10. Scaling up of cell culture

Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding

1. Animal cell science and Technology
10. Scaling of cell culture
ShailendraSingh Shera, Ph.D

2. LT.10. Scaling up of cell culture
Outline
1.Scaling up definition
2.Methods of scaling up
a.Scale-up in suspension
b.Scale-up in monolayer
c.Immobilized cell cultures
d.Microcarrierculture
3.Test your understanding

3. Definition: Scaling up
•Scalingupreferstoincreaseinsizeofcellculture
•Scale-upinvolvesthedevelopmentofculturesystemsinstagesfrom(smallscale)laboratoryto
(largescale)industry
•Themethodologyadoptedtoincreasethescaleofaculturedependsontheproliferationofcells
MethodsofScalingUpofcellculture
a.Scale-up in suspension
b.Scale-up in monolayer
c.Immobilized cell cultures
d.Microcarrierculture

4. Scale-upinsuspensionisthepreferredmethodasitissimpler.Scale-upofsuspensionculture
primarilyinvolvesanincreaseinthevolumeoftheculture.Smallscalegenerallymeanstheculture
capacitylessthan2litresvolume(orsometimes5litres).
Stirredsuspensioncultures:
Itisusuallynecessarytomaintaincellstrainsinstirredsuspensioncultures,byagitation(orstirring)
ofthemedium.Thestirringoftheculturemediumisachievedbyamagnetencasedinaglass
pendulumorbyalargesurfaceareapaddle.Thestirringisusuallydoneataspeedof30-100rpm.
Thisissufficienttopreventsedimentationofcellswithoutcreatingshearforcesthatwoulddamage
cells.
Static suspension cultures:
Some cells can grow in suspension cultures, without stirring or agitation of the medium, and form
monolayer cells. However, static suspension cultures are unsuitable for scale-up.
Scale up in suspension culture

5. •Scaling up in suspension culture involves following elements:
(i)An increase in the volume of the culture medium
(ii) Agitation is done when the depth exceeds 5mm and above 5-10 cm.
(iii) Spargingwith CO2 is done to maintain adequate gas exchange.
(iv) Stirring should be between 30-100rpm as:-
(v) Stirring at higher speed creates shear forces that would damage the cells.
(vi) It should be sufficient enough to prevent cell sedimentation.
(vii) Antifoam or PluronicF68 is added at conc. 0.01-0.1% when serum conc. is 2%.
(viii) Carboxymethylcellulose(1-2%) is added to inc. the viscosity of the medium.
Reactors used for large scale suspension culture
•Stirred Bioreactors
•Continuous flow reactors
•Air lift fermentors
Stirred Tank Bioreactors: These are glass (smaller vessels) or stainless steel (larger volumes)
vessels. These are closed systems with fixed volumes and are usually agitated with motor-
driven stirrers with considerable variations in design details

6. Continuous-Flow Cultures
•Themediumentersfrominletsideandthecellsalongwithproductexitfromoutletside.
•Continuouscycleismaintained.
•Itcanoperateatchemostat(constantchemicalconstituentinreactor)orturbidostatmode
(constantcelldensity)inreactor.
AirliftFermentersCulturesinsuchvesselsarebothaeratedandagitatedbyair(5%CO2inair)
bubblesintroducedatthebottomofvessels.Thevesselhasaninnerdrafttubethroughwhichthe
airbubblesandtheaeratedmediumrise.

7. MonolayerCultureThistypeofcultureisessentialforanchoragedependentcells.Scalingupof
suchculturesisbasedonincreasingthesurfaceareaofthesubstrateinproportiontothenumber
ofcellsandthevolumeofmediumandthereforetendstobemorecomplexthansuspension
cultures.Theavailablesurfaceareacanbeincreasedbyusingplates,spirals,ceramicsandmicro
carriers.
Followingstrategiesareusedforscalingupofcellcultureasmonolayer
a.Roux bottle
b.Plastic film
c.Roller bottle
d.Helicellvessels
e.Multitrayunit
f.Bead bed reactor
g.Synthetic hollow fibrecartridge
h.Heterogenousreactors
i.Opticellculture system

8. RouxBottleItiscommonlyusedinlaboratoryandiskeptstationarysothatonlyaportionofits
internalsurfaceisavailableforcellanchorage.EachbottleprovidesCa.175-200cm2surfacearea
forcellattachmentandoccupies750-1000cm3space.
RollerBottleThisvesselpermitsalimitedscaleupasitisrockedorpreferablyrolledsothatits
entireinternalsurfaceisavailableforanchorage.Severalmodificationsofrollerbottlefurther
enhancetheavailablesurface,
Roux bottleRoller bottle
https://www.sciencedirect.com/topics/engineering/roller-bottle
https://www.thomassci.com/Laboratory-Supplies/Culture-Bottles/_/ROUX-CULTURE-BOTTLE

9. MultitrayUnit
Astandardunithas10chambersstackedoneachother,whichhaveinterconnectingchannels;this
enablesthevariousoperationstobecarriedoutinonegoforallthechambers.
Eachchamberhasasurfaceareaof600cm
2
.
Thispolystyreneunitisdisposableandgivesgoodresultssimilartoplasticflasks.
NuNc Cell factories

10. Byimmobilizingthecellincultures,therestabilityandspecificityincreases.Twobasicmechanisms
usedforimmobilizationofanimalcellsare;ImmurementculturemethodsandEntrapmentculture
methods
Immurementculture–Inthistypeofculture,cellsareencapsulatedinapolymericmatrixby
adsorption.Matrixusedforimmobilizationaregelatin,polylysine,alginateandagarose.
Entrapmentculture–Inthistypeofculture,thecellsareheldwithinanopenmatrixthroughwhich
themediumflowsfreely.Thecellsmaybeentrappedwithintheporousceramicwallsoftheunitor
cellsalsocanbeenmeshedincellulosefibers.
Scale up as Immobilized culture

11. Monolayerscanbegrownonsmallsphericalcarriersormicro-beads(80-300pmdiameter)
referredtoasmicro-carriers.Thesesystemsuse90-300pmdiaparticlesassubstrateforcell
attachment.Initially,Dextranbeads(SephadexA-50)wereusedbyVanWezelin1967;these
werenotentirelysatisfactoryduetotheunsuitablechargeofbeadsandpossiblyduetotoxic
effects.Themicro-carriersaremadeupofanyonethefollowingmaterials(tradenamesgivenin
brackets).
i.Plastic(acrobeads,bioplas).
ii.Glass(bioglass,ventreglas).
iii.Gelatin(ventregel,cytodex-3).
iv.Collagen(biospex,biospheres)
v.Cellulose(DE-52/53).
vi.DEAEDextran(cytodexI,dormacell).
Scale up as microcarrier culture

12. *Practice questions: MCQ
1. For adherent culture scale up can be done as
a.Monolayer
b.Suspension
c.Immobilized
d.None of the above
2. For transformed cells can be scaled up as
a.Monolayer
b.Suspension
c.Immobilized
d.None of the above
3. What do you understand by microcarrierculture scale up
a.Scaling up in suspension
b.Scaling up as monomalyer
c.Scale up using Dextranor glass bead
d.None of the above
4. NUNC cell factories is used for scale up of
a.Adherent cells
b.Non-adherent cells
c.Transformed cells
d.Lymphoblast cells
*Questions adapted from: Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: ShailendraSingh Shera. Publisher: Amazon Kindle.

13. Suggested reading
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
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