Growing and Maintaining Cells in Culture

Cell culture is the process by which either prokaryotic or
eukaryotic cells are grown under controlled conditions.
In practice the term "cell culture" has come to refer to
the culturing of cells derived from multicellular
eukaryotes, especially animal cells. The historical
development and methods of cell culture are closely
interrelated to those of tissue culture and organ culture.
The growth of microorganisms such as bacteria and
yeast, or human, plant, or animal cells in the laboratory.
Cell cultures may be used to diagnose infections, to test
new drugs, and in research. The technique or process of
keeping tissue alive and growing in a culture medium.
Animal cell culture became a routine laboratory
technique in the 1950s, but the concept of maintaining
live cell lines separated from their original tissue source
was discovered in the 19th century.
1

Type of cell cultures:Type of cell cultures:
1.1. Primary cellsPrimary cells are cells taken directly from theare cells taken directly from the
live animal tissue (they typically are tough tolive animal tissue (they typically are tough to
keep alive in the Petri dish, and do not livekeep alive in the Petri dish, and do not live
very long; typical cell divides once and dies).very long; typical cell divides once and dies).
They only live if they can attach to bottom ofThey only live if they can attach to bottom of
Petri Dish (anchorage-dependent). ThePetri Dish (anchorage-dependent). The
bottom of the Petri Dish has a special layerbottom of the Petri Dish has a special layer
that cells can bind.that cells can bind.
2.2. Fetal cellFetal cell lines can live longer; cells dividinglines can live longer; cells dividing
20-80 times before they die.20-80 times before they die.
2

Cell culture techniques were advanced significantly
in the 1940s and 1950s to support research in
virology. Growing viruses in cell cultures allowed
preparation of purified viruses for the manufacture
of vaccines. The Salk polio vaccine was one of the
first products mass-produced using cell culture
techniques. This vaccine was made possible by the
cell culture research of John Franklin Enders,
Thomas Huckle Weller, and
Frederick Chapman Robbins, who were awarded a
Nobel Prize for their discovery of a method of
growing the virus in monkey kidney cell cultures
3

Concepts in mammalian cell culture
Isolation of cells
Cells can be isolated from tissues for ex vivo culture in
several ways. Cells can be easily purified from blood,
however only the white cells are capable of growth in
culture. Mononuclear cells can be released from soft
tissues by enzymatic digestion with enzymes such as
collagenase, trypsin, or pronase, which break down the
extracellular matrix. Alternatively, pieces of tissue can be
placed in growth media, and the cells that grow out are
available for culture. This method is known as
explant culture. A Cells that are cultured directly from a
subject are known as primary cells. With the exception of
some derived from tumours, most primary cell cultures
have limited lifespan. After a certain number of population
doublings cells undergo the process of senescence and
stop dividing, while generally retaining viability.
4

Extracellular matrix
In biology, extracellular matrix (ECM) is any material
part of a tissue that is not part of any cell. Extracellular
matrix is the defining feature of connective tissue.
1. Components
The ECM's main components are various
glycoproteins, proteoglycans and hyaluronic acid. In
most animals, the most abundant glycoproteins in the
ECM are collagens. In fact, collagen is the most
abundant protein in the human body.
5

ECM also contains many other components: proteins
such as fibrin, elastin, fibronectins, laminins, and
nidogens, and minerals such as hydroxylapatite, or
fluids such as blood plasma or serum with secreted
free flowing antigens.
In addition it sequesters a wide range of cellular growth
factors, and acts as a local depot for them. Changes in
physiological conditions can trigger protease
activities that cause local release of such depots. This
allows the rapid and local activation of cellular
functions, without de novo synthesis
6

Functions
Given this diversity, ECM can serve many functions,
such as providing support and anchorage for cells,
providing a way of separating the tissues, and
regulating intercellular communication. The ECM
regulates a cell's dynamic behavior.
3.Adhesion to matrix
Many cells bind to components of the extracellular
matrix. This cell-to-ECM adhesion is regulated by
specific cell surface cellular adhesion molecules (CAM)
known as integrins. The integrins transmit mechanical
stimuli from the ECM to the cytoskeleton
7

Senecence:
The word senescence is derived from the Latin word
senex, meaning "old man" or "old age."
(upper) Primary mouse embryonic
fibroblast cells (MEFs) before
senescence. Spindle-shaped. (lower)
MEFs became senescent after
passages. Cells grow larger, flatten
shape and expressed senescence-
associated β-galactosidase (SABG,
blue areas), a marker of cellular
senescence.
8

Cellular senescence is the phenomenon where normal
diploid differentiated cells lose the ability to divide. This
phenomenon is also known as "replicative senescence",
the "Hayflick phenomenon", or the Hayflick limit in
honour of Dr. Leonard Hayflick who was the first to
publish it in 1965. In response to DNA damage
(including shortened telomeres) cells either senesce or
self-destruct (apoptosis, programmed cell death) if the
damage cannot be repaired. In this 'cellular suicide', the
death of one, or more, cells may benefit the organism as
a whole
9

Maintaining cells in culture
Cells are grown and maintained at an appropriate temperature and gas
mixture (typically, 37°C, 5% CO2) in a cell incubator. Culture conditions
vary widely for each cell type, and variation of conditions for a
particular cell type can result in different phenotypes being expressed.
Aside from temperature and gas mixture, the most commonly varied
factor in culture systems is the growth medium. Recipes for growth
media can vary in pH, glucose concentration, growth factors, and the
presence of other nutrient components. The growth factors used to
supplement media are often derived from animal blood, such as calf
serum. These blood-derived ingredients pose the potential for
contamination of derived pharmaceutical products with viruses or
prions. Current practice is to minimize or eliminate the use of these
ingredients where possible.
10

Some cells naturally live without attaching to a surface,
such as cells that exist in the bloodstream. Others
require a surface, such as most cells derived from
solid tissues. Cells grown unattached to a surface are
referred to as suspension cultures. Other adherent
cultures cells can be grown on tissue culture plastic,
which may be coated with extracellular matrix
components (e.g. collagen or fibronectin) to increase
its adhesion properties and provide other signals
needed for growth.
11

Growth factor
The term growth factor refers to a naturally occurring
protein capable of stimulating cellular proliferation and
cellular differentiation. Growth factors are important for
regulating a variety of cellular processes.
Growth factors typically act as signaling molecules
between cells. Examples are cytokines and hormones
that bind to specific receptors on the surface of their
target cells.
They often promote cell differentiation and maturation,
which varies between growth factors. For example,
bone morphogenic proteins stimulate bone cell
differentiation, while vascular endothelial growth factors
stimulate blood vessel differentiation.
12

Example of growth factors
Individual growth factor proteins tend to occur as members of larger
families of structurally and evolutionarily related proteins. There
are dozens and dozens of growth factor families such as TGF-beta (
transforming growth factor-beta), BMP (bone morphogenic protein
), neurotrophins (NGF, BDNF, and NT3), fibroblast growth factor
(FGF), and so on.
Several well known growth factors are:
Transforming growth factor beta (TGF-β)
Granulocyte-colony stimulating factor (G-CSF)
Granulocyte-macrophage colony stimulating factor (GM-CSF)
Nerve growth factor (NGF)
Neurotrophins
Platelet-derived growth factor (PDGF)
13

Erythropoietin (EPO)
Thrombopoietin (TPO)
Myostatin (GDF-8)
Growth differentiation factor-9 (GDF9)
Basic fibroblast growth factor (bFGF or FGF2)
Epidermal growth factor (EGF)
Hepatocyte growth factor (HGF]
14

Cytokines are a group of proteins and peptides that
are used in organisms as signaling compounds.
These chemical signals are similar to hormones and
neurotransmitters and are used to allow one cell to
communicate with another. The cytokine family
consists mainly of smaller water-soluble proteins
and glycoproteins (proteins with an added sugar
chain) with a mass of between 8 and 30 kDa. While
hormones are released from specific organs into the
blood and neurotransmitters are released by nerves,
cytokines are released by many types of cells. They
are particularly important in both innate and
adaptive immune responses
15

Due to their central role in the immune system,
cytokines are involved in a variety of immunological,
inflammatory and infectious diseases. However, not all
their functions are limited to the immune system, as
they are also involved in several developmental
processes during embryogenesis.
Cytokines are produced by a wide variety of cell types
(both haemopoietic and non-haemopoietic) and can
have effects on both nearby cells or throughout the
organism. Sometimes these effects are strongly
dependent on the presence of other chemicals and
cytokines.
16

Effects
Each cytokine binds to a specific cell-surface receptor. Subsequent
cascades of intracellular signalling then alter cell functions. This
may include the upregulation and/or downregulation of several
genes and their transcription factors, in turn resulting in the
production of other cytokines, an increase in the number of surface
receptors for other molecules, or the suppression of their own
effect by feedback inhibition.
The effect of a particular cytokine on a given cell depends on the
cytokine, its extracellular abundance, the presence/abundance of
the complementary receptor on the cell surface, and downstream
signals activated by receptor binding; these last two factors can
vary by cell type. Interestingly, cytokines are characterized by
considerable "redundancy", in that many cytokines appear to share
similar functions.
17

Generalization of functions is not possible with
cytokines; nonetheless, their actions may be comfortably
grouped as:
autocrine, if the cytokine acts on the cell that secretes it
paracrine, if the action is restricted to the immediate
vicinity of a cytokine's secretion
endocrine, if the cytokine diffuses to distant regions of
the body (carried by blood or plasma) to affect different
tissues.
Nomenclature
Cytokines have been variously named as lymphokines,
interleukins and chemokines, based on their presumed
function, cell of secretion or target of action
18

Manipulation of cultured cellsManipulation of cultured cells
As cells generally continue to divide in culture, they generally grow to fillAs cells generally continue to divide in culture, they generally grow to fill
the available area or volume. This can generate several issues:the available area or volume. This can generate several issues:
Nutrient depletion in the growth mediaNutrient depletion in the growth media
Accumulation ofAccumulation of apoptoticapoptotic//necroticnecrotic (dead) cells.(dead) cells.
Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stopCell-to-cell contact can stimulate cell cycle arrest, causing cells to stop
dividing known asdividing known as contact inhibitioncontact inhibition..
Cell-to-cell contact can stimulate promiscuous and unwantedCell-to-cell contact can stimulate promiscuous and unwanted
cellular differentiationcellular differentiation..
These issues can be dealt with using tissue culture methods that rely onThese issues can be dealt with using tissue culture methods that rely on
sterile techniquesterile technique. These methods aim to avoid contamination with. These methods aim to avoid contamination with
bacteria or yeast that will compete with mammalian cells for nutrientsbacteria or yeast that will compete with mammalian cells for nutrients
and/or cause cell infection and cell death. Manipulations are typicallyand/or cause cell infection and cell death. Manipulations are typically
carried out in acarried out in a biosafety hoodbiosafety hood oror laminar flow cabinetlaminar flow cabinet to excludeto exclude
contaminating micro-organisms.contaminating micro-organisms. AntibioticsAntibiotics can also be added to thecan also be added to the
growth media.growth media.
Amongst the common manipulations carried out on culture cells areAmongst the common manipulations carried out on culture cells are
media changes, passaging cells, and transfecting cellsmedia changes, passaging cells, and transfecting cells
19

Media changes
The purpose of media changes is to replenish
nutrients and avoid the build up of potentially
harmful metabolic byproducts and dead cells.
In the case of suspension cultures, cells can be
separated from the media by centrifugation and
resuspended in fresh media. In the case of
adherent cultures, the media can be removed
directly by aspiration and replaced
20

Passaging cells
Passaging or splitting cells involves transferring a small
number of cells into a new vessel. Cells can be cultured
for a longer time if they are split regularly, as it avoids the
senescence associated with prolonged high cell density.
Suspension cultures are easily passaged with a small
amount of culture containing a few cells diluted in a
larger volume of fresh media. For adherent cultures, cells
first need to be detached; this was historically done with
a mixture of trypsin-EDTA, however other enzyme mixes
are now available for this purpose. A small number of
detached cells can then be used to seed a new culture.
21

Transfection and transduction
Another common method for manipulating cells
involves the introduction of foreign DNA by
transfection. This is often performed to cause cells to
express a protein of interest. More recently, the
transfection of RNAi constructs have been realised as a
convenient mechanism for suppressing the expression
of a particular gene/protein.
DNA can also be inserted into cells using viruses, in
methods referred to as transduction, infection or
transformation. Viruses, as parasitic agents, are well
suited to introducing DNA into cells, as this is a part of
their normal course of reproduction.
22

Transfection describes the introduction of foreign
material into eukaryotic cells. Transfection typically
involves opening transient pores or 'holes' in the cell
plasma membrane, to allow the uptake of material.
Genetic material (such as supercoiled plasmid DNA or
siRNA constructs), or even proteins such as antibodies,
may be transfected. Transfection is frequently carried
out by mixing a cationic lipid with the material to
produce liposomes, which fuse with the cell plasma
membrane and deposit their cargo inside.
23

The term transfection is most often used in reference to
mammalian cells, while the term transformation is used
for the same process in bacteria and, occasionally,
plants. The original meaning of transfection was
'infection by transformation', i.e. introduction of DNA (or
RNA) from a eukaryote virus or bacteriophage into cells,
resulting in an infection. Because the term transformation
had another sense in eukaryotic cell biology (a genetic
change allowing long-term propagation in culture, or
acquisition of properties typical of cancer cells), the term
transfection acquired, for animal cells, its present
meaning of a change in cell properties caused by
introduction of DNA.
Cell 2004 24

Methods
There are various methods of introducing foreign DNA into a
eukaryote cell. One of the cheapest (and least reliable) is
transfection by calcium phosphate, originally discovered by
S. Bacchetti and F. L. Graham in 1977. HEPES-buffered saline
solution (HeBS) containing phosphate ions is combined with
a calcium chloride solution containing the DNA to be
transfected. When the two are combined, a fine precipitate of
calcium phosphate will form, binding the DNA to be
transfected on its surface. The suspension of the precipitate
is then added to the cells to be transfected (usually a cell
culture grown in a monolayer). By a process not entirely
understood, the cells take up some of the precipitate, and
with it, the DNA. Other methods of transfection include
electroporation, heat shock, magnetofection, nucleofection
and proprietary transfection reagents such as Lipofectamine,
Fugene.
25

Other methods use highly branched organic compounds,
so-called dendrimers, to bind the DNA and get it into the
cell. A very efficient method is the inclusion of the DNA to
be transfected in liposomes, i.e. small, membrane-
bounded bodies that are in some ways similar to the
structure of a cell and can actually fuse with the
cell membrane, releasing the DNA into the cell. For
eukaryotic cells, lipid-cation based transfection is more
typically used, because the cells are more sensitive.
Another method is DEAE-Dextran transfection, in which a
mutant plasmid and Luciferase reporter are used. Dextran
is positively charged. A cell performs endocytosis to take
in the positively charged Dextran, which is attracted to the
DNA (negatively charged).
26

A direct approach to transfection is the gene gun, where
the DNA is coupled to a nanoparticle of an inert solid
(commonly gold) which is then "shot" directly into the
target cell's nucleus. DNA can also be introduced into cells
using viruses as a carrier. In such cases, the technique is
called viral transduction, and, the cells are said to be
transduced.
Stable and transient transfection
For most applications of transfection, it is sufficient if the
transfected gene is only transiently expressed. Since the
DNA introduced in the transfection process is usually not
inserted into the nuclear genome, the foreign DNA is lost
at the later stage when the cells undergo mitosis. If it is
desired that the transfected gene actually remains in the
genome of the cell and its daughter cells, a stable
transfection must occur.
27

To accomplish this, another gene is co-transfected, which
gives the cell some selection advantage, such as
resistance towards a certain toxin. Some (very few) of the
transfected cells will, by chance, have inserted the foreign
genetic material into their genome. If the toxin, towards
which the co-transfected gene offers resistance, is then
added to the cell culture, only those few cells with the
foreign genes inserted into their genome will be able to
proliferate, while other cells will die. After applying this
selection pressure for some time, only the cells with a
stable transfection remain and can be cultivated further.
A common agent for stable transfection is Geneticin, also
known as G418, which is a toxin that can be neutralized by
the product of the neomycin resistant gene.
28

Transformation .
In molecular biology, Transformation is the genetic
alteration of a cell resulting from the uptake and
expression of foreign genetic material (DNA). Separate
terms are used for genetic alterations resulting from
introduction of DNA by plasmid-encoded conjugation or by
viruses (transduction). Transformation of animal cells is
usually called transfection.
The term transformation is also used more generally to
describe mechanisms of DNA and RNA transfer in
molecular biology (i.e. not only the genetic consequences).
For example the production of transgenic plants such as
transgenic maize requires the insertion of new genetic
information into the maize genome using an appropriate
mechanism for DNA transfer; the process is commonly
referred to as transformation.
29

RNA molecules may also be transferred into cells using
similar methods, but this does not normally produce
heritable change and so is not true transformation.
Bacteria
In bacteria, transformation refers to a stable genetic
change brought about by taking up naked DNA (DNA
without associated cells or proteins), and competence
refers to the state of being able to take up exogenous DNA
from the environment. Two different Natural competence
Some bacteria (around 1% of all species) are naturally
capable of taking up DNA under laboratory conditions;
many more may be able to take it up in their natural
environments . Such species carry sets of genes
specifying machinery for bringing DNA across the cell's
membrane or membranes
30

Artificial competence
Artificial competence is not encoded in the cell's genes. Instead it is
induced by laboratory procedures in which cells are passively made
permeable to DNA, using conditions that do not normally occur in
nature. These procedures are comparatively easy and simple, and
are widely used to introduce plasmids and genetically engineered
bacteria. Artificially competent cells of standard bacterial strains
may also be purchased frozen, ready to use.
Chilling cells in the presence of divalent cations such as Ca2+ (in
CaCl2) prepares the cell walls to become permeable to plasmid DNA.
Cells are incubated on ice with the DNA and then briefly heat
shocked (42oC for 30-120 seconds), which causes the DNA to enter
the cell. This method works very well for circular plasmid DNAs. An
excellent preparation of competent cells will give ~108 colonies per
μg of plasmid. A poor preparation will be about 104/μg or less. Good
non-commercial preps should give 105 to 106 transformants per
microgram of plasmid.
31

The method does not work for linear molecules such
as fragments of chromosomal DNA, probably because
exonuclease enzymes in the cell rapidly degrade
linear DNAs. Cells that are naturally competent are
usually transformed more efficiently with linear DNA
than with plasmids.
Electroporation is another way to make holes in
bacterial (and other) cells, by briefly shocking them
with an electric field of 10-20kV/cm. Provided plasmid
DNA is present when the shock is applied, it can enter
the cell through these holes. Natural membrane-repair
mechanisms will rapidly close these holes after the
shock.
32

Electroporation: make transient holes in cell membranes
using electric shock; this allows DNA to enter as
described above for Bacteria.
Viral transformation (transduction): Packages the desired
genetic material into a suitable plant virus and is allow
this modified virus to infect the plant. If the genetic
material is DNA, it can recombine with the chromosomes
to produce transformant cells. However genomes of most
plant viruses consist of single stranded RNA which
replicates in the cytoplasm of infected cell. For such
genomes this method is a form of transfection and not a
real transformation, since the inserted genes never reach
the nucleus of the cell and do not integrate into the host
genome. The progeny of the infected plants is virus free
and also free of the inserted gene.
33

Transfection of mammalian cells by electrophoration
1. Grow cells to ~ 70-80 % confluence
2. Trypsinize and resuspend them in complete
medium ( 10 7 cells/ml)
3. Combine DNA (40ug) with resistance (2ug) plasmid
(neo or Hyg) and mix.
4. Electrophorate in a gene pulser@950uF and
o.22KV/cm(t=20-30ms)
5. Perform duplicate electrophorations.
6. Plate cells in six 10 cm plates as follows
1. 2000ul 5x10 5
2.1200ul 3x10 5
3. 800ul 2x 10 5
4. 400ul 1x 10 5
5. 200ul 5x 10 4
6. 100ul 2.5x 10 4 34

LIPOFACTAMINE:
1. Wash the cell once with serum free medium.
2. Seed the cell (2-3 x 106/well) in six well plate in 0.8
ml serum free medium.
3. Prepare the solutions in 12x 75 mm sterile tubes;
Solution A: For each tarnsfection dilute 1-3 ug of
DNA into 100ul serum free medium (OPTI-MEM).
Solution B: For each Transfection dilute 2-25 of
LIPOFECTAMINE reagent into 100 ul of serum free
medium.
4. Combine the two solutions, mix gently and incubate
2 hr at 37.C .
5. Following incubation add 4 ml complete medium to
each well and incubate at incubator and add DNA
mixture gently and incubate 24-72 hr
35

Basic Techniques - The "Do's
and Don'ts" of Cell Culture
Given below are a few of the essential
"do’s and don'ts" of cell culture.
Some of these are mandatory e.g.
use of personal protective equipment
(PPE). Many of them are common
sense and apply to all laboratory
areas. However some of them are
specific to tissue culture.
36

The Do’s
1. Use personal protective equipment, (laboratory
coat/gown, gloves and eye protection) at all times.
In addition, thermally insulated gloves, full-face
visor and splash-proof apron should be worn when
handling liquid nitrogen.
2. Always use disposable caps to cover hair.
3. Wear dedicated PPE for tissue culture facility and
keep separate from PPE worn in the general
laboratory environment. The use of different
colored gowns or laboratory coats makes this
easier to enforce.
4. Keep all work surfaces free of clutter.
37

5.Correctly label reagents including flasks, medium
and ampules with contents and date of
preparation.
6.Only handle one cell line at a time. This common-
sense point will reduce the possibility of cross
contamination by mislabeling etc. It will also
reduce the spread of bacteria and mycoplasma by
the generation of aerosols across numerous
opened media bottles and flasks in the cabinet.
7.Clean the work surfaces with a suitable
disinfectant (e.g. 70% ethanol) between operations
and allow a minimum of 15 minutes between
handling different cell lines.
8.Wherever possible maintain separate bottles of
media for each cell line in cultivation.
38

9. Examine cultures and media daily for evidence of
gross bacterial or fungal contamination. This
includes medium that has been purchased
commercially.
10. Quality Control all media and reagents prior to use.
11. Keep cardboard packaging to a minimum in all cell
culture areas.
12. Ensure that incubators, cabinet, centrifuges and
microscopes are cleaned and serviced at regular
intervals.
13. Test cells for mycoplasma on a regular basis.
39

The Don’ts
1. Do not continuously use antibiotics in culture
medium as this will inevitably lead to the
appearance of antibiotic resistant strains and
may render a cell line useless for commercial
purposes.
2. Don’t allow waste to accumulate particularly
within the microbiological safety cabinet or in
the incubators.
3. Don't have too many people in the lab at any
one time.
4. Don't handle cells from unauthenticated
sources in the main cell culture suite. They
should be handled in quarantine until quality
control checks are complete.
5. Avoid keeping cell lines continually in culture
without returning to frozen stock
40

The Don’ts
1. Avoid cell culture becoming fully confluent.
Always sub-culture at 70-80% confluency or
as advised on cell culture data sheet.
2. Do not allow media to go out of date. Shelf
life is only 6 weeks at +4ºC once glutamine
and serum is added.
3. Avoid water baths from becoming dirty .
4. Don’t allow essential equipment to become
out of calibration. Ensure microbiological
safety cabinets are tested regularly.
41

Aseptic Technique and Good
Cell Culture Practice
• Aim
To ensure all cell culture procedures are performed to a
standard that will prevent contamination from bacteria, fungi and
mycoplasma and cross contamination with other cell lines.
• Materials
• Chloros / Presept solution (2.5g/l)
• 1% formaldehyde based disinfectant e.g.Virkon,Tegador
• 70% ethanol in water .
• Equipment
• Personal protective equipment (sterile gloves, laboratory coat,
safety visor)
• Microbiological safety cabinet at appropriate containment level
42

Procedure
1. Sanitize the cabinet using 70% ethanol before commencing
work.
2. Sanitize gloves by washing them in 70% ethanol and allowing
to air dry for 30 seconds before commencing work.
3. Put all materials and equipment into the cabinet prior to
starting work after sanitizing the exterior surfaces with 70%
ethanol.
4. Whilst working do not contaminate gloves by touching
anything outside the cabinet (especially face and hair). If
gloves become contaminated re-sanitize with 70% ethanol as
above before proceeding.
5. Discard gloves after handling contaminated cultures and at
the end of all cell culture procedures.
43

Procedure
6. Equipment in the cabinet or that which will be taken into the
cabinet during cell culture procedures (media bottles, pipette
tip boxes, pipette aids) should be wiped with tissue soaked
with 70% ethanol prior to use.
7. Movement within and immediately outside the cabinet must
not be rapid. Slow movement will allow the air within the
cabinet to circulate properly.
8. Speech, sneezing and coughing must be directed away from
the cabinet so as not to disrupt the airflow.
9. After completing work disinfect all equipment and material
before removing from the cabinet. Spray the work surfaces
inside the cabinet with 70% ethanol and wipe dry with tissue.
Dispose of tissue by autoclaving.
44

Procedure
10. Cell culture discard in chloros must be kept in the
cabinet for a minimum of two hours (preferably
overnight) prior to discarding down the sink with
copious amounts of water.
11. Periodically clean the cabinet surfaces with a
disinfectant such as Presept,Tegador or Virkon or
fumigate the cabinet according to the
manufacturers instructions. However you must
ensure that it is safe to fumigate your own
laboratory environment due to the generation of
gaseous formaldehyde, consult your on-site Health
and Safety Advisor.
45

Resuscitation of Frozen Cell
Lines
• Aim
Many cultures obtained from a culture collection,
such as ATCC, will arrive frozen and in order to use
them the cells must be thawed and put into culture. It
is vital to thaw cells correctly in order to maintain the
viability of the culture and enable the culture to
recover more quickly. Some cryoprotectants, such as
DMSO , are toxic above 4ºC therefore it is essential
that cultures are thawed quickly and diluted in culture
medium to minimize the toxic effects.
46

Resuscitation of Frozen Cell
Lines
• Materials
• Media– pre-warmed to the appropriate temperature (refer to the ECACC Cell
Line Data Sheet for the correct medium and size of flask to resuscitation into.)
• 70% ethanol in water
• DMSO
• Equipment
• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• CO2
incubator
• Pre labeled flasks
• Marker Pen
• Pipettes
• Ampule Rack
• Tissue
47

Procedure
1. Read Technical data sheet to establish specific requirements
for your cell line.
2. Prepare the flasks as appropriate (information on technical
data sheet). Label with cell line name, passage number and
date.
3. Collect ampule of cells from liquid nitrogen storage wearing
appropriate protective equipment and transfer to laboratory in
a sealed container.
4. Still wearing protective clothing, remove ampule from
container and place in a water bath at an appropriate
temperature for your cell line e.g. 37ºC for mammalian cells.
Submerge only the lower half of the ampule. Allow to thaw
until a small amount of ice remains in the vial - usually 1-2
minutes. Transfer to class II safety cabinet.
48

Procedure
5. Wipe the outside of the ampule with a tissue
moistened (not excessively) with 70% alcohol hold
tissue over ampule to loosen lid.
6. Slowly, dropwise, pipette cells into pre-warmed
growth medium to dilute out the DMSO (flasks
prepared in Step 2).
7. Incubate at the appropriate temperature for species
and appropriate concentration of CO2
in
atmosphere.
8. Examine cells microscopically (phase contrast)
after 24 hours and sub-culture as necessary
49

Key Points
1. Most text books recommend washing the thawed cells in
media to remove the cryoprotectant. This is only necessary if
the cryoprotectant is known to have an adverse effect on the
cells. In such cases the cells should be washed in media
before being added to their final culture flasks.
2. Do not use an incubator to thaw cell cultures since the rate of
thawing achieved is too slow resulting in a loss of viability.
3. For some cultures it is necessary to subculture before
confluence is reached in order to maintain their
characteristics e.g. the contact inhibition of NIH 3T3 cells is
lost if they are allowed to reach confluence repeatedly
50

Subculture of Adherent Cell
Lines
• Aim
Adherent cell lines will grow in vitro until they have covered the
surface area available or the medium is depleted of nutrients. At
this point the cell lines should be sub-cultured in order to
prevent the culture dying. To subculture the cells they need to
be brought into suspension. The degree of adhesion varies from
cell line to cell line but in the majority of cases proteases, e.g.
trypsin, are used to release the cells from the flask. However,
this may not be appropriate for some lines where exposure to
proteases is harmful or where the enzymes used remove
membrane markers/receptors of interest. In these cases cells
should be brought into suspension into a small volume of
medium mechanically with the aid of cell scrapers
51

Subculture of Adherent Cell
Lines
• Materials
• Media– pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct
medium)
• PBS without Ca2+
/Mg2+
• 0.25% trypsin/EDTA in HBSS, without Ca2+
/Mg2+
• Trypsin
• Soybean trypsin Inhibitor
• Equipment
• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• CO2
incubator
• Pre-labeled flasks
• Marker Pen
• Pipettes
• Ampule Rack
• Tissue
52

Procedure
1. View cultures using an inverted microscope to assess the
degree of confluency and confirm the absence of bacterial
and fungal contaminants.
2. Remove spent medium.
3. Wash the cell monolayer with PBS without Ca2+
/Mg2+
using a
volume equivalent to half the volume of culture medium.
Repeat this wash step if the cells are known to adhere
strongly.
4. Pipette trypsin/EDTA onto the washed cell monolayer using
1ml per 25cm2
of surface area. Rotate flask to cover the
monolayer with trypsin. Decant the excess trypsin.
5. Return flask to the incubator and leave for 2-10 minutes.
53

Procedure
6. Examine the cells using an inverted microscope to ensure that
all the cells are detached and floating. The side of the flasks
may be gently tapped to release any remaining attached
cells.
7. Resuspend the cells in a small volume of fresh serum-
containing medium to inactivate the trypsin. Remove 100-
200uL and perform a cell count .
8. Transfer the required number of cells to a new labeled flask
containing pre-warmed medium (refer to Cell Line Data Sheet
for the required seeding density).
9. Incubate as appropriate for the cell line.
10. Repeat this process as demanded by the growth
characteristics of the cell line.
54

Key Points
1. Some cultures whilst growing as attached lines adhere only
lightly to the flask, thus it is important to ensure that the culture
medium is retained and the flasks are handled with care to
prevent the cells detaching prematurely.
2. Although most cells will detach in the presence of trypsin alone
the EDTA is added to enhance the activity of the enzyme.
3. Trypsin is inactivated in the presence of serum. Therefore, it is
essential to remove all traces of serum from the culture medium
by washing the monolayer of cells with PBS without Ca2+
/Mg2
.
4. Cells should only be exposed to trypsin/EDTA long enough to
detach cells. Prolonged exposure could damage surface
receptors.
5. Trypsin should be neutralized with serum prior to seeding cells
into new flasks otherwise cells will not attach
55

Key Points
6. Trypsin may also be neutralized by
the addition of soybean trypsin
inhibitor , where an equal volume of
inhibitor at a concentration of 1mg/ml
is added to the trypsinised cells. The
cells are then centrifuged,
resuspended in fresh culture medium
and counted . This is especially
necessary for serum-free cell culture.
56

Subculture of Semi-Adherent
Cell Lines
• Aim
Some cultures grow as a mixed
population (e.g. B95-8 ) where a
proportion of cells do not attach to the
tissue culture flask and remain in
suspension. Therefore to maintain this
heterogeneity both the attached cells
and the cells in suspension must be
subcultured.
57

Procedure
1. View cultures using an inverted phase contrast microscope to
assess the degree of confluency and confirm the absence of
bacterial and fungal contaminants. Give the flask a gentle
knock first, this may dislodge the cells from the flask and
remove the need for a trypsinisation step with the subsequent
loss of some cells due to the washings.
2. Decant spent medium into a sterile centrifuge tube and retain.
3. Wash any remaining attached cells with PBS without Ca2+
/Mg2+
using 1-2ml for each 25cm2
of surface area. Retain the
washings.
4. Pipette trypsin/EDTA onto the washed cell monolayer using
1ml per 25cm2
of surface area. Rotate flask to cover the
monolayer with trypsin. Decant the excess trypsin.
5. Return flask to incubator and leave for 2-10 minutes
58

Procedure
6. Examine the cells using an inverted microscope to ensure that
all the cells are detached and floating. The side of the flasks
may be gently tapped to release any remaining attached cells.
7. Transfer the cells into the centrifuge tube containing the
retained spent medium and cells.
8. Centrifuge the remaining cell suspension at 150g for 5 minutes.
Also centrifuge the washings from Number 3 above if they
contain significant numbers of cells.
9. Decant the supernatants and resuspend the cell pellets in a
small volume (10-20mls) of fresh culture medium. Pool the cell
suspensions. Count the cells.
10. Pipette the required number of cells to a new labeled flask and
dilute to the required volume using fresh medium (refer to
ECACC Cell Line Data Sheet for the required seeding density).
11. Repeat this process every 2-3 days as necessary.
59

Key Points
1. Although most cells will detach in the presence of trypsin alone
the inclusion of EDTA is used to enhance the activity of the
enzyme.
2. Trypsin is inactivated in the presence of serum. Therefore, it is
essential to remove all traces of serum from the culture medium by
washing the monolayer of cells with PBS without Ca2+
/Mg2+
.
Repeated warming to 37ºC also inactivates trypsin.
3. Cells should only be exposed to trypsin/EDTA long enough to
detach cells. Prolonged exposure could damage surface receptors.
In general a shorter time of exposure to trypsin is required for semi
adherent cell lines.
4. Trypsin should be neutralized with serum prior to seeding cells
into new flasks otherwise cells will not attach.
5. Trypsin may also be neutralized by the addition of Soybean trypsin
Inhibitor , where an equal volume of inhibitor at a concentration of
1mg/ml is added to the trypsinised cells. The cells are then
centrifuged, resuspended in fresh culture medium and counted as
above.
6. If a CO2
incubator is not available gas the flasks for 1-2 minutes
with 5% CO2
in 95% air filtered through a 0.25m filter. 60

Subculture of Suspension Cell
Lines
• Aim
In general terms cultures derived from blood
(e.g. lymphocytes) grow in suspension. Cells
may grow as single cells or in clumps (e.g.
EBV transformed lymphoblastoid cell lines).
For these types of lines subculture by dilution
is relatively easy. But for lines that grow in
clumps it may be necessary to bring the cells
into a single cell suspension by centrifugation
and resuspension by pipetting in a smaller
volume before counting.
61

Procedure
1. View cultures using an inverted phase contrast
microscope. Cells growing in exponential growth phase
should be bright, round and refractile. Hybridomas may
be very sticky and require a gentle knock to the flask to
detach the cells. EBV transformed cells can grow in very
large clumps that are very difficult to count and the
center of the large clumps may be non-viable.
2. Do not centrifuge to subculture unless the pH of the
medium is acidic (phenol red = yellow) which indicates
the cells have overgrown and may not recover. If this is
so, centrifuge at 150g for 5 minutes, re-seed at a slightly
higher cell density and add 10- 20% of conditioned
medium (supernatant) to the fresh media.
3. Take a small sample of the cells from the cell suspension
(100-200uL ). Calculate cells/ml and re-seed the desired
number of cells into freshly prepared flasks without
centrifugation just by diluting the cells. The data sheet
will give the recommended seeding densities.
4. Repeat this every 2-3 days.
62

Key Points
1. If the cell line is a hybridoma or other
cell line that produces a substance
(e.g. recombinant protein or growth
factor) of interest retain the spent
media for analysis.
63

Cell Quantification
• Aim
For the majority of manipulations using cell cultures,
such as transfections, cell fusion techniques,
cryopreservation and subculture routines it is necessary
to quantify the number of cells prior to use. Using a
consistent number of cells will maintain optimum growth
and also help to standardize procedures using cell
cultures. This in turn gives results with better
reproducibility.
• Materials
• Media– pre-warmed to appropriate temperature (refer
to the ECACC Cell Line Data Sheet for the correct
medium and temperature)
• 70% ethanol in water 0.4% Trypan Blue Solution
• Trypsin/EDTA
64

Cell Quantification
• Equipment
• Personal protective equipment (sterile gloves,
laboratory coat, safety visor)
• Microbiological safety cabinet at appropriate
containment level
• Centrifuge
• CO2
incubator
• Haemocytometer , Improved Neubauer, Camlab
CCH.AC1)
• Inverted phase contrast microscope
• Pre-labeled flasks
65

Procedure
1. Bring adherent and semi adherent cells into
suspension using trypsin/EDTA and resuspend in a
volume of fresh medium at least equivalent to the
volume of trypsin. For cells that grow in clumps
centrifuge and resuspend in a small volume and
gently pipette to break up clumps.
2. Under sterile conditions remove 100-200uL of cell
suspension.
3. Add an equal volume of Trypan Blue (dilution factor
=2) and mix by gentle pipetting.
4. Clean the haemocytometer.
5. Moisten the coverslip with water or exhaled breath.
Slide the cover-slip over the chamber back and forth
using slight pressure until Newton’s refraction rings
appear (Newton’s refraction rings are seen as
rainbow-like rings under the cover-slip). 66

Procedure
6. Fill both sides of the chamber (approx. 5-10uL) with
cell suspension and view under a light microscope
using x20 magnification.
7. Count the number of viable (seen as bright cells)
and non-viable cells (stained blue) . Ideally >100
cells should be counted in order to increase the
accuracy of the cell count . Note the number of
squares counted to obtain your count of >100.
8. Calculate the concentration of viable and non-viable
cells and the percentage of viable cells using the
equations below.
67

• A is the mean number of viable cells counted, i.e. Total
viable cells counted divided by Number of squares
• B is the mean number of non-viable cell counted, i.e.
Total non-viable cells counted divided by Number of
squares
• C is the dilution factor and
• D is the correction factor (this is provided by the
haemocytometer manufacturer).
• Concentration of viable cells (cells/ml) = A x C x D
Concentration of non-viable cells (cells/ml) = B x C x D
Total number of viable cells = concentration of viable
cells x volume
Total number of cells = number of viable + number of
dead cells
Percentage Viability = (No of viable cells x 100)
divided by Total No of cells
68

Key Points
1. Trypan Blue is toxic and is a potential carcinogen.
Protective clothing, gloves and face/eye protection
should be worn. Do not breathe the vapor.
2. The central area of the counting chamber is
1mm2.This area is subdivided into 25 smaller
squares (1/25mm2
). Each of these is surrounded by
triple lines and is then further divided into 16
(1/400mm2
).The depth of the chamber is 0.1mm.
3. The correction factor of 104
converts 0.1mm3
to 1ml
(0.1mm3
= 1mm2
x 0.1mm)
69

Key Points
4. There are several sources of inaccuracy:
o The presence of air bubbles and debris in the
chamber.
o Overfilling the chamber such that sample runs into
the channels or the other chamber
o Incomplete filling of the chamber.
o Cells not evenly distributed throughout the chamber.
o Too few cells to count. This can be overcome by
centrifuging the cells, resuspending in a smaller
volume and recounting.
o Too many cells to count. This can be overcome by
using a higher dilution factor in trypan blue e.g. 1:1070

Cryopreservation of Cell Lines
71

• Materials
• Freeze medium (commonly 70% basal
medium, 20% FCS, 10% DMSO or glycerol,
check data sheets for details).
• PBS without Ca2+ Mg2+
• 0.25% trypsin/EDTA in HBSS, without
Ca2+/Mg2+
• DMSO
• Trypsin/EDTA
72

• Equipment
Laboratory coat)
• Full-face protective mask/visor
• Waterbath set to 37ºC
containment level
• Centrifuge
• Haemocytometer , Improved Neubauer – Camlab
CCH.AC1)
• Pre labeled ampules/cryotubes
• Cell Freezing Device
73

Procedure
1. View cultures using an inverted microscope to
assess the degree of cell density and confirm the
absence of bacterial and fungal contaminants.
suspension using trypsin/EDTA as above (Protocol
3 and 4 – Subculture of adherent/attached and
semi-adherent cell lines) and re-suspend in a
volume of trypsin. Suspension cell lines can be
used directly.
3. Remove a small aliquot of cells (100-200uL) and
perform a cell count (Protocol 6 – Cell
Quantification). Ideally the cell viability should be in
excess of 90% in order to achieve a good recovery
after freezing.
4. Centrifuge the remaining culture at 150g for 5
minutes
74

Procedure
1. View cultures using an inverted microscope to
assess the degree of cell density and confirm the
absence of bacterial and fungal contaminants.
suspension using trypsin/EDTA as above (Protocol
3 and 4 – Subculture of adherent/attached and
semi-adherent cell lines) and re-suspend in a
volume of trypsin. Suspension cell lines can be
used directly.
3. Remove a small aliquot of cells (100-200uL) and
perform a cell count (Protocol 6 – Cell
Quantification). Ideally the cell viability should be
in excess of 90% in order to achieve a good
recovery after freezing.
4. Centrifuge the remaining culture at 150g for 5
minutes 75

Procedure
5. Re-suspend cells at a concentration of 2-4x106
cells per ml in freeze medium.
6. Pipette 1ml aliquots of cells into cyroprotective
ampules that have been labeled with the cell line
name, passage number, cell concentration and
date.
7. Place ampules inside a passive freezer e.g.
Nalgene Mr. Frosty . Fill freezer with isopropyl
alcohol and place at –80ºC overnight.
8. Frozen ampules should be transferred to the vapor
phase of a liquid nitrogen storage vessel and the
locations recorded
76

Key Points
• Culture cell line in the absence of antibiotics for 2
passages prior to testing.
• Bring attached cells into suspension with the use of
a cell scraper. Suspension cell lines may be tested
directly.
• Inoculate 2 x Thioglycollate Medium (TGM) and 2 x
Tryptone Soya broth (TSB) with 1.5ml test sample.
• Inoculate 2 (TGM) and 2 (TSB) with 0.1ml C.albicans
(containing 100 colony forming units, cfu).
• Inoculate 2 (TGM) and 2 (TSB) with 0.1ml B. subtilis
(containing 100cfu).
77

Key Points
5 An alternative to the Mr. Frosty system is the Taylor Wharton
passive freezer where ampules are held in liquid nitrogen vapor
in the neck of Dewar. The system allows the ampules to be
gradually lowered thereby reducing the temperature. Rate
controlled freezers are also available and are particularly useful
if large numbers of ampules are frozen on a regular basis.
6. As a last resort if no other devices are available ampules may
be placed inside a well insulated box (such as a polystyrene box
with sides that are at least 1cm thick) and placed at –80ºC
overnight. It is important to ensure that the box remains upright
throughout the freezing process. Once frozen, ampules should
be transferred to the vapor phase of a liquid nitrogen storage
vessel and the locations recorded.
7. If using a freezing method involving a -80ºC freezer it is
important to have an allocated section for cell line freezing so
that samples are not inadvertently removed. If this happens at a
crucial part of the freezing process then viability and recovery
rates will be adversely affected.
78

Testing for Bacteria and Fungi
• Aim
In cases of gross contamination the naked eye may identify
the presence of bacteria and fungi. However, it is necessary
to detect low-level infections by incubation of cell cultures
and/or their products in microbiological broth. Equally these
sterility tests can be used to confirm the absence of bacteria
and fungi from the preparation which is important when
preparing cell banks or cell culture products.
• Materials
• Soybean Casein Digest (Tryptone Soya Broth,TSB) (15ml
aliquots) TSB Powder
• Fluid Thioglycollate Medium (20ml aliquots) (TGM)
• Bacillus subtilis NCTC*
• Candida albicans NCTC*
• Clostridium sporogenes NCTC*
79

Procedure
1. Culture cell line in the absence of antibiotics for 2
passages prior to testing.
2. Bring attached cells into suspension with the use of
a cell scraper. Suspension cell lines may be tested
directly.
3. Inoculate 2 x Thioglycollate Medium (TGM) and 2 x
Tryptone Soya broth (TSB) ( with 1.5ml test
sample.
4. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml
C.albicans (containing 100 colony forming units,
cfu).
5. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml B.
subtilis (containing 100cfu).
80

Procedure
6. Inoculate 1 TGM with 0.1ml C. sporogenes
(containing 100cfu).
7. Leave 2 (TGM) and 2 (TSB) un-inoculated as
negative controls.
8. Incubate broths as follows:
o For TSB, incubate one broth of each pair at 32ºC the other
at 22ºC for 14 days
o For TGM, incubate one broth of each pair at 32ºC the
other at 22ºC for 14 days
o For the TGM inoculated with C.sporogenes incubate at
32ºC for 14 days
9. Examine Test and Control broths for turbidity after
14 days.
81

• Criteria for a Valid Result
All positive control broths show evidence of
bacteria and fungi within 14 days of
incubation and the negative control broths
show no evidence of bacteria and fungi.
• Criteria for a Positive Result
Test broths containing bacteria or fungi show
turbidity.
• Criteria for a Negative Result
Test broths should be clear and show no
evidence of turbidity
82

• Notes
• The positive controls should be handled in a
laboratory remote from the main tissue
culture laboratory.
• Control organisms (Bacillus subtilis,
Clostridium sporogenes and Candida
albicans) are also available from the National
Collection of Type Cultures (NCTC), UK*.
• This test procedure should be carried out in a
microbiology laboratory away from the cell
culture laboratory
83

Detection of Mycoplasma by Culture
• Aim
Detection of mycoplasma by culture is the reference
method of detection and has a theoretical level of
detection of 1 colony-forming unit (cfu). However there
are some strains of mycoplasma that are non-cultivable
(certain strains of Mycoplasma hyorhinis). The method
is suitable for the detection of mycoplasma in both cell
cultures and cell culture reagents and results are
obtained within 4 weeks. Mycoplasma colonies
observed on agar plates have a ‘fried egg’ appearance
• Materials
• Mycoplasma Pig Agar plates (in 5cm petri dishes)
• Mycoplasma Pig Agar broths (in 1.8ml aliquots)
• M. orale NCTC
• M. pneumoniae NCTC 84

• Equipment
containment level
• CO2 Incubator set at 32ºC
• Gas Jar (Gallenkamp)
• Gas Pak Anaerobic System (Gallenkamp)
• Gas Pak Catalyst (Gallenkamp)
• Gas Pak Anaerobic Indicator (Gallenkamp
85

Procedure
• Inoculate 2 agar plates with 0.1ml of test sample.
• Inoculate 1 agar plate with 100cfu M. pneumoniae.
• Inoculate 1 agar plate with 100cfu M. orale.
• Leave 1 agar plate un-inoculated as a negative control.
• Inoculate 1 broth with 0.2 ml of test sample.
• Inoculate 1 broth with 100cfu M. pneumoniae.
• Inoculate 1 broth with 100cfu M. orale.
• Leave 1 agar plate un-inoculated as a negative control.
• Incubate agar plates anaerobically for 14 days at 37ºC
using a gas jar with anaerobic gas pak and catalyst.
• Incubate broths aerobically for 14 days at 37ºC.
• Between 3 and 7 days and 10 and 14 days incubation,
subculture 0.1 ml of test broth onto an agar plate and
incubate plate anaerobically as above.
• Observe agar plates after 14 days incubation at x300
magnification using an inverted microscope for the
presence of mycoplasma colonies 86

All positive control agar plates and broths show
evidence of mycoplasma by typical colony formation
on agar plates and usually a color change in broths.
All negative control agar plates and broths show no
evidence of mycoplasma.
Test agar plates infected with mycoplasma show
typical colony formation.
The test agar plates show no evidence of
mycoplasma.
87

• Notes
• Mycoplasma colonies have a typical colony formation
commonly described as “fried egg” due to the opaque
granular central zone of growth penetrating the agar
surrounded by a flat translucent peripheral zone on the
surface. However in many cases only the control zone
will be visible.
• Positive controls may be included at a concentration to
give 100 colony-forming units. These controls should
obviously be handled in a laboratory remote from the
main tissue culture laboratory.
• Control organisms (M. pneumoniae, and M. orale) are
available from National Collection of Type Cultures (UK).
• Mycoplasma pneumoniae is a potential pathogen and
must be handled in a class II microbiological safety
cabinet operating to ACDP Category 2 Conditions.
• This test procedure should be carried out in a
microbiology laboratory away from the cell culture
laboratory.
88

Testing for Mycoplasma by Indirect DNA
Stain (Hoechst 33258 stain)
• Aim
DNA staining methods such as Hoechst staining
techniques are quick with results available within 24
hours, which compares favorably with 4 weeks for
detection by culture. However the staining of cultures
directly with a DNA stain, results in a much-reduced
sensitivity (~106cfu/ml). This may be improved by co-
culturing the test cell line in the presence of an indicator
cell line such as Vero (Prod.No. 84113001-1v1). This
enrichment step results in a sensitivity of 104 cfu/ml of
culture. This step also improves sensitivity by increasing
the surface area upon which mycoplasma can adhere.
Like detection by culture, DNA staining methods are
suitable for the detection of mycoplasma from cell cultures or cell
culture reagents.
89

• Materials
• Media– pre-warmed to 37ºC (refer to Cell Line Data
Sheet for the correct medium)
• Methanol
• Acetic Acid Glacial
• Hoechst 33258 stain solution
• Vero cells (Prod. No. 84113001-1v1)
• Mountant (Autoclave 22.2ml 0.2M citric acid with
27.8ml 0.2M disodium phosphate. Add 50ml glycerol.
Filter sterilize and store at 4ºC)
• Mycoplasma hyorhinis NCTC* 10112
90

• Equipment
• Microbiological safety cabinet of appropriate
containment level
• Centrifuge
• CO2 Incubator set at 37ºC
• Microscope (uv Epi-Fluorescent.)
• 35mm plastic tissue culture dishes
• Multidish 24 well
• Cell scraper
• Microscope slides and 22mm cover slips
• Aluminum foil
91

• Procedure Equipment
• For each sample and control sterilize 2 cover slips in a
hot oven at 180ºC for 2 hours or by immersing in 70%
ethanol and flaming in a blue Bunsen flame until the
ethanol has evaporated. Also sterilize 2 cover slips to
use as a negative control.
• Place the cover slips in 35mm culture dishes (1 per
dish).
• Store until needed.
• To prepare the Vero indicator cells add 2x104 cells in
2ml of antibiotic-free growth medium to each tissue
culture dish.
• Incubate at 37ºC in 5% CO2 for 2 – 24 hrs to allow the
cells to adhere to the cover slips.
• Bring attached test cell lines into suspension using a
cell scraper. Suspension cell lines may be tested
directly.
• Remove 1ml of culture supernatant from duplicate
dishes and add 1ml of test sample to each. Inoculate 2
dishes with 100cfu M. hyorhinis and 2 with 100cfu M.
orale.. org.uk
92

• Leave duplicate tissue culture dishes un-inoculated as negative
controls.
• Incubate dishes at 37ºC in 5% CO2 for 1-3 days.
• After 1 day observe one dish from each pair for bacterial or
fungal infection. If contaminated discard immediately. Leave the
remaining dish of each pair for a further 2 days.
• Fix cells to cover-slip by adding a minimum of 2ml of freshly
prepared fixative (1:3 glacial acetic acid: absolute methanol) to
the tissue culture dish and leave for 3 to 5 minutes.
• Decant used fixative to toxic waste bottle. Add another 2ml
aliquot of fixative to cover-slip and leave for a further 3 to 5 min.
Decant used fixative to toxic waste.
• Air dry cover-slip by resting it against the tissue culture dish for
30-120 min.
• Replace cover-slip in dish and add a minimum of 2ml Hoechst
stain . Leave for 5 minutes shielded from direct light by
aluminum foil .
• Decant used and unused stain to toxic waste.
• Add 1 drop of mountant to a pre-labeled microscope slide and
place cover-slip (cell side down) onto slide.
• Keep slide covered with aluminum foil , allowing it to set for at
least 15 min at 37ºC or for 30 min at room temperature.
• Observe slide under uv Epi-Fluorescence at x1000 93

Negative controls show no evidence of mycoplasma
infection
Positive controls show evidence of mycoplasma
infection
Vero cells clearly seen as fluorescing nuclei.
Samples infected with mycoplasma are seen as
fluorescing nuclei plus extra-nuclear fluorescence of
mycoplasma DNA (small cocci or filaments).
Uninfected samples are seen as fluorescing nuclei
against a dark background. There should be no
evidence of mycoplasma.
94

• Notes
• DNA stains such as Hoechst stain bind specifically to DNA.
In all cultures cell nuclei will fluoresce. Uncontaminated
cultures will show only fluorescent nuclei whereas
mycoplasma positive cultures contain small cocci or
filaments which may or may not be adsorbed onto the cells.
• Hoechst stain is toxic and should be handled and discarded
with care.
• Culture dishes should be placed in a sealed box or cultured
in large petri dishes to reduce evaporation.
• Positives should obviously be handled in a laboratory
remote from the main tissue culture laboratory.
• Control organisms (M. hyorhinis) are available from the
National Collection of Type Cultures (UK).
95

• In some instances results may be difficult to
interpret for the following reasons:
– Bacterial/yeast/fungal contamination
– Too much debris in the background
– Broken nuclei as cells are all dead
– Too few or no live cells
• Although this procedure recommends the setting
up of positive controls, this may not necessarily
be feasible nor desirable in a cell culture facility
with limited resources. If positive controls are to
be set up they should be done so in a separate
laboratory from the main tissue culture facility. If
this is not possible then positive slides can be
purchased from ECACC. If positive controls are
not being used then it is strongly recommended
that you get an independent testing laboratory to
periodically test your cell lines.
96

• For animal cell culture the level of risk is dependent
upon the cell line to be used and is based on whether
the cell line is likely to cause harm to humans. The
different classifications are given below:
• Low risk
• Non human/non primate continuous cell lines and
some well characterized human diploid lines of finite
lifespan (e.g. MRC-5).
• Medium risk
• Poorly characterized mammalian cell lines.
• High risk
• Cell lines derived from human/primate tissue or
blood.
• Cell lines with endogenous pathogens (the precise
categorization is dependent upon the pathogen) –
refer to ACDP guidelines, 1985, for details.
• Cell lines used following experimental infection where
the categorization is dependent upon the infecting
agent - refer to ACDP guidelines, 1985, for details*. 97

Waste Disposal
• Any employer has a ‘duty of care’ to dispose of all
biological waste safely in accordance with national
legislative requirements. Given below is a list of ways
in which tissue culture waste can be decontaminated
and disposed of safely. One of the most important
aspects of the management of all laboratory-
generated waste is to dispose of waste regularly and
not to allow the amounts to build up. The best
approach is ‘little and often’. Different forms of waste
require different treatment.
98

• Tissue culture waste (culture medium) - Inactivate
overnight in a solution of hypochlorite (10,000ppm)
prior to disposal to drain with an excess of water
• Contaminated pipettes should be placed in
hypochlorite solution (2500ppm) overnight before
disposal by autoclaving and incineration
• Solid waste such as flasks, centrifuge tubes,
contaminated gloves, tissues etc. should be placed
inside heavy duty sacks for contaminated waste and
autoclaved prior to incineration. These bags are
available from Bibby Sterilin and Greiner.
• If at all possible waste should be incinerated rather
than autoclaved
99

Sourcing of Cell Lines
• Large numbers of cell lines look identical. Cell lines
with very different origins and biological
characteristics typically cannot be separated on
grounds of morphology or culture characteristics.
Infection or contamination of a cell line with an
adventitious virus or mycoplasma may significantly
change the characteristics of the cells but again such
contamination will be inapparent. Cell lines will also
change with time in culture, and to add to all these
natural hazards it is all too easy to mis-label or cross-
contaminate different cell lines in a busy cell culture
laboratory
100

Main Types of Cell Culture
• Primary Cultures
• Primary cultures are derived directly from excised,
normal animal tissue and cultured either as an
explant culture or following dissociation into a single
cell suspension by enzyme digestion. Such cultures
are initially heterogeneous but later become
dominated by fibroblasts. The preparation of primary
cultures is labor intensive and they can be
maintained in vitro only for a limited period of time.
During their relatively limited life span primary cells
usually retain many of the differentiated
characteristics of the cell in vivo.
101

• Continuous Cultures
• Continuous cultures are comprised of a single cell type
that can be serially propagated in culture either for a
limited number of cell divisions (approximately thirty) or
otherwise indefinitely. Cell lines of a finite life are
usually diploid and maintain some degree of
differentiation. The fact that such cell lines senesce
after approximately thirty cycles of division means it is
essential to establish a system of Master and Working
banks in order to maintain such lines for long periods.
• Continuous cell lines that can be propagated
indefinitely generally have this ability because they
have been transformed into tumor cells. Tumor cell
lines are often derived from actual clinical tumors, but
transformation may also be induced using viral
oncogenes or by chemical treatments. Transformed
cell lines present the advantage of almost limitless
availability, but the disadvantage of having retained
very little of the original in vivo characteristics.
102

Commonly used cell lines of
each culture type
• Attached Cell Lines
• Name Species and tissue of origin Morphology
• MRC-5 Human lung Fibroblast
• HELA Human cervix Epithelial
• VERO African Green Monkey Kidney Epithelial
• NIH 3T3 Mouse embryo Fibroblast
• L929 Mouse connective tissue Fibroblast
• CHO Chinese Hamster Ovary Fibroblast
• BHK-21 Syrian Hamster Kidney Fibroblast
• HEK 293 Human Kidney Epithelial
• HEPG2 Human Liver Epithelial
• BAE-1 Bovine aorta Endothelial
103

• Suspension Cell Lines
• Name Species and tissue of origin Morphology
• NSO Mouse myeloma Lymphoblastoid-
like
• U937 Human Hystiocytic Lymphoma Lymphoblastoid
• Namalwa Human Lymphoma Lymphoblastoid
• HL60 Human Leukaemia Lymphoblastoid-like
• WEHI 231 Mouse B-cell Lymphoma Lymphoblastoid
• YAC 1 Mouse Lymphoma Lymphoblastoid
• U 266B1 Human Myeloma Lymphoblastoid
• SH-SY5Y Human neuroblastoma Neuroblast
104

Growing and Maintaining Cells in Culture

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