Tissue Culture Trouble-Shooting
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Courtesy of JRH
Many laboratories refer to tissue culture as "black art" or
voodoo, due to the numerous variables that make it difficult to determine
solutions when problems arise. A systematic approach that examines the
symptoms and meticulously retraces each step in the culture process will
help narrow a problem down to one material or critical procedure that
has created the issue.
Almost every problem encountered in cell culture can be identified as
one of the following:
- The cells are growing poorly or not at all.
- The cells have an abnormal morphology.
Each of these problems can usually be traced to one of the four following
- Materials: They were poor quality, inappropriate, compromised,
- Equipment: It may need to be calibrated or the laboratory
has gotten away from suggested manufacturer usage.
- Environment: The cultures were exposed to the wrong type of
- Cells: They were exposed to toxicity, contamination, or nutritional
- Technique: The procedures or protocols were not correct for
the cell type.
Retracing steps will be easier, if the laboratory has maintained accurate
and complete records of all materials, procedures, and culture work performed.
This not only helps the laboratory find critical mistakes, it also saves
time when dealing with manufacturers. A technician may be able to pinpoint
the problem on paper, illuminating the need for long trials. Also keeping
records of lot numbers, seeding densities, protocols, etc. will limit
your time on the phone with manufacturers, when trying to identify if
the problems lie outside the laboratory.
If the plastics are the reason for growth problems, it will generally
fall into one of two issues: manufacturers quality of plastics or cell
line. While most plastic manufacturers make quality plastics not every
plastic will work with every cell line. How each manufacturer treats their
plastics is different (and proprietary) and some cell lines may adhere
better to one plastic versus another. While most cell lines will grow
well with most plastics, there are a few plastics that offer better performance.
When identifying the plastics as a problem manufacturing is more likely
the problem versus cells adhering to the plastic. Generally they fall
into these two areas:
- Quality manufacturing practices.
- Handling issues.
In manufacturing there are several practices that affect the quality of
the end product, such as; the molding, the environment, quality control,
and packaging. Most TC plastics are injection molded. Problems can arise
from molds wearing down and not being replaced often enough or to overall
poor designs. A mold problem will most often show up in as an uneven growth
surface. If you experience "spotting" or "clumping"
of cells (this is when cells grow better in the middle or in spots) this
can be an uneven growth surface. Since TPP, Falcon, Nunc, Corning, and
Sarstedt are all ISO9001 certified this should not be a problem. What
is more likely the problem is poor design.
Again, since 5 of the 6 major TC plastic manufacturers are ISO9001 certified,
the clean-room environment should never be a problem. What is a consideration
is the certification that a manufacturer will give to their products.
Here is a chart that shows who will certify their products non-pyrogenic
and/or RNAse and DNAse free:
RNAse / DNAse Certification
If contamination, uneven growing surfaces, or variability in cell growth
are problems, utilizing a manufacturer that will certify their products
may be worth investigating. Most manufacturers or distributors of these
products are very helpful with sampling.
Another issue that can cause cross-contamination and/or variability in
cell culture is how a plastic is handled in the laboratory. Since no one
can guarantee perfect performance with cell culture, look for a manufacturer
that provides benefits for handling. TPP plates and dishes with gripping
edges for easier handling, there are pipettes that will leak less and
cause less arm fatigue, and there are even vacuum filters that operate
faster with less chance of tipping. TPP also offers black alpha-numerics
on their plates for easier reading and well identification under a microscope
or hood. Overall improvements like these can mean more success for cell
growth, less wasted media, serum, and plastics through less dropping,
cross contamination, spilling, and all around easier handling of materials.
If plastics are identified as the main problem, it is worthwhile to do
a side-by-side comparison of all the manufacturers to see if results vary
and ease of operation can improve. While many laboratories do not want
to take risks with their cells, the return of improved plastic performance
will pay off immediately and in the long term.
Picking the "right" culture medium is vital to the
cells' growth and vitality. The original supplier of a cell line can provide
the best recommendation to use. A media supplier will provide information
on storage, preparation, and disposal.
Storage: Incorrect media storage or expired media can make even
the best medium choice null and void. Media should always be stored as
directed: This always means in the dark and usually refrigerated at 2°
to 8°C. Media components can break down when exposed to light and
to temperature extremes. In some situations toxic by-products will form
due to poor storage practices. Disposal requirements of expired media
or media that appears cloudy or contains precipitates should always be
followed. Freezing of media may cause irreversible precipitation of critical
Preparation of Dry Powder Media: Media incorrectly prepared in
the lab from dry powder also can be a source of problems. Common mistakes
include not letting the powder fully dissolve, incorrect pH adjustment,
or using water above 30° C. Sterile filtering should be done with
a final 0.2 µm cellulose acetate or other low-protein binding filter.
Filters coated with wetting agents should not be used to avoid the inadvertent
addition of cytotoxic detergents to the media. Filters made of nylon or
glass may remove necessary lipids and fatty acids, while nitrocellulose
filters can strip proteins from the medium. Filtering sterile medium after
supplementing with serum or other growth factors also is not recommended.
Although there are exceptions, most media contain heat labile components
and should not be autoclaved. Serum-free medium should not be filtered
through membrane porosities of less than 0.2 µm as this may remove
To reduce the chances of microbial contamination or cross-contamination
of cell lines, use separate media containers for each cell line and do
not share the same bottle of material with co-workers. Aseptically dividing
material into a separate vessel for each person and for each cell line
is good laboratory practice. Never work with more than one cell line at
The two most common cell culture media supplements are serum and L-glutamine.
The formation of precipitates or contamination can happen by incorrect
handling of serum. When using a new serum (or even a new lot) expect a
slight lag in growth. One of the top errors made in cell culture is using
media that is deficient in L-glutamine. The half-life for L-Glutamine
in liquid form, at refrigerated temperatures, is an estimated 6 to 8 weeks.
To achieve optimum results, periodically replenish L-Glutamine levels
Always clean cell culture glassware with phosphate-free detergents. Thoroughly
rinsing glassware will remove residue from cleaning detergents that can
be toxic to cells. Also a film of surfactants and cell debris can develop
in spinner flasks over time. Stripping and recoating the vessels with
a silicon reagent will reduce shear and friction. Repeated use of glassware
may be a source of endotoxin. To eliminate glassware as a problem, schedule
periodic washing in sodium hydroxide (NaOH) or dry oven exposure at 250
C for three hours.
Some laboratories will look to their equipment as the last place to check,
but often a contamination problem can be as simple as cleaning the incubator.
All manufacturers of CO2 incubators and Bio-Safety cabinets supply helpful
trouble-shooting in their manuals and some even do it on their websites.
Here are a few common errors that can cause big problems:
Common Equipment Areas to Check to Limit
CO2 Incubator Calibration:
All manufacturers recommend a regular calibration (at least once
a year) using a FYRITE CO2 Gas
Monitor CO2 Tank Levels:
Letting a CO2 tank run empty will
compromise your cell lines and may kill them. Many auto tank switches
are not also alarms.
General Maintenance of Incubators and Bio-Safety Cabinets.
As the manufacturer recommends units need to cleaned and decontaminated,
CO2 supply lines checked for leaks,
replace filters, etc., on a regular basis.
General Operation Requirements: All general operation requirements
from manufacturers are designed for safety and proper operation
of the unit(s). For example, disconnecting the window alarm on a
bio-safety cabinet is a problem waiting to happen. . Also, on a
bio-safety cabinet, if the window is not used at its appropriate
height airflow will not operate properly and a safe clean environment
is drastically compromised.
Carbon Dioxide (CO2): Mammalian
cell culture media typically contain buffering systems that require a
CO2 atmosphere. Severe pH shifts can
result if adequate CO2 levels are not
maintained. 5% CO2 is usually sufficient
in most culture systems; however, this level may need to be adjusted to
accommodate or enhance the growth of certain cell lines. It is also important
to leave the flask caps of these cultures open slightly to allow adequate
gas exchange. Insect cell culture media, as well as a few mammalian media
such as L-15, contain buffering systems that are not dependent on CO2.
These cultures should be exposed to atmospheric conditions. As a general
rule, media and buffers containing Earle's Balanced Salts require CO2
for optimum performance, while those with Hanks' Balanced Salts are for
use under atmospheric conditions.
Humidity: For culture vessels that must have the caps loose for
gas exchange, humidity levels are also important. Low humidity can lead
to the evaporation of water from the medium resulting in concentrated
salt levels and conditions that cause cell lysis. A pan of water placed
in the bottom of the incubator usually is sufficient to supply the proper
humidity level. You may want to add clean water and change the water daily.
pH: When conditions are too acidic or basic, poor cell growth and
permanent cell damage can occur. For the best results keep mammalian cells
at pH range of 7.2 ± 0.2 and with insect cells approximately 6.2
± 0.1. Monitor cultures periodically and perform media changes
as needed, because the pH of a medium will change as cells take up nutrients
and as the levels of respiratory by-products increase.
Temperature: Fluctuation of temperature out of normal ranges can
lead to permanent damage of cells. For example, when incubating optimum
growth of mammalian cells is at 37 ± 1 C, while insect cultures
must be kept at 25 ± 2 C.
Toxicity: Cell toxicity, or cytotoxicity, is the inhibition of
cell growth or deterioration of cells due to excessive concentrations
of a particular agent or ingredient. Abnormal morphology is the main indicator
of cytotoxicity and can include one or more of the following: giant cells,
multi-nucleated cells, a granular or "bumpy" appearance, vacuoles
in the cytoplasm or in the nucleus, and/or ragged cell edges.
To identify which material (medium, serum, supplements, flasks, etc.)
is causing the cytotoxicity, substitute them one at a time with control
reagents, and screen for the signs of toxicity listed above. Serial dilutions
of a suspected material can be used to determine the level at which that
component becomes cytotoxic to the cultures.
Contamination: Microbial contamination comes in many forms including
gram positive and gram negative bacteria, mycoplasma, virus, mold, and
yeast. Indicators of contamination include turbid culture media, changed
growth rates, abnormally high pH, poor attachment, multi-nucleated cells,
grainy cellular appearance, vacuolization, inclusion bodies, and cell
One initial method to confirm suspicion of contamination is to transfer
fluids from the suspected culture to a "clean" culture, incubate,
and observe daily for cell deterioration. Specific identification assays
are available for determining the group or type of contamination present.
Positively identifying the contaminant is crucial to determining its source
and in planning corrective action.
Contaminated cultures should be autoclaved and discarded immediately to
reduce the risk of spreading the contamination to other cultures or other
areas of the lab.
Antibiotics such as gentamicin, streptomycin, and penicillin often are
used in cell culture media to control low levels of contamination. However,
some researchers feel that it is wise to avoid using antibiotics altogether.
It is better to detect a contamination right away and handle it immediately
than to mask it with antibiotics. Antibiotics also can be cytotoxic when
used at elevated concentrations, and low serum or serum-free cultures
are especially susceptible to this problem.
Nutritional Deficiencies: Because different cell types and cell
lines have different nutritional requirements, it is difficult to have
one medium system that will satisfy all of your cultures. Unsatisfactory
cell performance (poor growth, poor attachment, and/or monolayer sloughing)
often occurs if the cell's nutritional requirements are not being met.
Other indicators are giant cells, vacuoles, and ragged cell edges. Deficiencies
can arise from a certain component or components being absent altogether
or from levels being exhausted too quickly.
Supplementing medium with additional amino acids (particularly L-glutamine),
vitamins, glucose, or higher levels of serum and observing the cell's
response can help to identify the component(s) not present at sufficient
concentrations. Depending on the degree of the deficiency, the cultures
may be too thoroughly damaged to completely recover. It is often necessary
to start new cultures once this problem has been identified.
Common Problems Due To Technique
Check for bubbles in medium that have attached and displaced cells.
If this is not the problem it could be an uneven growth surface
due to the plastics or the incubator
Condensation buildup, which may not only be a technique problem,
but also an incubator problem. Some plastic manufacturer's, like
TPP, have features that help compensate for this problem.
"Clumping" of Cells
Cells were not totally re-suspended prior to seeding, cells were
damaged during sub-culturing, uneven growth surface due to an un-level
incubator or poor plastic manufacturing
Cells That Are Stringy
This mostly likely is due to an overexposure
to trypsin, but it could be that your pipettes have poor vacuums
and do not remove all the trypsin.
Not enough media was used or poor distribution of media over cells.
Poor distribution can sometimes be attributed to poor design of
flasks, dishes, and/or pipettes or the incubator not being level.
Peeling or Sloughing
Prolonged incubation or over seeding can cause overgrowth.
Can be caused by excessive foaming or uneven growth surfaces in
your flasks, dishes, or plates.
Circular Banding in Roller Bottles
Rollers turning too quickly.
Banding on One Side of Roller Bottles
Rollers turning too slowly.
Trypsinization: The misuse of trypsin can lead to immediate and
long-term problems with a set of cultures. Trypsin is a popular detachment
factor because it digests the proteins that keep the cell attached to
a surface. Prolonged exposure to trypsin can result in too much of the
cell being digested, which can cause cell damage or destruction. A few
keys to correctly using trypsin:
- Use the correct concentration for the cultures being used. Some cells
are easy to detach and others are much more difficult to dissociate
from the substrate. Different concentrations of trypsin are available
with or without EDTA. EDTA, also called Versene, increases a trypsin
solution's effect by taking up calcium and magnesium ions.
- Watch the clock. Only leave the trypsin on the cultures as long as
is absolutely necessary. Check the monolayer often to see if it is beginning
to come off and at that point, a gentle tap against the side of the
vessel may help to dislodge the cells. Be aware that forcing cells to
detach by harsh mechanical treatment such as smacking the flask can
cause the cells to "tear" off the surface and cause permanent
- Neutralize the trypsin after the cells have detached by adding serum
or gently centrifuging the cells into a pellet and re-suspending in
fresh media. Because serum contains trypsin inhibitors, it is important
to completely remove serum prior to trypsinization. This can be done
by first rinsing the monolayer with serum-free medium or a calcium-
and magnesium-free balanced salt solution such as DPBS.
- Control the trypsin by controlling the temperature. Cells that are
sensitive to trypsin should be trypsinized at room temperature or refrigerated
temperatures, while trypsinizing in a 37° C incubator can speed
up the process for more stubborn cells and reduce the total exposure
Seeding: Using an inappropriate seeding density can lead to everything
from overgrowth and cell sloughing to sparse cultures that may never become
fully confluent. The correct density is dependent on the cell type, the
medium and amount of serum used, the size of the vessel, and when the
cultures are to be used or sub-cultured again.
If you are unsure about how many cells to add to each vessel, try several
different concentrations and determine which grows to the appropriate
cell density in the time period that is applicable for your transfection,
assay, or cell stocks. Remember that high cell densities will use nutrients
quickly and may require additional supplementation to avoid deficiencies.
Adapting From Serum to Serum-Free: Adapting cells to serum-free
conditions can be a smooth transition, however, if it is done incorrectly,
many problems can arise. This transition should typically be done over
several passages to gradually select cells that can grow under serum-free
Other Important Factors to Consider:
- Use actively growing cells that are in their log phase of growth.
Use cells that are 80 - 90% viable.
- Keep exposure to trypsin at a minimum. Cells in the absence of serum
are very susceptible to trypsin, so use cold trypsinization procedures
or dilute trypsin with calcium- and magnesium-free DPBS.
- Handle the cells gently. Do not centrifuge at high speeds or roughly
re-suspend the cells.
- Use a serum-free media that is designed for your type of application.
- Feeding and sub-culturing the cells at more frequent intervals than
used with serum-containing conditions may be necessary.