Midterm 1B

Question 1

In vivo cell differentiation processes

Answer 1

tissue renewal
stress-induced

Question 2

tissue renewal cell differentiation + eg + flowchart

Answer 2

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

Question 3

stress induced tissue renewal

Answer 3

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

Question 4

in vitro cell differentiation

Answer 4

manipulation/experimentally induced
culture density

Question 5

manipulation into cell differentiation

Answer 5

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)

Question 6

culture density - effects on cell differentiation

Answer 6

cell differentiaiton is limited by cell density
high [cells] = low differentiation

therefore need to balance proliferation vs differentiation (or adjust based on the experiment)

Question 7

gauging degree of cell differentiation

Answer 7

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

Question 8

Types of loss of differentiation

Answer 8

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

Question 9

neoplasticity

Answer 9

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

Question 10

neoplasticity occurrence

Answer 10

spontaneous (random)
induced/experimental/manipulated

Question 11

testing for for successful neoplasticity - in vivo

Answer 11

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

Question 12

testing for successful neoplasticity - in vitro

Answer 12

growth characteristics
genome analysis
invasiveness capability

Question 13

Tumourigenic cells - growth characteristics

Answer 13

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

Question 14

Tumourigenic cells - genome analysis

Answer 14

check ploidy
check tumour suppressors
check oncogenes

Question 15

tumourigenic cells - invasiveness capability

Answer 15

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

Question 16

cell tissue selection

Answer 16

• 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

Question 17

feeder cells for selection of cell types

Answer 17

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

Question 18

Increasing culture homogeneity

Answer 18

cloning method or by FACS

Question 19

cloning to produce homogeneous culture

Answer 19

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

Question 20

cloning efficiency improvements

Answer 20

enriched/conditioned media
more serum
growth factors/hormones

Question 21

FACS to produce homogeneous culture

Answer 21

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)

Question 22

cell counting methods

Answer 22

hemocytometer
computer particle counter
indirect counting by standard curves

Question 23

hemocytometer

Answer 23

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

Question 24

electronic/computer particle counting

Answer 24

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

Question 25

cell counting by standard curves

Answer 25

• based on concentration of total proteins or glucose depletion
• where [x] change over time correlates with increasing #cells
• obtain results by spectrophotometry (wavelengths for the molecule being measured)
• compare to standard curve to estimate #cells/vol

Question 26

define cell viability

Answer 26

the ability or readiness for cells to proliferate and undergo further cell cycles

Question 27

cell viability assessment

Answer 27

colony counting
marker retention (LDH and CFDA)
dye exclusion
tetrazolium assay
resazurin assay

Question 28

colony counting for cell viability

Answer 28

more colonies = more cell viability

Question 29

marker retention for cell viability - LDH

Answer 29

cells contain LDH enzyme
less viability = dead cells = more LDH in suspension
test LDH activity –> more activity = more dead cells

Question 30

marker retention for cell viability - CFDA

Answer 30

CFDA = non pol non fluor dye
if viable cell –> dye is intaked –> converted to CF fluor dye
therefore more dye = more viability

Question 31

dye exclusion

Answer 31

trypan dye = bulky blue molecule
viable cells = intact CM = no dye
dead cells = trypan uptake –> dyed dark blue

more blue cells = lower culture viability

Question 32

tetrazolium (MTT) assay

Answer 32

tetrazolium –> mitochondrial activity –> reduction to formazan
tetrazolium = yellow
formazan = blue

therefore more blue wavelength absorbance = more viability

MITOCHONDRIAL metabolic activity measurement (direct measurement)

Question 33

resazurin

Answer 33

resazurin –> enzymatic activity –> reduction to resorufin
resazurin = dark blue/pruple
resorufin = pink

more pink wavelength absorbance = more viability

OVERALL metabolic activity measurement

Question 34

Industrial culturing - chemical constraints

Answer 34

O2 availability - limited by diffusion through media + causes foaming (wastes serum)
NH4 accumulation - glutamine = unstable – >degrades to NH4 –> impacts glycosylation
lactic acid accumulation - [acid] increases over time –> pH shift –> lethal

Question 35

culture types

Answer 35

batch
semi continuous
continuous

Question 36

bioreactor mixing methods

Answer 36

stir tank reactor (STR) = mechanical mixing (gentle to prevent cell dmg) using impellers

bubble column = bubbling in riser causes media to low density therefore rises. bubble pop at the surface causing return to heavy density. media falls back down to bottom in downcomer columns. non mechanical mixing –> lower risk of cell dmg + easier to maintain/clean

Question 37

cell immobilization purpose in industrial culturing

Answer 37

allows better protection of cells

Question 38

immobilization - non anchorage cells (methods and explanations)

Answer 38

entrapment - cells embedded in agarose microbeads –> agarose = porous therefore materials diffusion. Allows ease of separation of cells from dissolved products

encapsulation - cells embedded in Ca alginate microbeads –> covered semipermeable w/biopolymer membrane –> chelation to remove Ca therefore dissolve alginate –> creates biopolymer shells w/cells inside. allows modification of bead permeability to control what materials diffuse into suspension

Question 39

immobilization - anchorage dependent cells (methods and explanations)

Answer 39

• use large flasks/plates (increase SA)
• use cell factory - stacked plates to increase SA and overall density in vessel
• use roller flasks - cells grown on internal walls. flask in partially filled with media but rolled. Allows cells to get enough nutrients using less overall media (lower cost) and gas exchange in open air (cells will not desiccate fast enough when not submerged)

-microcarriers - cells grown on surface of dextran or glass microbeads –> allows very high [cell] in suspensionwhile also using STR or BC reactor methods. porous microcarriers further increase SA but make measuring [cell] and cell extraction more difficult