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Understanding and ManagingCell Culture ContaminationTechnical BulletinLifeSciencesJohn Ryan, Ph.D.Corning IncorporatedLife SciencesActon, MA 01720Table of ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1What Are the Major Cell CultureContaminants? . . . . . . . . . . . . . . . . . . . . . . . . . . 2What Are the Sources of BiologicalContaminants? . . . . . . . . . . . . . . . . . . . . . . . . . 8How Can Cell Culture ContaminationBe Controlled? . . . . . . . . . . . . . . . . . . . . . . . . 11A Final Warning . . . . . . . . . . . . . . . . . . . . . . . 20Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 21References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Cell Culture Protocols andTechnical Articles . . . . . . . . . . . . . . . . . . . . . . .22IntroductionNo cell culture problem is as universal as that ofculture loss due to contamination. All cell culturelaboratories and cell culture workers have experienced it. Culture contaminants may be biologicalor chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affectboth the use of your cell cultures and the qualityof your research. Contamination problems can bedivided into three classes: Minor annoyances — when up to several platesor flasks are occasionally lost to contamination;Serious problems — when contaminationfrequency increases or entire experiments orcell cultures are lost;Major catastrophes — contaminants are discoveredthat call into doubt the validity of your past orcurrent work.

Table 1. Some Consequences ofContamination Loss of time, money, and effort Adverse effects on the cultures Inaccurate or erroneous experimental results Loss of valuable products Personal embarrassmentThe most obvious consequence of cellculture contamination is the loss of yourtime, money (for cells, culture vessels,media and sera) and effort spent developing cultures and setting up experiments.However, the less obvious consequencesare often more serious (Table 1). Firstthere are the adverse effects on culturessuffering from undetected chemical orbiological contaminants. These hidden(cryptic) contaminants can achieve highdensities altering the growth and characteristics of the cultures. Worse yet are thepotentially inaccurate or erroneous resultsobtained by unknowingly working withthese cryptically contaminated cultures.Products, such as vaccines, drugs ormonoclonal antibodies, manufactured bythese cultures will probably be useless.For some researchers the most seriousconsequence of contamination is sufferingthe embarrassment and damage to theirreputation that results when they notifycollaborators or journals that their experimental results are faulty and must beretracted due to contaminants in theircultures.Preventing all cell culture contaminationhas long been the dream of manyresearchers, but it is an impractical, if notimpossible, dream. Contamination cannotbe totally eliminated, but it can be managedto reduce both its frequency of occurrence andthe seriousness of its consequences. The goalof this bulletin is to review the nature ofcell culture contamination and the problems it causes, and then to explore someof the key concepts and practical strategies for managing contamination to prevent the loss of valuable cultures andexperiments.2What Are the MajorCell Culture Contaminants?A cell culture contaminant can be definedas some element in the culture systemthat is undesirable because of its possibleadverse effects on either the system or itsuse. These elements can be divided intotwo main categories: chemical contaminants and biological contaminants.Chemical ContaminationChemical contamination is best describedas the presence of any nonliving substancethat results in undesirable effects on theculture system. To define further is difficult; even essential nutrients become toxicat high enough concentrations. Nor istoxicity the only concern since hormonesand other growth factors found in serumcan cause changes that, while not necessarily harmful to cultures, may beunwanted by researchers using thesystem. (Reviewed in references 1-3.)MediaThe majority of chemical contaminantsare found in cell culture media and comeeither from the reagents and water usedto make them, or the additives, such assera, used to supplement them. Reagentsshould always be of the highest qualityand purity and must be properly stored toprevent deterioration. Ideally, they shouldbe either certified for cell culture use bytheir manufacturer or evaluated by theresearcher before use. Mistakes in mediapreparation protocols, reading reagentbottle labels, or weighing reagents areother common sources of chemicalcontamination.SeraSera used in media have long been asource of both biological and chemicalcontaminants. Due to cell culture-basedscreening programs currently used bygood sera manufacturers, it is unusual tofind a lot of fetal bovine sera that is toxicto a majority of cell cultures. However, itis common to find substantial variationsin the growth promoting abilities ofdifferent lots of sera for particular cell

culture systems, especially for culturesthat have specialized or differentiatedcharacteristics. Uncontrollable lot-to-lotvariation in hormone and growth factorconcentrations makes this probleminevitable; careful testing of sera beforepurchase, or switching to serum-freemedia can avoid these problems.Table 2. Types and Sources ofPotential Chemical Contaminants Metal ions, endotoxins, and other impuritiesin media, sera, and water Plasticizers in plastic tubing and storagebottles Free radicals generated in media by thephotoactivation of tryptophan, riboflavinor HEPES exposed to fluorescent light Deposits on glassware, pipettes, instrumentsetc., left by disinfectants or detergents,antiscaling compounds in autoclave water,residues from aluminum foil or paper Residues from germicides or pesticides usedto disinfect incubators, equipment, and labs Impurities in gases used in CO2 incubatorsRemember also that serum proteins havethe ability to bind substantial quantitiesof chemical contaminants, especiallyheavy metals, that may have entered theculture system from other sources, rendering them less toxic. As a result, switching from serum-containing medium to aserum-free system can unmask these toxicchemical contaminants, exposing the cellsto their adverse effects.WaterThe water used for making media andwashing glassware is a frequent sourceof chemical contamination and requiresspecial care to ensure its quality. Traditionally, double or triple glass distillationwas considered to be the best source ofhigh quality water for cell culture mediaand solutions. Newer purification systemscombining reverse osmosis, ion exchangeand ultrafiltration are capable of removingtrace metals, dissolved organic compoundsand endotoxins and are increasingly popular. However, these systems must beproperly maintained and serviced to ensurecontinued water quality. Because of its3aggressive solvent characteristics, highlypurified water can leach potentially toxicmetal ions from glassware or metal pipes,and plasticizers from plastic storage vesselsor tubing. These contaminants can thenend up in media or deposited on storagevessels and pipettes during washing andrinsing. Water used to generate steam inautoclaves may contain additives to reducescale buildup in pipes; these potentiallytoxic additives can also end up onglassware.EndotoxinsEndotoxins, the lipopolysaccaridecontaining by-products of gram negativebacteria, are another source of chemicalcontaminants in cell culture systems. Endotoxins are commonly found in water, seraand some culture additives (especially thosemanufactured using microbial fermentation) and can be readily quantified usingthe Limulus Amebocyte Lysate assay (LAL).These highly biologically reactive molecules have major influences in vivo onhumoral and cellular systems. Studies ofendotoxins using in vitro systems haveshown that they may affect the growthor performance of cultures and are asignificant source of experimental variability (Reviewed in references 6 and 39).Furthermore, since the use of cell cultureproduced therapeutics, such as hybridomas and vaccines, are compromised byhigh endotoxin levels, efforts must bemade to keep endotoxin levels in culturesystems as low as possible.In the past, sera have been a major sourceof endotoxins in cell cultures. As improvedendotoxin assays (LAL) led to an increasedawareness of the potential cell cultureproblems associated with endotoxins,most manufacturers have significantlyreduced levels in sera by handling the rawproducts under aseptic conditions. Poorlymaintained water systems, especially systems using ion exchange resins, can harborsignificant levels of endotoxin-producingbacteria and may need to be tested ifendotoxin problems are suspected ordiscovered in the cultures.

Figure 1. Photomicrograph ofa low level yeast infection ina liver cell line (PLHC-1, ATCC# CRL-2406). Budding yeastcells can been seen in severalareas (arrows). At this lowlevel of contamination, nomedium turbidity would beseen; however, in the absenceof antibiotics, the culturemedium will probablybecome turbid within a day.Figure 2. Photomicrograph ofa small fungal colony growingin a cell culture. At this point,this colony would still beinvisible to direct visualobservation. If this culturewas subcultured at this point,all of the cultures or experiments set up from it wouldsoon be lost to fungalcontamination.Storage VesselsMedia stored in glass or plastic bottlesthat have previously contained solutionsof heavy metals or organic compounds,such as electron microscopy stains,solvents and pesticides, can be anothersource of contamination. The contaminants can be adsorbed onto the surfaceof the bottle or its cap (or absorbed intothe bottle if plastic) during storage of theoriginal solution. If during the washingprocess they are only partially removed,then once in contact with culture mediathey may slowly leach back into solution.Residues from chemicals used to disinfectglassware, detergents used in washing,or some aluminum foils and wrappingpapers for autoclaving or dry heat sterilization can also leave potentially toxicdeposits on pipettes, storage bottles andinstruments.Fluorescent LightsAn important but often overlookedsource of chemical contamination resultsfrom the exposure of media containingHEPES (N-[2-hydroxylethyl] piperazineN'-[2-ethanesulfonic acid]) — an organicbuffer commonly used to supplementbicarbonate-based buffers), riboflavin ortryptophan to normal fluorescent lighting. These media components can bephotoactivated producing hydrogen peroxide and free radicals that are toxic tocells; the longer the exposure the greaterthe toxicity (4,5). Short term exposureof media to room or hood lighting whenfeeding cultures is usually not a significant problem; but leaving media on labbenches for extended periods, storingmedia in walk-in cold rooms with thelights on, or using refrigerators with glassdoors where fluorescent light exposureis more extensive, will lead to a gradualdeterioration in the quality of the media.IncubatorsThe incubator, often considered a majorsource of biological contamination, canalso be a source of chemical contamination.The gas mixtures (usually containing carbon dioxide to help regulate media pH)perfused through some incubators maycontain toxic impurities, especially oils orother gases such as carbon monoxide, thatmay have been previously used in the4same storage cylinder or tank. This problem is very rare in medical grade gases,but more common in the less expensiveindustrial grade gas mixtures. Care mustalso be taken when installing new cylinders to make sure the correct gas cylinderis used. Other potential chemical contaminants are the toxic, volatile residues leftbehind after cleaning and disinfectingincubators. Disinfectant odors should notbe detectable in a freshly cleaned incubator when it is placed back into use.Keep in mind that chemical contaminantstend to be additive in cell culture; smallamounts contributed from several different sources that are individually nontoxic,when combined together in medium, mayend up overloading the detoxificationcapabilities of the cell culture resultingin toxicity-induced stress effects or evenculture loss.Biological ContaminationBiological contaminants can be subdivided into two groups based on the difficultyof detecting 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 seriousculture problems, — viruses, protozoa,insects, mycoplasmas and other celllines.For a comprehensive review, see references7 and 8. Ultimately, it is the length of time that aculture contaminant escapes detection thatwill determine the extent of damage itcreates in a laboratory or research project.Bacteria, Molds, and YeastsBacteria, molds and yeasts are found virtually everywhere and are able to quicklycolonize and flourish in the rich and relatively undefended environment providedby cell cultures. Because of their size andfast growth rates, these microbes are themost commonly encountered cell culturecontaminants. In the absence of antibiotics, microbes can usually be readilydetected in a culture within a few days ofbecoming contaminated, either by directmicroscopic observation. (See Figures 1and 2.) or by the effects they have on the

abFigures 3a and 3b. Photomicrographs of a winterflounder (Pseudopleuronectesamericanus) fibroblast-likecell culture. Figure 3a showsan apparently healthy earlypassage culture; Figure 3bshows the same cultureapproximately 24 hours later.Electron microscopy showedvirus-like particles in thesecells. Multiple attempts toestablish cell lines from thisspecies were unsuccessfuland showed cytopathiceffects that appeared to becaused by an unknown virus.culture (pH shifts, turbidity, and celldestruction). However, when antibioticsare routinel

monoclonal antibodies, manufactured by these cultures will probably be useless. For some researchers the most serious consequence of contamination is suffering the embarrassment and damage to their reputation that results when they notify collaborators or journals that their exper-imental results are faulty and must be retracted due to contaminants in their cultures. Preventing all cell .