cell culture techniques Flashcards
1
Q
the types of cells used
A
- primary cells
- primary cell lines
- cell strain
- immortal cell lines
2
Q
requirements for maintenance
A
- counted using haemocytometer/automatic counter
- seeded at required density in sterile plastic ware
- incubated in humidified, temperature and gas incubator
- inspected for density and contamination
- medium is refreshed every 48h-72hrs
3
Q
requirements for growth
A
- pH
- temperature: 37C
- gas: 5% CO2 in air
- nutrients: amino acids, lipids, vitamins, fatty acids
- sterile environment free of contamination
- cryopreservation
4
Q
passaging of adherent cells
A
- inspect cells
- remove spent medium
- rinse with phosphate-buffered saline
- remove PBS and add trypsin (yields single cell suspension)
- re-suspend cells in fresh medium and plate at required density
5
Q
passaging of suspension cells
A
- count cells/ml of spent medium
- calculate the volume to yield required number of cells
- centrifuge that volume to pellet the cells
- discard supernatant
- re-suspend cells in fresh medium and plate at required density
6
Q
contamination of mammalian cells
A
- dispose of contaminated cells
- review aseptic technique, media prep
- clean work area
- decontaminate incubator
- disinfect using hypochlorites, phenolics or alcohol
7
Q
long-term storage of mammalian cells
A
- pellet cells by centrigugation
- re-suspend in cell culture medium containing serum
- add DMSO to give a 10% solution
- aliquot into cryovials
- freeze cells overnight at -80C
- transfer to liquid nitrogen
8
Q
uses of cell culture
A
- vaccine production
- production of antibodies
- study of gene function
- study basic biological processes
9
Q
what are primary cells?
A
- derived from excised tissue or body fluids e.g. blood, urine)
- retention of in vivo characteristics
- divide in the cell culture vessel
- may contain other cell types
- mechanical + enzymatic to obtain single-cell suspension
10
Q
what are primary cell lines?
A
- split from original cell culture vessel and propagated
- retain in vivo characteristics for some time
- will lose characteristics e.g. phenotype
- limited lifestyle
11
Q
what are immortalised cell lines?
A
- consist of a single-cell type
- indefinite propagation
- naturally immortal are isolated from tumour tissue e.g. HeLa
- transformed cells are from spontaneous, deliberate genetic transformation and chemical treatment
- stem cells generate different cell types
