Introduction to animal Introduction to animal cell culturecell culture

CELL CULTUREWhy do it ?

GeneTherapy

Make proteins:commercial

scale

Stem and cancer

cells

Antibody production:

monoclonals

Embryo culture

Primary Humanand animal Cell culture

Tool for the study of animal cell biology using Tool for the study of animal cell biology using convenient convenient in vitroin vitro model of cell growthmodel of cell growth

Mimic of Mimic of in vivoin vivo cell behaviour (cell behaviour (e.ge.g cancer cells)cancer cells)

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

Highly selective and defined environment which Highly selective and defined environment which is easily manipulatedis easily manipulated (used to optimise cell (used to optimise cell signalling pathways)signalling pathways)

Cell Culture: why do it?Cell Culture: why do it?

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 culture Primary culture 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 immortalised skeletal muscle cell lineIn our lab: C2C12 immortalised skeletal muscle cell lineMyoblastsMyoblasts were extracted from the thigh muscle of C3H mice 70 h after a were extracted from the thigh muscle of C3H mice 70 h after a

crush injury and cultured. They became crush injury and cultured. They became immortalisedimmortalised. (. (YaffeYaffe and and Saxel,1977). Very useful tool to study effects of various factorSaxel,1977). Very useful tool to study effects of various factors on s on myoblastmyoblast proliferation and differentiation and proliferation and differentiation and myotubemyotube formation formation

Note: mouse cells readily Note: mouse cells readily immortaliseimmortalise whereas human cells do NOT. This whereas human cells do NOT. This is due to telomeraseis due to telomerase (discussed later under ageing)(discussed later under ageing)

Can STORE cells, Can STORE cells, cryopreservedcryopreserved in liquid nitrogen for yearsin liquid nitrogen for years

Passaging or subPassaging or sub--cultureculture

Cells dissociated from flaskusing enzymes

Split 1 into 2 flasks

Contact inhibitionContact inhibition

Therefore needto split them tomaintain growth

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 may In a mixed primary culture differences in growth rate may mean a loss of the cell type of interest mean a loss of the cell type of interest –– selection selection techniquestechniquesSome cells are prone to spontaneous transformationSome cells 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 that affect ECM)Enzymatic (proteases that affect ECM)

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

Can be a combinationCan be a combination

The cell culture The cell culture environmentenvironment

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.

Factors affecting cell behaviour in the Factors affecting cell behaviour in the complex complex in vivo in vivo environment environment

The local microThe local micro--environment: metabolites, environment: metabolites, local growth factors, ECM, architecturelocal growth factors, ECM, architecture

CellCell--cell interactionscell interactions

Circulating proteins, cytokines, hormonesCirculating proteins, cytokines, hormones

How to best mimic this in vitro?

(2) Culture Surface(2) Culture Surface

Most adherent cells require attachment to Most adherent cells require attachment to proliferateproliferateChange charge of the surfaceChange charge of the surface

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

PLUS SERUMPLUS SERUM

DMEMDMEM

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 following[Horse serum, foetal calf serum, [Horse serum, foetal calf serum,

chick embryo extract: all not fully defined]chick embryo extract: all not fully defined]

Basic nutrientsBasic nutrientsHormones and growth factorsHormones and growth factorsAttachment and spreading factorsAttachment and spreading factorsBinding proteins (albumin, Binding proteins (albumin, vitronectinvitronectin, , transferrin), hormones, vitamins, minerals, transferrin), hormones, vitamins, minerals, lipidslipidsProtease inhibitorsProtease inhibitorspH bufferpH buffer

Freshney.(1992) Animal Cell Culture.

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

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

Carbon dioxideCarbon dioxideBufferingBuffering

COCO22 IncubatorIncubator

Controlled COControlled CO22

HumidifiedHumidified3737ooCC

(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

You have the ingredientsYou have the ingredientsNow let us look at the Now let us look at the

procedures.procedures.

ContaminationContamination

Minimise 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

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 2 Biological

Safety Cabinet

HEPA filtersLaminar flow

Humans shed particles of skin, Humans shed particles of skin, bacteria, fungi etc all the timebacteria, fungi etc all the time

““Sitting or standing with Sitting or standing with no movementno movement, wearing , wearing cleanroomcleanroom garments, an individual will shed garments, an individual will shed approximately approximately 100,000 particles of 0.3um and 100,000 particles of 0.3um and larger per minutelarger per minute. The same person with only . The same person with only simple arm movement will emit 500,000 simple arm movement will emit 500,000 particles. particles. Average arm and body movements Average arm and body movements with some slight leg movement will produce over with some slight leg movement will produce over 1,000,000 particles per minute1,000,000 particles per minute; average walking ; average walking pace 7,500,000 particles per minute; and pace 7,500,000 particles per minute; and walking fast 10,000,000 particles per minutes. walking fast 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.’

Aseptic TechniquesAseptic TechniquesControlled environmentControlled environment

Traffic, air flowTraffic, air flowSterile media and reagentsSterile media and reagentsAvoid aerial contamination of solutionsAvoid aerial contamination of solutionsAvoid manual contamination of equipment Avoid manual contamination of equipment Avoid repeated opening of bottlesAvoid repeated opening of bottles70% ethanol swab70% ethanol swabUV irradiation before and afterUV irradiation before and afterOnly use disposable equipment onceOnly use disposable equipment once

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

Aseptic Technique

“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.

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 0.1 µ

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)

In the LAB you will be studying In the LAB you will be studying MyogenesisMyogenesis: : skeletalskeletal muscle muscle formationformation

MyoblastProliferation Differentiation Fusion

Myotubematuration

IN VIVOMuscle regeneration

- likeness to formation of myotubes in culture.

Haematoxylin and Eosin stained paraffin embedded section of mouse muscle 12 days after injury

IN VITROMyotubes formed by myoblasts

grown in culture for 7 days. Culture viewed on an inverted phase microscope. (phase microscopy)

Inverted MicroscopeInverted Microscope

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 Mouse Skeletal Muscle Cultured Cell Line

Cell CountingCell Counting -- haemocytometerhaemocytometer

Media FormulationMedia FormulationProliferation/maintenanceProliferation/maintenance

Hams F10 nutrient mix (for primary cell culture)Hams F10 nutrient mix (for primary cell culture)20% FCS (foetal calf serum)20% FCS (foetal calf serum)5ng/ml 5ng/ml bFGFbFGF

Differentiation and fusionDifferentiation and fusionDMEMDMEM2%2% horse serum (horse serum (Note: change in serum typeNote: change in serum type))InsulinInsulinLinoleicLinoleic aciudaciud

FusionFusion

Media 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 (become quiescent)Senesce (become quiescent)Differentiate Differentiate –– leading to fusion and myotubesleading to fusion and myotubes

AnalysisAnalysis

Adult Mouse Skeletal Muscle - Primary cultureNote: this also contains fibroblasts.

cultured (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

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

Bjanka Gebski