basic cell culture technique and the maintenance of cell lines - part i - boutee (slides)

7/29/2019 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


DNA assays

Colorimetric assays


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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



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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?

basic cell culture technique and the maintenance of cell lines - part i - boutee (slides)

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