Title: Seminar of Cell Culture Techniques
1Seminar of Cell Culture Techniques
- Tapodi Antal
- Department of Biochemistry and Medicinal
Chemistry, Faculty of Medicine, University of
Pecs, Hungary
2Contents
- I. Cells Types
- II. Introduction to Cell Culture Lab
- III. Techniques
3I. 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
41. 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
52. 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
6Hybridomas
- Cell fusion of
- HGPRT and TK-/- myeloma and B-cells from
immunized animal - Selection of hybridomas in HAT (Hypoxanthine,
Aminopterine and Thymidine) medium
7Hybrid 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.
8Effect of HAT-medium Selection
9 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
10Production of Polyclonal and Monoclonal antibodies
11Neuro Hybryds
- It works with adherent cells.
- Cell fusion of HGPRT and TK-/-, no secreting
neuoblastoma and neural cells. - Selection in HAT medium
12Cell lines
- Adherent (WRL-68, HepG2, HeLa etc.)
- Suspension (Jurkat)
- Cells from ATCC and ETCC
Jurkat
WRL-68
HeLa
HepG2
13Online Order of Cell Lines
14II. Introduction of Cell Culture Lab(Equipment)
- CO2-thermostats
- Airflow
- Solutions
- Dishes
- Freezers
- Liquid nitrogen
- Centrifuges
- Autoclave
- Vacuum ovens
- Cryotubes
- Microscopes
- ELISA-readers
15CO2 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.
16Laminar 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.
17Dishes
- Dishes
- Multiwell plates
- Flasks
- Flasks on slide
18Freezers
19Centrifuges
20Autoclaves
21Vacuum Ovens
22Microscopes
23ELISA readers
24FACS
25II. 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)
26III. 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
27Metabolic 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-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
28Effect of HO-3089 (Novel PARP-inhibitor) on
WRL-68 in Oxidative Stress
29Detection 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
30Apoptosis signalling
31Activation and inhibition of Apoptosis
32The Roll of mitochondria in apoptosis
33Caspase Cascade
34Fluorescent 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
35Fluorescent 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
36DNA Laddering
- To investigate the DNA fragmentation, the
extracted DNA has to run on 1,5 agarose gel. - DNA fragments show ladder-pattern.
37DNA Laddering
38Detection 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
39Induction 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
40PARP(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
41Reaction 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
42(No Transcript)
43III. 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
44Transfection I.
pEGFP with NLS
- Expression vector systems
- pcDNA
- pEGFP
pEGFP without NLS
45Transfection II.
- RNAi
- siRNA
- stRNA or Dicer RNAi
- shRNA Using vectors for RNAi analysis
- siRNA cassette
46Proposed mechanism for how siRNA works
47stRNA or Dicer RNAi
48Gene deletion (Demonstration)
49Clinical 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)
50Epithelial 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.
51FISH (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
52Detection 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)
53Flow Cytometric Methods
- Separation of labeled cells
- Clinical applications
54DNA Array technique
55Cell suspension by NMR