Cell Culture Contamination Continues To Be Major Problem Biology Essay

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bioproduct manufacturers. Contamination is truly what endangers the use of cell cultures as reliable reagents and tools. Each cell culture system is unique. Usually contamination is chronic because it is unseen, unrecognized and often benign within a given cell culture system.

If contamination is defined as a component of the cell culture systemthat has a negative impact on the cell culture and/or its use. Then we can categorize cell culture contamination in three groups,

1) Physical

2) Chemical

3) Biological

Chemical Contamination

Chemical contamination is best described as the presence of any nonliving substance that

results in undesirable effects on the culture system. To define further is difficult; even essen-

tial nutrients become toxic at high enough concentrations. Nor is toxicity the only concern

since hormones and other growth factors found in serum can cause changes that, while not

necessarily harmful to cultures, may be unwanted by researchers using the system. (Reviewed

in references 1-2.)


The majority of chemical contaminants are found in cell culture media and come either from

the reagents and water used to make them, or the additives, such as sera, used to supplement

them. Reagents should always be of the highest quality and purity and must be properly

stored to prevent deterioration. Ideally, they should be either certified for cell culture use by

their manufacturer or evaluated by the researcher before use. Mistakes in media preparation

protocols, reading reagent bottle labels, or weighing reagents are other common sources of

chemical contamination (3).


Sera used in media have long been a source of both biological and chemical contaminants.

Due to cell culture-based screening programs currently used by good sera manufacturers, it

is unusual to find a lot of fetal bovine sera that is toxic to a majority of cell cultures. However,

it is common to find substantial variations in the growth promoting abilities of different lots

of sera for particular cell culture systems, especially for cultures that have specialized or dif-

ferentiated characteristics. Uncontrollable lot-to-lot variation in hormone and growth factor

concentrations makes this problem inevitable; careful testing of sera before purchase, or

switching to serum-free media can avoid these problems.

Table 2. Types and Sources of Potential Chemical Contaminants

Metal ions, endotoxins, and other impurities in media, sera, and water

Plasticizers in plastic tubing and storage bottles

Free radicals generated in media by the photo activation of tryptophan, riboflavin

or HEPES exposed to fluorescent light

Deposits on glassware, pipettes, instruments etc., left by disinfectants or detergents, antiscaling

Compounds in autoclave water, residues from aluminum foil or paper

Residues from germicides or pesticides used to disinfect incubators, equipment, and labs

Impurities in gases used in CO2 incubators

Remember also that serum proteins have the ability to bind substantial quantities of chemical

contaminants, especially heavy metals, that may have entered the culture system from other

sources, rendering them less toxic. As a result, switching from serum-containing medium to

a serum-free system can unmask these toxic chemical contaminants, exposing the cells to

their adverse effects.


The water used for making media and washing glassware is a frequent source of chemical

contamination and requires special care to ensure its quality. Traditionally, double or triple

glass distillation was considered to be the best source of high quality water for cell culture

media and solutions. Newer purification systems combining reverse osmosis, ion exchange

and ultrafiltration are capable of removing trace metals, dissolved organic compounds and endotoxins are increasingly popular.


Endotoxins, the lipopolysaccaride-containing by-products of gram negative bacteria, are

another source of chemical contaminants in cell culture systems. Endotoxins are commonly

found in water, sera and some culture additives (especially those manufactured using micro-

bial fermentation) and can be readily quantified using the Limulus Amebocyte Lysate assay


These highly biologically reactive molecules have major influences in vivo on humoral and

cellular systems. Studies of endotoxins using in vitro systems have shown that they may

affect the growth or performance of cultures and are a significant source of experimental

variability (Reviewed in references 6 and 39). Furthermore, since the use of cell culture pro-

duced therapeutics, such as hybridomas and vaccines, are compromised by high endotoxin

levels, efforts must be made to keep endotoxin levels in culture systems as low as possible.

In the past, sera have been a major source of endotoxins in cell cultures. As improved

endotoxin assays (LAL) led to an increased awareness of the potential cell culture problems

associated with endotoxins, most manufacturers have significantly reduced levels in sera by

handling the raw products under aseptic conditions. Poorly maintained water systems, espe-

cially systems using ion exchange resins, can harbor significant levels of endotoxin-produc-

ing bacteria and may need to be tested if endotoxin problems are suspected or discovered

in the cultures.

Storage Vessels

Media stored in glass or plastic bottles that have previously contained solutions of heavy

metals or organic compounds, such as electron microscopy stains, solvents and pesticides,

can be another source of contamination. The contaminants can be adsorbed onto the sur-

face of the bottle or its cap (or absorbed into the bottle if plastic) during storage of the

original solution. If during the washing process they are only partially removed, then once

in contact with culture media they may slowly leach back into solution. Residues from

chemicals used to disinfect glassware, detergents used in washing, or some aluminum foils

and wrapping papers for autoclaving or dry heat sterilization can also leave potentially toxic

deposits on pipettes, storage bottles and instruments.

Fluorescent Lights

An important but often overlooked source of chemical contamination results from the

exposure of media containing HEPES (N-[2-hydroxylethyl] piperazine-N'-[2-ethanesul-

fonic acid]) - an organic buffer commonly used to supplement bicarbonate-based buffers),

riboflavin or tryptophan to normal fluorescent lighting. These media components can be

photoactivated producing hydrogen peroxide and free radicals that are toxic to cells; the

longer the exposure the greater the toxicity (4). Short term exposure of media to room or

hood lighting when feeding cultures is usually not a significant problem; but leaving media

on lab benches for extended periods, storing media in walk-in cold rooms with the lights on,

or using refrigerators with glass doors where fluorescent light exposure is more extensive,

will lead to a gradual deterioration in the quality of the media.


The incubator, often considered a major source of biological contamination, can also be a

source of chemical contamination. The gas mixtures (usually containing carbon dioxide to

help regulate media pH) perfused through some incubators may contain toxic impurities,

especially oils or other gases such as carbon monoxide, that may have been previously used

in the same storage cylinder or tank. This problem is very rare in medical grade gases, but

more common in the less expensive industrial grade gas mixtures (5). Care must also be

taken when installing new cylinders to make sure the correct gas cylinder is used. Other

potential chemical contaminants are the toxic, volatile residues left behind after cleaning and

disinfecting incubators. Disinfectant odors should not be detectable in a freshly cleaned

incubator when it is placed back into use.

Keep in mind that chemical contaminants tend to be additive in cell culture; small amounts

contributed from several different sources that are individually nontoxic, when combined

together in medium, may end up overloading the detoxification capabilities of the cell cul-

ture resulting in toxicity-induced stress effects or even culture.

Biological Contamination

Biological contaminants can be subdivided into two groups based on the difficulty of detect-

ing them in cultures:

those that are usually easy to detect - bacteria, molds and yeast;

- Those that are more difficult to detect, and as a result potentially more serious culture

problems, - viruses, protozoa, insects, mycoplasmas and other cell lines.

For a comprehensive review, see references 7 and 8.

Ultimately, it is the length of time that a culture contaminant escapes detection that will determine

the extent of damage it creates in a laboratory or research project.

Bacteria, Molds, and Yeasts

Bacteria, molds and yeasts are found virtually everywhere and are able to quickly colonize

and flourish in the rich and relatively undefended environment provided by cell cultures.

Because of their size and fast growth rates, these microbes are the most commonly encoun-

tered cell culture contaminants. In the absence of antibiotics, microbes can usually be readi-

ly detected in a culture within a few days of becoming contaminated, either by direct micro- scopic

observationor by the effects they have on the culture (pH

shifts, turbidity, and cell destruction). However, when antibiotics are routinely used in cul-

ture, resistant organisms may develop into slow growing, low level infections that are very

difficult to detect by direct visual observation. Similar detection problems can occur with

naturally slow growing organisms or very small or intracellular bacteria that are difficult to

see during routine microscopic culture observation. These cryptic contaminants may persist

indefinitely in cultures causing subtle but significant alterations in their behavior. By the

time these cryptic contaminants are discovered, many experiments and cultures may have

been compromised.


Due to their extremely small size, viruses are the most difficult cell culture contaminants

to detect in culture, requiring methods that are impractical for most research laboratories.

Their small size also makes them very difficult to remove from media, sera, and other solu-

tions of biological origin. However, most viruses have stringent requirements for their orig-

inal host species' cellular machinery (may also be tissue specific) which greatly limits their

ability to infect cell cultures from other species. Thus, although viruses may be more com-

mon in cell cultures than many researchers realize, they are usually not a serious problem

unless they have cytopathic or other adverse effects on the cultures. (Reviewed in Ref. 7, 40.)

Since cytopathic viruses usually destroy the cultures they infect, they tend to be self-limit-

ing. Thus, when cultures self-destruct for no apparent reason and no evidence of common

biological contaminants can be found, cryptic viruses are often blamed. (See Figures 4a and

4b.) They are perfect culprits, unseen and undetectable; guilty without direct evidence. This

is unfortunate, since the real cause of this culture destruction may be something else, possi-

bly mycoplasma or a chemical contaminant, and as a result will go undetected to become a

more serious problem.

A major concern of using virally infected cell cultures is not their effects on the cultures but rather the

potential health hazards they pose for laboratory personnel. Special safety precautions should always

be used when working with tissues or cells from humans or other primates to avoid possible transmission of viral infection (HIV, hepatitis B, Epstein-Barr, simian herpes B virus, among others) fromthe cell cultures to laboratory personnel (9). Contact your safety office for additional assistance if indoubt as to appropriate procedures for working with potentially hazardous tissues, cultures or viruses.


Both parasitic and free-living, single-celled protozoa, such as amoebas, have occasionally

been identified as cell culture contaminants. Usually of soil origin, amoebas can form spores

and are readily isolated from the air, occasionally from tissues, as well as throat and nose

swabs of laboratory personnel. They can cause cytopathic effects resembling viral damage

and completely destroy a culture within ten days. Because of their slow growth and mor-

phological similarities to cultured cells, amoebas are somewhat difficult to detect in culture,

unless already suspected as contaminants (7). Fortunately, reported cases of this class of

contaminants are rare, but it is important to be alert to the possibility of their occurrence.


Insects and arachnids commonly found in laboratory areas, especially flies, ants, cockroaches

and mites, can both be culture contaminants as well as important sources of microbial con-

tamination. Warm rooms are common sites of infestation. By wandering in and out of cul-

ture vessels and sterile supplies as they search for food or shelter, they can randomly spread

Insects and arachnids commonly found in laboratory areas, especially flies, ants, cockroaches

and mites, can both be culture contaminants as well as important sources of microbial con-

tamination. Warm rooms are common sites of infestation. By wandering in and out of cul-

ture vessels and sterile supplies as they search for food or shelter, they can randomly spread

a variety of microbial contaminants. Occasionally they are detected by the trail of "foot

prints" (microbial colonies) they leave behind on agar plates, but usually they don't leave

any visible signs of their visit other than random microbial contamination. Mites can be a

serious problem in plant cell culture facilities, especially those doing large scale plant propa-

gation. Although bacteria, molds and yeast may sometimes appear to 'jump' from culture to

culture, these multilegged contaminants really can. While not nearly as common as other

culture contaminants, it is important to be alert to the presence of these invertebrates in

culture areas.


Mycoplasmas were first detected in cell cultures by Robinson and coworkers in 1956. They

were attempting to study the effects of PPLO (pleuropneumonialike organisms - the

original name for mycoplasma) on HeLa cells when they discovered that the control HeLa

cultures were already contaminated by PPLO (10). In addition, they discovered that the

other cell lines currently in use in their laboratory were also infected with mycoplasma, a

common characteristic of mycoplasma contamination. Based on mycoplasma testing done by the

FDA, ATCC, and two major cell culture testing companies, at least 11 to 15% of the cell cultures in

the United States are currently infected by mycoplasmas (Table 3). Since many of these cultures

were from laboratories that test routinely for mycoplasma, the actual rates are probably

higher in the many laboratories that do not test at all (11-13). In Europe, mycoplasma con-

tamination levels were found to be even higher: over 25% of 1949 cell cultures from the

Netherlands and 37% of 327 cultures from former Czechoslovakia were positive (14). The

Czechoslovakia study had an interesting, but typical finding: 100% of the cultures from labs

without mycoplasma testing programs were contaminated, but only 2% of the cultures from labs that

tested regularly. Other countries may be worse: 65% of the cultures in Argentina and 80%

in Japan were reported to be contaminated by mycoplasma in other studies (11).

Unfortunately, mycoplasmas are not relatively benign culture contaminants but have the

ability to alter their host culture's cell function, growth, metabolism, morphology, attach-

ment, membranes, virus propagation and yield, interferon induction and yield, cause chro-

mosomal aberrations and damage, and cytopathic effects including plaque formation (12).

Thus, the validity of any research done using these unknowingly infected cultures is ques-

tionable at best. (See references 11, 12, and 15-18 for good overviews of this very serious

mycoplasma contamination problem.)

What gives mycoplasmas this ability to readily infect so many cultures? Three basic charac-

teristics: a) these simple, bacteria-like microbes are the smallest self-replicating organism

known (0.3 to 0.8 ┬Ám in diameter), b) they lack a cell wall, and c) they are fastidious in their

growth requirements. Their small size and lack of a cell wall allow mycoplasmas to grow to

very high densities in cell culture (107 to 109 colony forming units/mL are common) often

without any visible signs of contamination - no turbidity, pH changes or even cytopathic

effects. (See Figures 5a and 5b.) Even careful microscopic observation of live cell cultures

cannot detect their presence. These same two characteristics also make mycoplasmas, like

viruses, very difficult to completely remove from sera by membrane filtration. In addition,

their fastidious growth requirements (unfortunately, easily provided for by cell cultures)

make them very difficult to grow and detect using standard microbiological cultivation

methods. Thus, these three simple characteristics, combined with their ability to alter virtu-

ally every cellular function and parameter, make mycoplasmas the most serious, widespread,

and devastating culture contaminants.

Mycoplasmas have been described as the "crabgrass" of cell cultures, but this is too benign a

description for what are the most significant and widespread cell culture contaminants in

the world.