Introduction to animal cell culture - University of … Culture is a Fussy Discipline In the tissue culture laboratory: • bench tops should be kept clear and clean • wearing a

Introduction to animal Introduction to animal cell culturecell culture

CELL CULTUREWhy do it ?

GeneTherapy

Research proteinsof interest

Stemcells

Antibody production

Embryo culture

Primary HumanCell culture

Cell Culture: why do it?Cell Culture: why do it?Tool for the study of animal cell biology Tool for the study of animal cell biology in vitroin vitro model of model of cell growthcell growth

Mimic of Mimic of in vivoin vivo cell behaviourcell behaviour

Model for Model for optimising treatmentsoptimising treatments (for further (for further in vivoin vivostudies)studies)

Artificial (some cell types are thus difficult to culture)Artificial (some cell types are thus difficult to culture)

Highly selective environment which is easily manipulatedHighly selective environment which is easily manipulated(used to optimise cell signalling pathways)(used to optimise cell signalling pathways)

Cell Culture is a Cell Culture is a FussyFussy DisciplineDisciplineIn the tissue culture laboratory:

• bench tops should be kept clear and clean

• wearing a long sleeve lab coat: minimises contamination from street clothing (hair, etc)

• wearing gloves while doing tissue culture work: minimisescontamination from skin organisms

• Surfaces, gloves, solutions and plasticware sprayed with 70% alcohol before placed into the biological hood

• solutions, reagents and glassware used in tissue culturework should NOT be shared with non-tissue culture work

Primary application of animal cell Primary application of animal cell culture in the investigation of:culture in the investigation of:Mechanisms of cell cycle controlMechanisms of cell cycle control

Characteristics of cancer cellsCharacteristics of cancer cells

Detection, production and function of :Detection, production and function of :growth factors growth factors hormones hormones virusesviruses

The study of:The study of:differentiation processesdifferentiation processesspecialised cell functionspecialised cell functioncellcell--cell and cellcell and cell--matrix interactionsmatrix interactions

Primary Primary vsvs Cell lineCell linePrimary culturePrimary culture

freshly isolated from tissue source freshly isolated from tissue source

Cell lineCell lineFinite cell line: dies after several subFinite cell line: dies after several sub--culturesculturesContinuous cell line: transformed ‘immortal’Continuous cell line: transformed ‘immortal’

In our lab: C2C12 cell lineIn our lab: C2C12 cell lineYaffeYaffe and and SaxelSaxel (1977) (1977)

MyoblastsMyoblasts cultured from the thigh muscle of C3H mice 70 h after a crush cultured from the thigh muscle of C3H mice 70 h after a crush injuryinjury

Cells were shown to be capable of differentiationCells were shown to be capable of differentiationModel to studyModel to study factors controlling differentiation, fusion and maturationfactors controlling differentiation, fusion and maturation

into skeletal muscle cellsinto skeletal muscle cells

Passaging or subPassaging or sub--cultureculture

Cell dissociated from flask

Split 1 in 2

Contact inhibitionContact inhibition

Initiation, establishment Initiation, establishment and propagation of cell and propagation of cell

culturescultures

Cultures can be initiated fromCultures can be initiated fromtissue or organ fragmentstissue or organ fragmentssingle cell suspensionssingle cell suspensions

Choices to be madeChoices to be madeDisaggregation techniquesDisaggregation techniquesMediaMediaCulture conditionsCulture conditionsSelection proceduresSelection procedures

ConsiderationsConsiderationsSensitivity to mechanical dispersal or enzymes; cellSensitivity to mechanical dispersal or enzymes; cell--cell cell contact may be required for proliferationcontact may be required for proliferationDispersed cells in culture are vulnerableDispersed cells in culture are vulnerableMost primary cells require satisfactory adherenceMost primary cells require satisfactory adherenceSome cells are not normally adherent in vivo and can be Some cells are not normally adherent in vivo and can be grown in liquid suspensiongrown in liquid suspensionIn a mixed primary culture, differences in growth rate In a mixed primary culture, differences in growth rate may mean a loss of the cell type of interest may mean a loss of the cell type of interest –– selection selection techniques (techniques (e.ge.g fibroblasts fibroblasts vsvs myoblasts)myoblasts)Some cell are prone to spontaneous transformationSome cell are prone to spontaneous transformationLimited life span of some culturesLimited life span of some cultures

(1) Dispersal of tissues(1) Dispersal of tissues

MechanicalMechanicalMincing, shearing, sievesMincing, shearing, sieves

ChemicalChemicalEnzymatic (proteases)Enzymatic (proteases)

Trypsin, Trypsin, pronasepronase, collagenase, , collagenase, dispasedispase

Can be a combinationCan be a combination

The cell culture The cell culture environmentenvironment

Factors affecting cell behaviour Factors affecting cell behaviour in in vivovivo

The local microThe local micro--environmentenvironmentCellCell--cell interactionscell interactionsTissue architectureTissue architectureTissue matrixTissue matrixTissue metabolitesTissue metabolitesLocally released growth factor and Locally released growth factor and hormoneshormones

(2) Culture Surface(2) Culture Surface

Most adherent cells require attachment to Most adherent cells require attachment to proliferateproliferateChange charge of the surface: different Change charge of the surface: different kinds of plastic (originally used glass)kinds of plastic (originally used glass)

PolyPoly--LL--lysinelysineCoating with matrix proteinsCoating with matrix proteins

Collagen, Collagen, lamininlaminin, , gelatingelatin, fibronectin, fibronectin

(3) Media formulation(3) Media formulation

Initial studies used body fluidsInitial studies used body fluidsPlasma, lymph, serum, tissue extractsPlasma, lymph, serum, tissue extracts

Early basal mediaEarly basal mediaSalts, amino acids, sugars, vitamins Salts, amino acids, sugars, vitamins supplemented with serumsupplemented with serum

More defined mediaMore defined mediaCell specific extremely complexCell specific extremely complex

DMEMDMEM(Dulbecco’s Modified Essential Medium)(Dulbecco’s Modified Essential Medium)

Media FormulationMedia FormulationInorganic ionsInorganic ions

Osmotic balance Osmotic balance –– cell volumecell volumeTrace ElementsTrace Elements

CoCo--factors for biochemical pathways (Zn, Cu)factors for biochemical pathways (Zn, Cu)Amino AcidsAmino Acids

Protein synthesisProtein synthesisGlutamine required at high concentrationsGlutamine required at high concentrations

VitaminsVitaminsMetabolic coMetabolic co--enzymes for cell replicationenzymes for cell replication

Energy sourcesEnergy sourcesglucoseglucose

Serum provides the followingSerum provides the followingBasic nutrientsBasic nutrientsHormones and growth factorsHormones and growth factorsAttachment and spreading factorsAttachment and spreading factorsBinding proteins (albumin, transferring) carrying Binding proteins (albumin, transferring) carrying hormones, vitamins, minerals, lipidshormones, vitamins, minerals, lipidsProtease inhibitorsProtease inhibitorspH bufferpH buffer

Use rich foetal calf serum (FCS) or chick embryo extract Use rich foetal calf serum (FCS) or chick embryo extract (CEE) for best growth/proliferation of cells. (CEE) for best growth/proliferation of cells.

Use serum from adult (nonUse serum from adult (non--growing) animals, growing) animals, e.ge.g horse horse serum (HS), for cell maintenance.serum (HS), for cell maintenance.

Fully defined, serumFully defined, serum––free media are available: expensive free media are available: expensive but more reproducible. May be essential for clinical but more reproducible. May be essential for clinical transplantation of cells into humans (to avoid issues with transplantation of cells into humans (to avoid issues with animal products)animal products)

Freshney.(1992) Animal Cell Culture.

(4) The gas phase(4) The gas phase

OxygenOxygenAerobic metabolismAerobic metabolismAtmospheric 20%Atmospheric 20%Tissue levels between 1Tissue levels between 1--7%7%

Carbon dioxide (4%)Carbon dioxide (4%)BufferingBuffering

(5) pH Control(5) pH Control

Physiological pH 7Physiological pH 7pH can affectpH can affect

Cell metabolismCell metabolismGrowth rateGrowth rateProtein synthesisProtein synthesisAvailability of nutrientsAvailability of nutrients

COCO22 acts as a buffering agent in acts as a buffering agent in combination with sodium bicarbonate in combination with sodium bicarbonate in the mediathe media

(6) Temperature and Humidity(6) Temperature and Humidity

Normal body temperature 37Normal body temperature 37ooC C

Humidity must be maintained at saturating Humidity must be maintained at saturating levels as evaporation can lead to changes levels as evaporation can lead to changes inin

OsmolarityOsmolarityVolume of media and additivesVolume of media and additives

C2C12 Mouse Skeletal Muscle Cultured Cell Line

ProliferationScattered myoblasts 24h after

subculture.High Serum Media(20% FCS DMEM)

Differentiation and fusionMyotubes formed at 7 days in

fusion medium(2% HS DMEM)

C2C12 skeletal muscle culture stained with desmin (green) – to identify myotubes and Hoescht (blue) to identify cell nuclei

ContaminationContaminationMinimise the riskMinimise the risk

Sources of ContaminationSources of ContaminationBacteriaBacteriaFungiFungiMouldMouldYeastYeastMycoplasmaMycoplasmaOther cell typesOther cell types

Free organisms, dust particles or aerosolsFree organisms, dust particles or aerosolsSurfaces or equipmentSurfaces or equipment

Class 1 Cabinets:Preparation of primary cultures(removing muscle from mice)

protect the product only

Laminar Flow HoodLaminar Flow Hood

Class 2 Cabinets:Protection of personnel, environment and product

Laminar Flow Hood

Vertical Laminar Airflow

Air Barrier

Exhaust Fan

Exhaust HEPA Filter

Laminar Flow Fan

Laminar HEPA Filter

Class II Biological

Safety Cabinet

HEPA filtersLaminar flowNATA certified

““Sitting or standing with no movement, wearing Sitting or standing with no movement, wearing cleanroomcleanroom garments, an individual will shed garments, an individual will shed approximately 100,000 particles of 0.3um and approximately 100,000 particles of 0.3um and larger per minute. larger per minute.

The same person with only simple arm movement The same person with only simple arm movement will emit 500,000 particles. will emit 500,000 particles.

Average arm and body movements with some Average arm and body movements with some slight leg movement will produce over 1,000,000 slight leg movement will produce over 1,000,000 particles per minute; average walking pace particles per minute; average walking pace 7,500,000 particles per minute; and walking fast 7,500,000 particles per minute; and walking fast 10,000,000 particles per minutes. 10,000,000 particles per minutes.

Boisterous activity can result in the release of as Boisterous activity can result in the release of as many as 15x10many as 15x1066 to 30x10to 30x1066 particles per minute particles per minute into the into the cleanroomcleanroom environment.’environment.’

Establishing a Primary Establishing a Primary CultureCulture

Skeletal MuscleSkeletal Muscle

First have a5-10 minute break and stretch

Aseptic Technique 1Aseptic Technique 1

Controlled environmentControlled environmentTraffic, air flowTraffic, air flow

Sterile media and reagentsSterile media and reagentsAvoids aerial contamination of solutionsAvoids aerial contamination of solutionsAvoids manual contamination of Avoids manual contamination of equipmentequipment

Aseptic Technique 2Aseptic Technique 2

Minimise trafficMinimise trafficClear work areaClear work area70% ethanol swab70% ethanol swabMinimise work area (field of vision)Minimise work area (field of vision)Keep work area cleanKeep work area cleanDo not lean over open vesselsDo not lean over open vesselsUV irradiation before and afterUV irradiation before and afterOnly use disposable equipment onceOnly use disposable equipment once

Aseptic Technique 3Aseptic Technique 3Minimise exposure to airMinimise exposure to airFlame bottles if on open benchFlame bottles if on open benchAvoid repeated opening of bottlesAvoid repeated opening of bottlesAvoid liquid accumulation around necks and lips Avoid liquid accumulation around necks and lips of bottlesof bottlesAvoid excessive agitationAvoid excessive agitationOnly one cell type at a timeOnly one cell type at a timeDo not open contaminated solutionsDo not open contaminated solutionsNo burner in hoodNo burner in hood

Establishing a Primary Establishing a Primary CultureCulture

Skeletal MuscleSkeletal Muscle

Muscle regeneration in vivo- likeness to formation of myotubes in culture.

Haematoxylin and Eosin stained paraffin embedded section of mouse muscle 12 days after injury(viewed by bright field microscopy)

Myotubes formed by myoblastsgrown in culture for 7 days.

Culture viewed on an inverted phase microscope. (phase microscopy)

ConsiderationsConsiderationsHighly structured tissueHighly structured tissue

disaggregationdisaggregationHeterogeneousHeterogeneous

purificationpurificationMultiMulti--cellular versus single cellcellular versus single cell

mediamediaContaminationContamination

Sterile techniqueSterile techniqueProliferation versus differentiationProliferation versus differentiation

MediaMediasubstratesubstrate

CollectionCollectionSterilitySterility

Wash mouse skin in 70% ethanolWash mouse skin in 70% ethanolGentamicinGentamicinPenicillin/StreptomycinPenicillin/Streptomycin((fungizonefungizone))

SpeedSpeed45 minutes45 minutes

Tissue collected/cleanTissue collected/cleanRemoval of tendon, fat, nerveRemoval of tendon, fat, nerve

Primary Cell Culture Isolation

From a Solid Tissue Solid Tissue - need to separatecells of interest from connective tissue and extra cellular matrix• explant culture• mechanical and enzymic dissociation• washed in medium with serum• filtered and plated in final medium

•ALL this done in a Class 1 lamina flow hood

From a cell suspension tissue cell suspension tissue - blood or body fluid (ascites)• isolation by centrifugation (differential)• grown in large volume of tissue culture medium

DisaggregationDisaggregation

Mechanical mincingMechanical mincingscissorsscissors

CollagenaseCollagenaseDispaseDispase

TrypsinTrypsin

WashingWashing

Laminar Flow HoodLaminar Flow Hood

FilteringFiltering

100 micron100 micronRemoval of undigested materialRemoval of undigested materialLets through single cellsLets through single cells

Can count cells using haemocytometer Can count cells using haemocytometer to plate at a particular cell densityto plate at a particular cell density

Clarification

Microfiltration

Ultrafiltration

Reverse Osmosis

Micron = 10 -6 m

80

40

20

10

5

.8

.4

.2

.1

.05

2

.008

.004

.002

.001

.02

.0003

Human Hair DiameterSmallest Visible Particle

Erythrocyte

BacteriaMycoplasma

Polio Virus

S I Z E

0.8µ pre-filter0.22µ end filter 0.1µ

Tissue culture medium cannot be autoclaved. It is filtered through 0.2µ membrane filters.

There are different filter membrane types for sterilizing gases, solvents and aqueous solutions.

NOW many items are purchased sterile (expensive)

• Lab coat• Gloves• tip does not touch the tube• holding of the tube

Aseptic Technique

8 well culture dish. Allows comparison of 8 samples:can have different stains or are fixed at different times.THEN- remove wells and gasket. Leaves ONE slide with 8 separate samples for easy microscopic analysis of (stained) cells

96 well plateAllows comparison of many culture conditions.Samples often in triplicate.

Pipettes (glass or disposable)are plugged to minimise aerosol contamination, whensolutions are expelled from them.

“Pipette-Aid”- power or battery operated

Motorised intake and expellingof fluids transferred from one sterile container to another.

In line air filter.

“Transfer Pipette”Disposable Enclosed Plastic

Packaged as Sterile so their contained air is sterile.

Cell CountingCell Counting -- haemocytometerhaemocytometer

Filtering and PreFiltering and Pre--plateplate

100 micron100 micronRemoval of undigested materialRemoval of undigested materialLets through single cellsLets through single cellsCan count cells to plate at a particular densityCan count cells to plate at a particular density

ORPrePre--plateplate

60 mins60 minsDifferential attachment to culture plasticDifferential attachment to culture plastic

PrePre--platingplating –– to remove other cells, to remove other cells, e.ge.g fibroblastsfibroblasts

TimeSkeletal MuscleFrom Mouse 0 60min etc

Supernatant Transferred from flask to new flask after given time

Putative Muscle DerivedStem Cell (MDSC)

Media FormulationMedia FormulationGROWTH MEDIUMGROWTH MEDIUM

Proliferation/maintenanceProliferation/maintenanceHams F10 nutrient mixHams F10 nutrient mix20% FCS (foetal calf serum)20% FCS (foetal calf serum)5ng/ml 5ng/ml bFGFbFGF

FUSION MEDIUMFUSION MEDIUMDifferentiation and fusionDifferentiation and fusion

DMEMDMEM2% horse serum (Note: change in serum type)2% horse serum (Note: change in serum type)InsulinInsulinLinoleicLinoleic aciudaciud

MyogenesisMyogenesisGrowth mediumreplaced by Fusion medium at Time 0

FusionFusionMedia changed from nutrient rich to Media changed from nutrient rich to nutrient poornutrient poor

Induces withdrawal from the cell cycle giving Induces withdrawal from the cell cycle giving the cells 3 choicesthe cells 3 choices

Die (apoptosis)Die (apoptosis)Senesce (age/stop)Senesce (age/stop)DifferentiateDifferentiate

[Note: in vivo differentiation may result from contact [Note: in vivo differentiation may result from contact inhibition rather than decreased GF]inhibition rather than decreased GF]

COCO22 IncubatorIncubator

Controlled COControlled CO22

HumidifiedHumidified3737ooCC

Inverted MicroscopeInverted Microscope

Mouse Skeletal Muscle Cell Line C2C12

ProliferationScattered myoblasts 24h after

subculture.High Serum Media(20% FCS DMEM)

Differentiation and fusionMyotubes formed at 7 days in

fusion medium(2% HS DMEM)

AnalysisAnalysis

Primary muscle culture stained with desmin (green) – to identify myoblasts and Hoescht (blue) to identify cell nuclei

Adult Mouse Skeletal Muscle - Primary culturecultured (on fibronectin) in 8 well slide,

fixed and stained for desmin

Mouse Skeletal Muscle Cell line (H-2Kb)cultured (on poly-D-lysine) in 35mm dish,

fixed and stained for desmin

Housekeeping/MaintenanceHousekeeping/Maintenance

CONTUNE TO GROW / MAINTAIN CELLSCONTUNE TO GROW / MAINTAIN CELLSProliferation mediaProliferation mediaTrypsinisationTrypsinisation and splitting/passaging of culturesand splitting/passaging of cultures

Contact inhibitionContact inhibition

1% 1% gelatingelatin coated dishescoated dishes

STORAGESTORAGECryopreservation in liquid nitrogenCryopreservation in liquid nitrogen

10% FCS and 10% DMSO10% FCS and 10% DMSO