Z3221319 Cell Culture Techniques and Proceedures

From CellBiology

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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/

Disclaimer

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.

General Points

Cell separation from tissue

  • proteases/calcium chelators to break down ECM-cell adhesions

Isolating specific cells from mixed cell suspension

  • centrifuges
  • 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).

Growing/Maintaining cells

  • 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.

Practical

Cleaning/Disinfection

  • 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

Contamination sources

  • 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)

Waste Disposal

  • 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:

http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/ecacc-handbook/cell-culture-techniques-12.html

  1. Aseptic technique and good cell culture practice
  2. 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.
  3. 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
  4. Subculture of semi-adherent cell lines
  5. Subculture of suspension cell lines
  6. 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.
  7. Cryopreservation of cell lines. Note that cell viability should be in excess of 90% (on cell count) to achieve good recovery after freezing.
  8. Testing for bacterial and fungal contamination – includes production of negative and positive controls as well. Results are obtained within 2 weeks.
  9. Detection of Mycoplasma by culture – results obtained by 4 weeks. Mycoplasma colonies have a ‘fried egg’ appearance on agar plates.
  10. Detection of Mycoplasma by Indirect DNA Stain – much faster. Results available within 24 hours, but reduced sensitivity.