Introduction to Animal Cell Culture

This presentation presents an overview of definition, equipment, various cell culturing methods,
characterization, and applications of animal cell culture. This presentation also contains MCQs to acquaint reader about types of question asked in various competitive examinations.

1. Animal cell science & Technology
1. Introduction to Cell Culture
ShailendraSingh Shera, Ph.D

2. 1. Introduction to cell culture
Outline:
1.What is cell culture?
2.Cell culture equipment
3.Media and supplements for cell culture
4.Cell culture environment
5.Cell culture methods
6.Morphology of cells
7.Storage
8.Cell culture safety
9.Applications
10.MCQs for practice

3. What is cell culture?
The observation by Jolly ( 1903) that animal cell can be cultivated outside animal body in
an artificial environment is a significant discovery with respect to cell culture.
The Theactual beginning of animal cell culture and tissue culture was made by Harrison
(1907) and later by Carrel (1912).
Forcultivationofanimalcellsinlaboratorycellsarefirstlyremovedfromthebodythrough
excisionanddisaggregatedusingmechanicalorenzymaticmeddum.Thedisaggregated
tissuesarethencultivatedinsuitablemediumandfavorablegrowthmedia.
Definition:
“Animal cell culture is defined as the cultivation and maintenance of animal cells in an
artificial and controlled environment.”

4. Cell culture equipments
Animalcellcultureisasophisticatedtechniquesandrequireshighleveloftechnicalskills.Given
thecomplexityinvolvedinanimalcells,culturingrequiressomeadvancedandsophisticated
equipments.Further,thechoiceofenvironmentdependsuponthetypesofworkperformed---
routineorhighvalueresearchorteaching.But,allcellculturelaboratoryshouldhavefollowing
basicequipmenttostartwith:
Autoclave Deep freezer
( -80°C)
Cell culture micro-welltubes
Cellcounter Serological Pippetors Cell culture gradetubes and
other labware
Cell culture hood /
Biosafetycabinet
Media, sera, cell media
additives
Sodium Hypochlorite for
routine disinfection
CO2incubator Centrifuges
pH meter Gloves
Inverted and Fluorescence
microscope
Cryopreservation facility
Chemical balance Liquid Nitrogen
Freezer( -20°C) Pasteur pippetes

5. System of cell culture
•Variousmethodsareavailableforculturinganimalormammaliancellsinartificialenvironment.
•Cellscanbeeitherculturedasmonolayerorsuspensionculture.Monolayerculturearebest
suitedforgrowinganchoragedependentcellswhilesuspensionculturearebestsuitedfor
anchorageindependentcells.
Variousmethodsofculturingcellsare:
•Coversliporslideculturemethod(singlecoverslipwithplasmaclotanddoublecoverslip
method)
•Hangingdropmethods
•Flaskculturemethods(Usedforestablishingstrainsfromfreshexplantoftissues)

6. Media and supplements of cell culture
Thepurposeofmediaistoprovidefavorableconditionforgrowth,expansionandsurvivalof
cultivatedcell.
Themediaprovidesnutrientandenergy,maintainsrequiredpH(7.2-7.4),osmoticfactorsfor
cellulargrowthanddivision.
Commoncomponentsofanimalcellculturemediaincludes:aminoacids,vitamins,carbohydrate,
Inorganicsalts,bufferingsystem,antibiotics.
Animalcellculturemediacanbedividedintofollowingcategory:
i.NaturalMedia
ii.Syntheticmedia
(i)Naturalmedia:
Naturalmediaconsistsolelyofnaturallyoccurringbiologicalfluidse.g.Plasmaclot,serum,
aminioticfluid
(ii)Syntheticmedia:
Artificialorsyntheticmediaarepreparedbyaddingnutrients(bothorganicandinorganic),vitamins,
salts,O
2andCO
2gasphases,serumproteins,carbohydrates,cofactors.Eg.Ham'sF-10,
RPMI-1640,DMEM(DulbeccoModifiedEagleMedium),MEM(MinimumEssentialMedia).

7. Synthetic media
Serum
containing
media
Serum Free
media
Chemically
defined media
Protein free
media
Balanced salt
solution
Balance salt solution is used to keep cell alive only.
Types of synthetic media

8. Media Supplements
Media Supplements refers to thing, molecules or substance added in addition to enhance the quality of
media.
Supplements helps animal cells to produce proteins and biomoleculesrequired in high Concentration
for growth, maintenance and survival.
The advantages of supplements are:
i.Helps in customization of growth condition of cells
ii.Improves cell viability and growth
iii.Keeps cell healthier longer
Some examples of media supplements are:
•Amino Acid Solution * Fibroblast growth factors
•Bovine Serum Albumin * Cytokines
•Transferrin
•Insulin
•Lipid supplements
•Sodium pyruvate
•Yeast Solution

9. Cell culture environment
Theinvitrocultureofanimalcellsoutsidebodyrequiresexactlysameconditionas
availableinsidebody.Theinvitroculturehasrequiredtoolstomaintainandcontrolall
physiochemicalandphysiologicalfactorssuchasnutrientrequirements,pH,temperature,
bufferingandcontamination.
Media Containsnutrients,lipids,vitamins,antibiotics,traceelements,pH
indicators,hormones
pH 7.2-7.4;shouldbearoundneutral
Temperature Dependsupontheanimalcellsofhosts
Mammaliancells/human:37°C
Insectcells:27°C-30°C
CO2 Requiredforbuffering
5-10%iscommon;VeryminuteamountofO2isalsorequired
Phenolphthalein
red
•pHindicator;ChangescolorwhenpHofmediumchanged
ThechangeofpHmaybeduetoaccumulationoftoxicgrowthor
•Duetobacterial,yeastandfungalcontamination
Antibiotic •Usedtoporeventcontaminationofcellsinculture
•Frequentuseincellculturenotrecommended
•Penicillin-Streptomycinsolution=50-100µg/ml
•amphotericinB=2.5µg/ml

10. Morphology & Characterization
Cells in culture attains basically three types of morphology . The categorization of cells are based
on their shape and appearance under microscope.
Fibroblasticcells Epithelial cells Lymphoblast
Adherent cells Adherent cells Suspension
Spindle shaped Polygonal shaped and
appearance
Round, spherical shaped
Adapted from: https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/introduction-to-cell-
culture.html
Fibroblast Epithelial Lymphoblast

11. Characterization
Oncethecellsareculturedinvitro,thecellsshouldbeidentifiedfortheirknownpropertiesand
behavior.Characterizationofcelllineisthefirstindispensiblestepaftereachcelllineisgenerated
fordeterminingitsfunctionality,authenticity,contamination,originetc
Accordingtocurrentregulation,followingaspectsofcelllinecharacterizationshouldbe
considered
•Originandhistoryofcellline
•Cellularmorphologyandgrowthcharacteristics
•Purityofcelllines
•Tumorigenicity&oncogenecity
•Stability
•Itguaranteesagainsttheadvertentcontaminationofcelllineduringcellculturingwork

12. Methods of cell characterization
Various methods of cell line for identification of and cross contaminations and misidentifications
are outlined below:
•Morphological characteristics
•Karyotyping( Chromosome analysis)
•Viral susceptibility
•Specific antigenecity
•DNA fingerprinting
•MHC ( Major HistocompatibilityComplex)
•Flow Cytometry
•ConfoccalMicroscopy
•Staining ( GiemsaStaining)

13. Storage
•Storagefacilityinanimaltissuelaboratoryisofutmostimportance.Storageisrequiredforsafe
keepingofmedia,reagents,celllines,culturedishesandpreservingcellsandcelllines.
•Properstoragesavestime,expensesandmakesworkingwithculturestresslessandeasy.
•Labwares,chemicals,reagentsandsparescanbesafelykeptinproperlylabelleddedicated
cupboard.
•Biologicalandotherlabilesubstancescanbestoredinrefrigerator(4°and-20°C).
•Cellandcelllinesshouldbecryo-preservedandstoredinliquidN
2.
Cryopreservation:
Surpluscellsandcellsmarkedforfutureusearetreatedwithsuitablecryoprotectant(Dimethyl
sulphoxide,DMSOorglyccerol)andstoredattemperaturesbelow–130°C(cryopreservation)
untiltheyareneeded
Usuallyforlongtermstorage,cellsarestoredinliquidN
2at-196°C.

14. Cell culture safety
•Ensuringsafetyofyourself,yourcolleagues,familyandsocietyatlargeisofimmense
importancewhileworkingincellculturelaboratory.Peopleworkingincellculturelaboratoryare
continuouslyatriskofexposuretotoxic,mutagenicandinfectiouschemicalsandbiological
agentsrespectively.
•Whiledealingwithanimalandhumantissues,extraprecautionsareneededastheycontain
virusesandotherdangerousbiologicalagent.
•Byfollowinggoodlaboratorypractices,wecanavoidhealthyhazardwhileworking
Someofthemanyguidelinesthatshouldbefollowedareoutlinedbelow:
Wearsafetyclothes(gloves,closedshoes,labcoat).Ifworkingwithhighlyinfectiousand
contagiousvirussuchasCoronvirus-2,wearpersonalprotectiveequipments.
Noorlowaerosolcreation.
Decontaminateallsurfacesbeforeandaftertheexperiment
Avoidusingsharpobjects.
Washhandswhenenteringandbeforeleavingthelaboratory.
Workinaccordancewiththefacilityguidelines.

15. Applications
•Animal cell culture finds applications in basic, applied research and commercial and Industrial
scale.
•In basic research cells in culture are used to study basics about intracellular activity such
as DNA transcription, protein synthesis, cell cycle etc.; Intracellular flux; Genomic ; Proteomics,
cell-cell and cell-material interactions.
•In applied research , cell cultures are used for production of high value therapeutic compounds
such as mono clonal antibodies, vaccines etc; Toxicology studies; Immunology, pharmacology
and Tissue engineering.
•As a model system for diseases and drug screening.
•Helps to study normal cell homeostasis, cell biochemistry, metabolism,
•mutagenesis, diseases, and compound effects.
Cell culture are preferred for high value products due to batch to batch consistency and
reproducibility.

16. Testing your understanding ( MCQs)*
1.Animal cells grown in culture generally retain their functions even though they are growing in vitro.
( True / False)
2. When we grow cells in culture
a. we can only grow one cell type at a time
b. cells with a growth advantage can outgrow a cell culture given enough time
c. cells supply other cells everything they need to grow
d. serum must always be heat-inactivated
3. L-glutamine is an important amino acid because
a.It is an amino acid
b.Cells get much of their energy from the catabolism of L-glutamine
c.It is not important since it is made by some cells
d.It is an amine donor
4.When growing animal cells it is important to
a.use continuous vigilance to guard against contamination
b.wear sturdy work shoes
c.have lots of computer memory
d.drink plenty of water

17. 5. Antibiotics should be
a.always be used
b.used, only when necessary
c.never be used
d.only during a cross-contamination event
6. Which benchmark cell line verification is not recommended by ATCC
a.STR analysis
b.growth curve analysis
c.DNA sequencing
d.mycoplasmadetection
7. Characterization of cell line is necessary for
a.Identification of species
b.Detection of contamination
c.To check the stability of cells in culture
d.All the above
8. Cell culture are used to produce high value therapeutic product due to
a.Reproducibility
b.Batch to batch consistency
c.Both (a) and (b)
d.Only (a)
9. Cross-contamination is
a.not to much of a problem and never happens
b.the result of working with multiple cells at one time
c.can be controlled with antibiotics
d.cannot really happen if you follow good protocols
*Questions adapted from : Practice and Learn Animal cell Science and Technology: Multiple choice question for
learning. Author: ShailendraSingh Shera. Publisher: Amazon Kindle.

18. 1.https://www.labmanager.com/laboratory-technology/cell-line-characterization-methods-reviewed-
20877
2.https://www.thermofisher.com/in/en/home/life-science/cell-culture/mammalian-cell-culture/media-
supplements.html
3.https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/introduction-to-cell-
culture.html
4.https://www.ptglab.com/support/cell-culture-protocol/introduction-to-cell-culture/
References & Further reading
Further readings
1.Watson, J.D., Gilman, M., WitowskiJ.andZoller, M. Recombinant DNA, 2nd ed., Scientific American
Books, 1983
2.Glick, B.R. and Pasternack, J.J. Molecular Biotechnology, 3rd ed., ASM Press, 2003
3.Davis J.M. Basic Cell Culture: A Practical Approach, IRL Press, 1998
4.FreshneyR.I. Animal Cell Culture a practical approach, 1987
5.Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: ShailendraSingh Shera. Publisher: Amazon Kindle.