Download - Basic Cell Culture Technique and the Maintenance of Cell Lines - Part I - Boutee (SLIDES)
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Basic Cell Culture Technique
and the Maintenance of CellLinesRadona BoutteFall 2012
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Section 6: The culture of cells in suspension
Section 7:In vitro cell growth behaviour
Section 8: Determinations of cell growth data
Section 9: Cryopreservation and retrieval of cells from
frozen storage
Chapter Five
ec on : ranspor a on o ce s Section 11: Safety in the cell culture laboratory
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Non-adherent substrates for small scale culture
Cell substrate adherence is not desirable.
Mass Culture of cells in fluid suspension Cells can be cultured using spinner flasks and bioreactors
Micro-encapsulation
The culture of cells insuspension
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Adaption to culture
Survival of the fittest concept
Time consuming
Cells condition their environment
Phases of cell growth
In vitrocell growthbehaviour
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Phases of cell growth
Lag phase
Log phase
Plateau phase (stationary)
In vitrocell growthbehaviour
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Calculations of in vitro age
Number of passages (subcultures)
Number of cell generations
Multiplication rate and population doubling time
Multiplication rate (r)- number of generations that occur per
unit time
Determinations of cellgrowth data
Population doubling rate (PDT)-the time it takes the cell todouble and the reciprocal of (r).
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Counting cells in suspension
Hemocytometer
Electronic particle counting
Counting cells adherent to a substrate
Visual counting of cells or nuclei
Densitometry of fixed and stained cultures
Determinations of cellgrowth data
DNA assays
Colorimetric assays
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Determinations of cellgrowth data
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Phases of the cell cycle
Labeling mitosis method
S, G2, mitotic, G1
Determinations of cell growth data
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Cell cycle analysis by DNA content (flow cytometry)
Amounts of DNA changes as the cell goes through different
stages Cell generation time by time-lapse photomicrography
Visual inspection using a time-lapse microscopy
Determinations of cellgrowth data
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Growth fraction
Estimation of the percentage of cells active in the cell cycle
Expressions of culture efficiency
Attachment efficiency-percentage of cells that attach to the
culture substrate within a given period
Colony-forming efficiency-percentage of cells seeded that
Determination of cellgrowth data
form colonies
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Purpose of cell banking
If done properly, able to use later to perform
experimentation using same equivalency of cells from
previous study
Mechanism of cell freezing, and factors that affect
viability
Cryopreservation and retrievalof cells from frozen storage
DMSO Vial storage
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Supplies and equipment for cell freezing
Cryoprotectants
Freezing vials Freezing devices
Cell storage refrigerators
Cryopreservation and retrieval of
cells from frozen storage
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Transporting frozen cells
Frozen cells in vials or ampoules can be packaged in dry ice
and shipped by overnight courier
Transporting growing cells Suspension-cultured cells
Use of microcarrier beads during transport of substrate
Transportation of cells
Cultures growing in screw-capped flasks
Re-initiating incubation following transportation
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Potential risks in routine cell culture
Assess risk in laboratory
Awareness of increased risk associated with human cells
Classification of cell lines as
aetiolo ical a ents
Safety in the cell culture laboratory
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Precautions in handling pathogenic organisms and human
cells
Education and instruction
Good laboratory procedure for work involving potential
pathogens
Safety in the cell culturelaboratory
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ANY QUESTIONS?