BASIC ANIMAL CELL CULTURE HANDLING

HISYAM ABDUL HAMID IPoPS2013

Ice-breaking
Transfer cells from subjects/models into artificial

environment finite/continuous cell

Convenient tool to study animal cell biology Fussy discipline requires a lot of considerations

Getting started - environment

Safety
Certain human-derived cancer cell harbouring pathogen

(potentially harmful yet to be known)

Equipment and chemical hazard

eg: trypan blue, EtBr carcinogenic, UV irradiation, thin glass slide NO mouth pipetting! NO smoking, eating or drinking! DO label!

Getting started - environment

Contamination
Sources of contamination: bacteria, yeast, fungi, mycoplasma,

cross-contamination etc..

Sterile
Air circulation/Ventilation
Aseptic technique

Aseptic technique
Sterile media and reagents autoclave/filter

Avoid aerial contamination of solutions

Avoid repeated opening of bottles 70% ethanol swab UV irradiation before and after Disposable items one time usage only Wear proper and clean cloth, glove/mask Pipetting technique and cell handling

Getting started materials and instrumentation

1. Equipments

Biosafety cabinet

Getting started materials and instrumentation

1. Equipments

Centrifuge

Refrigerator

Getting started materials and instrumentation

1. Equipments

Incubator

Inverted microscope

Getting started materials and instrumentation

1. Equipments

Autoclave

Liquid nitrogen tank

Getting started materials and instrumentation

2. Accessories

Pipettor

Haemocytometer

Getting started materials and instrumentation

2. Accessories

Consumable items

Getting started materials and instrumentation

3. Materials

Reagents

Choosing the Cell

What type of study?
Cytotoxic preliminary study Mechanism of reaction Detection, production and function of hormones, growth

factors etc..

The study of interactions: cell-cell and cell-matrix

Choosing the Cell

What type of cell?
Primary?

- Freshly isolated from experimental model

- More morphologically and physiologically resemble the parent

cell

- Not dedifferentiated and no worry on difference karyotype

Disadvantages Higher difficulty to obtain and maintain (very susceptible towards contamination) Short life span Variation in population media preparation complexity

Choosing the Cell

What type of cell?
Cell line? - AKA immortalized/ transformed cells More morphologically

and physiologically resemble the parent cell proliferate infinitely

- Acquired stable and heritable mutation giving the cell ability to

Disadvantages Prone to mutation behavioral and physiological changes which may interfere the result of the experiment

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Maintaining the Cell Growth Media

INGREDIENTS
Sodium bicarbonate stabilize pH by regulating the level of CO2 Phenol red monitor media pH (can mimic certain steroid

hormone)

L-glutamine protein production, energy source and nucleic acid

metabolism.

Amino acid Vitamins + MEDIA SUPPLEMENTS such as antibiotic/antimycotic, animal sera

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Maintaining the Cell Culture Vessel and Surfaces

Glass vessel were originally used heavy, expensive, labor-

intensive cleaning and poor microscopic viewing developed for polystyrene incubator requirements

replaced by plastic vessel surface treatment techniques were Provide additional protection from contamination and simpler Plastic walls is slightly permeable to CO2 and O2 might interfere

with anoxia study and long-term storage media

Loose cap and vented cap

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Maintaining the Cell Culture Vessel and Surfaces

SELECTING YOUR VESSEL
3 types of cell culture:
Anchorage Suspension Anchorage/suspension

4 basic culture systems:

Stationary monolayer - flask, petri dish, well plate Moving monolayer roller bottles Stationary suspension flask, petri dish, well plate Moving suspension spinner flask (stirred), bioreactors

Maintaining the Cell Cell Growth

Growth cycle of culture

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Maintaining the Cell - Subculture

Defrost/stabilize the temperature of trypsin, growth media appropriate with the temparature of the cell (370C) Discard cell culture medium from the flask Rinse the with PBS cell

Examine the culture on the next day to monitor reattachment of the cell

Add 2-3mL trypin and incubate for 515 minutes

Count/simply divide the cell into new flask with new complete growth medium and incubate.

Add 6-8mL complete growth media to inactivate the trypsin

Observe the cell detachment under microscope gentle tap if necessary

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Maintaining the Cell Cell counting

1. Assemble a cleaned and dry haemacytometer with the cover slip 2. Transfer a small amount of cell suspension to each counting chambers 3. View under inverted microscope at 100X mag. 4. Focus on quadrant labeled 1,2,3 and 4. 5. Record number of cell in each section. Number of cells in sample Average number of cell in 1,2,3,4

Dilution factor

104

Hayflicks Phenomenon
Normal human embryonic cells can divide up to 50 times before entering

senescence phase

The more cell divides, the shorter the telomeres

It is advisable to use cell lines with range passages from 40-60 at most

Rubin H. (2002)

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Cryopreservation
Advantages of cryopreservation:
Generation of safety stocks Elimination of time, energy and materials required for

maintaining cultures

Preservation of finite cells

Insurance against phenotypic drift in culture Creating standard reagent to be used for a series of

experiments

Modified from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Cryopreservation - procedure
Identify cell needs to be stored Collect 1x106 to 5x106 viable cells/ml

Label cryovials properly (name, cell line, date)

Remove one vial, restore to determine viability and sterility 24 hours Liquid nitrogen tank

Resuspend the cell with complete growth medium and transfer into cryovials + 5% DMSO

24 hours
Put into -800C freezer 60 Transfer into -200C freezer

30

Put on ice

Seal

Adapted from ATCC animal cell culture guide; retrieved from https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_ Guide.pdf

Thawing
Remove vial from liquid nitrogen tank

+ 9ml complete growth medium

Thaw until ice melted (gentle agitation in waterbath)

Decontaminate the vial using 70% ethanol

Examine and subculture if necessary 24 hours

Centrifuge tube 125 x g, 10

Discard supernatant Resuspend the cell gently using complete growth medium

Culture vessel (should contain at least 10ml culture medium)

References
ATCC Animal Cell Culture Guide; retrieved from

https://www.atcc.org/~/media/PDFs/Culture%20Gui des/AnimCellCulture_Guide.pdf at 27 August 2013

Celis, J.E. Cell Biology (Third Edition) A Laboratory

Handbook. Elsevier Inc., 2006. Imprint.

Mehra A., McDonald I., Pillay T.S. (2007). Variability in

3T3-L1 adipocyte differentiation depending on cell culture dish. Analytical Biochemistry 362; 281-283 cellular senescence in vitro to aging in vivo. Archives of Gerontology and Geriatrics 34; 275-286

Rubin H. (2002). Promise and problems in relating

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Albert Einstein

TERIMA KASIH