Basic Cell Culture Technique and the Maintenance of Cell Lines

Radona Boutte Fall 2012

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 Section 10: Transportation of cells Section 11: Safety in the cell culture laboratory

Chapter Five

 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 in suspension

 Adaption to culture
Survival of the fittest concept Time consuming Cells condition their environment

Phases of cell growth

In vitro cell growth

behaviour

 Phases of cell growth

Lag phase Log phase Plateau phase (stationary) Decline phase (death)

In vitro cell growth

behaviour

 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 Population doubling rate (PDT)-the time it takes the cell to double and the reciprocal of (r).

Determinations of cell growth data

 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 DNA assays Colorimetric assays

Determinations of cell growth data

Determinations of cell growth data

 Phases of the cell cycle

Labeling mitosis method
S, G2, mitotic, G1

Determinations of cell growth data

 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 cell growth data

 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 form colonies

Determination of cell growth data

 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

DMSO Vial storage

Cryopreservation and retrieval of cells from frozen storage

 Supplies and equipment for cell freezing

Cryoprotectants Freezing vials Freezing devices Cell storage refrigerators

Additional comments regarding cryopreservation

Cryopreservation and retrieval of cells from frozen storage

 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 dependent cells Cultures growing in screw-capped flasks

Re-initiating incubation following transportation

Transportation of cells

 Potential risks in routine cell culture

Assess risk in laboratory

Awareness of increased risk associated with human cells Classification of cell lines as aetiological agents

Safety in the cell culture laboratory

 Precautions in handling pathogenic organisms and human cells

Education and instruction Good laboratory procedure for work involving potential pathogens

Safety in the cell culture laboratory

ANY QUESTIONS?