12. Cell synchronization and Immortalization

Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.

1. Animal cell science and Technology
12. Cell Synchronization
ShailendraSingh Shera, Ph.D

2. LT.12 Cell Synchronization & Transformation
Content Outline
1.Cell synchronization
2.Methods of cell synchronization
3.Cell synchronization using thymine double block method
4.Confirmation of cell synchronization
5.Transformation ( Cellular)
6.Immortalization
7.Mechanism of immortalization
8.Practice questions

3. Cell synchronization
•Cell synchronization is a process by which cells at different stages of the cell cycle in a culture are
brought to the same phase.
•Through synchronization, cells at distinct cell cycle stage could be obtained.
•Many cell-synchronization protocols involve placing cells under growth inhibitory conditions for
periods of time related to the duration of specific cell-cycle phases.
•Cell synchronisationis used to study the progression of cells through the cell cycle
Cell can be synchronized in any of the following main stages of cell cycle
G0/G1 phase –cell rest and recovery in preparation for subsequent rounds of cell division
S Phase –DNA replication (interphase)
G2/M phase –chromosome segregation and mitosis
Targeting the cell cycle stage:
•Release from GO arrest,
•release from M-and S-phase blocking agents,
•mitotic detachment
•centrifugal elutriation

4. Methods of cell synchronization
Cell
synchronization
Physical
Centrifugal
treatment
Flow Cytometry
Affinity of
antibodies
Chemical
Double
thymidineblock
Taxolarrested
cells with Cdk
inhibitors
Nocodazole
treatment

5. PhysicalFractionation
•Physicalfractionationorcellseparationtechniquescanbebasedoncelldensity,cellsize,
affinityofantibodiesoncellsurfaceepitopesandlightscatterorfluorescentemissionbylabeled
cells.
•Centrifugalseparationorfluoresecene-activatedcellsortingareprimarilyusedforphysical
fractionation.Centrifugalseparationenablestheseparationofcellsbasedonsizeand
sedimentationvelocity.Fluorescence-activatedcellsorting(FACS)sortscellsonbasedon
differenceswhichcanbedetectedbylightscatter(e.g.cellsize)orfluorescenceemission(by
penetratedDNA,RNA,proteins,antigens).Thiscanbecarriedoutusingaflowcytometeror
fluorescence-activatedcellsorter.
Physical methods

6. Chemical methods
ChemicalBlockade
•AsthenamessuggeststhismethoduseschemicalssuchasThymidinetoblockmetabolic
reactions.ThiscanbeachievedthroughinhibitionofDNAsynthesislargelyduringtheS-Phaseof
thecellcycle.Inhibitorssuchasthymidine,aminopterin,hydroxyureaandcytosinearabinosidecan
havevariableeffects.Serumstarvingyourcellsfor24hrswillresultinanaccumulationofcellsatG1
phase.Theeffectsofserumstarvationcanbereversedbytheadditionofserumtomediaoncecell
synchronyhasoccured.
•Differentchemicalscaninducecellsynchronisationatdifferentstagesinthecellcycle
Cell Cycle Stage TargetedTreatment used
G1 arrest Double Thymidineblock,Serumstarvation. Inhibition of cyclindependent
kinase(CDKs)
G2 arrest Inhibition of microtubules, Inhibition of cyclindependent kinase(CDKs)
M-Phase
Taxol, Nocodazole

7. •TosynchronisecellsattheG1/Sborderafreshlypreparedthymidinesolution(16mM)wasmade
upincompletemediumandfilter-sterilisedusinga0.22µmfilterdisc.
•Cells(1X10
7
)wereculturedin60mlcompletemediumcontainingthymidine(2mM)for16hina
175cm
3
cellcultureflask.
•Thecellswerethenresuspendedincompletemedium(52.5ml)for8htoallowcellsto
•reenterthecellcycle.
•Cellswereharvestedbycentrifugationat400xgfor5minandwashedX2incompletemedium
(10ml).Cells(1X10
7
)wereculturedin60mlcompletemediumcontainingthymidine(2mM)for16
htosynchronisecellsattheG1/Sborder.
•Cellsynchronisationwasconfirmedbyflowcytometry.
Cell synchronization using double thymidine block
Protocol adopted as it is from
https://www.elisagenie.com/cell-synchronisation-methods/
Also visit this site for other detailed protocol

8. •Cellsynchronisationcanbeconfirmedbymicroscopyorflowcytometry.Microscopyallowsyouto
seewhatisactuallygoingoninsideyoucells.
•Flowcytometryenablesyoutocompareyourtreatedsynchronisedcellsagainstaasynchronous
control.Brieflytheprotocolisasfollows;
1.Fixandpermeabilizeyourcellsin70%ethanol
2.Stainwith40µg/mlpropidiumiodide,andinclude25µg/mlofRnase(todegradeRNAand
ensurethatyoustainDNAonly).
3.Runyoursamplesontheflowcytometer.
Confirmation of cell synchronization

9. Transformation ( Cellular transformation)
•Transformationofculturedcellsimpliesaspontaneousorinducedpermanentphenotypicchange
resultingfromaheritablechangeinDNAandgeneexpression.
•Althoughtransformationcanarisefrominfectionwithatransformingvirus,suchaspolyoma,or
fromtransfectionwithgenessuchasmutantras,itcanalsoarisespontaneously,afterexposureto
ionizingradiation,oraftertreatmentwithchemicalcarcinogens
•Transformationisassociatedwithgeneticinstability
Phenotypicchangesassociatedwithtransformationare:
i.Immortalization
ii.Aberrantgrowthcontrol
iii.Malignancy
CharacteristicofTransformedcells
Geneticinstability:
•Cellshavehighrateofundergoingmutationsandchangestheirgeneticmakeupevenwith
slightinduction
•Continuouscelllines,particularlyfromtumorsofallspecies,areveryunstable.
•DeletionoralterationinDNAsurveillancegenes,suchasp53,areusuallyimplicated.

10. •Continuous cell lines are usually heteroploid, meaning they show a wide range in chromosome
number among individual cells in the population, implying substantial genetic diversity.
Therearetwomaincausesofgeneticheterogeneity:
(1)thespontaneousmutationrateappearstobehigherinvitro,associated,perhaps,withthehigh
rateofcellproliferationanddefectiveDNAsurveillancegenes,particularlyp53,
(2)mutantcellsarenoteliminatedunlesstheirgrowthcapacityisimpaired.
Chromosomalaberration
•Aberrationreferstoacharacteristicthatdeviatesfromthenormaltype
•Chromosomalabnormalitiesoccurwhenthereiseitheranexcessoralossofatotalchromosome
orwhenaportionofachromosomeisabsent(macrodeletionsandmicrodeletions).
•Mosttumorcultureshowsignsofaneuploidy,meaningdeviationsfromthenormalcomplementof
chromosomes.Somespecificaberrationsareassociatedwithparticulartypesofmalignancy
Asresultofchromosomalaberrations,thetransformedcellsshowanchorageindependence,loss
ofcontactinhibition,andlowserumrequirement.

11. Anchorageindependence
Thereoccurseveralchangesonthecellsurfacesoftransformedcells.Theseincludealterationsin
thecellsurfaceglycoproteinsandintegrin’s,andlossoffibronectin.Someofthetransformedcells
maytotallylackcelladhesionmolecules(CAMs).Themodificationsonthesurfaceoftransformed
cellsleadstoadecreaseincell—cell,andcell-substrateadhesion.
Contactinhibition:
Thetransformedcellsarecharacterizedbylossofcontactinhibition.Thiscanbeobservedbythe
morphologicalchangesinthedisorientedanddisorganizedmonolayercells.Thisresultina
reduceddensitylimitationofgrowth,consequentlyleadingtohighersaturationdensitycomparedto
normalcells.
Lowserumrequirement:
Ingeneral,transformedcellsortumorcellshavelowerserumdependencethanthenormalcells.
Thisismostlyduetothesecretionofautocrinegrowthfactorsbythetransformedcells

12. •Mostnormalcellshaveafinitelifespanof20to100generations,butsomecells,notablythose
fromrodentsandfrommosttumors,canproducecontinuouscelllineswithaninfinitelifespan.
•Thesedominantlyactinggenessynthesizeproductswhichinhibitthecellcycleprogression.
•Itisstronglybelievedthatimmortalizationoccursduetoinactivationofsomeofthecellcycle
regulatorygenese.g.Rb,p
53
genes.
•Outof10genesinvolvedinsenescence,someofgenesowingtomutationnegativelyregulate
theexpressionoftelomerase,requiredfortheterminalsynthesisoftelomericDNA,which
otherwisebecomesprogressivelyshorterduringafinitelifespan,untilthechromosomalDNAcan
nolongerreplicate.
Immortalization of cell

13. Mechanism of Immortalization
Ithasbeenassumedthatimmortalizationisamultistepprocessinvolvingtheinactivationofa
numberofcellcycleregulatorygenes,suchasRbandp53.TheSV40LTgeneisoftenusedto
induceimmortalization.Theproductofthisgene,Tantigen,isknowntobindRbandp53.Bydoing
so,itnotonlyallowsanextendedproliferativelifespanbutalsorestrictstheDNAsurveillance
activityofgeneslikep53,therebyallowinganincreaseingenomicinstabilityandanincreased
chanceofgeneratingfurthermutationsfavorabletoimmortalization(e.g.,theupregulationof
telomeraseorthedownregulationofoneofthetelomeraseinhibitors).Transfectionofthe
telomerasegenewitharegulatablepromoterissufficienttoimmortalizecells
MethodsofImmortalization:Makingcellsimmortal
Severalmethodsexistforimmortalizingmammalian cellsincultureconditions.
Withyearsofexperienceincellimmortalization.Recombinantlentiviral,retroviral(MMLV)and
adenoviralvirusesexpressingEBV,HPV-16E6/7andSV40Tantigens,hTERT,p53andRB
siRNAs,andras&mycmutants.Allthesetoolswillmakeyourcellimmortalizationprojectsimpler
andeasierthaneverbefore.
Followlinkforprocessofimmortalization:https://www.biocat.com/cell-biology/cell-immortalization

14. Test your Understanding
1.Cellular Transformation refers to
a.Transforming cells by inserting exogenous DNA
b.Transforming cells through alteration of endogenous DNA
c.Both (a) and (b)
d.None of the above
2.Centrifugal elutriation is used for
a.Cell synchronization
b.Sorting of cells
c.Both (a) and (b)
d.None of the above
3.What happens to cells if p53 and Rbgenes are inactivated
a.Cells dies instantaneously
b.Cell undergo apoptosis
c.Cell becomes immortal
d.None of the above
4.Anchorage independency is the characteristics of……………………..cells.
5. Starvation of cells of nutrient is one method of synchronizing cellular growth in culture. ( True / False)
*Questions adapted from: Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: ShailendraSingh Shera. Publisher: Amazon Kindle.

15. References
1.https://www.elisagenie.com/cell-synchronisation-methods/
2.https://link.springer.com/protocol/10.1007%2F978-1-4939-6603-5_1
3.https://experiments.springernature.com/articles/10.1007/978-1-4939-6603-5_1
4.https://www.sciencedirect.com/science/article/pii/S0091679X08615824
5.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156087/
6.https://info.abmgood.com/blog/your-basic-guide-to-cell-line-immortalization
7.https://www.biocat.com/cell-biology/cell-immortalization
MCQ Practice questions
1. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: ShailendraSingh Shera. Publisher: Amazon Kindle.
Available on: amazon.com