Z3221319 Cell Culture Techniques and Proceedures
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References and Other sources
Dr Hill’s website is gives a good list of references: 2009 Lab 8 - Tissue Culture 1
This website was compiled from the online text – Molecular Biology of the Cell, and also the Sigma-Aldrich website: http://www.sigmaaldrich.com/
It would be pointless regurgitating all the information on cell culture techniques here, so I have sufficed with putting up some important “trigger points”. In time, I will update more information specific to the experiments that I will be carrying out for the ILP.
Cell separation from tissue
- proteases/calcium chelators to break down ECM-cell adhesions
Isolating specific cells from mixed cell suspension
- antibody binding to glass (similar to ELISA)
- fluorescence-activated cell sorter
- If receiving cell lines from a cell bank, must undergo microbial quality control for viruses, fungi, bacteria (including mycoplasma).
- either as monolayer or suspension
- sterility/nutrients/pH + buffers + indicators/temperature/CO2 need to be considered.
- media – salts/carbohydrates/amino acids/fatty acids/proteins/vitamins
- serum containing media = liquid media + serum (often fetal bovine serum).
- chemically defined media = liquid media + specific proteins required by particular cells being grown.
- primary cultures: cells cultured after harvest, without multiplication step (either by explant or formation of single cell suspension). Short lifespan.
- continuous cultures: immortal or transformed cells.
- immortal cell lines – activated telomerase gene (usually inactivated in human somatic cells)
- transformed cells – cells transformed with tumour-inducing virus
Storage cell lines
- Cryopreservation – cool at rate of 1-3 degrees Celsius per minute; thaw in 37 degrees Celsius waterbath for 3-5 minutes. Need cryoprotectants.
- Storage at <= -135 degrees Celsius using electric freezers or liquid nitrogen.
- Hypochlorites (not on metals). 1000ppm surfaces, 2500ppm discard waste pots, washing pipettes, 10000ppm culture waste, spillage
- Alcohol (~70% ethanol) – not against non-enveloped viruses
- Aldehydes may be used for metals
- Reagents (depending on manufacturer quality controls) and Materials
- Sourced/supplied cell lines from outside the laboratory
- Bacteria/Fungi – contamination of culture usually changes colour of medium + turbidity. Gross examination of cultures should occur daily.
- Mycoplasma – more insidious effects. Detected by commercial kits, or isolation by culture and DNA staining (latter two more accurate/reliable).
- Viruses – usual source is serum ensure screening is in place (usually done by suppliers).
- If contaminated culture – dispose or antibiotics (sparingly)
- Culture waste – overnight inactivation in 10000ppm hypochlorite. Then wash down drain.
- Pipettes – 2500ppm hypochlorite solution overnight, the autoclaving and incineration
Sigma-Aldrich Cell Culture Protocols
The full steps for each protocol are given on the website below:
- Aseptic technique and good cell culture practice
- Thawing – should occur quickly and cells diluted in pre-warmed culture medium to prevent any toxic effects of cryoprotectants in super-zero temperatures. Incubate and examine (phase contrast) the next day. Cells may need to be washed in media if cryoprotectant has a known adverse cytopathic effect.
- Subculture of adherent cell lines (nutrients in medium are depleted – subculture to prevent cell death). Must first remove traces of serum with PBS (without calcium or magnesium), otherwise trypsin will be inactivated
- Subculture of semi-adherent cell lines
- Subculture of suspension cell lines
- Cell Quantification: Note that concentration of viable cells (cells/ml) = (viable cells/square) x dilution factor x correction factor (supplied by manufacturer). If too few cells to count – centrifuge cells and resuspend in a smaller volume. If too many, dilute more with trypan blue.
- Cryopreservation of cell lines. Note that cell viability should be in excess of 90% (on cell count) to achieve good recovery after freezing.
- Testing for bacterial and fungal contamination – includes production of negative and positive controls as well. Results are obtained within 2 weeks.
- Detection of Mycoplasma by culture – results obtained by 4 weeks. Mycoplasma colonies have a ‘fried egg’ appearance on agar plates.
- Detection of Mycoplasma by Indirect DNA Stain – much faster. Results available within 24 hours, but reduced sensitivity.