Animal Cell Culture_Basics in Animal Biotechnology.ppt

Basics of Animal Cell Culture

1. Animal Cell Culture –
Basics
P. Balaji
PG and Research Centre in Biotechnology
MGR College,
Dr. MGR Nagar, Hosur, Tamilnadu, India

2. Biotechn
ology
Microbiol
ogy
Genetics
Biochemi
stry
Food
Science
Chemistr
y
Electronic
s
Biotechnology isan
interdisciplinarylearning,
whichincludesmany
areasofLifeSciences

3. Biotechnology
Application of
Technology in
Living Organisms
Uses any organisms
or part of organisms
to make or modify
products

4. Morespecifically,AnimalBiotechnologyis
Toproducemedicine/vaccines
Toimprovetheanimalsortheirproductstheyproduce
Ex:Milk/meatandflesh/egg
Toreducethecostofanimalbreeding
Topreserveendangeredanimals

5. MilestonesinAnimalBiotechnology
1907–RossHarrison–FirstScientisttocultureanimal
cellsininvitrocondition
Cultivatedthefrognervecellsinlymphclotbyhangingdrop
methodandobservedthegrowthofnervefibresinvitro
1916–RousandJones–Trypsinizationandsubculture
ofexplants
1923–CarrelandEbeling–Subcultureoffibroblasticcell
lines
1943–Earle–Establishedfirstcellline
Mousefibroblastcellline
1952–Gey–Establishedfirsthumancellline
HeLafromcervicalcarcinoma

6. MilestonesinAnimalBiotechnology
1955–Eagle–Developmentofdefinedmedia
1990–GalasandWatson–HumanGenomeProject
1991–Kruse–Laminarairflowcabinets
1996–CampbellandWilmut–Firstclonedmammal
Femaledomesticsheep“Dolly”

7. Transgenic Animals

8. Various advancements in genetics allowed humans to directly
alter the DNA and therefore genes of organisms
In1972PaulBergcreatedthefirstrecombinantDNAmolecule–
DNAfromamonkeyvirus(x)lambdavirus
In1974RudolfJaenischcreatedatransgenicmouse-Introduced
foreignDNAintoitsembryo(world'sfirsttransgenicanimal)
Geneticallymodifiedmice-1984thatcarriedclonedoncogenes–
predisposingthemtodevelopingcancer
Thefirsttransgeniclivestock–1985
Firstanimaltosynthesizetransgenicproteinsintheirmilkweremice

9. Thefirstgeneticallymodifiedanimal–GloFish
AZebrafishwithafluorescentgene–glowinthedark–under
ultravioletlight.
ReleasedtotheUSmarketin2003

10. Thefirstgeneticallymodifiedanimaltobeapprovedforfooduse
wasSalmonin2015.
Thesalmonweretransformed–
Growth Hormone-regulating gene from a Pacific Chinook salmon
(x)
promoter from an ocean pout
Enabledtogrowyear-roundinsteadofonlyduringspringand
summer

11. Genetically modified animals for the beneficial products is
the main concern of Animal Biotechnology

12. Somatic cell nuclear transfer,
Cell nucleus from an adult cell
Transferred
Unfertilizedoocyte(developing egg cell in which
cell nucleus is removed).
Hybrid cell is stimulated to divide (electric shock)
Development
Blastocyst
Implanted in a surrogate mother.
Dolly was the first clone produced from a cell taken
from an adult mammal.
Mother 1 -Egg
Mother 2 -DNA
Mother 3 –
Carried the
cloned embryo
Dolly

14. MAJORDEVELOPMENT’SINCELLCULTURE
TECHNOLOGY
Firstdevelopmentwastheuseofantibioticswhichinhibits
thegrowthofcontaminants.
Secondwastheuseoftrypsintoremoveadherentcellsto
subculturefurtherfromtheculturevessel
Thirdwastheuseofchemicallydefinedculturemedium.

15. Models
Basiccellbiology/interactionsbetweendiseasecausing
agentsandcells/effectsofdrugsoncells/processand
triggeringofaging/nutritionalstudies
Toxicitytesting
Effectsofnewdrugs
Cancerresearch
Functionofvariouschemicals,virus&radiationto
convertnormalculturedcellstocancerouscells
Where cell cultures are used

16. Virology
Cultivationofvirusforvaccineproduction/tostudythere
infectiouscycle
GeneticEngineering
Productionofcommercialproteins/largescaleproductionof
virusesforuseinvaccineproduction
Ex:polio,rabies,chickenpox,hepatitisB&measles
Genetherapy
Cellshavingafunctionalgenecanbereplacedtocellswhich
arehavingnon-functionalgene

17. Invitrocultivationoforgans
(tissues&cellsatdefinedtemperatureusinganincubator&
supplementedwithamediumcontainingcellnutrients&growth
factorsiscollectivelyknownastissueculture)
Differenttypesofcellgrown(connectivetissueelementssuchas
fibroblasts,skeletaltissue,cardiac,epithelialtissue(liver,breast,
skin,kidney)andmanydifferenttypesoftumorcells)
Tissue Culture

18. Cellswhensurgicallyorenzymaticallyremovedfromanorganism
andplacedinsuitablecultureenvironmentwillattachandgrow
arecalledasprimaryculture
Primarycellshaveafinitelifespan
Primaryculturecontainsaveryheterogeneouspopulationofcells
Subculturingofprimarycellsleadstothegenerationofcelllines
Celllineshavelimitedlifespan,theypassageseveraltimesbefore
theybecomesenescent
Lineageofcellsoriginatingfromtheprimarycultureiscalleda
cellstrain
Primary Culture

19. Adherentcells
Thesecellsareanchoragedependentandpropagateasamonolayer.
Thesecellsneedtobeattachedtoasolidorsemi-solidsubstratefor
proliferation.
Suspensioncells
Suspensioncellsdonotattachtothesurfaceoftheculturevessels.
Thesecellsarealsocalledanchorageindependentornon-adherent
cellswhichcanbegrownfloatingintheculturemedium.
Confluentcultureandthenecessityofsub-culture
Afterthecellsareisolatedfromthetissueandproliferatedunder
theappropriateconditions,theyoccupyalloftheavailable
substrate
Types of Primary Cell Culture

20. Whenaprimarycultureissub-cultured,itisknownas
secondarycultureorcelllineorsub-clone.Theprocess
involvesremovingthegrowthmediaand
disassociatingtheadheredcells(usually
enzymatically).
Secondary Cell Culture and Cell lines

21. Finitecelllines
Thecelllineswhichgothroughalimitednumberofcelldivision
havingalimitedlifespanareknownasfinitecelllines.Thecells
passageseveraltimesandthenlosetheirabilitytoproliferate,
whichisageneticallydeterminedeventknownassenescence.Cell
linesderivedfromprimaryculturesofnormalcellsarefinitecell
lines.
Types of Cell Lines

22. Continuouscelllines
Whenafinitecelllineundergoestransformationandacquiresthe
abilitytodivideindefinitely,itbecomesacontinuouscellline.
Suchtransformation/mutationcanoccurspontaneouslyorcanbe
chemicallyorvirallyinducedorfromtheestablishmentofcell
culturesfrommalignanttissue.Cellculturespreparedinthisway
canbesub-culturedandgrownindefinitelyaspermanentcelllines
andareimmortal.
Types of Cell Lines

23. On the basis of morphology (shape & appearance) or on their
functional characteristics. They are divided into three.
Epitheliallike-attachedtoasubstrateandappearsflattenedand
polygonalinshape
Lymphoblastlike-cellsdonotattachremaininsuspension
withasphericalshape
Fibroblastlike-cellsattachedtoansubstrateappearselongated
andbipolar
Types of Cells

24. TYPES OF TISSUE CULTURE
(1)Organcultureimpliesthatthearchitecturecharacteristicofthetissuein
vivoisretained,atleastinpart,intheculture
(2)Inprimaryexplantcultureafragmentoftissueisplacedataglass(or
plastic)–liquidinterface,where,afterattachment,migrationispromotedin
theplaneofthesolidsubstrate
(3)Cellcultureimpliesthatthetissue,oroutgrowthfromtheprimaryexplant,
isdispersed(mechanicallyorenzymatically)intoacellsuspension,which
maythenbeculturedasanadherentmonolayeronasolidsubstrateorasa
suspensionintheculturemedium

25. Advantages of Animal Cell Culture
Controlledphysiochemicalenvironment(pH,temperature,
osmoticpressure,O
2,etc.)
Controlledanddefinedphysiologicalconditions
Homogeneityofcelltypes(achievedthroughserial
passages)
Economical,sincesmallerquantitiesofreagentsare
neededthaninvivo.
Legal,moralandethicalquestionsofanimal
experimentationareavoided.

26. Disadvantages of Animal Cell Culture
Expertiseisneeded,sothatbehaviourofcellsinculture
canbeinterpretedandregulated.
Tentimesmoreexpensiveforsamequantityofanimal
tissue;therefore,reasonsforitsuseshouldbecompelling.
Unstableaneuploidchromosomeconstitution.