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


Normal
Immortalized



Spontaneous
Transformation
Transfection
Somatic Cell Fusion
(Hybridomas, Hybrids)

Cell lines



Adherent
Suspension
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)
Immortalized

HeLa


Transfected with some sort
of oncogene; SV40
(Simian virus)Large T
antigen
(T IDBL)
Tumor cells (e.g.: Human
cervix carcinomas: 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

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
PP
Hypoxanthine
Inosine Monophosphate
Hypoxanthine Guanine
Phosphoribosyl Transferase
(HGPRT)
Guanine
Guanosine Monophosphate
(GMP)
PRPP
PP
Thymidine
Thymidine kinase
UDP
RNA
dTMP
dTDP
* Thymidylate
Synthetase
dUTP
dUMP
GDP
dGDP
GTP
dGTP
d TTP
dCTP
DNA
dATP
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: 37°C (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
Survival (%)
80
60
40
20
0
Ctrl.
0
0,1
0,5
M HO-3089
1
2
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
Protection:




BCL-2 family
IAP
Inhibition of PARP
HSP27,70,90
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
Ad
(NAD+)
Ad
R-P-P-R-R-P-P-R
Ad
Ad
Ad
N
PARP
Glu
-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
(Parkinson’s 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.
Péter Jakus
Cell suspension by NMR
 Dr.
Zoltán Berente