basic animal cell culture handling

7/27/2019 Basic Animal Cell Culture Handling

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BASIC ANIMAL CELLCULTURE HANDLINGHISYAM ABDUL HAMID IPoPS2013


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Ice-breaking

Transfer cells from subjects/models into artificialenvironment finite/continuous cell

Convenient tool to study animal cell biology

Fussy discipline requires a lot of considerations


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Getting started - environment

Safety

Certain human-derived cancer cell harbouring pathogen(potentially harmful yet to be known)

Equipment and chemical hazardeg: trypan blue, EtBr carcinogenic, UV irradiation, thin glassslide

NO mouth pipetting!

NO smoking, eating or drinking!

DO label!


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Getting started - environment

Contamination

Sources of contamination: bacteria, yeast, fungi, mycoplasma,cross-contamination etc..

Sterile

Air circulation/Ventilation

Aseptic technique


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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


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Getting started materials andinstrumentation

1. Equipments

Biosafety cabinet


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1. Equipments

Centrifuge Refrigerator


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1. Equipments

IncubatorInverted microscope


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1. Equipments

Liquid nitrogen tankAutoclave


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2. Accessories

Pipettor

Haemocytometer


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2. Accessories

Consumable items


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3. Materials

Reagents


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Choosing the Cell

What type of study?

Cytotoxic preliminary study

Mechanism of reaction

Detection, production and function of hormones, growthfactors etc..

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


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Choosing the Cell

What type of cell?

Cell line?

- AKA immortalized/ transformed cells More morphologically

and physiologically resemble the parent cell

- Acquired stable and heritable mutation giving the cell ability toproliferate infinitely

Disadvantages

Prone to mutation

behavioral and physiological changes whichmay interfere the result of the experiment

Adapted from ATCC animal cell culture guide; retrieved from


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Maintaining the Cell Growth Media

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 acidmetabolism.

Amino acid

Vitamins

INGREDIENTS


https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_

Guide.pdf

+ MEDIA SUPPLEMENTS such as antibiotic/antimycotic, animal sera



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Maintaining the Cell Culture Vesseland Surfaces

Glass vessel were originally used heavy, expensive, labor-intensive cleaning and poor microscopic viewing

replaced by plastic vessel surface treatment techniques were

developed for polystyrene

Provide additional protection from contamination and simplerincubator requirements

Plastic walls is slightly permeable to CO2 and O2 might interferewith anoxia study and long-term storage media

Loose cap and vented cap



Guide.pdf



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Maintaining the Cell Culture Vesseland Surfaces

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

SELECTING YOUR VESSEL



Guide.pdf


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Maintaining the Cell Cell Growth

Growth cycle of culture



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Maintaining the Cell - Subculture

Defrost/stabilize the

temperature of trypsin,

growth media

appropriate with the

temparature of the cell

(370C)

Discard cellculture medium

from the flask

Rinse the cell

with PBS

Add 2-3mL

trypin and

incubate for 5-

15 minutes

Observe the cell

detachment under

microscope gentle tap

if necessary

Add 6-8mL

complete growth

media to inactivate

the trypsin

Count/simply divide the

cell into new flask with

new complete growth

medium and incubate.

Examine the culture on

the next day to monitor

reattachment of the cell



Guide.pdf



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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 100Xmag.

4. Focus on quadrant labeled 1,2,3 and 4.

5. Record number of cell in each section.

Number of

cells insample

Average

number of

cell in

1,2,3,4

Dilution

factor104

p g ;


Guide.pdf


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Hayflicks Phenomenon

Normal human embryonic cells can divide up to 50 times before enteringsenescence phase

The more cell divides, the shorter the telomeres

Rubin H. (2002)

It is advisable to use cell lines with range passages

from 40-60 at most



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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

Cryopreservation

p g


Guide.pdf

Modified from ATCC animal cell culture guide; retrieved from


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Cryopreservation - procedure

Identify cell needs to be

stored

Resuspend the cell

with complete

growth medium

and transfer into

cryovials

Collect 1x106 to

5x106 viable

cells/ml

Label cryovials

properly (name,

cell line, date)

SealPut on

ice

Transfer into

-200

C freezer

Put into -800C

freezer


Guide.pdf

30

+ 5% DMSO

60

24 hours

Liquid nitrogen tank

Remove one vial,restore to determine

viability and sterility

24 hours



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Thawing

Decontaminatethe vial using 70%

ethanol

Remove vial

from liquid

nitrogen tank

Thaw until icemelted (gentle

agitation in

waterbath)

Centrifuge

tube

Discardsupernatant

Resuspend the cell

gently using complete

growth medium


Guide.pdf

+ 9ml complete

growth medium

Culture vessel (should

contain at least 10ml

culture medium)

Examine and subculture

if necessary

24 hours

125 x g, 10


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References

ATCC Animal Cell Culture Guide; retrieved fromhttps://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_Guide.pdf at 27 August 2013

Celis, J.E. Cell Biology (Third Edition) A LaboratoryHandbook. Elsevier Inc., 2006. Imprint.

Mehra A., McDonald I., Pillay T.S. (2007). Variability in3T3-L1 adipocyte differentiation depending on cellculture dish.Analytical Biochemistry362; 281-283

Rubin H. (2002). Promise and problems in relatingcellular senescence in vitro to aging in vivo.Archives ofGerontology and Geriatrics 34; 275-286


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basic animal cell culture handling

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