Laboratory of Molecular Genetics, KNU
Cell Culture
Laboratory of Molecular Genetics, KNU
Basics of Cell Culture
Laboratory of Molecular Genetics, KNU
Introduction
• Cell culture is the process by which prokaryo
tic, eukaryotic or plant cells are grown under
controlled conditions. But in practice it refers
to the culturing of cells derived from animal c
ells.
• Cell culture was first successfully undertaken
by Ross Harrison in 1907
• Roux in 1885 for the first time maintained em
bryonic chick cells in a cell culture
Laboratory of Molecular Genetics, KNU
Major development’s in cell culture technology
• First development was the use of antibi
otics which inhibits the growth of conta
minants.
• Second was the use of trypsin to remov
e adherent cells to subculture further fro
m the culture vessel
• Third was the use of chemically defined
culture medium.
Laboratory of Molecular Genetics, KNU
Why is cell culture used for?
Areas where cell culture technology is curr
ently playing a major role.
• Model systems for
Studying basic cell biology, interactions between di
sease causing agents and cells, effects of drugs on cells, pr
ocess and triggering of aging & nutritional studies
• Toxicity testing
Study the effects of new drugs
• Cancer research
Study the function of various chemicals, virus & r
adiation to convert normal cultured cells to cancerous cells
Laboratory of Molecular Genetics, KNU
Contd….
• Virology
Cultivation of virus for vaccine production,
also used to study there infectious cycle.
• Genetic Engineering
Production of commercial proteins, large
scale production of viruses for use in vaccine pr
oduction e.g. polio, rabies, chicken pox, hepatitis
B & measles
• Gene therapy
Cells having a functional gene can be repl
aced to cells which are having non-functional gene
Laboratory of Molecular Genetics, KNU
Culture media
• Choice of media depends
on the type of cell being c
ultured
• Commonly used Medium a
re GMEM, EMEM,DMEM et
c.
• Media is supplemented wit
h antibiotics viz. penicillin,
streptomycin etc.
• Prepared media is filtered
and incubated at 4 C
Laboratory of Molecular Genetics, KNU
Basic equipments used in cell culture
•
•
•
•
•
Laminar cabinet-Vertical are pre
ferable
Incubation facilities- Temperatur
e of 25-30 C for insect & 37 C f
or mammalian cells, co2 2-5%
& 95% air at 99% relative humidi
ty. To prevent cell death incubat
ors set to cut out at approx. 38.
5C
Refrigerators- Liquid media kept
at 4 C, enzymes (e.g. trypsin) &
media components (e.g. glutami
ne & serum) at -20 C
Microscope- An inverted micros
cope with 10x to 100x magnifica
tion
Tissue culture ware- Culture pla
stic ware treated by polystyrene
Laboratory of Molecular Genetics, KNU
Basic aseptic conditions
• If working on the bench use a Bunsen flame to heat t
he air surrounding the Bunsen
• Swab all bottle tops & necks with 70% ethanol
• Flame all bottle necks & pipette by passing very quick
ly through the hottest part of the flame
• Avoiding placing caps & pipettes down on the bench;
practice holding bottle tops with the little finger
• Work either left to right or vice versa, so that all mater
ial goes to one side, once finished
• Clean up spills immediately & always leave the work p
lace neat & tidy
Laboratory of Molecular Genetics, KNU
Working with cryopreserved cells
•
•
•
•
•
Vial from liquid nitrogen is plac
ed into 37 C water bath, agitate
vial continuously until medium i
s thawed
Centrifuge the vial for 10 mts at
1000 rpm at RT, wipe top of via
l with 70% ethanol and discard
the supernatant
Resuspend the cell pellet in 1
ml of complete medium with 20
% FBS and transfer to properly
labeled culture plate containing
the appropriate amount of medi
um
Check the cultures after 24 hrs
to ensure that they are attache
d to the plate
Change medium as the colour
changes, use 20% FBS until th
e cells are established
Laboratory of Molecular Genetics, KNU
Freezing cells for storage
• Remove the growth medium, wash the cells by PB
S and remove the PBS by aspiration
• Dislodge the cells by trypsin-versene
• Dilute the cells with growth medium
• Transfer the cell suspension to a 15 ml conical tub
e, centrifuge at 200g for 5 mts at RT and remove t
he growth medium by aspiration
• Resuspend the cells in 1-2ml of freezing medium
• Transfer the cells to cryovials, incubate the cryovi
als at -80 C overnight
• Next day transfer the cryovials to Liquid nitrogen
Laboratory of Molecular Genetics, KNU
Culturing of cells
• Cells are cultured as anchorage
dependent or independent
• Cell lines derived from normal ti
ssues are considered as anchor
age-dependent grows only on a
suitable substrate e.g. tissue cel
ls
• Suspension cells are anchorage
-independent e.g. blood cells
• Transformed cell lines either gro
ws as monolayer or as suspensi
on
Laboratory of Molecular Genetics, KNU
Adherent cells
•
•
•
•
•
•
•
•
•
Cells which are anchorage dependent
Cells are washed with PBS (free of ca & mg )
solution.
Add enough trypsin/EDTA to cover the mon
olayer
Incubate the plate at 37 C for 1-2 mts
Tap the vessel from the sides to dislodge th
e cells
Add complete medium to dissociate and disl
odge the cells
with the help of pipette which are remained t
o be adherent
Add complete medium depends on the subc
ulture
requirement either to 75 cm or 175 cm flask
Laboratory of Molecular Genetics, KNU
Suspension cells
•
•
•
•
Easier to passage as no need to detach them
As the suspension cells reach to confluency
Asceptically remove 1/3rd of medium
Replaced with the same amount of pre-warmed medi
um
Laboratory of Molecular Genetics, KNU
Common cell lines
•
•
•
•
•
•
•
•
•
Human cell lines
-MCF-7
breast cancer
HL 60
Leukemia
HEK-293
Human embryonic kidney
HeLa
Henrietta lacks
Primate cell lines
Vero
African green monkey kidney epithelial cells
Cos-7
African green monkey kidney cells
And others such as CHO from hamster, sf9 & sf21 from insect c
ells
Laboratory of Molecular Genetics, KNU
Contaminant’s of cell culture
Cell culture contaminants of
two types
• Chemical-difficult to detect
caused by endotoxins, plast
icizers, metal ions or traces
of disinfectants that are invi
sible
• Biological-cause visible effe
cts on the culture they are
mycoplasma, yeast, bacteri
a or fungus or also from cro
ss-contamination of cells fr
om other cell lines
Laboratory of Molecular Genetics, KNU
Cell viability
• Cell viability is determined by staining the cells w
ith trypan blue
• As trypan blue dye is permeable to non-viable c
ells or death cells whereas it is impermeable to t
his dye
• Stain the cells with trypan dye and load to haem
ocytometer and calculate % of viable cells
- % of viable cells= Nu. of unstained cells x 100
total nu. of cells
Laboratory of Molecular Genetics, KNU
Cell toxicity
• Cytotoxicity causes inhibition of cell growth
• Observed effect on the morphological alteration in
the cell layer or cell shape
• Characteristics of abnormal morphology is the gia
nt cells, multinucleated cells, a granular bumpy a
ppearance, vacuoles in the cytoplasm or nucleus
• Cytotoxicity is determined by substituting material
s such as medium, serum, supplements flasks etc
. at atime
Laboratory of Molecular Genetics, KNU
Transfection methods
•
•
•
•
•
•
Calcium phosphate precipitation
DEAE-dextran (dimethylaminoethyl-dextran)
Lipid mediated lipofection
Electroporation
Retroviral Infection
Microinjection
Transfection of eukaryotic cells
Transfection
What is Transfection?
Transfection is a method of transporting DNA,
RNA and/or various macromolecules into an
eukaryotic cell by using chemical, lipid or physical
based methods.
Methods: (just a few examples…)
Method
CaPO4, DEAE
Liposome Based
Polyamine Based
Application
DNA Transfection
DNA Transfection
DNA Transfection
Early Years
• Transfection =
“transformation” +
“infection”
• The infection of cells by
the isolated nucleic acid
from a virus, resulting in
the production of a
complete virus
1928: Frederick
Griffith transforms
Streptococcus
pneumoniae
1944: Avery, Macleod
and McCarty discover
DNA is the transforming
factor
1964: Foldes and
Trautner use the term
transfection
1965: Vaheri and Pagano
describe the DEAE-Dextran
transfection technique
Transfection vs. Transformation
Transformation: genetically altering
cells by transporting in foreign genetic
material.
Transfection: the process of
transporting genetic material and/or
macromolecules into eukaryotic cells
through typically non-viral methods.
Purpose/uses of Transfection
Study gene function
Study protein expression
Transfer DNA into embryonic stem cells
How it works
Utilize Chemical, lipid or physical
methods (direct microinjection,
electroporation, biolistic particle delivery)
for transportation of genetic materials
or macromolecules.
Transfection method 1: DEAEDextran
• Facilitates DNA binding to cell membranes and
entry via endocytosis
• DEAE-D associates with DNAcomplex binds
to cell surface
Add chloroquine to transfection medium
Add plasmid DNA to DEAE-Dextran medium
Incubate cells with mixture…efficient
transfection results in 25-75% death
DMSO “shock”
Transfection method 1: DEAEDextran
• Major variants: number of cells, concentration
of DNA and concentration of DEAE-Dextran
• Only method that cannot be used for stable
transfections
Transfection method 3: liposomemediated
• Use of cationic lipids
– chemical and physical similarities to biological
lipids
– spontaneous formation of complexes with
DNA, called lipoplexes
•
•
•
•
High efficiency for in vitro transfections
Can carry larger DNA than viruses
Safer than viruses
Low in vivo efficiency
http://homepage.usask.ca/~sdw132/images/lipid_nanoparticle.gif
How it works – Chemical and
lipid methods
Neutralize negative
charges on DNA
Chemical method:
Calcium phosphate;
creates precipitates that
settle on cells and are
taken in.
Lipid or Polymer
methods: interact with
DNA, promotes binding
to cells and uptake via
endocytosis.
Experimental method/process
(chemical methods)
HBS = HEPES-Buffered Saline
Transfection method 2:
electroporation
• Use of high-voltage electric shocks to introduce DNA into cells
• Cell membranes: electrical capacitors unable to pass current
• Voltage results in temporary breakdown and formation of
pores
Harvest cells and resuspend in electroporation buffer
Add DNA to cell suspension…for stable transfection DNA should be
linearized, for transient the DNA may be supercoiled
electroporate
Selection process for transfectant
Transfection method 2:
electroporation
• Variants: amplitude and length of electric pulse
• Less affected by DNA concentration-linear
correlation between amount of DNA present and
amount taken up
• Can be used for stable transfections
How it works – Physical methods
Electroporation: use of high
voltage to deliver nucleic
acids; pores are formed on
cell membrane.
Direct Microinjection: Use of
a fine needle. Has been used
for transfer of DNA into
embryonic stem cells.
Biolistic Particle delivery:
Uses high-velocity for
delivery of nucleic acids and
penetration of cell wall.
Advantages/Disadvantages
Advantages:
Provides the ability to
transfer in negatively
charged molecules into cells
with a negatively charged
membrane.
Liposome-mediated
transport of DNA has high
efficiency. Good for longterm studies requiring
incorporation of genetic
material into the
chromosome.
Disadvantages:
Chemical Reagents: not
useful for long-term studies.
Transfection efficiency is
dependent on cell health,
DNA quality, DNA quantity,
confluency (40-80%)
Direct Microinjection and
Biolistic Particle delivery is
an expensive and at times a
difficult method.
Laboratory of Molecular Genetics, KNU
Exploring protein function
1) Where is it localized in the cell?
Approaches:
a) Make antibodies - immunofluorescence
b) “Express” the protein in cells with a tag
 Fuse to GFP
2) What is it doing in the cell?
Approaches:
a) Reduce protein levels - RNA interference
b) Increase protein levels “over-express”
c) “Express” mutant versions
Transfection!!!!
Laboratory of Molecular Genetics, KNU
Transfection =
Introduction of DNA into mammalian cells
Gene is transcribed and translated into protein
= “expressed”
Laboratory of Molecular Genetics, KNU
Direct introduction of the DNA
Electroporation - electric field
temporarily disrupts plasma
membrane
Biolistics (gene gun)- fire
DNA coated particles into cell
Microinjection
Laboratory of Molecular Genetics, KNU
Virally-mediated introduction of the DNA
Infection:
Use recombinant viruses to deliver DNA
Retroviruses
Adenoviruses
Laboratory of Molecular Genetics, KNU
Carrier-mediated introduction of the DNA
Positively charged carrier molecules
are mixed with the DNA and added to
cell culture media:
Calcium Phosphate
DEAE Dextran
liposomes
micelles
Carrier-DNA complexes bind to plasma
membrane and are taken up
Laboratory of Molecular Genetics, KNU
Types of Transfection
Transient:
Expression assayed 24-48
hours post transfection
Stable:
Integration of the transfected DNA
into the cell genome - selectable
marker like neomycin resistance
required
“stably transfected” cell line
Laboratory of Molecular Genetics, KNU
DNA “expression” vector transfected:
For
expression
in cells
Insert gene
in here
Polyadenylation
site
To
generate
stable cell
line
pCMV/GFP
For
amplification
of the
plasmid in
bacteria
pUC
Polyadenylation
site
Bacterial origin of replication
Laboratory of Molecular Genetics, KNU
Three ways to make Green fluorescent
protein “GFP” fusion constructs:
PROTEIN X
GFP
PROTEIN
GFP
PROTEIN Y
GFP
Z
Laboratory of Molecular Genetics, KNU
EXPERIMENT:
Transfect unknown GFP fusion protein
Protein X, Y or Z
Visualize GFP protein fluorescence by fluorescence
microscopy in living cells
Counter-stain with known marker to compare
localization patterns in living cells
= “vital stain”
Laboratory of Molecular Genetics, KNU
Some Cellular Organelles
Laboratory of Molecular Genetics, KNU
•Compartments/organelles examined
•Protein sequences sufficient for localization
•Vital stains
Nuclei
Mitochondria
Secretory Pathway:
Endoplasmic Reticulum
Golgi Complex
Endocytotic Pathway:
Endosomes
Laboratory of Molecular Genetics, KNU
Nucleus
Transport through nuclear
pore
signal = basic amino acid
stretches
example: P-P-K-K-K-R-K-V
Laboratory of Molecular Genetics, KNU
Import of proteins into nucleus through nuclear pore
Laboratory of Molecular Genetics, KNU
Nuclear Stain:
Hoechst 33258
binds DNA
Laboratory of Molecular Genetics, KNU
Mitochondria
Transmembrane transport signal
Example: H2N-M-L-S-L-R-Q-S-I-R-F-F-KP-A-A-T-R-T-L-C-S-S-R-Y-L-L
Laboratory of Molecular Genetics, KNU
Protein being transported across mitochondrial membranes
Laboratory of Molecular Genetics, KNU
Mitochondrial dye =
MitoTracker Red
Diffuses through
membranes
Non-fluorescent until
oxidized
Accumulates in
mitochondria and oxidized
Mitotracker
DNA
Laboratory of Molecular Genetics, KNU
Cellular components of the secretory and endocytic pathways
lysosome
plasma
membrane
late
endosome
nuclear envelope
endoplasmic reticulum
early
endosome
CYTOSOL
cis
Golgi
network
Golgi
stack
trans
Golgi
network
Golgi apparatus
Laboratory of Molecular Genetics, KNU
Endoplasmic Reticulum
Entry into E.R.:
Transmembrane transport signal
= hydrophobic amino acid stretches
at amino terminus
Example: H2N-M-M-S-F-V-S-L-L-V-G-I-LF-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q
Retention in E.R. lumen:
Signal = K-D-E-L-COOH
at carboxy terminus
Laboratory of Molecular Genetics, KNU
Endoplasmic Reticulum marker
ER-Tracker Blue-White
Live bovine pulmonary artery
endothelial cells
Laboratory of Molecular Genetics, KNU
Mitotracker Red and ERblue/white
Laboratory of Molecular Genetics, KNU
nucleus
Golgi
From the ER, secreted and membrane proteins move to the Golgi, a
series of membrane-bound compartments found near the nucleus
Laboratory of Molecular Genetics, KNU
Golgi marker
BODIPY-TR ceramide
Ceramide = lipid
When metabolized,
concentrates in the
Golgi
Red fluorophore
Laboratory of Molecular Genetics, KNU
Cultured Epithelial Cells
DNA (Hoechst)
Golgi (ceramide)
Laboratory of Molecular Genetics, KNU
MDCK Cells
Madin-Darby Canine
Kidney
Polarized Epithelial
Cells
DNA (Hoechst)
Golgi (ceramide)
Lysosomes (LysoTracker)
Molecular Probes, Inc.
Laboratory of Molecular Genetics, KNU
Rhodamine transferrin
Does the fluorescent green protein co-localize?
Laboratory of Molecular Genetics, KNU
Immunofluorescence / confocal microscopy 원리
Laboratory of Molecular Genetics, KNU
Immunofluorescence / confocal microscopy 실험방법
Immunofluorescence / confocal microscopy
B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections
of unfixed, OCT embedded tissue:
1. wash cells 1x cold RPMI (no wash for cryostat sections).
2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **
3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.
4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.
5. wash 1x PBS/BSA.
6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.
7. wash 1x PBS/BSA.
8. 1° diluted in PBS/BSA 60 min RT; 100 l per tube or section.
9. wash 3x PBS/BSA.
10. Block 15 min 5% NGS in PBS/BSA.
11. wash 1x PBS/BSA.
12. 2° diluted in PBS/BSA 30 min RT; 100 l per tube or section.
13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer
if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes
Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass
just put two-three drops in each chamber after wash.
High-throughput loss-of-function screens using RNAi cell microarrays.
An example of a high-density, high-content RNAi cell-microarray screen in
cultured D. melanogaster Kc167 cells.
Laboratory of Molecular Genetics, KNU
Experimental Animals
Laboratory of Molecular Genetics, KNU
Model systems
E. coli
- easy and inexpensive to maintain
- 사람 장내 서식, 배설물의 주성분
- 해로운 박테리아의 생장 저해
Pathogenic은 sickness, death 유발
- 돌연변이 잘 유발 → Metabolism 연구에
이용
Eukaryote에서의 과정 일어나지 않음
Laboratory of Molecular Genetics, KNU
• Yeast
- Eukaryote와 prokaryote 사이의 차이점
연구에 이용
- Genetic study easy
- haploid, single-celled
- Grow very rapidly, inexpensive
- 2가지 종류 사용
→ Saccaromyces cerevisiae; budding
→ Schizosaccaromyces pombe; fission
Laboratory of Molecular Genetics, KNU
Cloning the yeast origin of replication
- yeast genome cut restriction enzyme
- fragments clone into plasmid vector
- recombinant plasmids growth and
select
- yeast survive in media lacking leucine
- selected yeast cells contain leu2+ gene
Laboratory of Molecular Genetics, KNU
Nematoda worm
- Small and easy to keep
- Three days to develop
Multicellular organism
Caenorhabditis elegans
Fruit fly
Identification of mutant
→ mutation 특징 확인이 쉬움
- 짧은 시간에 많은 자손 생산
- 알에서 성체가 되기까지 12일 소요
- Drosophila melanogaster
Laboratory of Molecular Genetics, KNU
Zebrafish
- Reproduction rapidly and high number
- vertebrate
- 발생 과정이 사람과 유사
- 알이 투명하여 발달 확인 가능
Danio rerio
Amphibians
- 알의 크기가 큼 →발생학 연구에 이용
- 1920년 슈페만 형성체 발견
- Xenopus leavis
Laboratory of Molecular Genetics, KNU
Chicken
배 발생 연구에서 알 사용됨
; 진화상 인간과 유사
- 알이 크고, 분화 관찰에 좋음
- Gallus gallus
Mouse
- vertebrate, mammals
Human과 더욱 가까움
; 생리학, 발생학적으로 유사
- Expensive to maintain, Reproduce
slowly
Knock out mouse 이용
→ 특정 gene의 기능 확인
Laboratory of Molecular Genetics, KNU
Human cell culture
Human blood나 tissue에서 분리
Primary culture
; derived directly from living tissue
- Grow for a short period time and stop
- 세포 분열 횟수 제한
Plants
- Grow slowly, long generation time
- 세포벽 때문에 형질전환이 어려움
- Large genome
- Transposon 연구에 이용 ; corn
- Arabidopsis thaliana