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Cell Culture

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Process by which cells are grown under controlled conditions ... Mechanical: rubber spatula, quick and easy but significant cell death ... – PowerPoint PPT presentation

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Title: Cell Culture


1
Cell Culture
  • Lisa OFlynn
  • 6th July 2006

Genome.tugraz.at//CellCulture.html
2
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
3
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
4
  • 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.

5
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

6
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

7
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
8
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

9
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

10
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

11
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

12
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/
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