Seminar of Cell Culture Techniques

Tapodi Antal Department of Biochemistry and Medicinal Chemistry, Faculty of Medicine, University of Pecs, Hungary

Contents
I. Cells Types II. Introduction to Cell Culture Lab III. Techniques

I. Cell Types

Primary cultures Secondary cultures

Cell lines

Normal Immortalized

Adherent Suspension

Spontaneous Transformation Transfection Somatic Cell Fusion (Hybridomas, Hybrids)

Cells from ATCC and ETCC

1. Primery Cultures

Tissue preparation from young animal, or isolation of cells from blood, intraperitoneal fluid, etc. Tissue dissociation

Dissection then Homogenization with Knife or Blender Enzymatic Digestion (collagenase, papain, trypsine)/cleaving of DNA of damaged cell with DNase Dissociation of cells in medium and selection of organic cell types

CO2 Incubator Knife Blender

2. Secondary cultures
H9c2

Normal cell lines

They were spontaneously immortalized.(e.g.: Cardiomyocytes from rat) Transfected with some sort of oncogene; SV40 (Simian virus)Large T antigen (T IDBL) Tumor cells (e.g.: Human cervix carcinomas: HeLa) Hybridomas

Immortalized

HeLa

Hybridomas

Cell fusion of HGPRT and TK-/myeloma and B-cells from immunized animal Selection of hybridomas in HAT (Hypoxanthine, Aminopterine and Thymidine) medium

Hybrid selection

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.

Effect of HAT-medium Selection

5-Amino Imidazole4-Carboxy Ribonucleotide *

5-Formido-Imidazole4-Carboxamine Ribonucleotide

PRPP
Hypoxanthine

PP
Inosine Monophosphate

Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) Guanine PRPP Thymidine Thymidine kinase RNA PP GDP GTP d TTP dCTP dGDP dGTP DNA dATP Guanosine Monophosphate (GMP)

UDP

dTMP dTDP * Thymidylate Synthetase dUTP dUMP

Production of Polyclonal and Monoclonal antibodies

Neuro Hybryds

It works with adherent cells. Cell fusion of HGPRT and TK-/-, no secreting neuoblastoma and neural cells. Selection in HAT medium

Cell lines

Adherent (WRL-68, HepG2, HeLa etc.) Suspension (Jurkat) Cells from ATCC and ETCC

WRL-68

Jurkat

HeLa

HepG2

Online Order of Cell Lines

II. Introduction of Cell Culture Lab (Equipment)

CO2-thermostats Airflow Solutions Dishes Freezers Liquid nitrogen Centrifuges

Autoclave Vacuum ovens Cryotubes Microscopes ELISA-readers

CO2 Incubators

Water Jacketed CO2 incubator 3 Gas/CO2 Incubator with RH Control

Precise control of Oxygen levels combined with CO2, N2 and RH ensure accurate conditions for applications such as, hypoxic cell studies and cancer research.

Laminar Flow Box

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

Dishes

Dishes Multiwell plates Flasks Flasks on slide

Freezers

Centrifuges

Autoclaves

Vacuum Ovens

Microscopes

ELISA readers

FACS

II. Introduction of Cell Culture Lab (Culture)

Growth of the cells in adequate media with serum (FCS/FBS) and antibiotics and antimycotics (chemically defined serum-free media) Environment:

Temperature: 37C (34 C, 41 C) High humidity 5% CO2

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

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

Metabolic activity (MTT, viability assay)

Seed the cells into 96-well plates at a starting density of 10 4 cell/well and culture overnight in humidified 5 % CO2 atmosphere at 37 C. Treat the cells modifying the their viability the following day. Remove medium from the wells containing 0,5% water suluble mitocondrial dye, (3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT+) Incubate 3 hours and solubilize the water insoluble blue formasan dye by 10% SDS in 10mM HCl Determine the optical density by an ELISA reder at 550 nm wavelength

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

100 80

Survival (%)

60 40 20 0 Ctrl. 0 0,1 0,5 1 2 M HO-3089

Detection of Apoptosis and Necrosis

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

Apoptosis signalling

Activation and inhibition of Apoptosis

The Roll of mitochondria in apoptosis

Caspase Cascade

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 of mitochondria: red

Fluorescent dyes II.

Propidium iodide: Latestage apoptotic and necrotic cells: red YO-PRO-1: Viable cell nuclei green Annexin V: early-stage apoptotic cells: green

DNA Laddering
To

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

DNA Laddering

Detection of Apoptosis and Necrosis

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

Induction and Protection of Apoptosis

Induction:

Hydrogen peroxide Etoposide Death domains: TNF, FAS, TRAIL BAD

BCL-2 family IAP Inhibition of PARP HSP27,70,90

Protection:

PARP(poly-ADP-rybose-polymerase)

Nuclear enzyme Structure of PARP 1st activator of PARP are ssDNA-breaks The roll of PARP in necrosis and apoptosis or repair-mechanism The roll of PARG

Reaction catalyzed by PARP

Ad
Nic-R-P-P-R (NAD+) Ad Ad

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

Ad PARP Glu

Ad

Ad
N

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

+
Nic
CONH2

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 FISH-probes Flow Cytometric Methods DNA Array

Transfection I.
pEGFP with NLS

Expression vector systems

pcDNA pEGFP
pEGFP without NLS

Transfection II.

RNAi siRNA stRNA or Dicer RNAi shRNA Using vectors for RNAi analysis siRNA cassette

Proposed mechanism for how siRNA works

stRNA or Dicer RNAi

Gene deletion
(Demonstration)

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 produce cultures and Characteristics of particular tissue. Possibility by damage and ageing (Parkinsons disease, diabetes)

Epithelial Stem Cell identification and isolation

First methods involved in the separation of an epithelial cell type from other cells will be examined, followed by ways in which the proliferative capacity of such a cell type can be assessed. Secondly, methods used for the maintenance of primery stem cells in culture and ways of caracterizing stem cells using immunocytochemistry will be described.

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

Detection of Bladder Cancer

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

Separation of labeled cells Clinical applications

DNA Array technique

Mr.

Pter Jakus

Cell suspension by NMR

Dr.

Zoltn Berente