Midterm 1B

tissue renewal stress-induced

continuous (constant) pluripotent –> progenitor –> precursor –> terminally diff’d eg bone marrow, GI lining

tissue renewal induced only by external stimuli partial de-differentiation (not completely back to pluripotency) –> proliferation –> re-differentiate

manipulation/experimentally induced culture density

mimicry of in vivo conditions to induce differentiation factors: - media composition (hormones/signals/factors) - coculturing (cell-cell comms to induce) - Extracell interactions –> ECM material (collagen, fibronectin)

cell differentiaiton is limited by cell density high [cells] = low differentiation therefore need to balance proliferation vs differentiation (or adjust based on the experiment)

lineage marker - look for cell characteristics unique to specific differentiation states (cumulative, more terminally differentiated cells inherit all previous characteristics) terminally differentiated markers - look for functional characteristics unique to the terminally diff’d state

deDIFFERENTIATION - irreversible loss of diff’d properties deADAPTATION - reversible loss –> can be reintroduced in vitro selection - accidental selection for less differentiated states due to passaging

the process of converting a normal/healthy cell into a tumourigenic cell –> usually to create a continuous culture

spontaneous (random) induced/experimental/manipulated

innoculation to SUITABLE host - if tumour forms –> success host must be immunocompromised or else no tumour is observed (transplant rejection) - nude host –> mutation to compromised - inbreeding to compromise - induced compromise state using meds/chems

growth characteristics genome analysis invasiveness capability

proliferate even at extreme [cell] no longer anchorage dependant immortal –> infinite # divs

check ploidy check tumour suppressors check oncogenes

well plate with conditioned media take neoplastic cells –> put on top of cultured media –> SEPARATED BY MATRIGEL if tumourigenic –> cells will migrate through matrigel and into cultured media

• differential adhesion - diff cell types –> diff readiness to adhere • selective detachment - diff cell types –> varied susceptibility to trypsin • substrata selection - selection based on substrata material/coating • feeder layer - selection for slow-growing cells (or those that otherwise wouldn’t grow) using feeder cells

cell type desired may struggle to proliferate alone –> needs to be cocultured feeder cells = supplementary coculturing cell –> mutated to be unable to proliferate therefore feeder cells =/=outcompete with desired cell type

cloning method or by FACS

method of producing extremely homogeneous culture explant –> chemical dissociation by trypsin serially dilute to ~1 cell/drop place drops in well plate microscopy to check 1 cell per well identify cell type of interest trypsinize to extract –> grow in fresh media –> confluency

enriched/conditioned media more serum growth factors/hormones

fluoroscent activated cell selection • diff cells –> diff immunofluour tag –> diff fluorophore • pass heterogenous mixture through flow chamber –> transducer separates to 1cell/droplet • laser excites fluorophore –> diff tag = diff emitted wavelength = diff cell type • deflection electrodes magnetically sort droplets (-/+ or no charged attraction)

hemocytometer computer particle counter indirect counting by standard curves

serially dilute cells –> place on hemocytometer grid slide 1 grid sqr = 0.1 ul count # cells per sqr –> avg extrapolate total #cells based on cells/0.1ul –> account or dilution tedious/slow method, but reliable

cell suspension of KNOWN volume suction tube in the suspension –> orifice small enough so only 1 cell at a time either side of orifice –> electrodes as cell passes through orifice –> interrupts electrical circuit –> “pulse” count # pulses = #cells issues cells clumping –> counted as only 1 cell partial cell loss (some deaths) therefore final #cells =/= true population in media