Cell Culture

Question 1

Cell culture definition

Answer 1

ability to grow cells in an artificial environment

Question 2

in vitro/viv/situ

Answer 2

• vitro=cell culture
• vivo=living animal
• situ=in place

Question 3

history

Answer 3

• 1900s: brain tissue put into a rudimentary cell culture
• 1951: george gey established HeLa cells in culture
• 1961: leonard hayflick hypothesis (for somatic not cancer cells)

Question 4

What is hayflicks hypothesis/limit

Answer 4

somatic cells will divide a maximum of 25-50x in culture and then die (apoptosis)

Question 5

Power of cell culture

Answer 5

• can work w a single cell type at a time
• can generate (not somatic) an unlimited # of cells in vitro
• can control the envionrment of the cells
• recombinant DNA technology (biologics-mAbs, FRET/GFP, novel cells to look at knock in/knock-out genes)
• tissue engineering
• stem cell therapy

Question 6

limits to cell culture

Answer 6

• cell culture conditions can direct the phenotype
• cells can change their phenotype over time bc of genetic drift (or not express the proper phenotype)

Question 7

HeLa Cells

Answer 7

genetic drift

Question 8

MDCK cells

Answer 8

• canine kidney cells
• fibroblast-like, not differntiated
• grown on cell culture insert and micro-porous device
• makes them grow like real kidney cells
• ex of how cells need certain conditions to express proper phenotype

Question 9

hepatocytes

Answer 9

• liver cells
• primary-freshly isolated-designed to detoxify
• problems: don’t divide in vitro and lose function over time

Question 10

Embryonic stem cells

Answer 10

needed to figure out how to grow them

Question 11

what are cell strains

Answer 11

• freshly isolated from tissue
• represent more closely than cell line what’s going on
• can divide 25-50 times
• advantage: similar functionality to in vivo
• limit: hayflick limit means only so many mortal cells

Question 12

steps to cell strain

Answer 12

• collect tissue of interest
• dissociaty cell from tissue w protease/EGTA
• place in cluture (primary culture
• subculture (expand) into two different cultures by dissociating them (ssecondary cultures)
• repeat process (reial passages) until cells wont divide anymore bc of hayflick limit

Question 13

what are cell lines

Answer 13

• immortal lineage (unlimited divisions)
• ex. hela and CHO

Question 14

advantages of cell lines

Answer 14

• unlimited # of cells
• good for collecting pOI when small amount exists in each cell
• cancer research/drug discovery

Question 15

types of cells from cell lines

Answer 15

• embryonic (ex. 3t3 mouse fibroblast)
• cancer cells
  a. tumor (buy)
  b. expose cell strain to a mutant (make ur own)
  c. spontaenously arise from strain (3t3 mutation)

Question 16

disadvantage of cell line

Answer 16

• cells are atypical/defective- don’t represent cells in vive

Question 17

telomerase-immortalized cell line/strain

Answer 17

• cell strains top producing bc telomere length gets too small
• so, transfect strains to make telomers longer/extended: immortalized compared to parent cell strains from which they were generated
• can make or buy

Question 18

growth media requirements-serum supplemental media

Answer 18

• ex 10% fetal calf (bovine) serum (FCS)- bc contains growth factors (many unknown) that cells need

parabiotic mouse experiment shows old mice have imparied regenration of muscle, possible due to less growth factors- why calves are used

Question 19

problems with FCS

Answer 19

• expensive
• unusable for cell therapy; cow preteins and prions cause issues

Question 20

serum free media/defined media

Answer 20

• less expensive
• okay for clinical use (cell therapy)

Question 21

growth substrates

cell culture dishes

Answer 21

• ex. multiwell plates, each contains x number of cells up to 1,536 wells/plate

Question 22

what are growth substrates

Answer 22

• what a cell grows on determines the phenotype they express

Question 23

growth substrates

microcarrier beads

Answer 23

• often used in big industrial purposes (batch cultures)
• large vates with beads, closed loop w collection manifold
• media circulates through, mAbs collect biologics

Question 24

growth substrates

roller bottles

Answer 24

• pharmaceutical industry
• cylindrical bottles slowly rotated

Question 25

growth substrates

tubes

Answer 25

• for vascular endothelial cells
• replicates what happens in vivo to help cells express correct phenotype

Question 26

cell culture inserts

Answer 26

• transcytosis-movement of antibodies across an endothelial layer
  -ex. lactation mom: antibodies from blood to mik through transytocis via mammary to cells
  -ex. neonates: antibodies form mother’s milk transcytoses through intestinal cells in blood

Question 27

neurospheres

Answer 27

• clusters of neural stem cells that can differentiate into neurons, glial cells, and oligodendrocytes

Question 28

what is tissue engineering

Answer 28

• multidimensional use of cells to gnerate lab grown constructs or organoids

Question 29

tissue engineering history

Answer 29

• started in 1980s
• 1991- vacanti mouse w human ear growing on its back

Question 30

3 pieces of tissue eng pie

Answer 30

• engineering materials: scaffollds, growth factor
• engineering tissue architect: 3D printing, decellularized organs
• engineering cells: crispr, stem cells

Question 31

scaffolds

Answer 31

• biodegradable: dissolve over time
• non-biodegradable: permanent

Question 32

big challenges w engineering tissues

Answer 32

• creating artificial blood vessels
• deceullularization (preserving extracellular matrix while removing cells)

Question 33

tissue engineered constructs: human bladder

Answer 33

implantable for children w spinal bifida grown from their own cells (2006)

Question 34

tissue engineered constructs: livers

Answer 34

• 4 types
• ELAD most important (extracororeal liver assist device)
• composed of cartridges with engineered hepatocytes (liver cells)
• externally compensates for liver damage while liver repairs itself
• fascimile (synthetic) primary hepatocyte: c3A cells-hepatoma cell line, Cyt p450-50% hepatocyte primary
• failed in clinical trails becuase cell filter contains C3A hepatoma cells, creating possibility of canccerous cells returning to pateint

Question 35

carticel

Answer 35

• knee cartilage
• genzyme corporation
• autologous: made from cells of patient

Question 36

pancreatic cancer turmoroid

Answer 36

• used to test drugs to fight pancreatic cancer

Question 37

beating heart patches

Answer 37

baindaid for heart tissue damaged in MCI

Question 38

making first human epidermis construct

Answer 38

• microporous membrane to culture skin cells (express the correct phenotype)
• seed the cells (NHEK): normal human epidermal keratinocytes
• Increase concentration of extracellular calcium in medium- ells differentiated
• change culture so its at air liquid interfae (stratum corneum)

Question 39

human epidermis analysis

Answer 39

• structural: TEM
• biocehmical: keratins w western blots
• functional analysis: test if it acts as barrier

Question 40

“Plants are mRNA Factories for edible vaccines”

Answer 40

• CHO and Hela preferred cells for clinical productions of mAbs
• but Univ of Ca Riverside researchers are trying to address the current problem of Modern and Pfizer’s requirement of keeping the current mRNA vaccines cold/frozen
• purpose of project: show that DNA coding for mRNA vaccines can be successfully delivered to plant cells, show that plants can produce enough mRNA to rival a traditional shot
• they are inserting genes into spinach and lettuce chloroplasts, not nuclear genome

Question 41

hayflick limit

Answer 41

• cells in culture have three phases
• human somatic cells can divide 50x max and then die
• indirect relationship between donor age and max number of potential doublings