Seminar of Cell Culture Techniques Tapodi Antal Department of Biochemistry and Medicinal Chemistry, Faculty of Medicine, University of Pecs, Hungary.

Seminar of Cell Seminar of Cell Culture Culture

TechniquesTechniquesTapodi Antal Tapodi Antal

Department of Biochemistry and Department of Biochemistry and Medicinal Chemistry, Faculty of Medicinal Chemistry, Faculty of

Medicine, University of Pecs, Medicine, University of Pecs, Hungary Hungary

ContentContentss

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

Culture LabCulture Lab IIIIII. Techniques. Techniques

I. I. CellCell Types Types

PrimPrimaarryy cultures cultures Secondary culturesSecondary 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

1. 1. PrimerPrimeryy CCulturesultures Tissue preparationTissue preparation

from young animal, or from young animal, or isolation of isolation of cells from cells from blood, blood, intraperitoneal intraperitoneal fluid, etc.fluid, 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 trypsine)/cleaving of DNA of damaged cell DNA of damaged cell with DNasewith DNase

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

Knife Blender

CO2 Incubator

2. 2. Secondary culturesSecondary cultures

Normal cell linesNormal cell lines They were spontaneousThey were spontaneouslyly

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.: Human Tumor cells (e.g.: Human

cervix carcinomas: HeLa) cervix carcinomas: HeLa) Hybridomas Hybridomas

H9c2

HeLa

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, aminopterin and thymidine (HAT medium).

When the main synthetic pathways are blocked with the folic acid analogue aminopterin (*), 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

5-Amino Imidazole- 4-Carboxy Ribonucleotide * 5-Formido-Imidazole- 4-Carboxamine Ribo- nucleotide PRPP PP

Hypoxanthine Inosine Monophosphate Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) Guanine Guanosine Monophosphate (GMP) PRPP PP Thymidine GDP dGDP Thymidine kinase RNA GTP dGTP

dTMP dTDP d TTP DNA * Thymidylate Synthetase UDP dUTP dUMP dCTP dATP

Production of Polyclonal Production of Polyclonal and Monoclonal antibodiesand Monoclonal antibodies

Neuro HybrydsNeuro Hybryds

It works with adherent cells.It works with adherent cells. Cell fusion of Cell fusion of HGPRTHGPRT and TK and TK-/--/-,, no no

secreting neuoblastomasecreting neuoblastoma and and neural neural cells.cells.

Selection in HAT mediumSelection in HAT medium

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

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

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 deletions (Demonstration)Gene deletions (Demonstration) 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 cell/well and culture overnight in density of 10 cell/well and culture overnight in humidified 5 % COhumidified 5 % CO22 atmosphere at 37 °C. atmosphere at 37 °C.

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

Remove mediumRemove medium from the wells containing from the wells containing 0,5% 0,5% water suluble mitocondrial dye, water suluble mitocondrial dye, (3-(4,5-(3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (bromide (MTTMTT+)+)

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

DetermineDetermine the optical density by an ELISA reder the optical density by an ELISA reder at 550 nm wavelengthat 550 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 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 signallingApoptosis signalling

Activation and inhibition of Activation and inhibition of ApoptosisApoptosis

The Roll of mitochondria in The Roll of mitochondria in apoptosisapoptosis

Caspase CascadeCaspase Cascade

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

Selective nuclear dyeSelective nuclear dye Chromatin Chromatin

condensation, condensation, fragnentationfragnentation

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

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

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

DNA LadderingDNA Laddering

DNA LadderingDNA Laddering

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

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

PARP(poly-ADP-rybose-PARP(poly-ADP-rybose-polymerase)polymerase)

Nuclear enzymeNuclear enzyme Structure of PARPStructure of PARP 1st activator of PARP are ssDNA-1st activator of PARP are ssDNA-

breaksbreaks The roll of PARP in necrosis and The roll of PARP in necrosis and

apoptosis or repair-mechanismapoptosis or repair-mechanism The roll of PARGThe roll of PARG

-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

NF-B/I-B

NF-B

módosult génexpresszió

MAP-kináz kaszkád

fokozott tirozinfoszforiláció

glutation /redox státusz

proteinfoszfatáz

gátlás

ROS

PARP-gátlók

PARP

(ADP-R)n

NAD+

NicA

ATP

CisplatinTaxol

ROS

Lipid peroxidáció

Protein oxidáció PARP-gátlók

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 deletions (Demonstration)Gene deletions (Demonstration) Clinical Application of cultured Human Clinical Application of cultured Human

Stem CellsStem Cells FISH-probesFISH-probes Flow Cytometric Methods Flow Cytometric Methods DNA ArrayDNA Array

TransfectionTransfection I. I.

Expression vector Expression vector systemssystems pcDNApcDNA pEGFPpEGFP

pEGFP with NLS

pEGFP without NLS

TransfectionTransfection II. II.

RNAiRNAisiRNAsiRNAstRNA or Dicer RNAistRNA or Dicer RNAishRNA Using vectors for shRNA Using vectors for RNAi analysis RNAi analysis

siRNA cassettesiRNA cassette

Proposed mechanism for Proposed mechanism for how siRNA workshow siRNA works

stRNA or Dicer RNAi

Gene Gene deletiondeletion

(Demonstratio(Demonstration)n)

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

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

But cells may be manipulated to But cells may be manipulated to produce cultures and produce cultures and Characteristics of particular tissue.Characteristics of particular tissue.

Possibility by damage and ageing Possibility by damage and ageing (Parkinson’s disease, diabetes)(Parkinson’s disease, diabetes)

Epithelial Stem Cell Epithelial Stem Cell identification and isolationidentification and isolation

First methods involved in the First methods involved in the separation of an epithelial cell type separation of an epithelial cell type from other cells will be examined, from other cells will be examined, followed by ways in which the followed by ways in which the proliferative capacity of such a cell type proliferative capacity of such a cell type can be assessed.can be assessed.

Secondly, methods used for the Secondly, methods used for the maintenance of primery stem cells in maintenance of primery stem cells in culture and ways of caracterizing stem culture and ways of caracterizing stem cells using immunocytochemistry will cells using immunocytochemistry will be described.be described.

FISH FISH (Fluorescence in situ (Fluorescence in situ

Hybridization)Hybridization) Application of FISH-probes Application of FISH-probes

Prenatal, Postnatal and Preimplantation Prenatal, Postnatal and Preimplantation GeneticsGenetics

Oncology, Cytology & Pathology Oncology, Cytology & Pathology Hematological Cancer Hematological Cancer Etc. Etc.

Equipments:Equipments: Fluorescence MicroscopeFluorescence Microscope Dye adequat filter setsDye adequat filter sets Sample and Reference DNASample and Reference DNA

Detection of Bladder Detection of Bladder CancerCancer

The probe was The probe was designed to detect designed to detect aneuploidy for aneuploidy for chromosomes 3, 7, 17 chromosomes 3, 7, 17 and loss of the 9p21 and loss of the 9p21 locus via fluorescence locus via fluorescence in situin situ hybridization hybridization (FISH) in urine (FISH) in urine specimens from specimens from subjects with subjects with transitional cell transitional cell carcinoma of the carcinoma of the bladder. bladder.

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)

Flow Cytometric MethodsFlow Cytometric Methods

Separation of labeled cells

Clinical applications

DNA Array techniqueDNA Array technique

Mr. Péter JakusMr. Péter Jakus

Cell suspension by Cell suspension by NMRNMR

Dr. Zoltán BerenteDr. Zoltán Berente