BIO345

Cell & Molecular Biology

Lab
• Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under
controlled conditions. But in practice it refers to
the culturing of cells derived from animal cells.

• Cell culture was first successfully undertaken by

Ross Harrison in .)erutluc llec fo rehtaf(1907
• First development was the use of antibiotics
which inhibits the growth of contaminants.

• Second was the use of trypsin to remove adherent

cells to subculture further from the culture vessel.

• Third was the use of chemically defined

culture medium.
• Model systems:
Studying basic cell biology, interactions between
disease causing agents and cells, effects of drugs
on cells…

• Toxicity testing:
Studying the effects of new drugs, cosmetics
and chemicals on survival and growth of
cells.

• Virology:
Replication of viruses in cell culture instead of
animals for vaccine production.
• Genetic Engineering:
The ability to reprogram cultured cells with new genetic
material has provided a major tool for studying the
expression of these genes(new proteins).
Production of new proteins in large quantities as well
as production of viruses for use in vaccine production.

• Cancer research:
Studying the basic differences between normal
and cancer cells.
Studying the function of various chemicals, viruses &
radiation to convert normal cultured cells to cancerous
cells.
Determining suitable drugs and methods for
selectively destroying different types of cancer.
Animal cell culture techniques are:
• Routinely applied in many laboratories
•Allow the use of many applications formerly
inapplicable or only possible by in vivo experiments.

Such applications include:

• Production of monoclonal antibodies
• Gene knock-out and over-expression studies
•Production of valuable proteins (i.e. enzymes and
hormones)
 What is animal cell culture?
• Cell culture, very
roughly speaking,
consists of:
Growing cells in vitro
(plastic dishes)
Harvesting cells from plates
Splitting and seeding cells
on fresh plates and
Storing them by freezing
when necessary
(preferably in liquid
nitrogen: -196o C).
 What is animal cell culture?
• Animal cells are much more “demanding” than
prokaryotic cells and thus their maintenance
requires:

 Tedious and careful work

Carefully chosen and prepared solutions
and equipment
 Sterile and well conditioned environment
Carefully designed culture medium that mimics
in vivo conditions
Selection of appropriate cell type among many is
also crucial for the outcome of the experiment
 Pros Cons
• Removal of cells from their in
• Use of animals reduced vivo environment means
removing the cells,
hormones, support
• In vitro models allow for structures and various other
control of the chemicals that the cells
extracellular interact with in vivo.
environment
• It is nearly impossible to
• Able to monitor various recreate the in vivo
environment.
elements and
secretions without
interference from other • The artificial conditions could
cause cells to behave
biological molecules
differently and produce
that occurs in vivo proteins other than it would in
vivo
 Classification of cell culture

• Cell cultures are described in two

ways:

Origin of cells

Manner of growth
 Classification by Origin
• Primary cells: Cells that are surgically or
enzymatically removed from an organism and placed
in suitable culture environment, where they attach and
grow.
• Primary cells have a finite life span.

• Continuous cell lines: They are well established

stable cell lines, mostly isolated from cancerous
tissues.
• There are also some cell lines that are produced by
manipulating their genome of a primary cell to
overcome senescence; these cells are called
“immortalized”.
Classification by Manner of Growth

• Cells are classified by the way they grow in

liquid culture or in semisolid medium.

• There are two types of growth:

 Suspension growth
 Adherent growth
 Suspension Cells
• Cells which are anchorage independent.
• Grow suspended in the growth medium.
• Cells cultured from blood, spleen, or bone marrow
tend to grow in suspension.
• Look like shiny little balls.
• Advantages: - Grow in large numbers
- Easy to harvest
 Adherent Cells
• Cells which are anchorage dependent
• Grow in monolayer, attached to the surface of
culture vessels.
• Derived from ectodermal or endodermal embryonic
cell layers (e.g. fibroblasts, epithelial cells)
• Mainly have a flat shape
• Advantage: Ability to adhere and spread on surfaces
such as coverslips, making microscopy, hybridization,
and functional assays more easily performed.
 Types of Cells
• On the basis of morphology (shape & appearance) or
on their functional characteristics.

• They are divided into three types:

Epithelial like; attached to a substrate and appears
flattened and polygonal in shape
Lymphoblast like; cells do not attach remain in
suspension with a spherical shape
Fibroblast like; cells attached to a substrate appears
elongated and bipolar
 Common Cell Lines
• Human cell lines:
MCF-7: Breast Cancer
HL 60: Leukemia
HEK-293: Human Embryonic kidney
HeLa: Cervical Cancer (Henrietta lacks, died in 1951)
• 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 cells
Solutions used in cell
culture
Phosphate Buffered Saline - (PBS)
• Used to wash cells

• To remove excess serum

that inhibits the function of
Trypsin- EDTA.

• Must be warmed in the

water bath before use so
cells are not shocked by
cold liquid.
 Trypsin EDTA

• An enzyme used to detach

the cells from a culture
dish.

• Trypsin cleaves peptide

bonds in fibronectin of the
extracellular matrix.

• EDTA chelates calcium ions in

the media that would normally
inhibit trypsin.
 Trypan Blue
• An exclusion dye

• Living cells cannot take up

the dye and will appear
bright.

• Dead cells with broken

membranes will absorb the
dye and appear blue.
 Trypan Blue Exclusion Assay
• Dilute your cell sample in Trypan Blue dye by
preparing a 1:1 dilution of the cell suspension
using a 0.4% Trypan Blue solution
• Clean the hemocytometer with 70% ethanol
• Center coverslip on top of the hemocytometer
• Fill the grid under the coverslip using a pipette with
10 ul of cell suspension.
• Count cells
 Hemocytometer
• Specialized chamber
with etched grid used
to count the number of
cells in a sample.

• Use of Trypan blue

allows differentiation
between living and
dead cells.
 Using the Hemocytometer
• Clean hemacytometer with
EtOH
• Center coverslip on
hemocytometer
• Barely fill the grid under the
coverslip via the pipette with
your cell suspension.
• Count cells in 4 squares
Trypan Blue Exclusion Assay
 Trypan Blue

• Stained cells (dark blue) indicate that they

incorporated the dye  DEAD
• Unstainedcells (white) indicated that they did
not incorporate the dye VIABLE
• Calculate your cells/ml:
Calculate the number of total cells in one ml of
your suspension.
Total cells counted x (dilution factor)
x 104 of squares

• Determine your percent viability:

 Viability is a measure of how many of your
cells survived your cell culture technique
% Cell Viability = # of viable (living) cells x
100
Total number of cells counted
 Cell Plating
• Different types of plates (6 well, 12 well, 96 well)

• Each one is used for different type of

experiments

• Depending on the confluency of the cells

needed for each type of experiment
 Culture Media
• The most commonly used formula of
an animal cell culture medium
contains a “basal medium” and some
“other components” that support the
cell growth.

• There are defined and undefined

media recipes for cell culture:
Defined media: contains only basal
media, whose composition is known.
Undefined media: contains basal
media and serum.
• Fetal bovine serum (FBS):
is the most common type of
serum used in cell culture and it
is obtained from the fetuses of
cows thus its composition is not
exactly known.

• Contains:
Growth factors
Albumin
Transferrin
Anti-proteases
Attachment factors
 Culture Media
• The selection of basal medium is crucial for the
establishment of a healthy and well growing cell
culture.
• Different cell types require different composition of
substances and therefore different kinds of basal
media for their optimal growth.

• There are many basal media types, such as:

Eagle’s medium and its derivatives (EMEM,
AMEM, DMEM, GMEM, JMEM)
Rosewell Park Memorial Institute medium
derivates (RPMI 1629, RPMI 1630, RPMI 1640).
 Components of basal media
1. Balanced salt solutions:
• They contain inorganic salts to maintain physiological
pH and osmotic pressure, and to provide necessary
ions that are used for key metabolic activities
(membrane potential, cofactors) (Na+, K+, Mg2+, Ca2+,
Cl -, SO44+, PO43+, and HCO3-).

2. Buffering system:
• Mostly used media are buffered with bicarbonate ions.
• HCO3-/CO2 is one the most important buffering
systems of the blood.
• It is also possible to use organic buffers such as HEPES
(N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid).
3. Energy source: ex. Glucose. Other sugars can be used
(i.e. maltose, sucrose, fructose, galactose and manose).
4. Amino acids
5. Vitamins
6. Hormones and growth factors: (they are normally
present in serum but in defined media they are added to
the basal medium)
7. Proteins and peptides
8. Fatty acids and lipids
9. Accessory factors: ex. zinc, iron, copper, selenium
10. Antibiotics: ex. penicillin, streptomycin etc.
 Why sub culturing?
• Once the available substrate surface is covered
by cells (a confluent culture) growth slows &
ceases.

• Cells to be kept in healthy & in growing state have

to be sub-cultured or passaged.

• Cells are passaged in flasks/dishes/plates when

they reach 80-90% confluency.

• Enzyme such as trypsin, collagenase in

combination with EDTA breaks the cellular glue
that attached the cells to the surface.
 Confluency
• How “covered” the growing
surface appears
• This is usually a guess
• Optimal confluency for moving
cells to a new dish is 70-80%
 Too low, cells will be in lag
phase and won’t proliferate
 Too high and cells may
undergo unfavorable
changes and will be difficult
to remove from plate
 Passage number
• The number of times the cells have been
removed (or “split”) from the plate and re-
plated.
• Always write this on your plate or flask as P#
Equipment Used in Cell
Culture
 Cell Culture Hood
• Laminar flow hoods protect the working
environment from dust and other airborn
contaminants by maintaining a constant,
unidirectional flow of filtered air over the work
area.
Cell culture hood layout
 CO2 Incubators
• The cells are grown in an atmosphere of 5-10%
CO2 .
• Humidity and 37 ºC are also maintained by
this incubator
 Water bath
• To warm media and
PBS before placing on
cells.

• Can harbor fungi and

bacteria, spray all items
with 70% ethanol before
placing in the hood.

• Usually takes 10 -15

minutes for media to
warm
 Inverted microscope
• Light source and condenser on the top, above the
stage pointing down, while the objectives and
turret are below the stage pointing up.
• Inverted microscopes are useful for observing
living cells or organisms at the bottom of a
large container (e.g., a tissue culture flask)
under more natural conditions than on a glass
slide.
 Liquid nitrogen tanks
• Liquid N2 is used to preserve
tissue culture cells, either in the
liquid phase (-196°C) or in the
vapor phase (-156°C).

• To minimize the lethal effects of

freezing, a cryoprotective agent
which lowers the freezing point,
such as DMSO (dimethyl
sulfoxide), is added.
– A typical freezing medium is
90% serum, 10% DMSO.
Problems Faced by Culture
Cells
 Contaminant’s of cell culture
• Cell culture contaminants are of two types:

 Chemical: Difficult to detect since its caused by

endotoxins, plasticizers, metal ions or traces of
disinfectants that are invisible

 Biological:
-Fast growing yeast, bacteria and fungi usually have
visible effects on the culture (changes in medium
turbidity and pH, thus easy to detect).
-Mycoplasma and viruses are not easy to detect
visually (require special detection methods).
- Cross-contamination of cells from other cell lines
• In general indicators of contamination are:

- turbid culture media

- change in growth rates
- abnormally high pH
- poor attachment
- multi-nucleated cells,
- graining cellular appearance
- vacuolization
- inclusion bodies
- cell lysis
Basic Cell Culture
Instructions
 Aseptic Technique
• Work in a culture hood set-aside for tissue culture
purposes

• Turn off the UV/antimicrobial light and turn on the

hood 30 minutes prior to entering the hood

• Wear gloves

• Clean your hands and all surfaces with 70% ethanol

• Use only sterilized pipets, plates, flasks and bottles in

the hood for procedures.
 Observing cells in culture
• Check color of media:
 Healthy growth usually leaves media
slightly orange
 Too yellow means bacterial growth
 Too purple means low carbon dioxide or
cell death
• Observe cells under phase microscope:
 Spread out or rounded?
 How confluent?
 What to do with growing cells
• If the cells are at least 70-80% confluent:
Subculture them (Also called passing or splitting)
Remove media, remove cells, re-suspend and
transfer some to a new plate

• If they are not very confluent

“Feed” them (Remove old media and replace with
fresh, warm media)