Introduction to animal Introduction to animal cell culturecell culture

CELL CULTURE

• Why do it ? - Research- Research- Diagnosis- Diagnosis

Tool for the study of animal cell biology In Tool for the study of animal cell biology In vitro model of cell growthvitro model of cell growth

Mimic of Mimic of in vivoin vivo cell behaviour cell behaviour

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

Highly selective environment which is Highly selective environment which is easily manipulatedeasily manipulated

Cell Culture is a Cell Culture is a FussyFussy Discipline Discipline

In the area of the laboratory assigned for tissue culture:

• benchtops should be kept clear and clean and not used for other work

• wearing a long sleeve gown when working in the tissue culture area minimises contamination from street clothing (hair, fur etc)

• wearing gloves while doing tissue culture work also minimises contamination from skin organisms

• solutions, reagents and glassware used in tissue culture work 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:The mechanisms of cell cycle controlThe mechanisms of cell cycle controlThe production of cells for biochemical analysisThe production of cells for biochemical analysisThe characteristics of cancer cellsThe characteristics of cancer cellsThe detection of stem cellsThe detection of stem cellsThe detection, production and function of growth The detection, production and function of growth factors and hormonesfactors and hormonesThe detection and production of virusesThe detection and production of virusesThe study of differentiation processesThe study of differentiation processesThe study of specialised cell functionThe study of specialised cell functionThe study of cell-cell and cell-matrix interactionsThe study of cell-cell and cell-matrix interactions

Primary vs Cell linePrimary vs Cell line

Primary culture – freshly isolated from Primary culture – freshly isolated from tissue sourcetissue source

Cell line – culture that has been passagedCell line – culture that has been passaged Finite cell line: dies after several sub-culturesFinite cell line: dies after several sub-cultures Continuous cell line: transformed ‘immortal’Continuous cell line: transformed ‘immortal’

Passaging or sub-culturePassaging or sub-culture

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 from tissue or organ fragmentstissue or organ fragments single cell suspensionssingle cell suspensions

Choices to be madeChoices to be made Disaggregation techniquesDisaggregation techniques MediaMedia Culture conditionsCulture conditions Selection proceduresSelection procedures

ConsiderationsConsiderations

Sensitivity to mechanical dispersal or enzymes; cell-cell Sensitivity to mechanical dispersal or enzymes; 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 – selection mean a loss of the cell type of interest – selection techniquestechniquesSome 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

MechanicalMechanical Mincing, shearing, sievesMincing, shearing, sieves

ChemicalChemical

Enzymatic (proteases)Enzymatic (proteases) Trypsin, pronase, collagenase, dispaseTrypsin, pronase, collagenase, dispase

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 micro-environmentThe local micro-environment

Cell-cell interactionsCell-cell interactions

Tissue architectureTissue architecture

Tissue matrixTissue matrix

Tissue metabolitesTissue metabolites

Locally 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 proliferateproliferate

Change charge of the surfaceChange charge of the surface Poly-L-lysinePoly-L-lysine

Coating with matrix proteinsCoating with matrix proteins Collagen, laminin, gelatin, fibronectinCollagen, laminin, gelatin, fibronectin

(3) Media formulation(3) Media formulation

Initial studies used body fluidsInitial studies used body fluids Plasma, lymph, serum, tissue extractsPlasma, lymph, serum, tissue extracts

Early basal mediaEarly basal media Salts, amino acids, sugars, vitamins Salts, amino acids, sugars, vitamins

supplemented with serumsupplemented with serum

More defined mediaMore defined media Cell specific extremely complexCell specific extremely complex

DMEMDMEM

Media FormulationMedia Formulation

Inorganic ionsInorganic ions Osmotic balance – cell volumeOsmotic balance – cell volume

Trace ElementsTrace Elements Co-factors for biochemical pathways (Zn, Cu)Co-factors for biochemical pathways (Zn, Cu)

Amino AcidsAmino Acids Protein synthesisProtein synthesis Glutamine required at high concentrationsGlutamine required at high concentrations

VitaminsVitamins Metabolic co-enzymes for cell replicationMetabolic co-enzymes for cell replication

Energy sourcesEnergy sources glucoseglucose

Serum provides the followingSerum provides the following

Basic nutrientsBasic nutrients

Hormones and growth factorsHormones and growth factors

Attachment and spreading factorsAttachment and spreading factors

Binding proteins (albumin, transferring) Binding proteins (albumin, transferring) carrying hormones, vitamins, minerals, carrying hormones, vitamins, minerals, lipidslipids

Protease inhibitorsProtease inhibitors

pH bufferpH buffer

Freshney.(1992) Animal Cell Culture.

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

OxygenOxygen Aerobic metabolismAerobic metabolism Atmospheric 20%Atmospheric 20% Tissue levels between 1-7%Tissue levels between 1-7%

Carbon dioxideCarbon dioxide BufferingBuffering

(5) pH Control(5) pH Control

Physiological pH 7Physiological pH 7pH can affectpH can affect Cell metabolismCell metabolism Growth rateGrowth rate Protein synthesisProtein synthesis Availability 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 OsmolarityOsmolarity Volume of media and additivesVolume of media and additives

Mouse Myogenic Cells (H-2Kb) - grown on fibronectinin 8 well slide in culture, fixed and stained for desmin

ContaminationContamination

Minimise the riskMinimise the risk

Sources of ContaminationSources of Contamination

BacteriaBacteriaFungiFungiMouldMouldYeastYeastMycoplasmaMycoplasmaOther cell typesOther cell types

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

Class II Biological

Safety Cabinet

Protection of • personnel• environment• product

Class 1 Cabinets protect the

product only

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 cleanroom garments, an individual will shed cleanroom garments, an individual will shed approximately 100,000 particles of 0.3um and approximately 100,000 particles of 0.3um and larger per minute. The same person with only larger per minute. The same person with only simple arm movement will emit 500,000 simple arm movement will emit 500,000 particles. Average arm and body movements particles. 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 minute; average walking 1,000,000 particles per minute; 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 30x10 to 30x1066 particles per minute particles per minute into the cleanroom environment.’into the cleanroom environment.’

Aseptic Technique 1Aseptic Technique 1

Controlled environmentControlled environment Traffic, air flowTraffic, air flow

Sterile media and reagentsSterile media and reagents

Avoids aerial contamination of solutionsAvoids aerial contamination of solutions

Avoids 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 3

Minimise 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