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
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
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
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
(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
(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
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
“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)
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
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
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