Midterm 1A

Question 1

Define cell culture

Answer 1

in vitro cell growth maintained in a culture medium

Question 2

cell culture requirements

Answer 2

sterile env
culture (abiotic) env
anchorage/substrata/cell support
nutrients

Question 3

sterile env

Answer 3

animal cells = slow growing –> easily outcompeted by contams
horizontal flow “clean” hood (class 1)
- blowing filtered air across top towards worker –> not suitable for high biohaz
- air is not recirculated

vertical flow “safety” hood (class 2)
- filtered air blown down onto work surface –> safe for high biohaz (eg primate cells)
- mostly recirculated air, some vented under sash

Question 4

HEPA

Answer 4

high efficiency particle air filter

Question 5

why is turbulent air flow bad

Answer 5

uneven airflow by turbulence causes air voxtexing to occur –> resulting in some unfiltered air to be pull into the hood therefore airborne contam of the work surface

Question 6

culture (abiotic) env

Answer 6

Temperature
pH
gas composition

T maintained by incubator
atmospheric O2 is usually enough
supply from CO2 tank –> tweaked gas composition for the cells
CO2 also aids in buffering pH when converted to bicarbonate

Question 7

anchorage/support

Answer 7

animal cells = anchorage dependant –> will not proliferate without adhesion to a substrata
need high SA to grow lots of cells –> grow in culture flasks
form confluent monolayer
cover cells in thin layer of media

coating the surface may allow stronger cell adhesion therefore encourage more growth (eg negative ozone)

Question 8

nutrients

Answer 8

complete media
defined basal media
serum
antibiotics
trace elements
other molecs (varies per culture)

Question 9

defined basal media

Answer 9

standard recipe
provides
- sugars (E and C sources)
- 13 essential AAs
- bulk ions

Question 10

serum

Answer 10

undefined - acquired from foetal/cattle bovine (blood but no cells)
provides
- heavy metal detox
- pH buffering
- low MW nutrients
- micronutrients

Question 11

antibiotics

Answer 11

animal cells susceptible to contam –> ABs
penecillin - targets CW synthesis –> restricts gram (+)
streptomycin + gentamycin –> target protein synth –> gram(+) and (-)
Amphotericin –> target yeast + fungi

Question 12

trace elements

Answer 12

micronutrients
vitamins
other compounds may be added later in the culture (eg low stability glutamine)

Question 13

cell types for culturing + what layer of blastula they form in

Answer 13

connective - mesoderm
muscle - mesoderm
nervous - ectoderm
epithelial - endo, meso and ectoderm

Question 15

embryonic stem cells

Answer 15

sourced from zygote blastula inner cell membrane
immortal
pluripotent

Question 16

adult stem cells + 2 types of division

Answer 16

tissue specific stem cells
not immortal –> undergo senescence
symmetric division - creates 2 spec’d cells
asymmetric division - creates 1 spec’d + 1 stem cell

Question 17

separation of cell types –> creating homogenous culture

Answer 17

physical selection ( blood only)
- blood sample –> ficoll –> separation
- ficoll separates the erythrocytes from the leukcocytes –> allows easier physical isolation of the cells

differential attachment
- diff cell types –> adhere to substrata more readily than others
- selection of cell type based on how readily the desired cell type adhere to substrata compared to others
- perform multiple rounds of diff attach selection to increase homogeneity

Question 18

initiating primary culture - Outgrowth method

Answer 18

explant –> mechanical dissociation (break it up)
place chunks in thin layer of media (not enough to float them, need adhesion)
incubate –> cells migrate out of explant
remove explant tissue
incubate to monolayer

Question 19

initiating primary culture - physical dissociation

Answer 19

only viable with unorganized tissues (eg nervous, marrow)
pass explant through sieve –> finely separates the cells
high pressure causes some cells to die
incubate to monolayer

Question 20

initiating primary culture - enzymatic method

Answer 20

explant –> enzymes to dissociate cells
- trypsin - attacks cell-cell adhesion proteins
- collagenase - ECM collagen breakdown
- EDTA - chelate Ca ions used for adhesion

Question 21

warning about trypsin use

Answer 21

prolonged exposure to trypsin (protease ) = cell damage
brief exposure to trypsin –> centrifuge cells –> move pellet to fresh media
serum proteins consume/neutralize remaining traces of trypsin

Question 22

Passaging/subculturing

Answer 22

grow primary to confluent monolayer
separate cells for passaging
- physically scrap cells off pruimary flask and into new media
- or enzymatically dissociate cells off flask (trypsin) –> aspirate cells –> pellet –> decant –> new media. original flask decant and add new media to neutralize trypsin traces

Question 23

finite cell line

Answer 23

limited # of divisions –> need cryopreservation to store over long time
maintain diploidy
passage 1 culture after between 10 to 30 generations to create cell line

Question 24

continuous cell line

Answer 24

infinite # divisions
prone to mutations –> unique to invitro (mutations that would not be seen invivo)
high ocogene presence
disabled tumour suppressors
RANDOM PLOIDY

Question 25

immortalizing cells

Answer 25

spontaneous (random) --> no intentional manipulation experimental --> induced by researcher (eg signalling, various factors)--> neoplastic transformation

Question 26

cryopreservation - possible damages

Answer 26

ice crystal formation/expansion protein denaturation freezing causes salts to ppt out --> increased salt concentration + change in pH ice expands --> some water leaves cells --> dehydration

Question 27

procedure for cryopreservation

Answer 27

- treat cells with cryoprotectant --> glycerol or DMSO - slow cooling --> allows water to slowly leave cells --. minimize ice crystalk damage when expanded - store under -130 C (liq N2) --> super cold retards new ice crystal formation - rapid thawing - most damage occurs btw -50 and 0 C --> speed past to minimize dmg

Question 28

mycoplasma

Answer 28

AKA PPLO smallest free-living organisms --> intermediate btw virus and bacteria lack CW --> renders most antibiotics useless (since most target CW synthesis) VERY easy to contaminate animal cultures --> easily out compete animal cells

Question 29

mycoplasma contamination (sources and detection)

Answer 29

sources - poor worker technique (sloppiness) - contam'd materials - original explant was contam'd detection - fluor staining --> very tiny dots = mycoplasma - electron microscopy - immunofluorescence --> tagged AB's specific to the mycoplasma

Question 30

cross contamination

Answer 30

UNINTENTIONAL contamination coculturing = intentional culturing of heterogenous culture caused by sloppy technique

Question 31

cross contamination detection + prevention

Answer 31

detection - karyotyping - DNA fingerprinting - isoenzyme analysis --> diff sp may release diff isometric structures of enzymes prevention - culture 1 cell at a time - do not share equipment/materials btw cell lines

Question 32

organoids

Answer 32

complex 3D cell tissue growth culture able to more closely mimic in vitro tissue structure and cell-cell interactions

Question 33

2.5D culture

Answer 33

culture forms physiologically relevant tissues

Question 34

3D culture

Answer 34

complex tissue culture embedded in the ECM

Question 35

mechanically supported culture

Answer 35

culture is grown on semipermeable membrane --> allows culture to be exposed to diff conditions simultaneously eg 1 side of membrane in open air, other side of membrane submerged in culture

Question 36

organoid requirements

Answer 36

- Source of cells - diff source --> diff cells. May be tissue derived (biopsy) or iPSC (induced pluripotency) - matrix/ECM. Matrigel (synthetic), collagen (natural), hydrogel (polymer based) - Soluble factors. growth factors/hormones to induce complex structure formation. May involve using spent medium (used media contains excreted factors) - physical cues. structurally mimic in vivo env