Techniques for isolating primary culture isolation Flashcards
Difference between primary cell culture and cell lines?
Primary cell culture-are cells directly from the tissues and has finite lifespan, similar morphology and expression markers to the parent cell, is primary culture till it’s subcultured.
While cell lines have an infinite number of doubling populations, it is different from the parent cell,have to continuosly divide for at least 6 months.
Cell lines have to be malignant
false
A cell line established from a patient with a tumour has to be a cancer cell line
false
What limits the cells growth when they are cultured?
cell cell contact, nutrient depletion, contact inhibition, accumulation of apoptotic cells
What do you use to take iut adherent cells when splitting a passage?
trypsin
What does dilution factor depend on primarily?
what type of cell line
Why shouldn’t cells be cultured continuously?
Becasue of genetic/phenotypic drift and to avoid contamination
What are the culture condition(o2,co2,temp)
o2-21%
co2-5%
37 degree
High humidity(reduce hypertonic)
What is in the growth media?
Foetal calf serum(contains growth factor and hormones)
Standard salt mix, bicarbonate with CO2
Defined cell lines use foetal calf serum
false, use EGF
What must you do consistently when culturing cells?
Replenish and replace media
What are the substrates for the cell that can be used?What do you co-culture with?
Microcarrier beads, treated plastic, glass, capillaries,collagen coated surface
Feeder cells and stromal cells
What are spheroid cells? What’s good about it?
cell cultures that are sphere shaped grwon in viscous scaffold. It mimics the environment found in in vivo, so there are difussion gradients for the cells that are inward and
Where do embryonic stem cells come from and what are their characteristics? How is it grown
4-5 days of inner cell mast of embryo.
Indefinitely proliferate and if didn’t differentiate after 6 months are pluripotent and can differentiate into any cell
Grown on feeder cells
What are the advantages and disadvantages of cell culture?
Can control physiochemical and physiological factors Homogenous culture economical unlimited supply of cell lines can store infinitely in liquid nitrogen Avoids use of animals -: expertise, no 3 environment, genetic and phenotypic drift, outgrowth of undifferentiated cells
What is confluence?
Reach maximum growth, reach senescence
How to produce primary cell culture?
Dissociation-mechanical or enzymes
suspension culture
explant method
What factors affect the viability of dissociated cells?
type of animal, tissue, age
concentration of Enzymes used, incubation time, impurities
What’s the strongest and the weakest enzyme used?
trypsin and deoxyribonuclease 1
What do if low cell yield and low viability?
decrease enzyme concentration
what if it’s low cell yield but high viability
increase enzyme concentration
high cell yield but low cell viability
decrease enzyme concentration
What are cells preserved in? How are they stored
cytoprotective agent such as DMSO( increased serum concentartion allows it to survive)
Liquid nitrogen -196
Cells are frozen rapidly and thawed slowly
false, they are frozen slowly and thawed rapidly
What are some ways to verify cell lines? Why is it important?
Cytogentic
mycoplasma detection
DNA/protein profiling
To make sure there is no contamination from cell lines or microbes or genetic or phenotype drift
How does mycoplasma infection dteection work?
By staining with hoechst stain or PCR or luminescence
What induces replicative senescence? What limits the replication? WHy don’t cancer cells have this
extrinsic and intrinsic factors
TTelomeres shorten witch each division and when it’s too short there will be genomic instability and will die. Because their telomerase are active and their p53 or RB might be inactive allowing cells to grow indefinitely
What the difference between immortalized and transformed cells?
Immortalized are able to replicate indefinitely through a spontaneous mutation or infection but will not cause cancer if injected in immunocompromised mice and they often gow as a single layer, However transformed cells can cause tumours if injected in mice and have loss of contact inhibition
What’s transfection? What does the frequency depend on?
Insertion of nucleic acid into eukaryotic cells through non viral methods cell number procedure toxicity concentration of nucleic acid
What are the ways to chemically transfect cells?
DEAE-dextran(positively charged) gets into cell through endocytosis and closely associates with DNA
Calcium chloride, DNA and phosphate buffer enter cell through phagocytosis or endocytosis
EXGEN500-polymer complexes with DNA and prevents its degradation in lysosome
osmotic shock
artificial liposomes(doxil)
What are physical transfection of the cells?
Microinjection Electroporation gene gun magentofection protein transfection(chariot peptide)
What selection antibody is usually used for transfection?
zeocin
What are pHook vectors?
they express single chanin antibodyagainst hapten then magnetic beads coated with hapten select for transfected cells
What reporters are used to detect transfected cells?
GFP, luciferase, CAT enzyme activity,bgal, secreted reported protein
How are genes quantitaviley regulated?
Tet on and tet off system, genes on in presence of TET but on in the absence of TET. Can be regulated differnetially depending on the concntrations
