Cell Culture Cell Culture TechniquesTechniques

Balazs VeresBalazs Veres

ContentContentss

II. Cell. Cell Types Types IIII. Introduction . Introduction toto Cell Cell

Culture LabCulture Lab IIIIII. Techniques. Techniques

I. I. CellCell Types Types

PrimPrimaarryy cultures cultures ContinuousContinuous cultures cultures

NormalNormal ImmortalizedImmortalized

SpontaneousSpontaneous TransformationTransformation

TransfectTransfectionion Somatic Cell FusionSomatic Cell Fusion

(Hybridomas, (Hybridomas, Hybrids)Hybrids)

Cell linesCell lines Adherent Adherent SuspensionSuspension

Cells from ATCC Cells from ATCC and ETCC and ETCC

PrimPrimaarryy CCultureulture Tissue preparationTissue preparation

directly directly from young from young animal, or isolation of animal, or isolation of cells from blood, cells from blood, intraperitoneal intraperitoneal fluid, fluid, etc.etc.

Tissue dissociation Tissue dissociation Dissection thenDissection then

HomogenizHomogenizationation with with Knife Knife or or BlenderBlender

Enzymatic Enzymatic DigestionDigestion ((collagenase, papain, collagenase, papain, trypsine)/cleaving of DNA trypsine)/cleaving of DNA of damaged cell with of damaged cell with DNaseDNase

DissociationDissociation of cells in of cells in medium and selection of medium and selection of organicorganic cell typescell types

Knife Blender

CO2 Incubator

Secondary culture, Secondary culture, extended cultureextended culture

Secondary culture:Secondary culture: From primary cultures, after 1 From primary cultures, after 1 passage.passage.

Extended culture (multipassage culture, cell Extended culture (multipassage culture, cell strain):strain): From primary cultures after several passages. From primary cultures after several passages.

Restricted lifespan (40-100 passages), l Restricted lifespan (40-100 passages), limited imited growth potential. growth potential.

ContinuousContinuous culture culture

Normal cell linesNormal cell lines They were They were

spontaneousspontaneouslyly immortalized.(e.g.: immortalized.(e.g.: Cardio-mCardio-myyocytocyteses from from rat)rat)

ImmortalizedImmortalized TransfectedTransfected with some with some

sort of oncogene; SV40 sort of oncogene; SV40 (Simian virus) Large T (Simian virus) Large T antigen antigen (T IDBL)(T IDBL)

Tumor cells (e.g.: Tumor cells (e.g.: Human cervix Human cervix carcinomas: HeLacarcinomas: HeLa 1952 1952) )

Hybridomas Hybridomas

H9c2

HeLa

Cell line: genetically homogeneous, everlasting lifespan

Growth-curve of cultured cells

HybridomasHybridomas

Cell fusion of Cell fusion of

HGPRTHGPRT and TK and TK-/--/- myeloma and myeloma and B-B-cells from cells from immunized animal immunized animal

Selection of Selection of hybridomas in hybridomas in HATHAT ((HHypoxanthine, ypoxanthine, AAminopterine and minopterine and TThymidine)hymidine) medium medium

Metabolic pathways relevant to hybrid selection in medium containing hypoxanthine, aminopterine and thymidine (HAT medium).

When the main synthetic pathways are blocked with the folic acid analogue aminopterine (*), the cell must depend on the “salvage” enzymes HGPRT and TK (thymidine kinase). HGPRT (-) cells cannot grow in HAT medium unless they are fused with HGPRT (+) cells.

Hybrid selectionHybrid selection

Effect of HAT-medium Effect of HAT-medium SelectionSelection

Ribose-5-P PRPP IMP

1) adeniloszukcinát xantilát (XMP)

adenilát (AMP) guanilát (GMP)

ADP GDP

ATP GTP

3) PRPP

adenin hipoxantin

(uracil) guanin

adenin-(uracil)

foszforibozil- HGPRT

transzferáz

AMP IMP

(UMP) PPi GMP

2)

HCO3- karbamil-P + Asp

karbamil-aszpartát

uridilát (UMP) UDP UTP

CTP

4) kinázok

uridin UMP

citidin CMP

adenozin AMP

guanozin GMP

timidin TMP

ATP ADP

Purine and pirimidine nucleotide biosynthesis

Production of Polyclonal Production of Polyclonal and Monoclonal antibodiesand Monoclonal antibodies

Cell linesCell lines

Adherent (WRL-68, Adherent (WRL-68, HepG2, HeLa etc.)HepG2, HeLa etc.)

Suspension Suspension (Jurkat)(Jurkat)

Cells from ATCC Cells from ATCC and ETCCand ETCC

JurkatWRL-68

HeLa HepG2

Online Order of Cell Online Order of Cell LinesLines

MediumMedium: : salt, glucose, essencial amino acids, vitamines, salt, glucose, essencial amino acids, vitamines,

buffer, phenol red indicator, serum, antibioticsbuffer, phenol red indicator, serum, antibiotics

EnvironmentEnvironment:: TemperatureTemperature: 37°C: 37°C High humidity High humidity 5% CO25% CO2

Cells in culture

II. Introduction of Cell II. Introduction of Cell Culture LabCulture Lab(E(Equipmentquipment))

COCO22-thermostats-thermostats AirflowAirflow SolutionsSolutions DishesDishes FreezersFreezers Liquid nitrogen Liquid nitrogen CentrifugesCentrifuges

AutoclaveAutoclave Vacuum ovensVacuum ovens Cryotubes Cryotubes Microscopes Microscopes ELISA-readersELISA-readers

COCO22 Incubators Incubators

Water Jacketed COWater Jacketed CO22 incubatorincubator

3 Gas/CO3 Gas/CO22 Incubator Incubator with RH Control with RH Control Precise control of Precise control of

Oxygen levels Oxygen levels combined with COcombined with CO22, N, N22 and RH ensure and RH ensure accurate conditions accurate conditions for applications such for applications such as, hypoxic cell studies as, hypoxic cell studies and cancer research.and cancer research.

Laminar Flow BoxLaminar Flow Box

HEPA filter rated HEPA filter rated at 99.99% efficient at 99.99% efficient for 0.3 micron for 0.3 micron particulates. The particulates. The HEPA filtered air is HEPA filtered air is then directed then directed verticvertically across ally across the work surface. the work surface.

DishesDishes

DishesDishes Multiwell platesMultiwell plates FlasksFlasks Flasks on slideFlasks on slide

FreezersFreezers

CentrifugesCentrifuges

AutoclavesAutoclaves

Vacuum OvensVacuum Ovens

MicroscopesMicroscopes

Cell growth

Contamination Contamination

Cell countingCell counting

DyeDye

Transfection efficiencyTransfection efficiency

ELISA readersELISA readers

FACSFACS

II. Introduction of Cell II. Introduction of Cell Culture LabCulture Lab(Culture) (Culture)

Growth of the cells in adequatGrowth of the cells in adequatee media media with serumwith serum (FCS(FCS/FBS/FBS) and antibiotics ) and antibiotics and and antimycotics (chemically defined serum-antimycotics (chemically defined serum-free media)free media)

Environment:Environment: Temperature: 37°CTemperature: 37°C (34 °C, 41 °C) (34 °C, 41 °C) HighHigh humidityhumidity 5% CO5% CO22

Split: Trypsin-EDTASplit: Trypsin-EDTA Count of Cells (Thrypan Blue)Count of Cells (Thrypan Blue)

III. TechniquesIII. Techniques Metabolic activity (MTT)Metabolic activity (MTT) Detection of Apoptosis and NecrosisDetection of Apoptosis and Necrosis Western blot from cells Western blot from cells TransfectionTransfection Gene deletionsGene deletions Clinical Application of cultured Human Stem Clinical Application of cultured Human Stem

CellsCells Flow Cytometric Methods Flow Cytometric Methods FISH-probes FISH-probes DNA ArrayDNA Array

Metabolic activityMetabolic activity(MTT, viability assay)(MTT, viability assay)

Seed the cellsSeed the cells into 96-well plates at a starting into 96-well plates at a starting density of 10 celldensity of 10 cellss/well and culture overnight in /well and culture overnight in humidified 5 % COhumidified 5 % CO22 atmosphere at 37 °C. atmosphere at 37 °C.

Treat the cellsTreat the cells modifying their viability the modifying their viability the following day.following day.

ReReplaceplace medium medium to mediumto medium containing containing 0.5% water 0.5% water soluble mitochondrial dye, soluble mitochondrial dye, (3-(4,5-dimethylthiazol-2-(3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (yl]-2,5-diphenyl-tetrazolium bromide (MTTMTT+)+). .

IncubateIncubate 3 hours and 3 hours and solubilizesolubilize the water insoluble the water insoluble blue formasan dye by 10 % SDS in 10mM HCl.blue formasan dye by 10 % SDS in 10mM HCl.

DetermineDetermine the optical density by an ELISA reader the optical density by an ELISA reader at at 550 nm wavelength550 nm wavelength..

4

0

20

40

60

80

100

Ctrl. 0 0,1 0,5 1 2

M HO-3089

Su

rviv

al

(%)

Effect of HO-3089 (Novel PARP-inhibitor) on WRL-68 in Oxidative

Stress

Detection of ROSDetection of ROS

Count cellsCount cells Treatment (eg: HTreatment (eg: H22OO22, PARP inhibitor), PARP inhibitor) C-400 dye binds to ROS: fluorescence signalC-400 dye binds to ROS: fluorescence signal ELISA readerELISA reader

Detection of Apoptosis and Detection of Apoptosis and NecrosisNecrosis

Activity of CaspaseActivity of Caspase 3 3 and Caspase 8and Caspase 8 Release of Cytochrome c and AIFRelease of Cytochrome c and AIF Fluorescence dyesFluorescence dyes

Hoechst 33342Hoechst 33342 Annexin VAnnexin V PropidPropidiium iodideum iodide RhodamineRhodamine

DNA LadderingDNA Laddering Induction and protection Induction and protection PARP PARP

Apoptosis signalling via the Fas Apoptosis signalling via the Fas receptorreceptor

Activation and inhibition of Activation and inhibition of ApoptosisApoptosis

The RolThe Rolee of mitochondria in of mitochondria in apoptosisapoptosis

Caspase CascadeCaspase Cascade

FIGURE 3

- + - + - - + +

cyt-c

cyt-c

Ca2+

CsA

AIF

AIF

EndoG

EndoG

P

S

S

P

S

P

Protein release from Protein release from mitochondriamitochondria

To investigate the DNA To investigate the DNA fragmentation, the fragmentation, the extracted DNA has to extracted DNA has to run on 1run on 1..5% agarose gel.5% agarose gel.

DNA fragments show DNA fragments show ‘ladder-pattern’.‘ladder-pattern’.

DNA LadderingDNA Laddering

DNA LadderingDNA Laddering

Fluorescent dyesFluorescent dyes I. I. Hoechst 33342:blueHoechst 33342:blue

Selective nuclear dyeSelective nuclear dye Chromatin Chromatin

condensation, condensation, fragfragmmentationentation

Rhodamine 110: Rhodamine 110: greengreen

Bis-L-asparic acide Bis-L-asparic acide amide (substrate by amide (substrate by caspase 3): greencaspase 3): green

TMRE: polarization TMRE: polarization of mitochondria: redof mitochondria: red

Fluorescent dyesFluorescent dyes II. II.

Propidium iodidePropidium iodide: : Late-stage Late-stage apoptotic and apoptotic and necrotic cells: necrotic cells: redred

YO-PRO-1YO-PRO-1: Viable : Viable cell nuclei cell nuclei greengreen

Annexin VAnnexin V: early-: early-stage apoptotic stage apoptotic cells: cells: greengreen

Induction and Protection of Induction and Protection of ApoptosisApoptosis

Induction: Induction: Hydrogen peroxideHydrogen peroxide EtoposideEtoposide Death domainDeath domains: s: TNFTNF, , FASFAS, TRAIL, TRAIL BADBAD

Protection:Protection: BCL-2 familyBCL-2 family IAPIAP Inhibition of PARPInhibition of PARP HSP27, 70, 90HSP27, 70, 90

PARPPARP poly-poly-((ADP-ryboseADP-rybose))--polymerasepolymerase

Nuclear enzymeNuclear enzyme Structure of PARPStructure of PARP 11stst activator of PARP are ssDNA- activator of PARP are ssDNA-

breaksbreaks The rolThe rolee of PARP in necrosis and of PARP in necrosis and

apoptosis or repair-mechanismapoptosis or repair-mechanism The rolThe rolee of of poly(ADP-ribose) poly(ADP-ribose)

glycohydrolase (glycohydrolase (PARGPARG) )

-R-P-P-R-R-P-P-R-R-P-P-R-R-P-P-R

Ad

PARP Glu

AdN

CONH2

Ad

R-P-P-R-R-P-P-R

Ad Ad

+

Nic

Nic-R-P-P-R

Ad

(NAD+)

Reaction catalyzed by

PARP

Gene transfer Gene transfer 1. Calcium phosphate: DNA-calcium phosphate precipitatum

complex

2. Alkali complex: DNA-protein complex

3. Liposome: DNA in lipid membrane-bounded bodies

4. Electroporation

5. Magnetofection: magnetic particles-DNA complex

6. Transient (transfected DNA does not incorporate to the genomic DNA); stable transfection (selection; G418-neomycin).

7. Viral: Adeno, retro, lenti and adeno-associated viruses; „new” cell-line, incorporated DNA.

TransfectionTransfection Plasmide: double stranded circular Plasmide: double stranded circular

DNADNA 3-20 kb., hundreds of copies3-20 kb., hundreds of copies Heavy metal, antibiotic resistencyHeavy metal, antibiotic resistency MCS: Multiple Cloning SiteMCS: Multiple Cloning Site Gene of interestGene of interest

pEGFP without NLS

pEGFP with NLS

RNA interferencyRNA interferency

RNAiRNAi: : RNA-guided regulation of RNA-guided regulation of genegene expressionexpression in which in which double-strandeddouble-stranded ribonucleicribonucleic acidacid inhibits the inhibits the expressionexpression of of genesgenes with with complementarycomplementary nucleotidenucleotide sequences.sequences. DNADNA→ transcription→ → transcription→ mRNAmRNA→ translation→ protein→ translation→ protein

siRNAsiRNA Dicer RNADicer RNA (RNase III.) (RNase III.) siRNA cassettesiRNA cassette

Mechanism for how siRNA Mechanism for how siRNA worksworks

Therapeutic approaches

• Chromosoma methylation• Diabetes• Viral infection• HIV• Tumor therapy:

β-cathenine: increased cell growth (tumors)Supressed β-cathenine: tumor inhibition

SIDE EFFECTS!!!

Clinical Application of Clinical Application of cultured Human Stem Cellscultured Human Stem Cells

Not only human embryonic stem Not only human embryonic stem cells can be cultured in the cells can be cultured in the laboratory.laboratory.

But cells could be manipulated to But cells could be manipulated to produce cultures and characteristics produce cultures and characteristics of particular tissue.of particular tissue.

Possibile solution for damage and Possibile solution for damage and ageing (Parkinson’s disease, ageing (Parkinson’s disease, diabetes)diabetes)

FISH FISH (Fluorescence in situ (Fluorescence in situ

Hybridization)Hybridization) To detect and localize the presence or To detect and localize the presence or

absence of specific absence of specific DNADNA sequencessequences on on chromosomeschromosomes. .

ApplicationsApplications

Two copies of chromosome 3 (red), four copies of chromosome 7 (green), five copies of chromosome 17 (aqua) and one copy of p16 gene (gold).

A metaphase cell positive for the bcr/abl rearrangement using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (up left) is the one where the wrong rearrangement is present.

Flow CFlow Cyytometrtometryy Counting, examining and sorting microscopic particles suspended in a stream of fluid. Physical and/or chemical characteristics of single cells. Forward Scatter or FSC correlates with the cell volume. Side Scatter or SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

Flow Cytometric MethodsFlow Cytometric Methods

Separation of labeled cells

Clinical applications

Analysis of a marine sample of photosyntetic picoplankton by flow cytometry showingthree different populations (Prochlorococcus, Synechococcus, picoeukaryotes).