UNIT 1: GENERAL INTRODUCTION

SYLLABUS: History; Definition of primary cell cultures; Diploid cell lines; Established or
immortal cell lines; Suspended and anchor dependent cell cultures; Morphological
differentiation of cell cultures; Animal Cell Culture Laboratory - lay out, equipment, media,
glass and plastic wares; Cell culture techniques – enzymatic disaggregation and explant cell
culture techniques, open and closed system, sub-culturing techniques, preservation and
revival of cell lines, enumeration of viable cells, cell line characterization

What is cell culture?

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
tissues. Cell culture refers to the removal of cells from a tissues and their subsequent growth
in a favourable artificial environment. This process occurs "in vitro" ("in glass") as opposed
to "in vivo" ("in life").

History

In 1907, Ross Harrison, was able to show the development of nerve fibres from frog
embryo tissue, cultured in a blood clot held by the hanging drop method. He was considered
by some as the father of cell culture. Interest in culturing human tissues started in 1950’s
after it was demonstrated by George Otto Gey that human tumor cells (HeLa) could give
rise to continuous cell lines. Major developments in cell culture technology include use of
antibiotics which inhibits the growth of contaminants, use of trypsin to remove adherent
cells to subculture further from the culture vessel and use of chemically defined culture
medium.

Important terms

 Primary culture

The culture produced by the freshly isolated cells or tissues taken from an organism is the
primary culture.

There are two basic methods for doing this.

First, for Explant Cultures, small pieces of tissue are attached to a glass or treated plastic
culture vessel and bathed 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.

The second, more widely used method is called as Enzymatic dissociation, using 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.

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Primary cells have a finite life span, slow growing and contain a very heterogeneous
population of cells. They represent the tissue of their origin with regard to their properties.

 Subculture/ passage

When the cells in the primary culture vessel have grown and filled up all of the available
culture substrate, their growth slows & ceases. They must be subcultured to keep the cells
healthy and give room for continued growth. This is usually done by removing them as
gently as possible from the substrate with enzymes. Most commonly used enzyme is trypsin
(other enzymes, e.g., collagenase, papain,dispase, and pronase, are also used), which in
combination with EDTA, breaks the cellular glue that attached the cells to the surface. Some
cell lines can be harvested by gently scraping the cells off the bottom of the culture vessel.
Once released, the cell suspension can then be subdivided and placed into new culture
vessels.

 Secondary cell cultures

When a primary culture is sub-cultured, it becomes known as secondary culture or cell line.
If a subpopulation of a cell line is positively selected from the culture by cloning or some
other method, this cell line becomes a cell strain. A cell strain often acquires additional
genetic changes subsequent to the initiation of the parent line.

A Cell Line or Cell Strain may be finite or continuous depending upon whether it has
limited culture life span or it is immortal in culture. On the basis of the life span of culture,
the cell lines are categorized into two types:

a) Finite cell Lines - The cell lines which have a limited life span and go through a limited
number of cell generations (usually 20-80 population doublings) are known as Finite cell
lines. These cell lines exhibit the property of contact inhibition, density limitation and
anchorage dependence. As they are passaged, cells with the highest growth capacity
predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.
The growth rate is slow and doubling time is around 24-96 hours. These cells that have the
normal number of chromosomes are called Diploid cells

b) Continuous Cell Lines - Cell lines transformed under laboratory conditions or in vitro
culture conditions give rise to continuous cell lines. When a “normal” finite cell line
becomes immortal, it has undergone a fundamental irreversible change or
“transformation”. Immortalization can be spontaneous or intentional using drugs, radiation
or viruses. The cell lines show the property of ploidy (aneupliody or heteroploidy), absence
of contact inhibition and anchorage dependence. They are grown in monolayer or
suspension form. The growth rate is rapid and doubling time is 12-24 hours.

Fig 1.1: Growth curve of finite and continuous cell lines

Fig 1: Initially, cells are in a lag phase, usually no more than 1-2 days in length, during which there
is little or no increase in cell number. During this time, cells are "conditioning" the medium,
undergoing internal cytoskeletal and enzyme changes and adjusting to the new medium. Secondly,

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the cells undergo a period of rapid division, so-called log phase growth. Then, as they approach
confluency and form contacts with one another, their rate of division slows. Finite cell lines undergo
senescence and death after this stage, whereas continuous cells undergo transformation, resulting in
indefinite growth.

 Cell culture systems

Two basic culture systems are used for growing cells, based primarily upon the ability of the
cells to either grow attached to a glass or treated plastic substrate (Monolayer culture
system) or floating free in the culture medium (suspension culture system).

a) Anchorage Dependent /Adherent cells- Cells shown to require attachment for growth
are set to be Anchorage Dependent cells. The Adherent cells are usually derived from
tissues of organs such as kidney where they are immobile and embedded in connective
tissue. They grow adhering to the cell culture flask/ plate surface. Transformed cells can
grow either in monolayer or in suspension. They need to be dissociated from the substrate
mechanically or enzymatically for subculture. Monolayer cultures are usually grown in
tissue culture treated dishes, T-flasks, roller bottles, Culture Chambers, or multiple well
plates, the choice being based on the number of cells needed, the nature of the culture
environment, cost and personal preference.

b) Suspension Culture/Anchorage Independent cells - Cells which do not require

attachment for growth or do not attach to the surface of the culture vessels are anchorage
independent cells/suspension cells. All suspension cultures are derived from cells of the
blood system because these cells are also suspended in plasma in vivo e.g. lymphocytes.
Suspension cultures are usually grown either in magnetically rotated spinner flasks or
shaken Erlenmeyer flasks where the cells are kept actively suspended in the medium or in
stationary culture vessels such as T-flasks and bottles where, although the cells are not kept
agitated, they are unable to attach firmly to the substrate.
 Morphological differentiation of cell cultures

Cultured cells are usually described based on their morphology (shape and appearance).
There are three basic morphologies:
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1. Epithelial-like: They are polygonal in shape with more regular dimensions, and grow
attached to a substrate in discrete patches.

2. Lymphoblast-like: They are spherical in shape and usually grown in suspension without
attaching to a surface.

3. Fibroblastic or Fibroblast-like: They are bipolar or multi-polar, have elongated shapes and
grow attached to a substrate.

4. Endothelial like: They are very flat, have a central nucleus

Fig 1.2: Different types of cell lines based on morphology

Fibroblastic cells Epithelial-like cells Lymphoblast-like cells

Advantages and limitations of animal cell culture

Advantages

1. Control of physicochemical environment- pH, temperature, dissolved gases (O2 and CO2),
osmolarity.

2. Regulation of physiological conditions-nutrient concentration, cell to cell interactions,

hormonal control.

3. The cultured cell lines become homogenous (i.e. cells are identical) after one or two
subcultures. This is in contrast to the heterogenous cells of tissue samples. The homogenous
cells are highly useful for a wide range of purposes.

4. The consistency and reproducibility of results that can be obtained from using a batch of
clonal cells.

5. It is easy to characterize cells for cytological and immunological studies.

6. Cultured cells can be stored in liquid nitrogen for several years and can be revived back
when required

7. Due to direct access and contact to the cells, biological studies can be carried out more
conveniently. The main advantage is the low quantities of the reagents required in contrast

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to in vivo studies where most of the reagents (more than 90% in some cases) are lost by
distribution to various tissues, and excretion.

8. Utility of tissue cultures will drastically reduce the use of animals for various
experiments.

Limitations:

1. Need of expertise and technical skill for the development, and regular use of tissue
culture.

2. Cost factor is a major limitation. Establishment of infrastructure, equipment and other

facilities are expensive.

3. It is estimated that the cost of production of cells is about 10 times higher than direct use
of animal tissues.

4. Control of the environmental factors (pH, temperature, dissolved gases, disposal of

biohazards) is not easy.

5. The native in vivo cells exist in a three- dimensional geometry while in in vitro tissue
culture, the propagation of cells occurs on a two dimensional substrate. Due to this, the cell
to cell interactive characters are lost.

6. The cell lines may represent one or two types of cells from the native tissue while others
may go unrepresented.

7. The components of homeostatic in vivo regulation (nervous system, endocrine system,

metabolic integration) are lacking in vitro cultures. Addition of hormones and growth
factors has been started recently.

Applications of cell culture

1. Model System:

Cell cultures are used as excellent model system to study basic cell biology, molecular
biology, normal physiology and biochemistry of cells (e.g., metabolic studies, aging); to
study the interaction between cell and disease causing agents like bacteria, virus; to study
the effect of drugs.

2. Cancer Research

The basic difference between normal cell and cancer cell can be studied using animal cell
culture technique, as both cells can be cultured in laboratory. Normal cells can be converted
into cancer cells by using radiation, chemicals and viruses. Thus, the mechanism and cause
of cancer can be studied. Cell culture can be used to determine the effective drugs for
selectively destroy only cancer cells.

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 3. Virology and vaccine production

Animal cell cultures are used to replicate the viruses instead of animals, for the production
of vaccine. For example vaccines for deadly diseases like polio, rabies, chicken pox,
measles and hepatitis B are produced using animal cell culture. Cell culture can also be used
to detect and isolate viruses, to study growth and development cycle and mode of infection
of viruses.

4. Toxicity Testing:

Animal cell culture is used to study the effects of new drugs, cosmetics and chemicals on
survival and growth of a number of types of cells. Especially liver and kidney cells.
Cultured animal cells are also used to determine the maximum permissible dosage of new
drugs.

5. Genetically Engineered Protein:

Animal cell cultures are used to produce commercially important genetically engineered
proteins such as monoclonal antibodies, insulin, hormones, and much more.

6. Replacement Tissue or Organ:

Animal cell culture can be used as replacement tissue or organs. For example artificial skin
can be produced using this technique to treat patients with burns and ulcers. However
research is going on artificial organ culture such as liver, kidney and pancreas. Organ
culture techniques and research are being conducted on both embryonic and adult stem cell
culture. These cells have the capacity to differentiate into many different types of cells and
organs. It is believed that by learning to control the development and differentiation of these
cells may be used to treat variety of medical conditions.

7. Genetic Counseling:

Fetal cell culture extracted from pregnant women can be used to study or examine the
abnormalities of chromosomes, genes using karyotyping, and these findings can be used in
early detection of fetal disorders.

8. Genetic Engineering:

Cultured animal cells can be used to introduce new genetic material like DNA or RNA into
the cell. These can be used to study the expression of new genes and its effect on the health
of the cell. Insect cells are used to produce commercially important proteins by infecting
them with genetically altered baculoviruses.

9. Gene Therapy:

Cultured animal cells can be genetically altered and can be used in gene therapy technique.
First cells are removed from the patient lacking a functional gene or missing a functional

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gene. These genes are replaced by functional genes and altered cells are culture and grown
in laboratory condition. Then these altered cells are introduced into the patient.

10. Drug Screening and Development:

Animal cell cultures are used to study the cytotoxicity of new drug. This is also used to find
out the effective and safe dosage of new drugs. Now these tests are being conducted in 384
and 1536 well plates. Cell-based assay plays an important role in pharmaceutical industry. A
large number of drugs can be screened in a single experiment and a single drug can be tested
using different cell lines representing different culture conditions.

Animal Cell Culture Laboratory - lay out, equipment, glass and plastic wares

All cell culture laboratories have the common requirement of being free from pathogenic
microorganisms (i.e., asepsis), and share some of the same basic equipment that is essential
for culturing cells.

Aseptic work area

The major requirement of a cell culture laboratory is the need to maintain an aseptic work
area that is restricted to cell culture work. Although a separate tissue culture room is
preferred, a designated cell culture area within a larger laboratory can still be used fort
sterile handling, incubation, and storage of cell cultures, reagents, and media. The simplest
and most economical way to provide aseptic conditions is to use a cell culture hood. The
cell culture room should be free of through traffic and, if possible, equipped with an air flow
cabinet which supplies filtered air around the work surface. A HEPA (High Efficiency
Particle Air Filter) filtered air supply is desirable but not always affordable. Primary animal
tissue and micro-organisms must not be cultured in or near the cell culture laboratory. Clean
laboratory coats should be kept at the entrance and should not be worn outside of this
laboratory and brought back in. All work surfaces, benches and shelves and the base of the
airflow cabinets must be kept clean by frequent swabbing with 70% ethanol or an alternative
disinfectant.

Cell Culture Hood

The cell culture hood provides an aseptic work area while allowing the containment of
infectious splashes or aerosols generated by many microbiological procedures. Three kinds
of cell culture hoods, designated as Class I, II and III, have been developed to meet varying
research and clinical needs. Class I cell culture hoods offer significant levels of protection
to laboratory personnel and to the environment when used with good microbiological
techniques, but they do not provide cultures protection from contamination. They are similar
in design and air flow characteristics to chemical fume hoods. Class II cell culture hoods
are designed for work involving bio safety level (BSL)-1, 2, and 3 materials, and they also
provide an aseptic environment necessary for cell culture experiments. A Class II biosafety
cabinet should be used for handling potentially hazardous materials (e.g., primate-derived
cultures, virally infected cultures, radio isotopes, carcinogenic or toxic reagents). Class III
biosafety cabinets are gas-tight, and they provide the highest attainable level of protection

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to personnel and the environment. A Class III biosafety cabinet is required for work
involving known human pathogens and other BSL-4 materials.

Cell culture hoods protect the working environment from dust and other airborn
contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the
work area. The flow can be horizontal, blowing parallel to the work surface, or it can be
vertical, blowing from the top of the cabinet onto the work surface. Depending on its design,
a horizontal flow hood provides protection to the culture (if the air flowing towards the
user) or to the user (if the air is drawn in through the front of the cabinet by negative air
pressure inside). Vertical flow hoods, on the other hand, provide significant protection to
the user and the cell culture.

Cell culture hood usually have U.V light which ensures sterility of the work space and the
things kept in it. The work area should be large enough to be used by one person at a time,
be easily cleanable inside and outside, have adequate lighting, and be equipped with Bunsen
burner. Keep the work space in the cell culture hood clean and uncluttered, and keep
everything in direct line of sight. Disinfect each item placed in the cell culture hood by
spraying them with 70% ethanol and wiping clean. The arrangement of items within the cell
culture hood usually adheres to the following right-handed convention, which can be
modified to include additional items used in specific applications.

Incubator

The purpose of the incubator is to provide the appropriate environment for cell growth. The
incubator should have forced air circulation and should have temperature control to within.
Stainless steel incubators allow easy cleaning and provide corrosion protection, especially if
humid air is required for incubation. Although the requirement for aseptic conditions in a
cell culture incubator is not as stringent as that in a cell culture hood, frequent cleaning of
the incubator is essential to avoid contamination of cell cultures. There are two basic types
of incubators, dry incubators and humid CO2 incubators. Dry incubators are more
economical, but require the cell cultures to be incubated in sealed flasks to prevent
evaporation. Placing a water dish in a dry incubator can provide some humidity, but they do
not allow precise control of atmospheric conditions in the incubator. Humid CO2 incubators
are more expensive, but allow superior control of culture conditions. It has been designed to
allow CO2 to be supplied from a main supply or gas cylinder so that an atmosphere of
between 2-5% CO2 is maintained in the incubator. In general, many cell lines can be
maintained in an atmosphere of 5% CO2: 95% air at 99% relative humidity. The incubation
temperature will depend on the type of cells being cultivated. Insect cells will grow best at
around 30 °C while mammalian cells require a temperature of 37 °C. Cultured cells can
generally survive lower temperatures, but rarely survive temperatures greater than 2 °C
above normal, and therefore the incubator should be set to cut out at approximately 38.5 °C
to prevent cell death. Incubators are designed to regulate an even temperature and this is
more important than accuracy, i.e., temperature should be ±0.5 °C. Most incubators have
areas of differing temperature; therefore fan assisted incubators are preferable to help
maintain even temperature distribution.

Microscopes

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It is vital to be able to recognize morphological changes in cultures since these may be the
first indication of deterioration of a culture. This makes microscopes an integral part of cell
culture labs. An upright light microscope with x100 magnification will suffice for routine
cell counts in a hemocytometer, cell staining or chromosome analysis. Additional features
such as a camera, CCD video camera, adapter and attachments and fluorescence analysis
facility may also be required for some purposes.

 Inverted microscopes

Inverted phase contrast microscope is one of the basic requirements of a cell culture lab. A
simple inverted microscope is essential so that cultures can be examined in flasks and dishes
from underneath. Inverted microscopes are constructed with the tip of the objective pointing
upward so as to view the specimen from below. The objective is underneath the stage and
light is directed on the specimen from above.

Fig 1.3: Inverted microscope

 Phase-Contrast Microscope

Light passing from one object into another object of a slightly different refractive index or
thickness undergoes a change in phase. In a phase-contrast microscope, this difference in
phase is translated into variation in brightness of the image and hence is detectable by eye.
With a phase-contrast microscope, the differences among various cells with different
refractive indices or thickness can be seen in unstained condition.

Microscopic objects can be seen in unstained condition, due to the difference in the
refractive index of the object and its surrounding medium. Unstained structures within cells,
not discernible by other microscopic methods can also be observed due to the slight
differences in their refractive indices or thickness. A phase-contrast microscope is a
compound microscope fitted with a phase-contrast condenser and a phase-contrast objective.
An annular aperture in the diaphragm placed in the focal plane of the sub-stage condenser
controls the illumination of the object.
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The image of the aperture is formed at the rear focal plane of the objective. In this plane,
there is a phase-shifting element or phase- plate. The phase plate also has an annular ring of
phase altering pattern, which can increase the wavelength of light passing through it. Light
coming through the annular aperture of condenser passes through the object. Those rays,
which are not deviated by the object (solid lines in figure), pass through the phase-altering
pattern of the phase plate and acquire longer wavelength. Those rays, which are deviated by
the object structures (broken lines in figure), due to different refractive index, pass through
the phase-plate not covered by the phase altering pattern. Thus, their wavelength remains
unchanged. The difference in phase (wavelength) gives the contrast for clear visibility of the
object.

Fig 1.4: Phase contrast microscope

 Fluorescent microscope

The "fluorescence microscope" refers to any microscope that uses fluorescence to generate
an image, whether it is a more simple set up like an epifluorescence microscope, or a more
complicated design such as a confocal microscope, which uses optical sectioning to get
better resolution of the fluorescent image. It is required in order to detect the expression of
fluorescent proteins in cells, such as green fluorescent protein (GFP) in transfected cells, or
to monitor the internalization of fluorescent labelled drugs (for ex:- FITC conjugated
peptides or nanoparticles).

The fluorescence microscopes used in cell culture labs, are of the epifluorescence design.
Light of the excitation wavelength is focused on the specimen through the objective lens.
The fluorescence emitted by the specimen is focused to the detector by the same objective
that is used for the excitation which for greater resolution will need objective lens with
higher numerical aperture. Since most of the excitation light is transmitted through the
specimen, only reflected excitatory light reaches the objective together with the emitted
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light and the epifluorescence method therefore gives a high signal-to-noise ratio. An
additional wavelength specific filter between the objective and the detector can filter out the
remaining excitation light from fluorescent light.

Fig 1.5: Fluorescent microscope mechanism

Refrigerator and freezer

For small cell culture laboratories, a domestic refrigerator (preferably one without
autodefrost freezer- to avoid self-thawing) is an adequate and inexpensive piece of
equipment for storing media, trypsin and other reagents at 2–8°C. For larger laboratories, a
cold room restricted to cell culture is more appropriate. Make sure that the refrigerator or the
cold room is cleaned regularly to avoid contamination. A freezer will be needed for keeping
pre-aliquoted stocks of serum, glutamine, vitamin stocks, other nutrients and antibiotics may
be stored at a temperature of -20 °C

Other general equipment

 Water bath- to bring the refrigerated medium and serum back to room temperature
before using it on cells
 Double distillation units or MilliQ apparatus- to make extra pure water to be used
for media and reagent preparation
 Liquid nitrogen storage vessel or -80°C deep freezer- for the cryopreservation of
cells
 Neubauer chamber or Hemocytometer- to count the number of cells before
seeding for assays or to take the viable cell count
 Cooling centrifuge with sealed buckets- to collect trypsinized cells or cells in
suspension for subculture/ cryopreservation/ assays

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  Cytocentrifuge- to concentrate cells in suspension and spread monolayerly on to a
focal area on microscopic slide in one operation, preserving morphological details of
the cells
 pH meter and Osmometer- to monitor the pH and osmolarity of the cell culture
medium and maintain the optimum levels according to the cell lines used.

Tissue culture vessels

Culture vessels provide a contamination barrier to protect the cultures from the external
environment while maintaining the proper internal environment. For anchorage-dependent
cells, the vessels provide a suitable and consistent substrate for cell attachment. Other
characteristics of vessels include easy access to the cultures and optically clear viewing
surfaces. Originally all culture vessels were glass. Drawbacks for glass include the heavy
weight, expense, labor intensive cleaning, and poor microscopic viewing compared to
plastic. By the 1960s, surface treatment techniques were developed for polystyrene,
allowing plastic vessels to replace glass for most cell culture applications.
The right vessels were selected based upon the culture system being used.
• Stationary monolayer cultures which are grown in undisturbed flasks, dishes, and multi-
well plates. These are the easiest culture systems to use and require the least amount of
equipment. However, these systems are very labor intensive for producing large quantities
of cells.
• Moving monolayer cultures which are grown primarily in roller bottles. These vessels are
slowly rotated (approximately 0.5 rpm to 1 rpm) on motorized racks or drums and are
widely used for producing large quantities of cells. Roller bottles employ simple technology
but require an investment in the appropriate equipment.
• Stationary suspension cultures which are grown without agitation in untreated dishes and
flasks. These are best for growing small volumes of anchorage-independent cells that grow
poorly in traditional stirred suspension cultures.
• Moving suspension cultures which are grown in mechanically stirred vessels (spinner
flasks), bioreactors, or fermentors. These systems are the most economical in terms of
space, labor and media; as a result, stirred suspension cultures are usually the method of
choice for producing large volumes of cells both in the lab and in industry. Many
anchorage-dependent cells can be adapted to grow on microcarriers to take advantage of
these systems.
Another criterion for culture vessel selection is based on whether the cells grow in an open
or closed system. Open-system plastic dishes are less expensive than closed-system flasks,
but require more expensive incubators that can regulate the CO₂ and humidity in the
atmosphere.
All dishes and multiwell plates are open systems. All other culture vessels can be used in
either mode by leaving caps loose for an open system or tightened for a closed system. The
plastic walls of culture vessels are slightly permeable to carbon dioxide and oxygen,
permitting a very small amount of gas exchange. Vented caps that allow gas exchange when
the cap is fully tightened are available to reduce opportunities for flask spills and
contamination in open systems.

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Alexis Carrel developed the first glass flasks in the 1920s. Harry Earle developed the more
traditional straight neck rectangular (also hexagonal) glass T-flasks in the 1940s. Today,
plastic flasks are available with a range of growing areas, a variety of shapes, with several
different neck designs. The most common sizes are 12.5, 25 and 75 cm2. Cell culture dishes
offer the best economy and access to the growth surface. This makes them the vessels of
choice for cloning or other manipulations such as scraping that require direct access to the
cell monolayer. Most commonly available dishes are in 35 mm, 60 mm, 100 mm and 150
mm diameter size. Cell culture dishes are available with either specially treated surface for
growing anchorage-dependent cells, or untreated (native) surfaces for growing suspension
cultures where attachment is not desired.
Multi-well plates are widely used vessels were originally designed for virus titration, but
have since become popular in many other applications, especially hybridoma production,
high-throughput screening, and toxicity testing. Multiwell plates offer significant savings in
space, media, and reagents when compared to an equal number of dishes. They are more
convenient to handle, especially if the pipettors, plate washers, readers, and other equipment
for processing these plates are used.
The roller bottle is developed for cultivating large numbers of anchorage-dependent cells.
Today they provide a more economical means for cultivating large volumes of cells using
essentially the same culture techniques as with flasks but with considerably less labor.
Besides the traditional smooth wall design, roller bottles are available with small ridges that
approximately double the surface area available for growing cells without increasing the
dimensions of the bottles.
Most tissue culture work uses disposable polystyrene vessels. The vessel surface is treated
to render it hydrophilic (wettable). Some fastidious cell lines require further treatment of the
growth surface before they will attach and proliferate. The most common techniques include
coating the surface with serum, collagen, laminin, gelatin, poly-L-lysine, or fibronectin.
Beyond simple attachment, some cells require specialized surface treatment in order for
them to differentiate into more tissue-like formations. For example, endothelial cells will
form tubules and neuronal cells will extend neurite processes when cultured on a surface of
extracellular matrix (ECM). These ECM proteins closely resemble the basal lamina
membrane surrounding cells in tissue and not only provide attachment points, but modulate
signal transduction from external growth factors and hormones, influence the permeability
of ions and nutrients, and actively “communicate” with intracellular processes through
integrins. Finally, some cells, particularly when seeded at low densities as for cloning,
require the support of living cells. Feeder layer cells support growth by conditioning the
medium through metabolic leakage and/or the active secretion of growth and other factors.
They also provide a support matrix for cell attachment and proliferation. To prevent feeder
layer cells from overgrowing the cells of interest, they are treated to prevent division.
Common methods include irradiation with X-rays or gamma rays or treatment with
mitomycin C. Each of these treatments damages cellular DNA so that the cells continue to
metabolize but can no longer proliferate. Some of the well-characterized feeder cells are
mouse embryonic endothelial cells, 3T3 cells (mouse embryonic fibroblast), multidrug
resistant mouse fibroblasts and mitomycin C treated neonatal human fibroblast cells. Micro-
carriers are mostly used for the propagation of anchorage-dependent cells in suspension.
They are in bead form and are made up of collagen, gelatin, polyacrylamide and
polystyrene.
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Cell culture media

The culture medium is the most important component of the culture environment, because it
provides the necessary nutrients, growth factors, and hormones for cell growth, as well as
regulating the pH and the osmotic pressure of the culture. Although initial cell culture
experiments were performed using natural media obtained from tissue extracts and body
fluids, the need for standardization, media quality, and increased demand led to the
development of defined media. Since 1950s, tissue culture media were developed and
conditions were worked out which closely simulate the situation in vivo.

Cell culture media are complex mixtures of nutrients. The requirements for these
components vary among cell lines, and these differences are partly responsible for the
extensive number of medium formulations. The major media constitutes are-

 Carbohydrates- mostly glucose, and in some instances glucose is replaced with

galactose to decrease lactic acid build up, but it is metabolized slowly. They are the
natural energy source for media. Pyruvate is used as another carbon/ energy source.
 Vitamins- essential for cell proliferation and differentiation and not synthesized by
cells during culture.
 Inorganic Salt - salts like potassium and calcium in media help to retain osmotic
balance of cells.
 Amino Acids- like L-glutamine are included in media for proteins synthesis by cells.
 Antibiotics- used to avoid microbial contamination.
 Trace Elements- like copper, selenium, zinc etc. Used mostly for serum free media.
 Proteins and Peptides- most proteins and peptide like transferrin, fibronectin and
albumin are used in cell culture media.
 Buffers- like CO₂/sodium bicarbonate, phosphate, and HEPES which regulate pH
level. Sera can also buffer a complete media. Phenol red work as pH indicator in
medium.
 Sterile milliQ water– all the above components should be added in appropriate
quantity in sterilized milliQ water and sterilized by autoclaving or filter sterilization.

There are mainly two types of media; Natural and artificial.

 Natural Media

These are biological fluids rich in nutrients that support growth of cells in culture. They can
be fluid (serum), tissue extract (embryo, bone marrow, spleen, liver etc.) or coagulant
(liquid plasma or plasma clots) in nature. Natural media are highly beneficial and produce
higher rates of cell growth and proliferation. But lack of knowledge of the exact
composition, poor reproducibility and contamination risk are major limiting factors.

 Artificial media/ defined media

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These contain fully defined media components and maintain a physiological pH of 7. The
most basic media are balanced salt solutions (BSS), e.g., phosphate-buffered saline (PBS),
which may be used for washing cells and for short incubations in suspension. More complex
defined media are used for growth and maintenance. Defined media can also vary in
complexity, by the addition of a number of constituents, e.g., from Eagle’s minimum
essential medium (MEM) which contains essential amino acids, vitamins and salts, to
McCoy’s medium, which contains a larger number of different amino acids, vitamins,
minerals and other extra metabolites (such as nucleosides). There are two types of defined
media; serum containing media and serum free media

 Serum Containing Media- which have some quantity of serum with other defined
nutrients. Under this category, when media + serum is called as complete media or
growth media and that without serum is called as incomplete media or maintenance
media. The majority of cell lines grow well in basal media, which contain amino
acids, vitamins, inorganic salts, and a carbon source such as glucose, but these basal
media formulations must be further supplemented with serum. Reduced-serum
media are basal media formulations enriched with nutrients and animal-derived
factors, which reduce the amount of serum that is needed.
 Serum Free Media- media formulated such as to avoid the use of serum and the
problems associated with it. They are supplemented with factors required for growth
and proliferation. Serum-free media formulations exist for many primary cultures
and cell lines, including recombinant protein producing lines of Chinese Hamster
Ovary (CHO), various hybridoma cell lines and for cell lines that act as hosts for
viral production (e.g., VERO, MDCK (Madin-Darby Canine Kidney), MDBK), and
others. Serum free media have many advantages like increased definition, selective
formulation for specific cell line, consistent performance, precise evaluation of
cellular functions, and better control over physiological responses. But, they have
disadvantage like slower growth and proliferation.

Important media ingredients

Animal serum

Although there is much research aimed at attempting to reduce the requirement of cells for
serum, by alternative supplementation of the media, it is apparent that most cell lines still
require serum for adequate growth. Serum is beneficial in many ways such as:
 source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as
A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace
elements.
 serum buffers the culture medium
 inactivates proteolytic enzymes
 increases medium viscosity (which reduces shear stress during pipetting or stirring)
 conditions the growth surface of the culture vessel
 act as albumin carrier factor that provide many essential hormones and proteins
 also found to regulate cell membrane permeability and serves as a carrier for lipids,
enzymes, micronutrients and trace elements to cells
15
  reduces toxicity of medium

Sera from fetal and calf bovine sources are commonly used to support the growth of cells in
culture. Fetal serum is a rich source of growth factors and is appropriate for cell cloning and
for the growth of fastidious cells. Calf serum, because of its lower growth-promoting
properties, is used in contact-inhibition studies with NIH/3T3 cellsIn contrast to fetal or calf
sera, horse serum is collected from a closed herd of adult animals ensuring lot-to-lot
consistency. Horse serum is less likely to carry the contaminants found in bovine sera such
as viruses and less likely to metabolize polyamines which may be mitogenic for some cells.
Horse and bovine calf sera are less expensive and more readily available than fetal bovine
serum. The pricing and availability of fetal serum fluctuates considerably. Unfortunately,
naturally derived products from bovine sources may contain adventitious viruses such as
bovine viral diarrhea virus (BVDV), bovine parvovirus, bovine adenovirus, and blue tongue
virus.

The exact composition is unknown and varies from lot to lot, although lot-to-lot consistency
has improved in recent years. All reputable suppliers test their products for infectious virus
by several methods including fluorescent antibody, cytopathic effect, and hemadsorption.
These products are also screened for the standard microbial contaminants such as bacteria,
fungi, and mycoplasma. At one time animal serum was a major source of mycoplasma
contamination of tissue culture cells. However, nearly all sera today are filtered through
several 0.1-μm pore (or smaller) filters which effectively remove this organism. Filtered
sera should have a pH of 7.2- 7.8, osmolality of 290-310 mOsm/l and haemoglobin content
less than 20 mg

Sera can be stored at −20°C or colder for storage over 30 days. It should not be stored at
temperatures above −20°C for any length of time. Sera need to be stored in aliquots to avoid
repeated freeze-thaws. Serum can be thawed in a 37°C water bath with gentle agitations at
intervals to mix the solutes that tend to concentrate at the bottom of the bottle. It should not
be thawed at 37°C for a prolonged time or at temperatures above 40°C. All sera may retain
some fibrinogen. Because external factors may initiate the conversion of fibrinogen to
fibrin, flocculent material or turbidity may be observed after serum is thawed. The presence
of this material does not alter the serum’s performance. If the presence of flocculent
material or turbidity is a concern, it can be removed by filtration through a 0.45-μm filter.

However using serum in media has a number of disadvantages, such as:

 high cost
 problems with standardization
 exact composition unknown
 specificity
 variability
 unwanted effects such as stimulation or inhibition of growth and/ or cellular
function, on certain cell cultures
 can be a possible source of bacterial or viral contamination, if not from a reliable
source

16
Buffering capacity

Cell cultures have an optimum pH for growth, generally between pH 7.4-7.7. The type of
buffering that is used for the media depends on the growth conditions. In tissue culture, cells
are grown either in open systems (where there is free exchange of the atmosphere
immediately above the medium with the atmosphere of the incubator) or in closed systems
(where the two atmospheres are kept separate). The buffering system employed in the
medium needs to be matched to the culture system. Otherwise the cells may be subject to
metabolic stress which will impair their performance.

In closed systems the level of CO₂ is regulated by the metabolism of the cells. The culture
vessel must be sealed (flasks tightly capped) to retain any CO₂ generated by the cells.
Consequently, closed systems provide additional protection against contamination and have
simpler incubator requirements than open systems. Closed systems usually require media
with buffers based on Hanks’ balanced salt solution having relatively low levels of sodium
bicarbonate.

In open systems, humidity (to reduce evaporation) and a means of regulating CO₂ levels (if
the culture medium contains sodium bicarbonate) are required during incubation to maintain
the pH of the culture medium. Open systems usually require the higher levels of sodium
bicarbonate found in Earle’s salt solution combined with a 5 to 10% CO₂ atmosphere
supplied by the incubator. In general, 1.2 g/L to 2.2 g/L of sodium bicarbonate is used with
5% CO₂ whereas 3.7 g/L sodium bicarbonate is used with 10% CO₂. The exact amount will
depend upon the medium formulation.

In some cases, researchers “gas” the atmosphere of the culture vessel with a stream of
sterile-filtered 5% CO₂/95% air mixture and then tightly seal the flask prior to incubation in
a non-humidified and non-CO₂ incubator. While these culture vessels work with simpler
non-humidified, non-CO₂ incubators, the medium requirements are those of an open system.

All cell culture media, with the exception of Leibovitz’s L-15, are designed to be used with
5% CO₂ levels. Most have a sodium bicarbonate concentration of 1.5 g/L and are
supplemented with extra sodium pyruvate. While most commercial formulations of liquid
media do contain the appropriate amount of sodium bicarbonate, it is generally omitted from
the powdered form and needs to be added before use. Some medium formulations
incorporate other buffering systems such as phosphate or HEPES in addition to CO₂/sodium
bicarbonate. These media have the advantage of maintaining optimal pH in an open system
when the culture vessel is removed from the enriched CO₂ atmosphere of the incubator.

 Sodium bicarbonate and buffering

Cells produce and require small amounts of carbon dioxide for growth and survival. In
culture media, dissolved CO₂ is in equilibrium with bicarbonate ions and many medium
formulations take advantage of this CO₂/bicarbonate reaction to buffer the pH of the
medium. CO₂ dissolves freely into the medium and reacts with water to form carbonic acid.
As the cells metabolize and produce more CO₂, the pH of the medium decreases. The
17
optimal pH range can be maintained by supplementing the medium with sodium bicarbonate
and regulating the level of CO₂ in the atmosphere above the medium.

 HEPES buffer

HEPES and other organic buffers can be used with many cell lines to effectively buffer the
pH of the medium. Indeed, some standard medium formulations include HEPES. HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic chemical
buffering agent. However, this compound can be toxic, especially for some differentiated
cell types, so evaluate its effects before use. HEPES has been shown to greatly increase the
sensitivity of media to the phototoxic effects induced by exposure to fluorescent light.

 Phenol red

Phenol red is used to monitor the pH of media. During cell growth, the medium changes
colour as it changes pH due to metabolites released by the cells. At low pH levels, phenol
red turns the medium yellow, while at higher pH levels it turns the medium purple. For most
tissue culture work (pH 7.4), the medium should be bright red. Unfortunately, phenol red
can mimic the action of some steroid hormones, particularly estrogen. For studies with
estrogen-sensitive cells, such as mammary tissue, use media without phenol red.
Additionally, the sodium-potassium ion homeostasis is upset when phenol red is included in
some serum-free formulations; this effect is neutralized by the inclusion of serum or bovine
pituitary hormone in the medium.

Amino acids

The requirement of amino acids may vary with cell culture type. Commonly the necessary
amino acids include cysteine and tyrosine, but some non-essential amino acids may be
needed.

L-Glutamine is an essential amino acid required by virtually all mammalian and insect cells
grown in culture. It is used for protein production, as an energy source, and in nucleic acid
metabolism. It has been suggested that cultured cells use glutamine as an energy and carbon
source in preference to glucose, although glucose is present in most defined media. It is also
more labile in liquid cell culture media than other amino acids. Once added to the medium
the glutamine is only stable for about 3 weeks at 4 °C. L-Glutamine concentrations for
mammalian cell culture media can vary from 0.68 mM in Medium 199 to 4 mM in
Dulbecco’s Modified Eagle’s Medium. Use caution when adding more L-glutamine than is
called for in the original medium formulation. L-Glutamine degradation results in the build-
up of ammonia which can have a deleterious effect on some cell lines. For most cell lines,
ammonia toxicity is more critical for cell viability than L-glutamine limitation.

All medium formulations contain the ten essential amino acids as well as cysteine,
glutamine, and tyrosine. The inclusion of the other non-essential amino acids (alanine,
asparagine, aspartic acid, glycine, glutamic acid, proline, and serine) in some media

18
formulations reduces the metabolic burden on the cells allowing for an increase in cellular
proliferation.

Sodium pyruvate

Pyruvate is an intermediary organic acid metabolite in glycolysis and the first component of
the Embden-Meyerhof pathway. It can pass readily into or out of the cell. It provides both
an energy source and a carbon skeleton for anabolic processes. Pyruvate may help in
maintaining certain specialized cells, in clonal selection, in reducing the serum
concentration of the medium, and in reducing fluorescent light-induced phototoxicity.
Cellular metabolism of pyruvate produces carbon dioxide which is given off into the
atmosphere and becomes bicarbonate in the medium. Sodium pyruvate is added to give a
final concentration of 1 mM in most media, but is increased to 5 mM in Leibovitz’s L-15
medium primarily to facilitate use in CO₂-free environments.

Media Supplements

The complete growth media recommended for some cell lines requires the addition of
components not already available in the base media and serum. These components include
hormones, growth factors and signaling substances that sustain proliferation and maintain
normal cell metabolism. Supplements are usually prepared as 100× (or higher) stock
solutions in serum-free medium. The addition of supplements can change the final
osmolality of the complete growth medium, which may have a negative effect on the growth
of cells in culture. It is best to recheck the osmolality of the complete growth medium after
small volumes of supplement stock solutions are added. After supplements have been added
to a base medium, the shelf life of the complete growth medium should be determined on a
case-by-case basis. Complete media containing protein supplements (e.g., epidermal growth
factor, bovine serum albumin, etc.) tend to degrade faster than base media alone. Most
complete growth media can be stored in aliquots at 2°C to 8°C for about a month. Some
fastidious cell lines may require that components be added immediately before use. Do not
freeze complete growth medium. Freezing cell culture media at –70°C or below causes
some of the growth factors and/or vitamins to precipitate out of solution. It can be very
difficult to get these components to go back into solution after thawing, even if warmed to
37°C.

Osmolality

The osmolality of cell culture media for most vertebrate cells is kept within a narrow range
from 260 mOsm/kg to 320 mOsm/kg, even though most established cell lines will tolerate a
rather large variation in osmotic pressure. In contrast, the osmolality requirements for some
invertebrate cell lines fall outside of this range. For example, the snail embryo requires
medium of about 155 mOsm/kg, while some insect cells prefer 360 mOsm/kg to 375
mOsm/kg. Most commercially available liquid media report osmolality and it is advisable to
check the osmolality of any medium after the addition of saline solutions, drugs or
hormones dissolved in an acid or base solution, or large volumes of buffers.

19
Antibiotics and Antimycotics

Antibiotics and/or antimycotic agents are added to cell culture media as a prophylactic to
prevent contamination, as a cure once contamination is found, to induce the expression of
recombinant proteins or to maintain selective pressure on transfected cells. Most commonly
used is penicillin-streptomycin solution with final concentration of 50 to 100 IU/mL
penicillin and 50 to 100 μg/mL streptomycin. Gentamicin sulfate, another antibiotic is used
at 50 to 100 μg/mL. The antimycotic amphotericin B is used at 2.5 μg/mL. These
concentrations apply to media that contain serum. For serum-free media, reduce the
concentrations by at least 50%.

Routine use of antibiotics or antimycotics for cell culture is not recommended unless they
are specifically required, such as G418 for maintaining selective pressure on transfected
cells. Antibiotics can mask contamination by mycoplasma and resistant bacteria. Further,
they can interfere with the metabolism of sensitive cells. Avoid antimycotics as they can be
toxic to many cell lines. In some cases, antibiotic use for short periods of time can serve as a
valuable prophylactic. For example, antibiotic use is recommended when developing and
working with primary culture and when using flow cytometry to isolate subpopulations.

Some of the commonly used culture media are:

 Eagle’s Minimum Essential Medium (EMEM) was among the first widely used
media and was formulated by Harry Eagle from his earlier and simpler basal
medium (BME). EMEM contains Earle’s balanced salt solution, nonessential amino
acids, and sodium pyruvate. It is formulated with a reduced sodium bicarbonate
concentration (1,500 mg/l) for use with 5% CO₂. Because EMEM is a simple
medium, it is often fortified with additional supplements or higher levels of serum.

 Dulbecco’s Modified Eagle’s Medium (DMEM) has roughly twice the

concentration of amino acids and four times the amount of vitamins as EMEM, as
well as ferric nitrate, sodium pyruvate, and some supplementary amino acids (though
not all nonessential amino acids). This formulation contained 1,000 mg/L of Glucose
and a reduced sodium bicarbonate concentration (1,500 mg/L) for use with 5% CO₂.

 McCoy’s 5A and RPMI-1640 were developed at Roswell Park Memorial Institute

(RPMI) in Buffalo, New York. McCoy’s 5A was originally used to grow Novikoff
hepatoma cells and will support the growth of primary cultures. RPMI-1640 is a
modification of McCoy’s 5A and was developed for the long-term culture of
peripheral blood lymphocytes. RPMI-1640 will support the growth of a wide variety
of cells in suspension as well as a number of cells grown as monolayers. It contains
higher amounts of glucose (4,500 mg/L), sodium pyruvate, and HEPES buffer. It
also contains a reduced concentration of sodium bicarbonate (1,500 mg/L) for use
with 5% CO₂.

 Kaighn’s modification of Ham’s F-12 (Ham’s F-12K) was designed to support the
growth and differentiation of primary cells with or without serum. F-12K has
20
 increased amounts of amino acids, pyruvate, biotin, calcium, magnesium, putrescine,
and phenol red in addition to other modifications from the F-12 formula.

 DMEM/F12 Medium is a 1:1 mixture of DMEM and Ham’s F-12. It is an

extremely rich and complex medium that supports the growth of a broad range of
cell types in both serum and serumfree formulations.

 Leibovitz’s L-15 Medium is formulated for use without CO₂ incubation as is found
in teaching laboratories or when collecting biopsy samples. The standard sodium
bicarbonate/CO₂ buffering system is replaced by a combination of phosphate
buffers, free-base amino acids, higher levels of sodium pyruvate, and galactose. Cell
cultures can be grown in CO₂ incubators with L-15 medium provided there is no
exchange between the air in the culture vessel with that of the incubator (i.e., caps of
flasks are tightly closed).

 Hybri-Care Medium is a combination and modification of DMEM and NCTC 135

medium supplemented with insulin, oxalacetic acid, and HEPES. It is used for the
propagation of hybridomas and other fastidious cell lines.

 Subculturing techniques

Adherent cells Suspension cells

 Subculture of adherent/monolayer cultures

Anchorage-dependent cell lines growing in monolayers need to be subcultured at regular

intervals to maintain them in exponential growth. It should be passaged when they are in the

21
log phase, before they reach confluence. A drop in the pH of the growth medium usually
indicates a buildup of lactic acid, which is a by-product of cellular metabolism. Lactic acid
can be toxic to the cells, and the decreased pH can be sub-optimal for cell growth. Cells
should be subcultured if a drop in pH is indicated through a change in media colour.
Subcultivation of monolayers involves the breakage of both intercellular and intracellular
cell-to-surface bonds. For some cells that are loosely attached, a sharp blow with the palm
of your hand against the side of the flask or gentle pipetting can dislodge them. Many
require the digestion of their protein attachment bonds with proteolytic enzymes such as
trypsin/EDTA. For some cell lines mechanical forces such as scraping to dislodge the cells
is preferred. After the cells have been dissociated and dispersed into a single-cell
suspension, they are diluted to the appropriate concentration and transferred into fresh
culture vessels with the appropriate growth medium where they will reattach, grow and
divide.

 Subculture of suspension cultures

Cell propagation in suspension has several advantages over propagation in monolayer.

Subculturing is a simple matter of dilution. There is little or no growth lag after splitting a
suspension culture as there is with a monolayer culture, because there is none of the trauma
associated with proteolytic enzyme dispersal. Suspension cultures require less lab space per
cell yield, and scale-up is straightforward. Cells can be propagated in bioreactors similar to
the fermentors used for yeast or bacteria cultures. Depending upon the cell type, suspension
cultures are seeded at densities from 2 × 10⁴ to 5 × 10⁵ viable cells/mL and can attain
densities of 2 × 10⁶ cell/mL. If cells are seeded at too low a density they will go through a
lag phase of growth, grow very slowly, or die out completely. If cell densities are allowed to
become too high, the cells may exhaust the nutrients in the medium and die abruptly.

Cryopreservation and cell revival

Techniques for cell-line preservation which enable reliable and reproducible recovery of
material with unchanged, defined characteristics are essential and cryopreservation is one
such technique. Cryopreservation is a method whereby cells are frozen, maintaining their
viability, until they are defrosted months or years later. Cells are cryopreserved to minimize
genetic change and avoid loss through contamination. Spermatozoa were the first
mammalian cells to be cryopreserved successfully (Polge et al., 1949). This success was due
to the serendipitous discovery of the cryoprotective effect of glycerol. Since then, many
methods have been developed for various types of cells, tissues and organs. Most cell
cultures can be stored for many years, if not indefinitely, at temperatures below –130°C
(cryopreservation). ATCC has recovered cells from cultures cryopreserved for more than 40
years.

The many advantages of cryopreservation far outweigh the required investment in

equipment and reagents. These advantages include:

• Generation of safety stocks to ensure against loss of the culture from equipment failures or
contamination by microorganisms or other cell lines.

22
• Elimination of the time, energy, and materials required to maintain cultures not in
immediate use.

• Preservation of cells with finite population doublings (that will ultimately senesce).

• Insurance against phenotypic drift in the culture due to genetic instability and/or selective
pressure.

Phenomena which can cause damage to cells during cryopreservation mainly occur during
the freezing stage, and include: solution effects, extracellular ice formation, dehydration and
intracellular ice formation. As ice crystals grow in freezing water, solutes are excluded,
causing them to become concentrated in the remaining liquid water. High concentrations of
some solutes can be very damaging. When tissues are cooled slowly, water migrates out of
cells and ice forms in the extracellular space. Too much extracellular ice can cause
mechanical damage to the cell membrane due to crushing. Migration of water, causing
extracellular ice formation, can also cause cellular dehydration. The associated stresses on
the cell can cause damage directly. While some organisms and tissues can tolerate some
extracellular ice, any appreciable intracellular ice is almost always fatal to cells.

Intracellular ice can be minimized if water within the cell is allowed to escape by osmosis
during the cooling process. A slow cooling rate, generally –1°C per minute, facilitates this
process. However, as the cells lose water, they shrink in size and will quickly lose viability
if they go beyond a minimum volume. The addition of cryoprotectant agents such as
glycerol or dimethylsulfoxide (DMSO) will mitigate these effects. Cryopreservation media
always contain 5 to 15% DMSO or glycerol, along with complete media. Polyols, such as
glycerol and several sugars, provide cryoprotection by stabilizing lipid membranes by
hydrogen bonding with the polar head groups of membrane lipids which is especially
important under severely dehydrated conditions. DMSO penetrates the cell preventing the
formation of ice crystals that could result in membrane rupture. Water freezes at 0 °C, and
DMSO freezes at 19 °C. Mixtures of the two have lower freezing points and likely to form
less disruptive crystals when frozen. DMSO can penetrate the cells much faster than
glycerol and yields more reproducible results. Unfortunately, DMSO can cause some cells
to differentiate (e.g., HL-60 promyeloblast cells) and may be too toxic for some cells (e.g.,
HBE4-E6/E7 lung epithelial cells). Glycerol should be used in these instances, but glycerol
can cause osmotic problem, especially after thawing. Glycerol can be sterilized by
autoclaving whereas DMSO must be sterilized by filtration.

The addition of 10% to 20% cell culture-grade bovine serum albumin to freezing medium
may also increase post freeze survival. For cells grown in serum-free medium, adding 50%
conditioned medium (serum-free medium in which the cells were grown for 24 hours) to
both the cell freezing and the recovery medium may improve recovery and survival.
Replacing standard medium-cryoprotectant mixtures with 95% serum and 5% DMSO may
be superior for some overly sensitive cell lines, especially hybridomas.

Cryopreserved samples are stored in glass or polypropylene vials called as cryovials. Glass
vials are more difficult to work with; they need to be sealed with a hot flame, and they can
be difficult to open. However, they are preferred for long-term storage of valuable seed
cultures. Plastic vials are used for the storage of distribution stocks.
23
An important instrument in cryopreservation is computer controlled, programmable
electronic freezing unit (Controlled rate freezing chambers) which maintains a cooling rate
of –1°C per minute.

The ultra-low temperatures (below –130°C) required for long-term storage can be
maintained by specialized electric freezers or more commonly by liquid nitrogen freezers.
Liquid nitrogen freezers permit storage either in the vapor phase above the liquid at
temperature between -140°C and -180°C, or submerged in the liquid at a temperature below
-196°C. Using vapor phase storage greatly reduces the possibility of leaky vials or ampules
exploding during removal (This occurs when liquid nitrogen leaks into the vials and rapidly
expands when removed from the tank).

So, the success of the freezing process depends on four critical areas: Proper handling and
gentle harvesting of the cultures, correct use of the cryoprotective agent, a controlled rate of
freezing and storage under proper cryogenic conditions

The recovery of cryopreserved cells is straightforward: Cells are thawed rapidly in a water
bath at 37°C, removed from the freeze-medium by gentle centrifugation and/or diluted with
growth medium, and seeded in a culture vessel in complete growth medium. Viability for
most cells declines and reaches the lowest point at 24 hours post-thaw. Most, if not all, of
this decline appears to be due to apoptosis (as opposed to necrosis) induced by the stress of
the cryopreservation process. After this time point, cells begin to recover and enter
exponential growth. Some cell lines, such as hybridoma cultures, take several days before
they fully recover from cryopreservation. There are numerous factors which affect the
viability of recovered cells such as the composition of the freeze medium, the growth phase
of the culture, the stage of the cell in the cell cycle and the number and concentration of
cells within the freezing solution.

Contamination and sterilization

Contamination of cell cultures is easily the most common problem encountered in cell
culture laboratories, sometimes with very serious consequences. It can arise from many
sources including other cell lines, reagents, supplies such as pipettes and culture vessels,
equipment such as tissue culture hoods and incubators, and laboratory personnel. The media
used to grow the cells also provide excellent nutrition for unwanted organisms.
Contamination often results in the loss of a great deal of time and money.
Cell culture contaminants of two types;
• Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of
disinfectants that are invisible.
• Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or
fungus or also from cross-contamination of cells from other cell lines. Biological
contaminants affect cell cultures in different ways. They competes for nutrients with host
cells, their secreted acidic or alkaline by-products ceases the growth of the host cells, they
degrase arginine & purine, thereby inhibiting the synthesis of histone and nucleic acid and
some of them also produce H2O2 which is directly toxic to cells.

24
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 and cell lysis.
When most bacterial contamination occurs, it usually occurs within a few days and is
typically obvious to the naked eye. Distinct changes to the medium such as turbidity,
presence of particles visible in suspension, a rapid decline in pH (yellow color, indicating
acidity) and sometimes presence of thin film on surface, are all indicators of bacterial
contamination. Under microscope, bacteria can be observed as tiny, moving granules with
characteristic morphology. Fastidious bacteria species that grow very slowly can be difficult
to detect.
Fungal contaminants may or may not cause a change in the pH of the medium and can be
distinguished from bacteria by checking for the presence of filamentous structures in the
suspension. The pH of the culture remains stable in the initial stages of contamination, then
rapidly increases as the culture become more heavily infected and becomes turbid. Under
microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as
denser clumps of spores. Spores of many mold species can survive extremely harsh and
inhospitable environments in their dormant stage, only to become activated when they
encounter suitable growth conditions.
Yeast cells are larger than bacteria, but may not appreciably change the pH of the medium,
and will appear as separate round or ovoid particles. Similar to fungal contamination, media
turbidity and colour change are observed, in advanced stages of infection.
Mycoplasmas are more insidious, the medium is not cloudy, the organisms cannot be seen
under an ordinary microscope (extremely small size, less than 1 μm), and most mycoplasma
species are very difficult to grow on agar plates. Mycoplasma are very difficult to detect
until they achieve extremely high densities and cause the cell culture to deteriorate; until
then, there are often no visible signs of infection. Some slow growing mycoplasma may
persists in culture without causing cell death, but they can alter the behaviour and
metabolism of the host cells in the culture. Chronic mycoplasma infections might manifest
themselves with decreased rate of cell proliferation, reduced saturation density, and
agglutination in suspension cultures. Testing for mycoplasma is time consuming but must be
done regularly because cross contamination is very easy. Fluorescent DNA stains (Hoechst)
or molecular probes may be used to detect their presence in cells, those cells which turn out
to contain DNA in the cytoplasm must be discarded. Some may have reported that
antibiotics Plasmocin (Novagen) and Ciproflaxin are effective against mycoplasmas.
Their extremely small size makes viruses very difficult to detect in culture, and to remove
them from reagents used in cell culture laboratories. Because most viruses have very
stringent requirements for their host, they usually do not adversely affect cell cultures from
species other than their host. However, using virally infected cell cultures can present a
serious health hazard to the laboratory personnel, especially if human or primate cells are
cultured in the laboratory. Viral infection of cell cultures can be detected by electron
microscopy, immunostaining with a panel of antibodies, ELISA assays, or PCR with
appropriate viral primers.
Cross-contamination of one cell line with another can sometimes lead to the replacement
of the original cell with the contaminant, particularly when the contaminant grows faster
than the original line. HeLa cells are perhaps the most famous example of a cross-
contaminating cell line overtaking and then masquerading as the original. In the 1950s and
25
1960s, many continuous lines were unknowingly cross-contaminated with other cell lines
including HeLa cells. This cross-contamination was only uncovered with the development
of suitable genetic markers beginning in 1967. More recently, cell repositories have used
DNA polymorphisms in addition to enzyme polymorphisms, HLA typing, and karyotyping
to confirm the identity of their cell lines. One of the most reliable methods to study DNA
polymorphisms is the profiling of short tandem repeats (STR) by PCR.

While the potential for contamination is constant, the risk can be reduced or eliminated by
proper precautions. Some basic preventive measures may be taken; such as using only
reagents of known quality and sterility, quarantining new cell lines until they are tested to be
free from contamination, performing routine maintenance and cleaning of all equipment,
properly training cell culture personnel, checking sterilizing procedures (autoclave and oven
procedures), checking sterility of laminar flow hoods, regular checks on cultures, each
worker having his or her own set of media and disposal of contaminated cultures rather than
attempting to decontaminate them. Microbiological media which can be used to test for
bacterial and fungal contamination include blood agar, thioglycollate broth, tryptic soy
broth, BHI broth, Sabouraud broth, YM broth, and nutrient broth with 2% yeast extract.
However, some microbial contamination is not apparent. For example, the use of antibiotics
can suppress bacterial growth and thus mask contamination.
Eliminating contamination from a cell line is time consuming and does not always work.
Discarding the culture and starting over is preferred. However, if the cells are unique and
irreplaceable, one should first identify the contaminant and select a suitable antibiotic for
treatment. It is best to test the contaminating microbe for its antibiotic sensitivity prior to
treatment; this allows for a shorter treatment time and limits exposure of the cell line to
potentially damaging reagents. The cells are cultured for 1 to 2 weeks in the presence of the
antibiotic, and then cultured without antibiotic for 1 to 2 months. At this point, the line
should be retested with a very sensitive test method to make sure that the culture is clean.
Periodic retesting should be employed to make sure that the contaminant does not reappear.

Much of the contamination comes from the culture medium and its components or from
inadequately cleaned glassware. Routine sterility checks on the medium, serum and
nutrients are recommended. To do this, a small aliquot of the medium should be incubated
at room temperature and another similar aliquot simultaneously incubated at 37 °C for up to
one week before the medium is used for culturing cells. If these small samples are found to
be contaminated, the medium should be autoclaved and discarded. Generally, if
contamination occurs, affected cultures should be disposed of into 2.5% hyperchlorite
solution. Media bottles known, or suspected, to be contaminated should be disposed of as
well.

Given below is a list of basic aseptic techniques to be followed compulsorily in a cell

culture lab:

 Ensure that the work area is clear, swabbed down regularly with 70% ethanol and
that all the equipment used has been sterilized. Clean laboratory coats are also
essential.

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  Use a Bunsen flame to heat the surrounding air. This causes the movement of air and
contaminants upwards and reduces the chance of contamination entering open
vessels. Open all bottles and perform all manoeuvres in this area only.
 Swab all bottle tops and necks with 70% ethanol to clean them before opening;
Flame all bottle necks and pipettes by passing very quickly through the hottest part
of the flame. This is not necessary with sterile, individually wrapped, plastic flasks
and pipettes.
 Avoid placing caps and pipettes down on the bench; practice holding bottle tops with
the little finger while holding the bottles for pouring or pipetting; Work either left to
right or vice versa, so that all material to be used is on one side and, once finished, is
placed on the other side of the Bunsen burner. (This may also stop the operator using
the same reagent twice).
 Manipulate bottles and flasks carefully. The inside of tops of bottles and flasks must
not be touched by the operator. Touching of open vessels should also be prevented
when pouring. If necessary practice pouring from one container to another, keeping a
distance of 5 mm between the two vessels.
 Never use the same media bottle for different cell lines. If caps are dropped or
bottles or pipette tips are touched unconditionally, replace them with new ones.
 Clear up spills immediately and always leave the work area clean and tidy.
 Dispose of glassware in appropriate bins and discard used plasticware in marked
polythene bags for autoclaving or incineration. All glassware or plasticware used for
infectious work must always be decontaminated by autoclaving before incineration.
Re-usable glassware should be immersed in disinfectant whilst awaiting transfer to
an autoclave.
 Always wear appropriate personal protective equipment. Change gloves when
contaminated, and dispose of used gloves with other contaminated laboratory waste.
 Take care to minimize the creation of aerosols and/or splashes.
 Switch on the laminar flow cabinet and its UV light 20 minutes prior to start
working.

Sterilization

Sterilization is an important part of aseptic cell culture practices. Sterilization procedures

are designed to kill the microorganisms, besides destroying the spores.

 Sterilization by heat

It can be by dry heat or moist heat. Dry heat sterilization is carried out at a minimum
temperature of 160°C for about one hour. Glassware, such as pipettes, conical flasks,
beakers (covered with aluminum foil) are sterilized by this method in a hot air oven. Certain
fluids and heat stable items can be sterilized in an autoclave at 121°C, 15 lbs pressure for
15-20 minutes. For effective moist heat sterilization, it is necessary that the steam penetrates
to all the parts of the sterilizing materials.

 Sterilization by filters

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The use of filters for sterilization of liquids often becomes necessary, since the heat labile
constituents (enzymes, hormones, of these liquids may get degraded at higher temperatures.
Sterile filtration is a novel technique for heat- labile solutions. The size of micropores of the
filters is 0.1-0.2 µm. Filters, made from several materials are in use. These materials include
nylon, cellulose acetate, cellulose nitrate, polycarbonate, polyethersulfone (PES) and
ceramics. The filters are made in different designs-disc filters, cartridges and hollow fiber.
In fact, many commercial companies (e.g. Millipore, Durapore) supply reusable and
disposable filters, designed for different purposes of sterilization.

Enumeration of cells

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell
concentrations ensure experimental reproducibility and accuracy. Cell counts are important
for monitoring cell health and proliferation rate, assessing immortalization or
transformation, seeding cells for subsequent experiments, transfection or infection, and
preparing for cell-based assays. It is important that cell counts be accurate, consistent, and
fast, particularly for quantitative measurements of cellular responses.
There are direct methods and indirect methods. In the direct method, cell numbers are
determined directly either by counting using a counting chamber or by using an electronic
particle counter. In the indirect methods measurement of some parameters such as DNA
content or protein content related to cell number is used as the method of estimating
biomass.

 Direct methods

 Haemocytometer

A hemocytometer (Improved Neubaur chamber) is the simplest and cheapest method of

counting. A haemocytometer has two grids and a full grid contains nine squares, each of
which has a volume of 0.1mm3 (with coverslip). The central counting area of the
hemocytometer contains 25 large squares and each large square has 16 smaller squares. The
corner 4 squares have 16 small squares. Corner squares are used for counting larger cells
and centre square for smaller blood, yeast or sperm cells. When counting, count only those
cells on the lines of two sides of the large square to avoid counting cells twice. Suspensions
should be dilute enough so that the cells or other particles do not overlap each other on the
grid, and should be uniformly distributed.
Fig 1.6: Haemocytometer

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 % Viability= No. of live cells/ total
number of cells (live and dead) X
100

Cell concentration (No. of cells/ml)

= Average number of viable cells X
dilution factor X 104

Viability determinations can be easily carried out after diluting cells with dye. Trypan blue
staining is commonly used to count the cells using hemocytometer and to check cell
viability. A live cell with intact plasma membrane proteins can expel trypan blue, which is a
membrane impermeant dye. Therefore live cells appear colourless. But dead cells with
damaged plasma membrane retain the stain and appear blue in color.
This counting method is open to error in a number of ways. Errors may be introduced by
incorrect preparation of the chamber, incorrect sampling of cells, and aggregation of cells.
However, it has the advantage of allowing visual inspection of the morphology of the cells.

 Coulter counters

Electronic particle counters consist of two electrodes separated by a small orifice. If a

potential is applied to the electrodes, current will pass between them through the buffer in
the orifice. Cells in suspension are drawn through a fine orifice in the Coulter counter and,
as each cell passes through, it produces a change in the current flowing across the orifice.
The size of the change in current flow depends on the size of the particle and the difference
in the dielectric constant (conductivity) of the particle and the suspending buffer. Each
change is recorded as a pulse and it is these pulses that are sorted and counted. The size of
the pulse is proportional to the volume of the particle passing through, so signals of varying
size are produced. The pulse height threshold can be set, therefore, to eliminate electronic
noise and weak pulses produced by debris.
Electronic counters, of which the Coulter counter is the most widely used, provide rapid
results, but high cell numbers are required to give an accurate count. Another disadvantage
is that they cannot distinguish between dead and viable cells and clumps of cells may
register as a single pulse thus leading to inaccurate counts.
Fig 1.7: Coulter counter mechanism

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  Flow Cytometry

It is an expensive but accurate method of counting. The basic principle of flow cytometry is
the passage of cells in single file in front of a laser beam, a detector pickup to light that
reflated through cells, so they can be detected, counted and their structure and shape can be
measured.

Fig 1.8: Flow cytometry mechanism

Indirect methods for determining cells in culture

Indirect methods such as the estimation of total DNA or protein are used less frequently.
They are useful when cells are grown in microwell plates or as hanging drop cultures. DNA
and protein assays are inaccurate, particularly if cells are multinucleated. They do not
distinguish viable and non-viable cells and are expensive.

Cell line characterization

It is the process of defining / outlining the characteristic traits of a cell line. It is important in
cell line authentication, confirming its origin and lineage (stem/ precursor/ differentiated),
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detection of transformation and cross-contamination, monitoring genetic instability and
phenotypic variations. Cells lines have been characterized at the DNA, RNA, and protein
level which allow the choice of correct cell line for further research or uses. Cell line
authentication is the sum of the processes by which a line’s identity is verified and shown to
be free of contamination from other cell lines and microbes. So it is necessary to carry out a
list of observations or assays in order to characterize a cell line. They are as follows

 Cell morphology; whether the cells are fibroblastic, epithelial or lymphoblastic. It

can be done by microscopic observation and immune fluorescence analysis using
cell type specific markers.
 Growth properties; Whether the cell line grow as adherent or in suspension
 Chromosome number analysis by karyotyping can confirm the species of origin and
chromosomal ploidy associated with transformation.
 Chromosome banding pattern analysis can reveal the structural rearrangements
and mutations in chromosome.
 Isoenzyme analysis to detect interspecific cell line cross contamination
 DNA barcoding using specific target genes such as mitochondrial Cytochrome C
Oxidase subunit I (COI), 16S rRNA and 18S rDNA for prokaryotes and eukaryotes
respectively.
 DNA fingerprinting using molecular markers such as RFLP (restriction fragment
length polymorphism), AFLP (amplified fragment length polymorphism), STR
(short tandem repeats) and SNPs (single nucleotide polymorphism.
 Cultural characteristics are analysed from cell growth curves plotted against
different media, pH, temperatures, FBS concentrations etc.
 Cryopreservation efficiency has to be checked by reviving cells cryopreserved at
different passage level. An average viability of 80% for the revived cells indicates
efficient cryopreservation.
 Quality control tests for viability, sterility and mycoplasma contamination have to
be performed before depositing the cell line to cell bank.

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