Seminar of Cell Culture Techniques

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Seminar of Cell Culture Techniques

• Tapodi Antal
• Department of Biochemistry and Medicinal
  Chemistry, 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 and ETCC

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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
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2. Secondary cultures
H9c2

• Normal cell lines
• They were spontaneously immortalized.(e.g.
  Cardio-myocytes from rat)
• Immortalized
• Transfected with some sort of oncogene SV40
  (Simian virus)Large T antigen
• (T IDBL)
• Tumor cells (e.g. Human cervix carcinomas HeLa)
  
• Hybridomas

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 and Thymidine) medium

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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.

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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 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
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Production of Polyclonal and Monoclonal antibodies
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Neuro Hybryds

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

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Cell lines

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

Jurkat
WRL-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 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.

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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.

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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
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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
  (chemically defined 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)
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• Seed the cells into 96-well plates at a starting
  density of 10 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-diphenyl-tetrazo
  lium 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

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

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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 33342blue
• Selective nuclear dye
• Chromatin condensation, fragnentation
• Rhodamine 110 green
• Bis-L-asparic acide amide (substrate by caspase
  3) green
• TMRE polarization of mitochondria red

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Fluorescent dyes II.

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

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DNA Laddering

• To investigate the DNA fragmentation, the
  extracted DNA has to run on 1,5 agarose gel.
• DNA fragments show ladder-pattern.

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DNA Laddering
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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

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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-breaks
• The roll of PARP in necrosis and apoptosis or
  repair-mechanism
• The roll of PARG

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Reaction catalyzed by PARP
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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
• FISH-probes
• Flow Cytometric Methods
• DNA Array

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Transfection I.
pEGFP with NLS

• Expression vector systems
• pcDNA
• pEGFP

pEGFP without NLS
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Transfection II.

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

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

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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.

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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 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)
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Flow Cytometric Methods

• Separation of labeled cells
• Clinical applications

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DNA Array technique

• Mr. Péter Jakus

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Cell suspension by NMR

• Dr. Zoltán Berente