Cell Culture

Master Program in Biomedical Sciences

Faculy of Medicine
Universitas Brawijaya

Main Points
Learn how to culture cells
Cell culture as a research tool

DEFINITION
Cell culture: Culture of cell from chemical or
enzymatic desegregation of tissue slice or from
suspension of liquid body.
Explants culture : tissue slice (1-3 mm) place in
substrate solid to form 3 dimension structure
called Histolytic culture.
Tissue culture: non desegregated tissue, 3
dimension structure still intact.

DEFINITION
1. Primary culture: Directly remove from organ,
and place in culture dish, never sub-culture.
2. Cell line: Primary culture which has been sub
cultured
- Finite cell line 20-80 generations, dies after
several sub culture
- Continuous cell line >100 generation.
transformed immortal

Primary cultures
Remove from organ, trypsin
digest and place in culture dish
Advantages
Similar chromosome
number as parent tissue
- Perform specialized
biochemical properties a
parent tissue
Mouse Embryonic Fibroblasts (MEF)

Disadvantages
Limited lifespan
- Low survival rate

Established cultures/ Cell line

Proliferate indefinitely through random mutation or
deliberate modification
Advantages
Many kinds of cell lines
Generally easy to grow and manipulate
Proliferate indefinitely
- Homogen
- Selection Cell strain
Disadvantages
Ploidy problems
Loss of biochemical properties of parent tissue
- In vitro transformation
ATCC (Amerika), DSM (Jerman), Japan Cell Bank

Established cell lines

Cell line

Meaning

Organism

HEK-293
HeLa
CHO
Sf-9

human embryonic kidney

Henrietta Lacks
Chinese hamster ovary
Spodoptera frugiperda

human
kidney (embryonic)
human
Cervical cancer
hamster
Ovary
Insect -Spodoptera frugiperda (Moth) Ovary

NIH-3T3
bEnd.5
MCF-10A
HMEC
MDCK II
COS-7
aethiops
HL-60
Jurkat

NIH, 3-day transfer, inoculum 3 x 105 cells

brain endothelial
mouse
Michigan Cancer Foundation human
human mammary epithelial cell human
Madin Darby canine kidney
dog
Cercopithecus aethiops, origin-defective SV-40
kidney
human leukemia
human
human

Origin tissue

mouse
embryo
brain
mammary gland
mammary gland
kidney
Ape -Cercopithecus
Myeloblast
T-Cell-Leukemia

Most adherent cells require attachment to

proliferate
Monolayer culture : adherent to substrate
(for survive and proliferation), form 1 layer
cells
Confluent culture: homogen and well
distributed
Suspension culture: came from cell that
can live and proliferate without adherent to
substrate

Common Cell Culture Surfaces

Biological Coatings
Collagen
Gelatin
Fibronectin
Laminin
Nonbiological Coatings
Poly-D-Lysine (PDL)
Polymer based plastics (treatments not coatings)
Surface modified polystyrene
Polycarbonate membranes

Progression of Cell Attachment & Adherence

Polystyrene Surface Treatment for Cell Culture

Untreated Polystyrene

--------------------

Untreated polystyrene surface

neutral
can be sterilized by irradiation
uncharged
hydrophobic
i.e. bacterial plates / Petri-dishes

Treated polystyrene surface

negatively charged
Tissue Culture Treated Polystyrene hydrophilic
(TCT)
regular cell culture plates

Surface Treated Polystyrene Oxygen Incorporation

Polystyrene a long carbon chain
polymer with benzene rings
attached to every other carbon
Polystyrene can be surface
modified by the addition of a
variety of different chemical
groups, breaking the carbon
chain backbone or opening the
benzene ring
Cell attachment correlates with
oxygen content
Treated polystyrene
~ increase oxygen content
~ addition of hydroxyl groups
~ addition of carboxyl groups
Result from treatments
~ more hydrophilic surface
~ negatively charged surface

HUVECs culture (mono layer, Fitri

LE 2004)

Stem Cells Culture

Self-renewing cells that with proper growth
conditions can differentiate into different cell
types with specific biological functions
Advantages
Can induce multiple cell types
Potential for therapeutics
Disadvantages
Ethical considerations
Most stem cells need to grow on feeder cells

Surfaces for Stem Cell Research

1. Ultra-Low Attachment Surface (ULA)
Embryoid body formation
Adult stem cell isolation and culture
2. Transwell Permeable Supports
Stem cell - co-culture and migration
Applications
3. CellBIND treatment
Improving Cell Attachment

Corning Ultra-Low Attachment Surface

Untreated polystyrene surface
Untreated Polystyrene

-----------------TC-treated Polystyrene

~ uncharged / neutral
~ hydrophobic

TC-treated polystyrene surface

~ negatively charged

~ hydrophilic

Ultra Low Attachment surface

Polystyrene with ULA Surface

~ uncharged / neutral covalent hydrogel

~ hydrophilic
~ low cell and protein attachment

Glioma Cell Growth on Ultra

Low Attachment

C6 rat glioma cell

colony (arrow) on the
Corning Ultra Low
Attachment surface

Glioma cell colonies on Ultra Low

Attachment surface (left panel) and on a
tissue culture treated polystyrene surface
(right panel)

In-vitro differentiation of embryonic stem

cells via embryoid body formation
Embryonic
stem cells
(ESC)

In vitro differentiation of Embryonic

Stem Cells
Isolation of undifferentiated cells in
suspension

Embryoid
Bodies (EB)

Formation of suspension aggregate

cells known as embryoid bodies
Embryoid Bodies (EB)
Dense, multilayered cell aggregates
Can differentiate into all three germ
layers
brain, neurons
hair, skin,
teeth,
ears, nose,
mouth,
pigment cells.

muscles, blood,

blood
vessels,
connective
tissues,
heart.

pancreas,
stomach,
liver, lungs,
bladder

Embryoid body (EB) formation is dependent on

surface chemistry
ULA Surface

Petri dish

EB Formation of R1 ES cells in suspension culture

Type of Vessel
Adherence
Standard 100 mm Petri dish
Corning ULA 6 well plate

Estimated
<5%
50 - 60%

Neural stem cells (NSC)

Potential therapeutic applications for spinal cord/brain injury
and neurodegenerative diseases (Parkinsons, Alzheimer's,
ALS, Multiple Sclerosis)
Early 1990s neural stem cells were isolated from adult and
fetal brain tissue from subventricular zone, ventricle zone,
and hippocampus
Neurosphere assay (NSA) is the most common method of
isolating and expanding neural stem cells
Aggregate in suspension to form a mixed population of
cells
Neurospheres retain pluripotency and can differentiate
into astrocytes, oligodendrocytes, and neurons

Neurosphere Assay (NSA) on ULA surface

Dissociate
embryonic
brain tissue

Mechanical or enzymatic
methods are used

Plate in serum-free
media+ EGF in
ULA vessels

Many cell types are initially

viable but die from anoikis
(inability to attach)

After 6 -8 days,
mechanically harvest
neurospheres

About 1% divide and form

neurospheres

Stem cell isolation and propagation from brain:

neurosphere assay
Neurosphere assay (NSA) a valuable tool for isolating and
understanding the biology of embryonic and adult CNS stem cells.

Adapted from:
Reynolds,
Nature Methods 5:333,
2005

Stem cell applications using Transwell

permeable supports

Differentiation of stem cells

Co-culture for paracrine effects
Migration assay (Homing of stem cells)

Transwell Permeable Supports

Polycarbonate (PC)
Membrane

Transwell Insert
Upper
Compartment
Porous Membrane
Lower Compartment

Collagen-coated
Polytetrafluoroethylene (PTFE)
Membrane

Polyester
(PET)
Membrane


CellBIND

Surface Review

Unique patented novel treatment for improved cell attachment

Developed to improved
conventional TC-treatment

attachment

of

cells

Non-biological treatment, not a coating

No special handling or storage requirements
Consistent and stable polymer surface

Electron Spectroscopy Chemical Analysis (ESCA)

60% increased oxygen incorporation
Lower contact angle, increased surface wettability

over

CellBIND Surface Review: Contact Angle

Contact Angle: Industry measure for surface consistency, hydrophobicity, and

wettability
CellBIND Surface exhibits higher and more consistent pre- to post-wash
wettability and increased stability

Pre-Wash 22

Pre-Wash 43

Post Wash 24

Post Wash 58

CellBIND

Standard TCT

Low Contact Angle

High Wettability

High Contact Angle

Low Wettability

CellBIND Surface Review

HEK-293 adaptation to reduced or serum free Media


CellBIND

Surface Review

More uniform cell attachment surface

Improves cell health and viability
Can replace expensive coatings
Can allow cell adaptation for reduced serum conditions
or serum free media
Improves cell retention for cell-based assay
Enhances primary cell attachment

The difference between in vitro culture system

and in vivo system:
1. Decrease matrix interaction among cells cause by cell
heterogenisity and 3 dimensional structure.
2. Environment of in vitro culture system does not have
homeostatic system component (endocrine system)
Cellular metabolism more stable
3. Energy metabolism occurs via glikolisis process
although citrate cycle still function.

FACTORS THAT INFLUENCE THE

GROWTH OF CELL CULTURE
Culture environment : including substrate,
medium, gas phase and temperature.
Substrate: a surface for cell adhesion, growth
and spread out. Example : hydrophobic
polystyrene
Medium: physical characteristic of the medium
(PH, buffer system , osmolarity, liquidity) and
medium composition (MEM-TCM 199)
Gas phase : depend on type of medium, culture
container, and buffer
Temperature: optimal 36,5 - 37oC

Cell culture medium

Inorganic ions
Osmotic balance cell volume
Trace Elements
Co-factors for biochemical pathways (Zn, Cu)
Amino Acids
- Protein synthesis
- Glutamine required at high concentrations
Vitamins
Metabolic co-enzymes for cell replication
Energy sources : glucose

Cell culture medium

Amino acids 2%
Inorganic salts
11%
Vitamins
1%
Sugars 5%
Other 3%
Serum 10%
Water 68%

Cell culture medium

Serum provides the following

(Horse serum, foetal calf serum, chick
embryo extract: all not fully defined)
Basic nutrients
Hormones and growth factors
Attachment and spreading factors
Binding proteins (albumin, vitronectin,
transferrin), vitamins, lipids
Protease inhibitors
pH buffer

pH Control
Physiological pH 7
pH can affect
Cell metabolism
Growth rate
Protein synthesis
Availability of nutrients
CO2 acts as a buffering agent in
combination with sodium bicarbonate
in the media

Essential equipment

Laminar flow hood (biological safety cabinet)

CO2 incubator (for most cells)
Inverted microscope
Pipette aid
Aspiration pump
Centrifuge
Water bath
Cold storage (refrigerator)
Cryopreservation equipment

Additional equipment

Sterilization equipment
Balance(s)
Vortex
Water purification
pH meter
Magnetic stirrer
Micropipettor(s)
Cell counter
Video camera and monitor

Hoods
No hood necessary if you have an
isolated clean room
Horizontal
Vertical or biological safety cabinets
Usually equipped with UV light for
sterilization of the work surface use it
BEFORE and AFTER not during work
Hood is not a storage area!

Horizontal vs. vertical hood

Horizontal
Maintains sterile working area
Air blows directly at the
investigator
Enough to protect your
cultures but not you

Horizontal vs. vertical hood

Vertical
Air is blown vertically in front of the investigator
Usually there is additional glass barrier in front of
the investigator
Air is recycled and goes through HEPA filter
before being returned to the room

Incubator
For most of mammalian cells
Temp = 36oC - 37oC
Humidity 95%
CO2 5%
All mammalian cells require CO2
incubator
Non mammalian cells might require
different culture conditions

Inverted microscope
It is vital to look at cultures regularly
A morphological change is often the
first sign of deterioration in culture
Inverted because it is not good to open
tissue culture dishes
It should have phase contrast
Most cells are not dense enough to be
visible in a regular light without staining

Choice of culture container

Majority of vertebrate cells in vitro grow as
mono-layers on an artificial substrate
Most cells need to spread out on a substrate in
order to proliferate
Overcrowding will inhibit proliferation but cells
dont like to be lonely
Cell yield is proportional to the available
surface
Equipment for cryopreservation
Liquid nitrogen
Liquid phase (-196oC) or vapor phase (-156oC)

Basic lab safety

Assume all cultures are hazardous since they
may harbor latent viruses or other organisms
that are uncharacterized
Use pipette aids to prevent ingestion and keep
aerosols down to a minimum
No eating, drinking, or smoking
Wash hands after handling cultures and before
leaving the lab
Decontaminate work surfaces with disinfectant
(before and after)
Properly dispose of biological and chemical
waste

Aseptic technique
Sterile Hood - All manipulations must be carried out in a
sterile cabinet
10-20 minutes prior to experiment turn on UV light and
blower
Turn the UV light off (you dont want to kill the cells or
sunburn your hands)
Open the cabinet
Wipe down with 70% ethanol
Bring materials into the hood
Wash all items inserted into the hood with 70% ethanol
when appropriate
Begin your work
Tightly close all bottles and caps
Remove materials from the hood
Clean after yourself !!!

Sterilization Techniques

Metal -A
Glass - A, R
Plastics A, R
Medium -R, F
Serum -R, F
Salt solutions - A, R, F

A-autoclaving, R-radiation, F-filtration

Filtration
Always filter through 0.2 m filter to remove
bacteria (bacteria are 1-2 m)
0.45 m filters remove only particulates
Mycoplasma and viruses will still filter through
Filters come in many sizes, all are
Cellulose, polycarbonate, PTFE, best are low
protein binding

Contamination
Happens to even the best
Fungal - yeast
Bacterial
Mycoplasma filterable
bacteria which will pass
across 0.2 m filter. Big
problem. It is treated with
cephalosporin antibiotics.
Can take over laboratories.

Procedure on Cell Culture

Preparing and aseptic technique (Sterilisation)
Material preparation (purify of the material/tissue
culture grade and water quality)
Initiation of Primary culture
Culture maintenance , cell propagation and cell
preservation

Initiation of Primary culture

Technique:
Mechanic (scalpel, syringe)
Chemistry (cation)
Enzymatic (tripsin, collagenase, pronase,
hyaluronidase)

Cell maintenance and cell

preservation
Medium change (decrease PH, cell concentration,
cell type and cell morphologic)
Subculture (Passage) and Propagation related to
proliferate and differentiated.
Cell preservation: the aim to minimal the change
of gene from continuous cell line

Passaging or sub-culture

Cells dissociated from flask

using enzymes

Split 1 into 2 flasks

Cell Culture: why do it?

Tool for the study of animal cell biology

using convenient in vitro model of cell growth.
Mimic of in vivo cell behaviour (e.g cancer cells)
Artificial (some cell types are thus difficult
to culture)
Highly selective and defined environment
which is easily manipulated (used to
optimize cell signaling pathways)

Cell Culture: why do it?

Advantages

Physical and chemical factors can be controlled

Characteristic and homogeneity of sample

Disadvatages

Does not have haemostatic system component

Need skill

Need special equipment

Relative expensive

Limited cell production

Cells are not stable

Culture cell in research

Make proteins:commercial scale
Intra-celullar activity (DNA, RNA, protein
sintesis) and Intra-celullar flow(RNA from
nucleus to cytoplasm,protein translocation)
Effect of environment ( nutrition, infection)
Communication and induction between cell
(contact inhibition, adhesion)
Antibody production: monoclonals
Gene Therapy
Pharmaceutical and toxicity.
Cell line production, transfection
Stem and Embryo culture production
Cancer cells and anti cancer

PRACTICAL CELLULAR IMMUNOLOGY

I. BASIC KNOWLEDGE OF CULTURE CELLS
II. BASIC CELL TECHNIQUES
2.1 Preparation of Lymphocytes from Blood
2.2 Measurement of Proliferative Responses
of Cultured Lymphocytes
2.3 Preparation of Phagocytic Cells from Blood
2.4 Variable Mononuclear Cell Count

III. ADVANCED CELL TECHNIQUES

3.1 Immunohistochemistry/
Immunophenotyping
3.2 Flowcytometry
3.3 Measurement of Cytokine
- Elisa
- Bioassay
3.4 Measurement of Surface Receptor

2.1 Preparation of Lymphocytes from Blood

PRINCIPLE:

DIFERENTIAL CENTRIFUGATION
ON A DENSITY GRADIENT GIVES
HIGH-PURITY LYMPHOCYTE

PERIPHERAL BLOOD

(Primary Source of Lymphoid Cells for Investigations of

Human Immunity System)

FICOLL-HYPAQUE DENSITY
GRADIENT CENTRIFUGATION

(A Simple and Rapid Method of Purifyng PBMC)

Mononuclear + Platelet Lower Density

Collect on The Top Layer
RBC + Granulocytes Higher Density
Collect on The Bottom

PROCEDURE:
CULTURE FOR MITOGEN-INDUCED
PROLIFERATION OF PERPHERAL BLOOD
MONONUCLEAR CELLS

Count PBMC 1x106 cells/ml

In RPMI 10 medium

Dispense 100 l cell suspesion into each well

Of 96 well round bottom microtiter plates

Add PHA 10 g/ml

Incubate 3 days in 37oC, 5% CO2 incubator

[3H] Thymidine

MTT-Assay

Gamma counter

ELISA Reader

Blast Transformation of Spleen Lymphocytes

Culture from Mice Immunized with Crude vaccine
of Plasmodium berghei (Fitri LE, 1996)

2.2 Measurement Of Proliferative Responses

Of Cultured Lymphocytes

FUNDAMENTAL TECHNIQUES FOR THE

ASSESMENT OF THEIR BIOLOGICAL
RESPONSES TO VARIOUS STIMULI

CELL PROLIFERATION
Radioisotope-Assays
Estimating
Incorporating
Of [3H] Thymidine
Into DNA

Measures the Number

Of Cells Synthesizing
DNA

Colorimetric-Assay
Estimating
Metabolism of the TetraZolium Salt (MTT) to The
Colored Formazan Product
By Mitochondrial Succine
Dehydrogenase Activity in
Viable Cells

Measures Energy
Metabolism of Living Cells

2.3 Preparation Of Phagocytic Cells From Blood

PRINCIPLE
MACROPHAGES CAN BE REMOVED FROM
A CELL SUSPENSION USING EITHER
THEIR ADHERENCE OR
PHAGOCYTIC PROPERTIES

2.3.1 Adherence to Sephadex/ Plastic/ Glass

PROCEDURE:
Pack 10 ml of sterile Sephadex into a 20 ml Syringe
Barrel fitted with a sintered plastic disc

Add a maximal 3x108 cells/ml allow to become

Included into collum bed

Wash out the non adherence cell with

20 ml of warm medium

ANALYSIS
I. MORPHOLOGICAL ALTERATION
II. FUNCTIONAL TEST
- Phagocytosis Assay (Latex Beads)
- Reactive Oxygen Intemediate Assay
- Cytokine Production Assay