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Seminar of Cell Culture Techniques. Tapodi Antal Department of Biochemistry and Medicinal Chemistry, Faculty of Medicine, University of Pecs, Hungary . I . Cells Types II . Introduction to Cell Culture Lab III . Techniques. Content s. Prim a r y cultures Secondary cultures Normal

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seminar of cell culture techniques

Seminar of Cell Culture Techniques

Tapodi Antal

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

content s
I. Cells Types

II. Introduction to Cell Culture Lab

III. Techniques

Contents
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

I. Cell Types
1 primer y c ultures
1. PrimeryCultures
  • 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 organiccell types

CO2 Incubator

Knife Blender

2 secondary cultures
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

hybridomas
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
Hybrid selection

Metabolic pathways relevant to hybrid selection in medium containing hypoxanthine, aminopterin andthymidine (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.

slide9

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

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

WRL-68

Jurkat

HeLa

HepG2

ii introduction of cell culture lab e quipment
CO2-thermostats

Airflow

Solutions

Dishes

Freezers

Liquid nitrogen

Centrifuges

Autoclave

Vacuum ovens

Cryotubes

Microscopes

ELISA-readers

II. Introduction of Cell Culture Lab(Equipment)
co 2 incubators
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
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
  • Dishes
  • Multiwell plates
  • Flasks
  • Flasks on slide
ii introduction of cell culture lab culture
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: 37°C (34 °C, 41 °C)
    • Highhumidity
    • 5% CO2
  • Split: Trypsin-EDTA
  • Count of Cells (Thrypan Blue)
iii techniques
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
Metabolic activity(MTT, viability assay)

4

  • 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-tetrazolium 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
detection of apoptosis and necrosis
Detection of Apoptosis and Necrosis
  • Activity of Caspase3 and Caspase 8
  • Release of Cytochrome c and AIF
  • Fluorescence dyes
    • Hoechst 33342
    • Annexin V
    • Propidium iodide
    • Rhodamine
  • DNA Laddering
  • Induction and protection
  • PARP
fluorescent dyes i
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
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
dna laddering
DNA Laddering
  • To investigate the DNA fragmentation, the extracted DNA has to run on 1,5% agarose gel.
  • DNA fragments show ‘ladder-pattern’.
slide38

Detection of Apoptosis and Necrosis

  • Activity of Caspase3 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 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
parp poly adp rybose polymerase
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
slide41

Ad

Nic-R-P-P-R

(NAD+)

Ad

Ad

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

Reaction catalyzed by PARP

Ad

Ad

Ad

N

+

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

PARP

Glu

CONH

2

Nic

slide43

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
Transfection I.

pEGFP with NLS

  • Expression vector systems
    • pcDNA
    • pEGFP

pEGFP without NLS

transfection ii
Transfection II.
  • RNAi
    • siRNA
    • stRNA or Dicer RNAi
    • shRNA Using vectors for RNAi analysis
    • siRNA cassette
clinical application of cultured human stem cells
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 (Parkinson’s disease, diabetes)
epithelial stem cell identification and isolation
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
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
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
Flow Cytometric Methods
  • Separation of labeled cells
  • Clinical applications
dna array technique
DNA Array technique
  • Mr. Péter Jakus
cell suspension by nmr
Cell suspension by NMR
  • Dr. Zoltán Berente