Cell culture and production of cell lines Flashcards
How are cells isolated from blood?
Density centrifugation and fluorescence activated cell sorts.
How are cells isolated from tissue?
Mechanical and enzymatic disruption.
What are primary cells?
derived directly from tissues and unmodified.
Negative aspects of primary cells?
aberrant expression of some genes, variable contamination, poor growth characteristic, inter patient variability, phenotypic instability, molecular manipulation is difficult.
What is the ideal model of cell cultures?
Good growth characteristics, defined population and molecular manipulation.
How are cell lines produced?
Spontaneously - from tumours of mutations.
Genetic manipulation.
When generations cell lines, what processes do you target?
Growth and ageing processes- p53, Rb and telomerase.
What does p53 and Rb do? and how?
regulate the cell cycle
p53 in a normal cell when the telomeres are shortened so much they can not replicate, p53 binds in the place of telomere binding proteins, this causes activation of p53 - stimulates apoptosis or growth arrest.
p53 on damaged DNA - levels of p53 rise, and act as a transcription factor- transcript p21 - this inhibits cyclin CDKs and prevents phosphorylation of Rb and release of E2F - inhibiting S phase.
Rb - inhibits DNA synthesis arresting cell cycle in G1.
How does telomerase work?
Binds to the end of telomeres and prevents erosion from cell replication. Some cells require telomerase and inhibition od RB to become immortal.
How can viruses stimulate cell lines to grow?
SV40 - T antigens interact with p53 and Rb - cause growth without loss of function.
HPV E6/7 - target p53 for degradation and bind to Rb.
How are cell lines grown?
Plasmid + gene for selection + growth promoting gene placed in growth medium.
Transfection of the gene for selection occurs and its uptaken into the plasmid.
selection pressures kill off all plasmids without gene for selection, leaving a colony of gene lines wanted.
What are the methods of transfection? and how do they work?
CaPO4 co precipitation - uses Ca++ to reduce negative negative irritation.
Lipofection - negative DNA is wrapped in a lipid bilayer and thus able to cross cell surface membrane, once inside structure is broken down and some DNA may integrate into host DNA.
Electroporation - electric field open pores in the cell and nuclear membrane allowing DNA to enter.
Viral transfection - retro/adeno/adeno associated viruses insert the genes directly after being genetically modified. bind to receptors on target cell and release contents - genes are reverse transcribed and DNA is integrated into hosts.
Nucleofection - combination of electroporation and liposuction.
How are the cell lines stored?
Cryo - preservation in liquid nitrogen, slow freezing, fast thawing as water is replaced with DMSO
Disadvantages of cell lines?
Lose their differentiation function when rapidly growing - balance between function and growth.
What is the ideal cell line?
cells that divide when required and stop when function is established.
