Principles of Cell Culture

Istilah
• Tissue culture: a generic term to include rgan culture and cell culture
• Organ culture: a three-dimensional culture of undisaggregated
tissue retaining some of all of histological features of the tissue in
vivo
• Cell culture: a culture derived from dispersed cells taken from
original tissue, from a primary culture, or from a cell line or cell strain
by enzymatic, mechanical, or chemicaldisaggregation.
• Histotypic culture: the cells have been reaggregated to re-create a
three-dimensional tissue like structure e.g. By cultivation at high
density in a filter wall ...
• Organotypic: the same procedures but recombining cells of different
lineages e.g. Epidermal keratinocytes in combined culture with
dermal fibroblast.
 Penelitian yang menggunakan kulture jaringan:

1. Aktivitas intraseluler: Replikasi dan transkripsi DNA,

sintesis protein, metabolisme energi, metabolisme obat,
2. Intracellular flux: RNA, translokasi hormon receptors dll
3. Interaksi lingkungan: gizi, infeksi, toksisitas,
karsinogenesis dll
4. Interaksi antar sel: morpogenesis, kinetika proliferasi
sel dll
5. Genetik: analisis genom, manipulasi genetic dll
6. Produk sel: sekresi produk, bioteknologi, disain
bioreaktor dll.
 What is tissue culture?
• In vitro culture (maintain and/or proliferate)
of cells, tissues or organs
• Types of tissue culture
– Organ culture
– Tissue culture
– Cell culture

Experimental Methods 2006 4

 Organ culture

• The entire embryos or organs are excised

from the body and culture
• Advantages
– Normal physiological functions are maintained.
– Cells remain fully differentiated.
• Disadvantages
– Scale-up is not recommended.
– Growth is slow.
– Fresh explantation is required for every
experiment.
Experimental Methods 2006 5
 Tissue Culture
• Fragments of excised tissue are grown in culture
media
• Advantages
– Some normal functions may be maintained.
– Better than organ culture for scale-up but not ideal.
• Disadvantages
– Original organization of tissue is lost.

Experimental Methods 2006 6

 Cell Culture
• Tissue from an explant is dispersed, mostly
enzymatically, into a cell suspension which may
then be cultured as a monolayer or suspension
culture.
• Advantages
– Development of a cell line over several generations
– Scale-up is possible
• Disadvantages
– Cells may lose some differentiated characteristics.

Experimental Methods 2006 7

EMP04 8
What is Cell & Tissue Culture
• Tissue culture is the general name for the removal of
cells, tissues or organs from an animal or plant and
their subsequent placement into artificial environment
conductive to growth
• This environment usually consists of a suitable glass
or plastic culture vessel containing a liquid or semi-
solid support medium that supplies the nutrients
essential for survival and growth
• When the cells are removed from the organ
fragments, thus disrupting their normal relationship
with neighboring cells, it is called cell culture
 Experimental Methods 2006

Why do we need Cell culture?

• Research
• To overcome problems in studying cellular behavior
such as:
• confounding effects of the surrounding tissues
• variations that might arise in animals under experimental stress
• Reduce animal use
• Commercial or large-scale production
• Production of cell material: vaccine, antibody, hormone

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 Experimental Methods 2006

Cell culture application

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 Experimental Methods 2006

Advantages of Cell culture

• Advantages:
• Absolute control of physical environment
• Homogeneity of sample
• Less compound needed than in animal models
• Disadvantages:
• Hard to maintain
• Only grow small amount of tissue at high cost
• Dedifferentiation
• Instability, aneuploidy

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Classes of Culture Cells
• Cultures of animal cells are usually divided into 3
classes:
1. Primary cells
2. Cell strains
3. and cell lines
1- Primary Culture
• When cells are surgically removed from an
organism and placed into a suitable culture
environment they will attach, divide and grow
• Most of the primary culture cells have a finite
lifespan of 5-10 divisions in vitro
• Due to their limited lifespan, one cannot do long-
term experiments with these cells
• Primary cells are considered by many
researchers to be more physiologically similar to
in vivo cells
 1- Primary Culture
• There are two basic methods
for obtaining primary culture:
1. Explant cultures:
• Small pieces of tissue are
attached (using plasma clots or
fibrinogen) to a glass or treated
plastic culture vessel and
immersed in culture medium
• After a few days individual cells
will move from the tissue explant
out onto the culture vessel surface
or substrate where they will begin
to divide and grow
 1- Primary Culture
2. Enzymatic dissociation:
• More widely used
• speeds up the process by adding
digesting (proteolytic) enzymes
such as trypsin or collagenase to
the tissue fragments to dissolve
the cement holding the cells
together
• This creates a suspension of
single cells that are then placed
into culture vessels containing
culture medium and allowed to
grow and divide
 Hayflick’s Phenomenon
• Cells will continue to grow and divide
normally for a limited number of
passages
• When they get to a certain point
even if they are given the
appropriate nutrients, they simply
stop dividing and will eventually die
• There appears to be a correlation
between the maximal number of
passages and aging
• The number of passages decreases
when cells are harvested from older
individuals
2- Cell Strains
• Cell strains are cells that have been adapted to
culture but, unlike cell lines, have a finite division
potential
• Upon serial transfers of primary cells, a gradual
selection may occur until a particular cell type
becomes predominant
• If these cells continue to grow at a constant rate over
successive passages, these primary cells are
referred to as a cell strain
• These cells have a finite lifespan of 40-60 divisions in
vitro
• They are useful in vaccine production
3- Cell Lines
• If the cells in a cell strain undergo a transformation
process (spontaneous or induced changes in karyotype,
morphology or growth properties) that makes them
"immortal“ (able to divide indefinitely) they are called a cell
line
• It is not known how a diploid cell strain becomes a cell
line, although this event may be mimicked by infection
with oncogenic viruses or by exposure to chemical
carcinogens
• Cell Lines often have abnormal chromosome numbers
and maybe tumorigenic when inoculated into susceptible
animals
• Cell lines that have been derived from tumors often do not
exhibit contact-inhibition (inhibition of growth under
crowded conditions), but rather continue to pile-up
 Isolation of cell lines for in vitro culture

Resected Tissue

Cell or tissue culture in vitro

Primary culture
Sub-culture
Secondary culture
Sub-culture
Cell Line
Single cell isolation Successive sub-culture
Immortalization
Loss of control
Clonal cell line of cell growth Senescence
Transformed cell line
Immortalised cell line
 Cell morphologies vary depending on cell type

Fibroblastic Epithelial

Endothelial Neuronal
Cell Culture Morphology

• Morphologically cell cultures take one of two forms:

– growing in suspension (as single cells or small free-floating clumps)
• cell lines derived from blood (leukaemia, lymphoma)
– growing as a monolayer that is attached to the tissue culture flask.
• cells derived from solid tissue (lungs, kidney), endothelial, epithelial,
neuronal, fibroblasts
Hela-Epithelial BAE1-Endothelial

Experimental Methods 2006 22

MRC5-Fibroblast SHSY5Y-Neuronal
 Cell culture environment (in vitro)

What do cells need to grow?

• Substrate or liquid (cell culture flask or scaffold material)

chemically modified plastic or coated with ECM proteins
suspension culture

• Nutrients (culture media)

• Environment (CO2, temperature 37oC, humidity)

Oxygen tension maintained at atmospheric but can be varied

• Sterility (aseptic technique, antibiotics and antimycotics)

Mycoplasma tested
 Peralatan

Laminar
flow Hood

24 Dr.Saba
 Cell culture environment (in vitro)

Basal Media
• Maintain pH and osmolarity (260-320 mOsm/L).
• Provide nutrients and energy source.

Components of Basal Media

Inorganic Salts
• Maintain osmolarity
• Regulate membrane potential (Na+, K+, Ca2+)
• Ions for cell attachment and enzyme cofactors
pH Indicator – Phenol Red
• Optimum cell growth approx. pH 7.4
Buffers (Bicarbonate and HEPES)
• Bicarbonate buffered media requires CO2 atmosphere
• HEPES Strong chemical buffer range pH 7.2 – 7.6 (does not require CO2)
Glucose
• Energy Source
 Cell culture environment (in vitro)

Components of Basal Media

Keto acids (oxalacetate and pyruvate)

• Intermediate in Glycolysis/Krebs cycle
• Keto acids added to the media as additional energy source
• Maintain maximum cell metabolism

Carbohydrates
• Energy source
• Glucose and galactose
• Low (1 g/L) and high (4.5 g/L) concentrations of sugars in basal media

Vitamins
• Precursors for numerous co-factors
• B group vitamins necessary for cell growth and proliferation
• Common vitamins found in basal media is riboflavin, thiamine and biotin

Trace Elements
• Zinc, copper, selenium and tricarboxylic acid intermediates
Cell culture environment (in vitro)
Supplements

L-glutamine
• Essential amino acid (not synthesised by the cell)
• Energy source (citric acid cycle), used in protein synthesis
• Unstable in liquid media - added as a supplement

Non-essential amino acids (NEAA)

• Usually added to basic media compositions
• Energy source, used in protein synthesis
• May reduce metabolic burden on cells

Growth Factors and Hormones (e.g.: insulin)

• Stimulate glucose transport and utilisation
• Uptake of amino acids
• Maintenance of differentiation

Antibiotics and Antimycotics

• Penicillin, streptomycin, gentamicin, amphotericin B
• Reduce the risk of bacterial and fungal contamination
• Cells can become antibiotic resistant – changing phenotype
• Preferably avoided in long term culture
 Cell culture environment (in vitro)

Foetal Calf/Bovine Serum (FCS & FBS)

• Growth factors and hormones

• Aids cell attachment
• Binds and neutralise toxins
• Long history of use

• Infectious agents (prions)

• Variable composition
• Expensive
• Regulatory issues (to minimise risk)

Heat Inactivation (56oC for 30 mins) – why?

• Destruction of complement and immunoglobulins
• Destruction of some viruses (also gamma irradiated serum)

Care! Overdoing it can damage growth factors, hormones & vitamins

and affect cell growth
 How do we culture cells in the laboratory?

Revive frozen cell population

Isolate from tissue
Containment level 2
cell culture laboratory

Maintain in culture (aseptic technique)

Typical
cell culture flask

Sub-culture (passaging)

Count cells
‘Mr Frosty’
Used to freeze cells
Cryopreservation
 Growth Conditions
• 37°C Body temperature
• 20% O2 Optimal respiration
• 5% CO2 Works with bicarbonate
buffer to maintain pH of medium
• Humidification – Helps prevent
evaporation of culture media
from flasks, which would result in
an increase in osmotic pressure -
stresses or damages cells.
 Are your cells happy?
• Morphology – phase
contrast microscopy
• Expression of specialised
functions or markers
• Cell number, growth rate,
viability
– Haemocytometer
– Trypan blue
 Passaging Cells

Why passage cells?

• To maintain cells in culture (i.e. don’t overgrow)
Check confluency of cells • To increase cell number for experiments/storage

Remove spent medium How?

• 70-80% confluency
• Wash in PBS to remove dead cells and serum
Wash with PBS • Trypsin digests protein-surface interaction to
release cells (collagenase also useful)
• EDTA enhances trypsin activity
Incubate with • Resuspend in serum (inactivates trypsin)
trypsin/EDTA • Transfer dilute cell suspension to new flask
(fresh media)
• Most cell lines will adhere in approx. 3-4 hours

Resuspend in serum
containing media

Transfer to culture flask 70-80% confluence 100% confluence