Subculturing Monolayer Cell

Cultures

Maulik P. Suthar

© 2009, Maulik Suthar

 Introduction
• Most animal cell lines and primary cultures grow as a single
thickness cell layer or sheet attached to a plastic or glass substrate.
• Once the available substrate surface is covered by cells (a confluent
culture), growth slows and then ceases.
• Why subculture? : to keep the cells healthy and actively growing
• Subcultivation process involves breaking the bonds or cellular ‘glue’
that attaches the cells to the substrate and to each other by using
proteolytic enzymes such as trypsin, dispase, or collagenase.
• Combined with divalent cation chelators such as EDTA (binds calcium
and magnesium ions).
• The loosened cells are then removed from the culture vessel,counted,
diluted and subdivided into new vessels.
• Cells then reattach, begin to grow and divide, and, after a suitable
incubation period (depending on the initial inoculum size, growth
conditions and cell line), again reach saturation or confluency. At this
point, the subcultivation cycle can be repeated.

© 2009, Maulik Suthar

 Sterile requirements
• Flask of actively growing cells that are 80 to 90% confluent
• Culture medium. This should contain all of the additives (fetal bovine
serum, glutamine etc.) required by the above cell line.
• Phosphate-Buffered Saline (Free from Calcium- and Magnesium)-- is
used to maintain proper pH and osmotic balance while the cells are being
washed to remove protease inhibitors that are found in most animal sera.
• 0.1% Trypsin solution: Trypsin is normally used in concentrations ranging
from 0.05% to 0.25%. Working concentrations are usually determined by
using the lowest trypsin concentration that can remove the cells from the
substrate and give a single cell suspension in a relatively short time (5 to 10
minutes). Trypsin solutions are often supplemented with other enzymes
(collagenase) or chelating agents (EDTA) to improve its performance.
• Centrifuge tubes - 15mL disposable screw cap
• Culture vessels : Appropriate type and size
• Pipettes -1, 5, 10 and 25mL -
• 0.04% Trypan blue (Sterile) solution for viablity staining
• Pipette tips
• Laminar flow hood © 2009, Maulik Suthar
 Procedure
1. Examination:
examine cultures daily and always prior to subcultivation.
Using an inverted phase contrast microscope (100 to 200x), quickly check
the general appearance of culture.
Look for signs of microbial contamination.
Many cells round up during mitosis, forming very refractile (bright)
spheres that may float free of the surface when the culture is disturbed.
Dead cells often round up and become detached but are usually not bright
or refractile.

2. Cell harvesting:
This step removes the cells from the plastic substrate and breaks cell-to-
cell bonds as gently as possible. When using enzymatic dissociation:
• the old medium is removed and discarded;
• the cell monolayer is gently rinsed;
• the enzyme solution is added and the culture incubated until the cells
are released.

© 2009, Maulik Suthar

 Procedure
a) Add 5mL of the trypsin solution (in CMF-PBS) to the flask and place the flask
back in an incubator at 37ºC to increase the activity of the enzyme solution.
(Prewarming of the enzyme solution to 37ºC will decrease the required exposure
period.)
b) Check the progress of the enzyme treatment every few minutes with an inverted
phase contrast microscope. Once most of the cells have rounded up, gently tap
the side of the flask to detach them from the plastic surface. Then add 5mL of
growth medium to the cell suspension and, using a 10mL pipette, vigorously
wash any remaining cells from the bottom of the culture vessel. At this point a
quick check on the inverted microscope should show that the cell suspension
consists of at least 95% single cells. If this is not the case, more vigorous
pipetting may be necessary
c) Collect the suspended cells in a 15mL centrifuge tube and place on ice. Some
dissociating agents should be removed at this point by centrifugation to prevent
carry over which can cause poor cell attachment or toxicity. However, the trypsin
in the cell suspension will be inactivated by the serum and does not absolutely
need to be removed. If removal is desired, spin the cell suspension at 100xg for
5 minutes. Then remove the trypsincontaining medium and replace with fresh
medium.

© 2009, Maulik Suthar

 Cell counting:

To determine growth rates or set up cultures at known concentrations it is necessary

to count the cell suspension. Hemacytometers or electronic cell counting
devices can be used.
The hemacytometer has the added advantages of both being less expensive and
allowing cell viability determinations to be made during counting.
Vortex the cell suspension and remove a 0.5mL sample and place in a tube for
counting. To this add 1mL of the vital stain trypan blue (0.04%).
Mix well by vortexing, withdraw a 20μL sample with a wide tip pipettor and carefully
load a clean hemacytometer. (Do not overfill!)
Do a viable cell count and calculate the number of viable cells/mL and the total cell
number.

© 2009, Maulik Suthar

 Plating
: How much and how many?
• After making the appropriate dilutions, add the correct amount of cells to each
culture vessel. Then add fresh medium to bring the culture vessel to its
recommended working volume.
• label all vessels accurately; write on the sides of flasks and around the outer
edge of the dish tops so as not to interfere with microscopic observation.

© 2009, Maulik Suthar

 Incubation
Most mammalian cell cultures – optimum temperature : between 35º and 37ºC.
High humidity levels and CO2 concentrations. The high humidity cuts down
evaporation losses in open systems such as petri dishes and microplates that
would otherwise result in hypertonic culture medium and stressed cells. The
elevated CO2 concentrations (usually 5% to 10%, depending on bicarbonate
concentrations in the medium) help maintain the proper pH (7.4 ± 0.2) when
used with the correct bicarbonate buffer system.
In order for this type of buffer system to work it is necessary to allow gas exchange
by using unsealed dishes and plates or flasks with gas permeable (vented)
caps.
Leave caps on flasks slightly loosened (or use vented caps on the flasks for extra
protection against spillage and contamination) and place on a shelf in a 37ºC,
humidified CO2 incubator.
Examine cultures daily and change medium as needed.

© 2009, Maulik Suthar

Trypsinization Procedure

© 2009, Maulik Suthar

 Requirements

• Culture medium: all of the additives (sera, glutamine, etc.) required by the
chosen cell line. 2. Calcium- and Magnesium-Free Phosphate-Buffered Saline
-PBS (10mL). This simple salt solution is used to maintain proper pH and
osmotic balance while the cells are being washed to remove protease inhibitors
that are found in FBS.
• 0.05% Trypsin/EDTA (0.05%) solution: Trypsin is normally used in
concentrations ranging from 0.05% to 0.25%. Working concentrations are
usually determined by using the lowest trypsin concentration that can
remove the cells from the substrate and give a single cell suspension in a
relatively short time (5 to 10 minutes). Trypsin solutions are often supplemented
with other enzymes (collagenase) or chelating agents (EDTA) to improve its
performance.
• 15mL disposable screw cap centrifuge tubes
• Hemacytometer
• Centrifuge
• Phase contrast microscope
• 1, 5, 10 and 25mL pipettes

© 2009, Maulik Suthar

 Procedure
• Aseptically aspirate spent media from the bottom well of the plate
• Rinse the top and bottom of the well insert 2 to 3 times with PBS to remove any serum
(contains trypsin inhibitors). Review chart on left for recommended buffer volumes.
• Add Trypsin/EDTA to the bottom of the well. Use the same liquid volumes as above.
Incubate for 10 minutes in a 37°C incubator, containing 5% CO2.
• After 5 minutes, view the cells under a phase contrast microscope for detachment.
Generally, the lower the trypsin concentration and the less time the cells stay in trypsin the
better.
• Once the cells look rounded or detached (approximately 10 minutes), remove the
Trypsin/EDTA containing cell suspension and add to a 15mL centrifuge tube containing
5mL of serumcontaining culture medium to inactivate the trypsin. (For serum-free culture
use a trypsin inhibitor.)
5. With the cover on the plate of the Transwell inserts, sharply hit the side of the plate with your
hand to detach cells from the membrane. Rinse the top and bottom of the Transwell insert
with Trypsin/EDTA to remove any remaining cells. Add the cell suspension to the centrifuge
tube.
6. Observe Transwell inserts under a phase contrast microscope to ensure that all of your cells
have been removed. Perform a second trypsinization if there are large numbers of cells still
remaining on the membrane by repeating the steps 3 to 5.
7. Gently vortex the cells to resuspend and then remove an aliquot of cell suspension and count
with a clean hemacytometer. Centrifuge cells for 5 to 10 minutes at 100xg. Remove media
from the centrifuge tube, leaving the cell pellet, and resuspend cells in the appropriate
amount of growth media.

© 2009, Maulik Suthar

Fixation and Staining

© 2009, Maulik Suthar

 Requirements

• Reagents
• Phosphate Buffered Saline (for cell culture)
• 2.5% Glutaraldehyde, EM Grade 1.06 (Polysciences)
• 0.5% Triton X-100
• Gill's Hematoxylin No.1 (Polysciences) Filter through a vacuum filter with a
• 0.2μm membrane.
• Acid alcohol: 0.5% Hydrochloric Acid in 70% ethanol
• 0.04%Ammonium Hydroxide
• Materials
• Fume hood
• Pipette aid and pipettes
• Modified cluster dishes (holes in the cover and base )
• Small basin or container

© 2009, Maulik Suthar

 Procedure

1. Rinse the Transwell membrane with cells by adding PBS (37°C) to the bottom of
the well plate until it reaches the membrane, and then add PBS to the top of the
membrane. The rinse should be added slowly and then aspirated. Do not touch
either surface of the membrane. Repeat the rinse procedure.
2. Pipette enough PBS to cover surface of the membrane. PBS will remove any
residual growth media. Remove the PBS from the Transwell insert.
3. Pipette enough 2.5% EM grade glutaraldehyde to cover the surface of the
membrane. Leaving the glutaraldehyde in the Transwell insert, incubate at room
temperature for 15 minutes. Remove glutaraldehyde by carefully aspirating it or
pouring it off.
4. Pipette enough 0.5% Triton X-100 to cover the surface of the membrane.
Leaving the Triton X-100 in the Transwell insert, incubate for three minutes at
room temperature. Remove Triton X-100 from the Transwell insert in the manner
described in step 3.

© 2009, Maulik Suthar

1. Pipette enough Gill's hematoxylin No.1 to cover the surface of the membrane.
Leaving the hematoxylin in the Transwell insert, incubate at room temperature
for 15 minutes. Remove the hematoxylin from the Transwell insert in the manner
described in step 3.
2. Rinse Transwell insert 3 to 4 times in a basin filled with distilled water to remove
the excess stain.
3. Pipette enough acid alcohol to cover the surface of the membrane and leave it
for 2 to 3 minutes to remove any residual stain (destain).
4. Rinse Transwell insert twice in a basin filled with fresh distilled water.
5. Pipette enough 0.04% NH4OH to cover the surface of the membrane and leave
it until a blue color is observed on the membrane (2-3 minutes).
6. Rinse Transwell insert twice in a basin filled with distilled water.
7. Air dry at an angle on a clean paper towel overnight.

© 2009, Maulik Suthar

Clonal Growth of Cells in Semisolid Media

© 2009, Maulik Suthar

 Introduction

• Established cell lines such as HeLa and L-cells, as well as normal cells transformed
by viruses, can form colonies suspended in soft agar-based media. Most normal
cells will not grow under these conditions although there are exceptions (e.g.
cartilage).
• The baby hamster kidney line, BHK-21, will not grow in agar, but will after
transformation by polyoma virus (1, 2). In contrast, if agarose is used, (a purified
agar, free of sulfated polysaccharides) BHK-21 will grow in suspension. If all these
factors are taken into consideration and standardized, this system can be used to
measure “transformation." It must be remembered, however, that morphological
transformation and ability to grow in suspension are not necessarily correlated with
the ability to form tumors in appropriate hosts.
• Growth of cells in semisolid medium, whether agar, agarose, or methylcellulose
offers a second advantage. The bacteria like colonies that form from monodispersed
cell suspensions offer a means of isolating clones with a minimal amount of effort.
• Using a finely drawn pipette, single, well isolated colonies can be removed from the
suspended state and subcultured. However, there are variations that must be used
depending upon cell types. Clones in which there is loose intercellular bonding can
be dissociated into a monodispersed population through gentle pipetting. However,
many cell types require further enzymatic treatment to disperse them or must be
treated as explants.

© 2009, Maulik Suthar

 Requirements
1. 20mL of 2.5% Bacto-Agar (Difco) in distilled water in 100mL glass bottle - Corning Cat. #
1395-100 or 1396-100. Solution should be sterilized by autoclaving. Agar needs to be
melted at 100°C prior to use and kept at 45°C until mixed with Nutrient Mix.
2. 50mL 1X base medium - sterile. This should be made from the standard medium (no
serum) used to grow the cells that will be cloned.
3. 20mL 2X base medium - sterile. Reconstituting 10X-powdered standardmedium with only
half the required water (no serum) is used to make the 2X medium.
4. 50mL complete growth medium – sterile. (This should be made from base medium plus
10% fetal bovine serum) for dilutions. Base medium should be the same medium that is
normally used to grow the cell culture.
5. Fetal bovine serum (10mL)
6. 80mL Nutrient Mix in 100mL glass bottle - Corning Cat. # 1395-100 or 1396-100 Make up
by combining:
2X base medium (20mL)
Fetal bovine serum (10mL)
1X base medium (50mL)
7. 60mm plastic dishes - Corning Cat. # 430166 (8)
8. 15mL plastic centrifuge tubes - Corning Cat. # 430055 or 430789 (16)
9. 10mL pipettes - Corning Cat. #4488 or 4101 (1 bag)
10. 1mL pipettes - Corning Cat. #4485, 4011 or 4012 (1 bag)
11. Water baths at 45°C and 100°C
12. Cell suspension for plating (1mL at 106 cells/mL)

© 2009, Maulik Suthar

 Procedure
Label and prepare all plates and dilution tubes in advance.
1. Prepare 1X nutrient agar medium:
a) Melt 2.5% agar in autoclave, microwave oven or boiling water bath, then place in
45°C water bath. The agar temperature must be allowed to cool to 45°C
before proceeding.
b) Warm nutrient mix in 45°C water bath. The temperature of the nutrient mix
must be allowed to reach 45°C before proceeding.
c) Pour contents of 2.5% agar into nutrient mix to create the osmotically balanced
1X nutrient agar medium with a 0.5% agar concentration. Mix gently but AVOID
BUBBLES. Do not allow mixture to cool. Keep nutrient agar medium in the
45°C water bath when not being used.
2. Pipette 7mL of the 1X nutrient agar medium per 60mm dish. Allow agar to cool
and harden. Once hardened, return the plates to the incubator. This agar layer
will provide a base nutrient layer to support cell growth for at least one week. It
will also keep the cells from reaching and attaching to the plastic on the bottom
of the dish.

© 2009, Maulik Suthar

3. Distribute 1mL aliquots of 1X nutrient agar medium in eight 15mL centrifuge
tubes. Keep tubes at 45°C in water bath and do not allow mixture to cool or
it will begin to harden and develop clumps.
4. Prepare the cell suspension in complete growth medium. When first plating a new
cell type, we recommend that tubes be set up with the following cell
concentrations: 1x105, 1x104, 1x103, and 1x102 cells/mL. Use 0.5mL cell
suspension added to 4.5mL complete growth medium (no agar) to make these
1:10 dilutions.
a) Add 0.5mL of each dilution to individual tubes of the nutrient agar mixture. Mix
gently (but avoid bubbles) and immediately pour contents of the tube (0.33%
agar) on top of the bottom agar layer in one of the dishes. Work rapidly. If the
nutrient agar is lower than 45°C prior to mixing, then the cell suspension may
form clumps when plated. If the medium is too warm, the cells will be heat-
shocked and may not survive.
b) Repeat the process for the seven remaining tubes and plates, setting up each cell
concentration in duplicate.
5. Allow agar in plates to harden for 15 to 30 minutes on the bench top and then
place them in a CO2 incubator. If the resulting medium is too soft, try increasing
the initial agar concentration to 3.5%. This will give a final agar concentration in
the base layer of 0.7%.
6. Examine plates every two or three days until colonies are large enough to see
with the unaided eye.

© 2009, Maulik Suthar

 Suspension Culture Issues
• Reduce Shearing Damage Because animal cells lack a protective cell wall, they can
be easily damaged by the shear forces that develop if they are spun too fast or shaken
too vigorously. Some cell lines, especially those grown in serum-free media, are very
sensitive to shear forces and a difference of only 30 to 50 RPM in mixing speeds can
lead to serious growth problems. For many cell lines, shaking them is often gentler
than stirring them and is a better approach when trying to suspension-adapt cells.
• For shaker flasks, it is recommended to start with a shaking rate of 75 to 150 RPM on
an orbital shaker with a medium volume of 30 to 40% of the nominal flask capacity (1L
of medium in a 3L flask). For Corning spinner flasks a starting speed of 50 to 150 RPM
is suggested.
• The use of baffled spinner and shake flasks can reduce the required mixing rates
considerably. Some cell types, such as insect cells, require higher oxygen levels and
may benefit from more vigorous mixing and the use of vented flasks or continuous
gassing. It is strongly recommended to empirically determine the optimum stirring or
mixing conditions.
• Start by choosing the lowest speed that appears to give an even cell distribution from
the top to the bottom of the flask. However, to get adequate gassing, higher speeds
may be necessary, Shearing damage from these higher speeds can be reduced by
increasing the medium viscosity by adding carboxymethylcellulose (1 to 2%), BSA
(100 μg/mL) or Pluronic® F-68 (0.1%) to the medium (Mather, 1998b). This is
especially important when using reduced serum or serum-free medium.

© 2009, Maulik Suthar

• Avoid Cell Clumping and Sticking: Some cell lines tend to form large clumps when grown in
suspension. These larger clumps tend to settle to the bottom of the flask or may attach to the
flask side walls and can result in lower cell viability and growth. Using a calcium-free medium
(Joklik’s MEM; S-MEM) will reduce cell clumping as these divalent cations are very important in
cell to cell binding (McLimans; 1979). Coating the surface of glass suspension flasks with
siliconizing solutions before sterilization will reduce cell clumps forming on the flask surface.
Sigmacote®, AquaSil™, and Siliclad® are some commercially available siliconizing solutions
suitable for cell culture applications. It is important to carefully follow the use and safety
directions for these products to avoid culture toxicity.
• Use Appropriate Seeding Densities: Using the correct initial seeding density is very important
when growing cells in suspension. It is always better to add too many cells rather than too few.
Start with a seeding density of 1 x 105 to 5 x 105 cells/mL; the higher concentration is better
when cells are adapting to serum-free conditions. An alternative approach for spinner and shake
flasks is to start with half the normal volume of medium. This reduces the number of cells
required to reach the optimum seeding densities by 50%. After the cells are actively growing
(after 24 to 48 hours), additional medium can be added to bring the vessel to its final operating
volume.
• Avoid Overheating Cultures: Flask cultures placed directly over magnetic stirrers or shaker
motors may overheat as the result of excessive heat transfer from the motors to the flasks. In
walkin warm rooms this may affect only flasks placed directly over the motor but in small
incubators it may cause the entire incubator to overheat. Check for this problem in advance by
placing identical culture flasks filled with water in the incubator and monitoring the temperature
for at least 48 hours. Sometimes heat transfer from the stirrer to the flask can be reduced by
elevating the flask a few millimeters above the stirrer surface so that air can flow beneath it. Also
make sure that any stirrers or shakers used in humidified CO2 incubators are designed to
withstand the corrosive atmosphere. Placing shakers in incubators will usually generate
vibrations which may prevent cells from attaching to culture vessels in the same or adjoining
incubator chambers.

© 2009, Maulik Suthar

Attachment-Dependent Culture Issues

• Use Appropriate Seeding Densities: Starting with the correct initial seeding density is
also very important when growing attachment-dependent cells. It is always better to add too
many cells than too few; seeding densities can always be lowered later. Start with a
seeding density of 104 to 2 x 104 cells/cm2; the higher concentration is better when using
difficult to grow cells or cells are adapting to serum-free conditions
• Help Cells Attach Quickly: Cell attachment problems are often a serious problem,
especially when growing cells in reduced- or serum-free medium. Prewarming the medium
used for the initial cell seeding and pregassing larger culture vessels so the medium will
reach its correct pH sooner will help cells attach more quickly. Pregassing larger vessels
before seeding is highly recommended and should be done with filtered medical grade 5%
CO2/95% air mixtures. For cells that have attachment problems on traditional cell culture
vessel surfaces, Corning recommends trying the patented Corning® CellBIND® surface on
flasks, roller bottles and CellSTACK® chambers This surface is created by a novel
microwave plasma process that improves cell attachment by incorporating significantly
more oxygen into the cell culture surface than traditional plasma or corona discharge
treatments, rendering it more hydrophilic (wettable) and increasing the stability of the
surface.

© 2009, Maulik Suthar

• Rotate Roller Bottles Slowly: The constant movement of the medium across the surface of the bottle,
slow though it appears, can make it more difficult for cells to attach and grow in roller bottles compared to
stationary vessels such as flasks and dishes (Figure 17). A recommended starting speed for initiating roller
bottle cultures is 0.5 to 1.0 revolutions per minute (rpm) to start. However, if cells have difficulty attaching
(or staying attached), slower speeds (0.1 to 0.4 RPM) should be used until the cells are attached (Clark et
al.; 1990). The constant motion of the medium can also lead to a more stressful cell environment than is
found in stationary culture systems. Consequently, any techniquerelated issues that reduce the attachment
ability of cells are magnified and clearly stand out. Using prewarmed medium and pregassing the bottles
with CO2 so that pH shifts are minimized when inoculating cells will make it easier for the cells to quickly
attach.
• Keep the Cells Happy: Maintaining optimal cell to medium ratios is important for obtaining good cell
growth. As a starting point, use 0.2 to 0.3 mL medium for each square centimeter of culture vessel growth
surface area (i.e., 5 to 7.5 mL for a 25 cm2 flask). Using more medium may reduce the need for feeding
(changing the medium) in the cultures but, due to the increased medium depth and the static nature of the
environment, will also slow the diffusion of oxygen to the cells. (See also Feed cultures appropriately, page
11.) Sometimes gassing the cultures will increase cell yields and viability. This is not practical on dishes,
flasks and roller bottles because of the large number of vessels involved but is useful with CellSTACK
chambers. It is also important to subculture cells before they reach confluency to keep them actively
growing and healthy. In addition, epithelial-like cells will often form very strong cell-cell bonds at confluency
making them much harder to remove from the substrate when subculturing them.
• Harvest Cells Gently: Don’t over dissociate cells when subculturing or harvesting. Too long exposure to
harsh dissociating agents can reduce viability and make it very difficult for cells to reattach. This often
occurs when attempting to harvest too many vessels simultaneously. It is better to harvest vessels a few at
a time rather than attempt to harvest many at once. If using serumfree medium, make sure the dissociating
agents are either inactivated or removed by gentle centrifugation. Keep harvested cells chilled until ready
to reseed new vessels. This will maintain viability and reduce cell clumping. There are a variety of
dissociating enzymes and agents available; experimenting with different combinations may improve both
harvesting efficiency and cell viability (Freshney; 2000).

© 2009, Maulik Suthar

 Cell Culture: Applications
• Cell culture has become one of the major tools used in cell and molecular
biology. Some of the important areas where cell culture is currently playing a
major role are briefly described below:
• Model Systems : Cell cultures provide a good model system for studying 1)
basic cell biology and biochemistry, 2) the interactions between disease-
causing agents and cells, 3) the effects of drugs on cells, 4) the process and
triggers for aging, and 5) nutritional studies.
• Toxicity Testing: Cultured cells are widely used alone or in conjunction with
animal tests to study the effects of new drugs, cosmetics and chemicals on
survival and growth in a wide variety of cell types. Especially important are liver-
and kidney-derived cell cultures.
• Cancer Research: Since both normal cells and cancer cells can be grown in
culture, the basic differences between them can be closely studied. By the use
of chemicals, viruses and radiation, to convert normal cultured cells to cancer
causing cells. Thus, the mechanisms that cause the change can be studied.
Cultured cancer cells also serve as a test system to determine suitable drugs
and methods for selectively destroying types of cancer.
• Virology : One of the earliest and major uses of cell culture is the replication of
viruses in cell cultures (in place of animals) for use in vaccine production. Cell
cultures are also widely used in the clinical detection and isolation of viruses, as
well as basic research into how they grow and infect organisms.

© 2009, Maulik Suthar

 Cell Culture: Applications
• Cell-Based Manufacturing: While cultured cells can be used to produce many important
products, three areas are generating the most interest. The first is the large-scale
production of viruses for use in vaccine production. These include vaccines for polio, rabies,
chicken pox, hepatitis B and measles.
• Second, is the large-scale production of cells that have been genetically engineered to
produce proteins that have medicinal or commercial value. These include monoclonal
antibodies, insulin, hormones, etc.
• Third, is the use of cells as replacement tissues and organs. Artificial skin for use in treating
burns and ulcers is the first commercially available product. However, testing is underway
on artificial organs such as pancreas, liver and kidney. A potential supply of replacement
cells and tissues may come out of work currently being done with both embryonic and adult
stem cells. These are cells that have the potential to differentiate into a variety of different
cell types. It is hoped that learning how to control the development of these cells may offer
new treatment approaches for a wide variety of medical conditions.
• Genetic Counseling: Amniocentesis, a diagnostic technique that enables doctors to
remove and culture fetal cells from pregnant women, has given doctors an important tool
for the early diagnosis of fetal disorders. These cells can then be examined for
abnormalities in their chromosomes and genes using karyotyping, chromosome painting
and other molecular techniques.

© 2009, Maulik Suthar

 Cell Culture: Applications
• Genetic Engineering: The ability to transfect or reprogram cultured cells with new genetic
material (DNA and genes) has provided a major tool to molecular biologists wishing to
study the cellular effects of the expression of theses genes (new proteins). These
techniques can also be used to produce these new proteins in large quantity in cultured
cells for further study. Insect cells are widely used as miniature cells factories to express
substantial quantities of proteins that they manufacture after being infected with genetically
engineered baculoviruses.
• Gene Therapy: The ability to genetically engineer cells has also led to their use for gene
therapy. Cells can be removed from a patient lacking a functional gene and the missing or
damaged gene can then be replaced. The cells can be grown for a while in culture and then
replaced into the patient. An alternative approach is to place the missing gene into a viral
vector and then “infect’’ the patient with the virus in the hope that the missing gene will then
be expressed in the patient’s cells.
• Drug Screening and Development: Cell-based assays have become increasingly
important for the pharmaceutical industry, not just for cytotoxicity testing but also for high
throughput screening of compounds that may have potential use as drugs. Originally, these
cell culture tests were done in 96 well plates, but increasing use is now being made of 384
and 1536 well plates.

© 2009, Maulik Suthar

 What is Cell and Tissue Culture?

Tissue Culture is the general term for the removal of cells, tissues, or organs from
an animal or plant and their subsequent placement into an artificial environment
conducive to growth. This environment usually consists of a suitable glass or
plastic culture vessel containing a liquid or semisolid medium that supplies the
nutrients essential for survival and growth.
Organ Culture: The culture of whole organs or intact organ fragments with the
intent of studying their continued function or development is called Organ
Culture.
Cell Culture.: When the cells are removed from the organ fragments prior to, or
during cultivation, thus disrupting their normal relationships with neighboring
cells, it is called Cell Culture.

© 2009, Maulik Suthar

How Are Cell Cultures Obtained?
• Primary Culture: When cells are surgically
removed from an organism and placed into a
suitable culture environment, they will attach,
divide and grow. This is called a 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, speeds up this
process by adding digesting (proteolytic)
enzymes, such as trypsin or collagenase, to the
tissue fragments to dissolve the cement holding
the cells together. This creates a suspension of
single cells that are then placed into culture
vessels containing culture medium and allowed to
grow and divide. This method is called Enzymatic
Dissociation.

© 2009, Maulik Suthar

 Subculturing
• When the cells in the primary culture vessel have grown and filled up all of the
available culture substrate, they must be Subcultured to give them room for
continued growth. This is usually done by removing them as gently as possible
from the substrate with enzymes. These are similar to the enzymes used in
obtaining the primary culture and are used to break the protein bonds attaching
the cells to the substrate.
• 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. Once a surplus of cells is available, they
can be treated with suitable cryoprotective agents, such as dimethylsulfoxide
(DMSO) or glycerol, carefully frozen and then stored at cryogenic temperatures
(below -130°C) until they are needed.
• Buying And Borrowing; An alternative to establishing cultures by primary
culture is to buy established cell cultures from organizations such as the
American Type Culture Collection (ATCC; www.atcc.org)
• More frequently, researchers will obtain (borrow) cell lines from other
laboratories. While this practice is widespread, it has one major drawback. There
is a high probability that the cells obtained in this manner will not be healthy,
useful cultures. This is usually due to previous mix-ups or contamination with
other cell lines, or the result of contamination with microorganisms such as
mycoplasmas, bacteria, fungi or yeast.

•
© 2009, Maulik Suthar
 What Are Cultured Cells Like?

• Once in culture, cells exhibit a wide range of behaviors, characteristics and

shapes.
• Cell Culture Systems: Two basic culture systems are used for growing cells.
These are based primarily upon the ability of the cells to either grow attached to
a glass or treated plastic substrate (Monolayer Culture Sytems) or floating free
in the culture medium (Suspension Culture Systems). Monolayer cultures are
usually grown in tissue culture treated dishes, T-flasks, roller bottles, or multiple
well plates, the choice being based on the number of cells needed, the nature of
the culture environment, cost and personal preference. Suspension cultures are
usually grown either:
• 1. In magnetically rotated spinner flasks or shaken Erlenmeyer flasks where the
cells are kept actively suspended in the medium; 2. 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. Many cell lines, especially those
derived from normal tissues, are considered to be Anchorage-Dependent, that
is, they can only grow when attached to a suitable substrate. Some cell lines that
are no longer considered normal (frequently designated as
• Transformed Cells) are frequently able to grow either attached to a substrate or
floating free in suspension; they are Anchorage-Independent. In addition, some
normal cells, such as those found in the blood, do not normally attach to
substrates and always grow in suspension.

© 2009, Maulik Suthar

 Types of Cells
• Cultured cells are usually described based on their morphology (shape and appearance) or
their functional characteristics. There are three basic morphologies:
2. Epithelial-like: cells that are attached to a substrate and appear flattened and polygonal in
shape.
3. Lymphoblast-like: cells that do not attach normally to a substrate but remain in suspension
with a spherical shape.
4. Fibroblast-like: cells that are attached to a substrate and appear elongated and bipolar,
frequently forming swirls in heavy cultures. It is important to remember that the culture
conditions play an important role in determining shape and that many cell cultures are capable
of exhibiting multiple morphologies.
Functional Characteristics
The characteristics of cultured cells result from both their origin (liver, heart, etc.) and how well
they adapt to the culture conditions. Biochemical markers can be used to determine if cells are
still carrying on specialized functions that they performed in vivo (e.g., liver cells secreting
albumin). Morphological or ultrastructural markers can also be examined (e.g., beating heart
cells). Frequently, these characteristics are either lost or changed as a result of being placed
in an artificial environment. Some cell lines will eventually stop dividing and show signs of
aging. These lines are called Finite. Other lines are, or become immortal; these can continue
to divide indefinitely and are called Continuous cell lines. When a “normal” finite cell line
becomes immortal, it has undergone a fundamental irreversible change or “transformation”.
This can occur spontaneously or be brought about intentionally using drugs, radiation or
viruses. Transformed Cells are usually easier and faster growing, may often have extra or
abnormal chromosomes and frequently can be grown in suspension. Cells that have the
normal number of chromosomes are called Diploid cells; those that have other than the
normal number are Aneuploid. If the cells form tumors when they are injected into animals,
they are considered to be Neoplastically Transformed.

© 2009, Maulik Suthar

 Finding A “Happy” Environment

• it means an environment that, at the very least, allows cells to increase in number by undergoing cell division
(mitosis). Even better, when conditions are just right, some cultured cells will express their “happiness” with
their environment by carrying out important in vivo physiological or biochemical functions, such as muscle
contraction or the secretion of hormones and enzymes.
• To provide this environment, it is important to provide the cells with the appropriate temperature, a good
substrate for attachment, and the proper culture medium and incubator that maintains the correct pH and
osmolality
• Temperature is usually set at the same point as the body temperature of the host from which the cells were
obtained. With cold-blooded vertebrates, a temperature range of 18° to 25°C is suitable; most mammalian
cells require 36° to 37°C. This temperature range is usually maintained by use of carefully calibrated, and
frequently checked, incubators.
• Anchorage-dependent cells also require a good substrate for attachment and growth. Glass and specially
treated plastics (to make the normally hydrophobic plastic surface hydrophilic or wettable) are the most
commonly used substrates. However, Attachment Factors, such as collagen, gelatin, fibronectin and laminin,
can be used as substrate coatings to improve growth and function of normal cells derived from brain, blood
vessels, kidney, liver, skin, etc. Often normal anchoragedependent cells will also function better if they are
grown on a permeable or porous surface. This allows them to polarize (have a top and bottom through which
things can enter and leave the cell) as they do in the body.
• The culture medium is the most important and complex factor to control in making cells “happy”. Besides
meeting the basic nutritional requirement of the cells, the culture medium should also have any necessary
growth factors, regulate the pH and osmolality, and provide essential gases (O2 and CO2). The ‘food’ portion
of the culture medium consists of amino acids, vitamins, minerals, and carbohydrates. These allow the cells to
build new proteins and other components essential for growth and function as well as providing the energy
necessary for metabolism.

© 2009, Maulik Suthar

• The growth factors and hormones help regulate and control the cells’ growth rate and
functional characteristics. Instead of being added directly to the medium, they are often
added in an undefined manner by adding 5 to 20% of various animal sera to the medium.
Unfortunately, the types and concentration of these factors in serum vary considerably from
batch to batch. This often results in problems controlling growth and function. When
growing normal functional cells, sera are often replaced by specific growth factors.
• The medium also controls the pH range of the culture and buffers the cells from abrupt
changes in pH. Usually a CO2- bicarbonate based buffer or an organic buffer, such as
HEPES, is used to help keep the medium pH in a range from 7.0 to 7.4 depending on the
type of cell being cultured. When using a CO2-bicarbonate buffer, it is necessary to
regulate the amount of CO2 dissolved in the medium. This is usually done using an
incubator with CO2 controls set to provide an atmosphere with between 2% and 10% CO2
(for Earle’s salts-based buffers). However, some media use a CO2-bicarbonate buffer (for
Hanks’ salts-based buffers) that requires no additional CO2, but it must be used in a sealed
vessel (not dishes or plates).
• Finally, the osmolality (osmotic pressure) of the culture medium is important since it helps
regulate the flow of substances in and out of the cell. It is controlled by the addition or
subtraction of salt in the culture medium. Evaporation of culture media from open culture
vessels (dishes, etc.) will rapidly increase the osmolality resulting in stressed, damaged or
dead cells. For open (not sealed) culture systems, incubators with high humidity levels to
reduce evaporation are essential.

© 2009, Maulik Suthar