Basic Cell Culture Protocols.pdf

Edited byCheryl D.HelgasonCindy L.MillerBasicCell CultureProtocolsTHIRD EDITIONVolume 290METHODS IN MOLECULAR BIOLOGYTMMETHODS IN MOLECULAR BIOLOGYTMEdited byCheryl D.HelgasonCindy L.MillerBasicCell CultureProtocolsTHIRD EDITIONBasic Cell Culture Techniques11From:Methods in Molecular Biology,vol.290:Basic Cell Culture Protocols,Third EditionEdited by:C.D.Helgason and C.L.Miller Humana Press Inc.,Totowa,NJ1Culture of Primary Adherent Cellsand a Continuously Growing Nonadherent Cell LineCheryl D.HelgasonSummaryCell culture is an invaluable tool for investigators in numerous fields.It facilitatesanalysis of biological properties and processes that are not readily accessible at the levelof the intact organism.Successful maintenance of cells in culture,whether primary orimmortalized,requires knowledge and practice of a few essential techniques.The pur-pose of this chapter is to explain the basic principles of cell culture using the mainte-nance of a nonadherent cell line,the P815 mouse mastocytoma cell line,and the isolationand culture of adherent primary mouse embryonic fibroblasts(MEFs)as examples.Procedures for thawing,culture,determination of cell numbers and viability,andcryopreservation are described.Key Words:Cell culture;nonadherent cell line;adherent cells;P815;primary mouseembryonic fibroblasts;MEF;hemocytometer;viability;subculturing;cryopreservation.1.IntroductionThere are four basic requirements for successful cell culture.Each of thesewill be briefly reviewed in this introduction.However,a more detaileddescription is beyond the scope of this chapter.Instead,the reader is referred toone of a number of valuable resources that provide the information necessaryto establish a tissue culture laboratory,as well as describe the basic principlesof sterile technique(14).The first necessity is a well-established and properly equipped cell culturefacility.The level of biocontainment required(Levels 14)is dependent on thetype of cells cultured and the risk that these cells might contain,and transmit,infectious agents.For example,culture of primate cells,transformed humancell lines,mycoplasma-contaminated cell lines,and nontested human cellsrequire a minimum of a Level 2 containment facility.All facilities should be01/Helgason/1-128/26/04,9:09 AM12Helgasonequipped with the following:a certified biological safety cabinet that protectsboth the cells in culture and the worker from biological contaminants;a centri-fuge,preferably capable of refrigeration and equipped with appropriate con-tainment holders that is dedicated for cell culture use;a microscope forexamination of cell cultures and for counting cells;and a humidified incubatorset at 37C with 5%CO2 in air.A 37C water bath filled with water containinginhibitors of bacterial and fungal growth can also be useful if warming of mediaprior to use is desired.Although these are the basic requirements,there arenumerous considerations regarding location of the facility,airflow,and otherdesign features that will facilitate contamination-free culture.If a new cell cul-ture facility is being established,the reader should consult facility requirementsand laboratory safety guidelines that are available from your institutionsbiosafety department or the appropriate government agencies.The second requirement for successful cell culture is the practice of steriletechnique.Prior to beginning any work,the biological safety cabinet should beturned on and allowed to run for at least 15 min to purge the contaminated air.All work surfaces within the cabinet should be decontaminated with an appro-priate solution;70%ethanol or isopropanol are routinely used for this purpose.Any materials required for the procedure should be similarly decontaminatedand placed in or near the cabinet.This is especially important if solutions havebeen warmed in a water bath prior to use.The worker should don appropriatepersonnel protective equipment for the cell type in question.Typically,thisconsists of a lab coat with the cuffs of the sleeves secured with masking tape toprevent the travel of biological contaminants and Latex or vinyl gloves thatcover all exposed skin that enters the biosafety cabinet.Gloved hands shouldbe sprayed with decontaminant prior to putting them into the cabinet and glovesshould be changed regularly if something outside the cabinet is touched.Careshould be taken to ensure that anything coming in contact with the cells ofinterest,or the reagents needed to culture and passage them,is sterile(eitherautoclaved or filter-sterilized).The biosafety office associated with your insti-tution is a valuable resource for providing references related to the discussionof required and appropriate techniques required for the types of cells you intendto use.A third necessity for successful cell culture is appropriate,quality controlledreagents and supplies.There are numerous suppliers of tissue culture media(both basic and specialized)and supplements.Examples include Invitrogen(),SigmaAldrich(),BioWhittaker(),and StemCell Technologies Inc.().Unless otherwise specified in the protocols accompanying your cells of inter-est,any source of tissue-culture-grade reagents should be acceptable for mostcell culture purposes.Similarly,there are numerous suppliers of the plasticware01/Helgason/1-128/26/04,9:09 AM2Basic Cell Culture Techniques3needed for most cell culture applications(i.e.,culture dishes and/or flasks,tubes,disposable pipets).Sources for these supplies include Corning( Falcon( cautionary notes are essential.First,sterile culture dishes can be purchased as either tissue culture treated orPetri style.Although either can be used for the growth of nonadherent cells,adherent cells require tissue-culture-treated dishes for proper adherence andgrowth.Second,it is possible to use glassware rather than disposable plasticfor cell culture purposes.However,it is essential that all residual cleaningdetergent is removed and that appropriate sterilization(i.e.,121C for at least15 min in an autoclave)is carried out prior to use.If the three above-listed requirements have been satisfied,the final neces-sity for successful cell culture is the knowledge and practice of the fundamen-tal techniques involved in the growth of the cell type of interest.The majorityof cell culture carried out by investigators involves the use of variousnonadherent(i.e.,P815,EL-4)or adherent(i.e.,STO,NIH 3T3)continuouslygrowing cell lines.These cell lines can be obtained from reputable supplierssuch as the American Tissue Type Collection(ATCC;www.atcc.org)or DSMZ(the German Collection of Microorganisms and Cell Cultures)(www.dsmz.de/mutz/mutzhome.html).Alternatively,they can be obtained from collaborators.Regardless of the source of the cells,it is advisable to verify the identity of thecell line(refer to Chapters 4 and 5)and to ensure that it is free of mycoplasmacontamination(refer to Chapters 2 and 3).In addition to working with immor-talized cell lines,many investigators eventually need or want to work withvarious types of primary cells(refer to Chapters 621 for examples).Bacterialcontaminations,as a consequence of the isolation procedure,and cell senes-cence are two of the major challenges confronted with these types of cell.The purpose of this chapter is to explain the basic principles of cell cultureusing the maintenance of a nonadherent cell line,the P815 mouse mastocytomacell line,and adherent primary mouse embryonic fibroblasts(MEF)asexamples.Procedures for thawing,subculture,determination of cell numbersand viability,and cryopreservation are described.2.Materials2.1.Culture of a Continuously Growing Nonadherent Cell Line(see Note 1)1.P815 mastocytoma cell line(ATCC,cat.no.TIB-64).2.High-glucose(4.5 g/L)Dulbeccos Modified Essential Medium(DMEM).Storeat 4C.3.Fetal bovine serum(FBS)(see Note 2).Sera should be aliquoted and storedat 20C.01/Helgason/1-128/26/04,9:09 AM34Helgason4.Penicillinstreptomycin solution.100X stock solution.Aliquot and store at 20C(see Note 3).5.L-Glutamine,200 mM stock solution.Aliquot and store at 20C.6.DMEM+growth medium:high-glucose DMEM(item 2)supplemented with10%FBS,4 mM glutamine,100 IU penicillin,and 100 g/mL streptomycin.Prepare a 500-mL bottle under sterile conditions and store at 4C for up to 1 mo(see Note 4).7.Trypan blue stain(0.4%w/v trypan blue in phosphate-buffered saline PBS fil-tered to remove particulate matter)or eosin stain(0.14%w/v in PBS;filtered)fordetermination of cell viability.8.Tissue-culture-grade dimethyl sulfoxide(DMSO)(i.e.,Sigma)stored at roomtemperature.9.Freezing medium,freshly prepared and chilled on ice,consisting of 90%FBSand 10%DMSO(see Note 5).2.2.Culture of Primary Mouse Embryonic Fibroblasts1.High-glucose(4.5 g/L)DMEM(see Subheading 2.1.).2.FBS(see Subheading 2.1.).3.Penicillinstreptomycin solution(100X)(see Subheading 2.1.).4.MEF culture medium.DMEM supplemented with 10%FBS and 1X(100 IUpenicillin and 100 g/mL streptomycin)antibiotics.5.Dulbeccos Ca2+-and Mg2+-free PBS(D-PBS).D-PBS can be purchased as 1Xor 10X stocks from numerous suppliers or a 1X solution can be prepared in thelab as follows:Dissolve the following in high-quality water(see Note 6):8 g/LNaCl,0.2 g/L KCl,0.2 g/L KH2PO4,2.16 g/L Na2HPO47H2O;adjust pH to 7.2.Filter-sterilize using a 0.22-m filter and store at 4C.6.0.25%Trypsin0.5 mM EDTA(T/E)solution(see Note 7).Store working stocksat 4C.7.Freezing medium(see Subheading 2.1.).8.Timed pregnant female mouse(see Note 8).9.70%Ethanol solution or isopropanol.10.Two sets of forceps and scissors;one set sterilized by autoclaving at 121C for15 min.11.Fine forceps(sterile)(Fine Science Tools,cat.no.11272-30).12.Small fine scissors(sterile).13.18-Gage blunt-end needles(sterile)(StemCell Technologies Inc.).3.MethodsPrior to the initiation of any cell culture work,it is essential to ensure that allequipment is in optimal working condition.Moreover,if cell culture is tobecome a routine technique utilized in the laboratory,scheduled checks andregular maintenance of the equipment are required.A partial checklist of thingsto consider includes the following:check to ensure that the temperature andCO2 levels in the incubator are at the desired levels;check to be sure that the01/Helgason/1-128/26/04,9:09 AM4Basic Cell Culture Techniques5water pan in the incubator is full of clean water and that it contains coppersulfate to inhibit bacterial growth;check to ensure that the water bath is at therequired temperature and contains adequate amounts of clean water;check toensure that the biological safety cabinet to be used is certified and operatingcorrectly;ascertain that the centrifuge is cleaned and decontaminated.3.1.Culture of a Continuously Growing Nonadherent Cell Line3.1.1.Thawing Cryopreserved P815 Cells1.In the biological safety cabinet,prepare one tube containing 9 mL of DMEM+growth medium warmed to at least room temperature.2.Remove one vial of cells from the storage container(liquid nitrogen or ultralowtemperature freezer)(see Note 9).3.Transfer the vial of cells to a 37C water bath until the suspension is just thawed(see Note 10).4.In the cell culture hood,use a sterile glass or plastic pipet to transfer the contentsof the vial slowly into the tube containing the growth medium.5.Centrifuge the cells at 1200 rpm(300g)for 7 min to obtain a pellet.6.Aspirate the supernatant containing DMSO and suspend the cell pellet in 10 mLof DMEM+growth medium(see Note 11).7.Transfer the cells to a tissue culture dish(100 mm)and incubate at 37C,5%CO2.8.Examine cultures daily using an inverted microscope to ensure that the culturewas not contaminated during the freezethaw process and that the cells aregrowing.3.1.2.Determination of Cell Number and Cell ViabilityEvery cell line has an optimal concentration for maintaining growth andviability.Until sufficient experience is gained with a new cell line,it is recom-mended to check cell densities and viability every day or two to ensure thatoptimal health of the cultures is maintained.1.Gently swirl the culture dish to evenly distribute the cell suspension.2.Under sterile conditions,remove an aliquot(100200 L)of the evenly distrib-uted cell suspension.3.Mix equal volumes of cells and viability stain(eosin or trypan blue);this willgive a dilution factor of 2.4.Clean the hemocytometer using a nonabrasive tissue.5.Slide the cover slip over the chamber so that it covers both sides.6.Fill the chamber with the well-mixed cell dilution and view under the lightmicroscope.7.Each 1-mm2square should contain between 30 and 200 cells to obtain accurateresults(see Note 12).01/Helgason/1-128/26/04,9:09 AM56Helgason8.Count the numbers of bright clear(viable)and nonviable(red or blue dependingon the stain used)cells in at least two of the 1-mm2 squares,ensuring that twonumbers are similar(i.e.,within 5%of one another).Count all five of the 1-mm2squares if necessary to ensure accuracy(see Note 13).9.Calculate the numbers of viable and nonviable cells,as well as the percentage ofviable cells,using the following formulas where A is the mean number of viablecells counted,B is the mean number of nonviable cells counted,C is the dilutionfactor(in this case,it is 2),D is the correction factor supplied by the hemocytom-eter manufacturer(this is the number required to convert 0.1 mm3 into milliliters;it is usually 104).Concentration of viable cells(per mL)=A C DConcentration of nonviable cells(per mL)=B C DTotal number of viable cells=concentration of viable cells volumeTotal number of cells=number of viable+number of dead cellsPercentage viability=(number of viable cells 100)/total cell number3.1.3.Subculture of Continuously Growing Nonadherent CellsMaintenance of healthy,viable cells requires routine medium exchanges orpassage of the cells to ensure that the nutrients in the medium do not becomedepleted and/or that the pH of the medium does not become acidic(i.e.,turnyellow)as a result of the presence of large amounts of cellular waste.1.View cultures under an inverted phase-contrast microscope.Cells growing inexponential growth phase should be round,bright,and refractile.If necessary,determine the cell density as indicated in Subheading 3.1.2.2.There is no need to centrifuge the cells unless the medium has become too acidic(phenol red=yellow),which indicates the cells have overgrown,or if low viabil-ity is observed.3.Transfer a small aliquot of the well-mixed cell suspension into a fresh dish con-taining prewarmed DMEM+growth medium(see Note 14),ensuring that theresulting cell density is in the optimal range for the particular cell line.4.Repeat this subculture step every 23 d to maintain cells in an exponential growthphase.3.1.4.Cryopreservation of Continuously Growing Nonadherent CellsContinuous culture of cell lines can lead to the accumulation of unwantedkaryotype alterations or the outgrowth of clones within the population.In addi-tion,continuous growth increases the possibility of cell line contamination bybacteria or other unwanted organisms.The only in