s5_cellculture

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Seminar of Cell Culture

TechniquesTapodi Antal

Department of Biochemistry and MedicinalChemistry, Faculty of Medicine, University

of Pecs, Hungary


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Contents

I. Cells Types

II. Introduction to Cell Culture Lab III. Techniques


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I. Cell Types

Primary cultures

Secondary cultures

Normal

Immortalized Spontaneous

Transformation

Transfection

Somatic Cell Fusion(Hybridomas, Hybrids)

Cell lines

Adherent

Suspension

Cells from ATCC andETCC


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1. Primery Cultures

Tissue preparation fromyoung animal, or isolation ofcells from blood,intraperitoneal fluid, etc.

Tissue dissociation Dissection then

Homogenization with Knife orBlender

Enzymatic Digestion(collagenase, papain,trypsine)/cleaving of DNA ofdamaged cell with DNase

Dissociation of cells inmedium and selection oforganic cell types

Knife Blender

CO2 Incubator


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2. Secondary cultures

Normal cell lines

They were spontaneously

immortalized.(e.g.: Cardio-

myocytes from rat)

Immortalized Transfected with some sort

of oncogene; SV40 (Simianvirus)Large T antigen

(T IDBL) Tumor cells (e.g.: Human

cervix carcinomas: HeLa)

Hybridomas

H9c2

HeLa


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Hybridomas

Cell fusion of

HGPRT and TK-/-myeloma and B-cells

from immunized animal Selection of hybridomas

in HAT (Hypoxanthine,Aminopterine andThymidine) medium


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Metabolic pathways relevant to hybrid selection inmedium containing hypoxanthine, aminopterin andthymidine (HAT medium).

When the main synthetic pathways are blocked with thefolic acid analogue aminopterin (*), the cell mustdepend on the salvage enzymes HGPRT and TK

(thymidine kinase). HGPRT (-) cells cannot grow inHAT medium unless they are fused with HGPRT (+)cells.

Hybrid selection


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Effect of HAT-medium Selection


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5-Amino Imidazole-4-Carboxy Ribonucleotide

*

5-Formido-Imidazole-

4-Carboxamine Ribo-nucleotide

PRPP PP

Hypoxanthine Inosine MonophosphateHypoxanthine GuaninePhosphoribosyl Transferase

(HGPRT)Guanine Guanosine Monophosphate

(GMP)PRPP PP

Thymidine GDP dGDP

Thymidine kinase RNA GTP dGTP

dTMP dTDP d TTP DNA* Thymidylate

SynthetaseUDP dUTP dUMP dCTP dATP


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Production of Polyclonal and

Monoclonal antibodies


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Neuro Hybryds

It works with adherent cells.

Cell fusion ofHGPRT and TK-/-, no secretingneuoblastoma and neural cells.

Selection in HAT medium


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Cell lines

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

Suspension (Jurkat)

Cells from ATCC andETCC

JurkatWRL-68

HeLa HepG2


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Online Order of Cell Lines


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II. Introduction of Cell Culture Lab

(Equipment)

CO2-thermostats

Airflow

Solutions

Dishes

Freezers

Liquid nitrogen

Centrifuges

Autoclave

Vacuum ovens

Cryotubes

Microscopes

ELISA-readers


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CO2 Incubators

Water Jacketed CO2incubator

3 Gas/CO2 Incubator

with RH Control Precise control of Oxygen

levels combined withCO2, N2 and RH ensure

accurate conditions forapplications such as,hypoxic cell studies andcancer research.


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Laminar Flow Box

HEPA filter rated at99.99% efficient for 0.3

micron particulates. TheHEPA filtered air is thendirected vertically acrossthe work surface.


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Dishes

Dishes

Multiwell plates

Flasks

Flasks on slide


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Freezers


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Centrifuges


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Autoclaves


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Vacuum Ovens


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Microscopes


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ELISA readers


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FACS

II Introd ction of Cell C lt re Lab


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II. Introduction of Cell Culture Lab

(Culture)

Growth of the cells in adequate media with serum(FCS/FBS) and antibiotics and antimycotics (chemicallydefined serum-free media)

Environment: Temperature: 37C (34 C, 41 C)

High humidity

5% CO2

Split: Trypsin-EDTA

Count of Cells (Thrypan Blue)


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III. Techniques

Metabolic activity (MTT)

Detection of Apoptosis and Necrosis

Western blot from cells

Transfection Gene deletions (Demonstration) Clinical Application of cultured Human Stem Cells

Flow Cytometric Methods

FISH-probes

DNA Array


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Metabolic activity

(MTT, viability assay)

Seed the cells into 96-well plates at a starting density of 10cell/well and culture overnight in humidified 5 % CO2atmosphere at 37 C.

Treat the cells modifying the their viability the following day.

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

Incubate 3 hours and solubilize the water insoluble blueformasan dye by 10% SDS in 10mM HCl

Determine the optical density by an ELISA reder at 550 nmwavelength

4


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0

20

40

60

80

100

Ctrl. 0 0,1 0,5 1 2

M HO-3089

Survival(%

)

Effect of HO-3089 (Novel PARP-inhibitor) on

WRL-68 in Oxidative Stress


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Apoptosis signalling


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Activation and inhibition of

Apoptosis


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The Roll of mitochondria in

apoptosis


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Caspase Cascade


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Fluorescent dyes I.

Hoechst 33342:blue Selective nuclear dye

Chromatin condensation,fragnentation

Rhodamine 110: green

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

TMRE: polarization ofmitochondria: red


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Fluorescent dyes II.

Propidium iodide: Late-stage apoptotic andnecrotic cells: red

YO-PRO-1: Viable cellnuclei green

Annexin V: early-stageapoptotic cells: green


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To investigate the DNA

fragmentation, the extracted

DNA has to run on 1,5%agarose gel.

DNA fragments show ladder-pattern.

DNA Laddering


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DNA Laddering


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Activity of Caspase 3 and Caspase 8

Release of Cytochrome c and AIF

Fluorescence dyes Hoechst 33342

Annexin V

Propidium iodide

Rhodamine

DNA Laddering

Induction and protection

PARP


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Induction and Protection of

Apoptosis

Induction: Hydrogen peroxide

Etoposide

Death domains: TNF, FAS, TRAIL

BAD

Protection: BCL-2 family

IAP Inhibition of PARP

HSP27,70,90


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PARP(poly-ADP-rybose-polymerase)

Nuclear enzyme

Structure of PARP

1st activator of PARP are ssDNA-breaksThe roll of PARP in necrosis and apoptosis or

repair-mechanism

The roll of PARG


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-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


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III. Techniques

Metabolic activity (MTT)


Western blot from cells

Transfection Gene deletions (Demonstration)

Clinical Application of cultured Human Stem Cells

FISH-probes


DNA Array


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Transfection I.

Expression vectorsystems

pcDNA

pEGFP

pEGFP with NLS

pEGFP without NLS


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Transfection II.

RNAi

siRNA

stRNA or Dicer RNAi

shRNA Using vectors for RNAi

analysis

siRNA cassette

d f


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Proposed mechanism for how siRNA

works


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stRNA or Dicer RNAi


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Gene deletion(Demonstration)


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Clinical Application of cultured Human

Stem Cells

Not only can human embryonic stem cells

be cultured in the laboratory.

But cells may be manipulated to producecultures and Characteristics of particular

tissue.

Possibility by damage and ageing(Parkinsons disease, diabetes)

E i h li l S C ll id ifi i


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Epithelial Stem Cell identification

and isolation

First methods involved in the separation of anepithelial cell type from other cells will beexamined, followed by ways in which the

proliferative capacity of such a cell type can beassessed.

Secondly, methods used for the maintenance ofprimery stem cells in culture and ways of

caracterizing stem cells usingimmunocytochemistry will be described.


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FISH

(Fluorescence in situ Hybridization)

Application of FISH-probes Prenatal, Postnatal and Preimplantation Genetics

Oncology, Cytology & Pathology

Hematological Cancer Etc.

Equipments:

Fluorescence Microscope Dye adequat filter sets

Sample and Reference DNA


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Detection of Bladder Cancer

The probe was designedto detect aneuploidy forchromosomes 3, 7, 17

and loss of the 9p21locus via fluorescence insituhybridization (FISH)in urine specimens from

subjects with transitionalcell carcinoma of thebladder.

two copies of chromosome 3 (red),four copies of chromosome 7(green), five copies of chromosome17 (aqua) and one copy of p16 gene(gold)


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Separation of labeledcells

Clinical applications


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DNA Array technique

Mr. Pter Jakus


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Cell suspension by NMR

Dr. Zoltn Berente

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