Cell Culture

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Cell Culture

• Lisa OFlynn
• 6th July 2006

Genome.tugraz.at//CellCulture.html
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Cell Culture Defined

• Process by which cells are grown under controlled
  conditions
• 19th Century Sydney Ringer developed salt
  solution to maintain animal heart cells in vitro
• 1907-1910 Ross Granville Harrison methodology
  of tissue culture
• 1950s technique developed

Ross Granville Harrison
yalemedicine.yale.edu/ym_wi03/faces.html
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Types of cell culture

• Primary Cell Culture
• Derived directly from excised normal animal
  tissue heterogeneous, later dominate by
  fibroblasts
• Continuous Cultures
• Suspension (as a single cell or small free
  floating clumps) e.g. blood lines - lymphoma
• or Monolayer that is attached to the tissue
  culture flask e.g. CHO (Chinese Hamster Ovary)

CHO Cells
www.essen_instruments.com/IncuCyte_Movies.htm
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• Cells lines of human origin- controversial in
  bioethics due to the fact cells may out live
  parent organism or they maybe used in the
  discovery of lucrative medical treatments
• 1990 Supreme Court of California human patients
  have no property rights on the cell lines derived
  from organs removed with their consent.

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Culture Maintenance

• Key is to mimic the environment in which they
  were found
• Cells maintained at
• - 37C
• - 5-10 CO2
• - pH controlled with buffered sodium
  bicarbonate/carbonic acid
• - humidity maintained

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Culture Maintenance
www.usak.ca

• Laminar Flow Hood
• -UV light
• -on 10-20mins before use
• -surfaces wiped with 70 ethanol before and
  after use
• Cultures examined daily

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Harvesting Cells

• Cells must be passaged (split) regularly to
  maintain life span
• Population density semi-confluent state log
  phase
• Methods
• Mechanical rubber spatula, quick and easy but
  significant cell death
• Proteolytic enzymes Trypsin, collagenase, fast
  reliable but may damage cell surface proteins
• EDTA gentler method to detach cells

http//www.bdbiosciences.com/discovery_labware/Pro
ducts/cell_cultureware/cell_culture_flasks/images/
flask_family.jpg
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Advantages of Cell Culture

• Minimize variability among experiment samples
• Reproducible results can be obtained by using
  clonal cell batch
• Cells can be stored in liquid nitrogen and
  returned to culture at a later date

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Disadvantages of Cell Culture

• After a period of continuous growth cell
  characteristics can change and may become quite
  different from those at the start
• Animal cells require 18-24hrs to double this
  leaves cells vulnerable to contamination from
  bacteria which would out grow the cells

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Precautions

• Risk assessment low, medium or high
• Containment reduces risk restrict staff movement
• Work areas must be uncluttered, reagents
  correctly stored and labeled
• Disinfection of work surfaces, equipment and
  waste
• Waste-inactivated overnight in a solution of
  hypochlorite

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Applications
http//tsmb.cryst.bbk.ac.uk

• Investigate normal physiology / biochemistry of
  cells e.g. cell metabolism
• Study the effects of various chemical compounds
  or of drugs on specific cell types
• Manufacture of viral vaccines many other
  products of biotechnology e.g. rDNA technology in
  animal cell culture including enzymes, hormones
  and immunobiologicals

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References

• http//en.wikipedia.org/wiki/Cell_culture_techniqu
  es
• http//www.biotech.ubc.ca/Bioengineering/CellCultu
  re/index.htm
• www.research.umbc.edu/jwolt/method5.htm
• CloneticsTM Airway Epithelial Cell Systems
  Instructions for Use
• Billie Ruth Bird, Francis T. Forrester Basic
  Laboratory Techniques in Cell Culture
• Images
• yalemedicine.yale.edu/ym_wi03/faces.html
• www.usak.ca
• Genome.tugraz.at//CellCulture.html
• www.essen_instruments.com/IncuCyte_Movies.htm
• http//www.bdbiosciences.com/discovery_labware/Pro
  ducts/cell_cultureware/cell_culture_flasks/images/
  flask_family.jpg
• http//tsmb.cryst.bbk.ac.uk/