confirm anticipated toxicity by minimal exposure, confirm absence of toxicity by prolonged exposure
- call density during exposure. media ration effects toxicity seen
- cell density during cloning. colonies may decrease at high toxin, seed more cells
- medium constituents (eg serum)
- colony size. cytostatic makes smaller colony size
- solvents. keep conc minimum due to toxic nature
describe survival assays
measures effects of toxic exposures after several hours or days. demonstrated survival instead of short term toxicity.
Assays measure the survival by demonstrating proliferative capacity for several population doublings
usually measured as plating efficiency (clonogenic assay)
describe survival assays
measures effects of toxic exposures after several hours or days. demonstrated survival instead of short term toxicity.
Assays measure the survival by demonstrating proliferative capacity for several population doublings
usually measured as plating efficiency (clonogenic assay). important to note that plating efficiency only applies to the clonogenic fraction of the cell population, often 20%
describe metabolic cytotoxicity assay
use microtitration for economical high-throughput analysis.
Analysis estimates viable cells by metabolic activity, not actual cell numbers. surviving cells have retention of metabolic or proliferative ability by the cell population as a whole. measured some time after removing toxic influence. measures: protein (labelled Met molecules), DNA (labelled T bases), ATP, NADH
limitation: not direct measurement of cell number. confirm results by clonogenic survival assay
describe MTT assay
uses yellow MTT which forms purple formazan product in living cells. measure absorbance (increase in purple product) to indicate living cells
describe genotoxicity and transformation assays
measure damage to DNA (mutagenesis) as toxicity.
Assays for DNA damage and stress and apoptosis. increased p53 measured. COMET assays directly measure DNA strand breaks
measure transformation via anchorage independent growth, reduced density limitation.
measure mutagenesis by sister chromatid exchange (SCEs) which increase with carcinogens
how to test for carcinogens
difficult, no universally accepted criterion for malignant transformation in vitro. Current assumption is that carcinogens are mutagenic, so assay for mutagenic capability:
Ames test
SCE
increased oncogene expression
how to test for inflammation
no good culture test. current process is animal testing. organ-typically assays need to be developed further to study complex interactions of multiple cell types, esp skin and cornea (cosmetic testing). Can measure for cytokines in organotypic assay
