The ability of tumor cells of the lymphoreticular system to grow in vitro.

JoseM.Trujillo,BenjaminDrewinko,andMichaelJ.AhearnDepartmentofClinicalPathology,TheUniversityofTexasM.D. AndersonHospitalandTumorInstituteatHouston,Houston,Texas77025SUMMARYFifty-eightculturesoflymphoreticularcells(7normal,51pathological)werestudied.Whilealllines,regardlessoforiginordiagnosis,wereabletogrowinvitroforshortterms,cellsderivedfrommalignanttumorshadthegreatestabilitytogrowforlongperiods.Thisproliferativecapacitywasalsopresentinamixedlymphocyticculturederivedfromtwohealthydonorsandinlymphnodescontainingmetastatictumors. Fourlineswereestablishedincontinuous culture.Twoofthesesynthesizeimmunoglobulinandhaveshownevidenceofviralparticles. Allofthelong-termcultureshaveaneuploidkaryotypesandsimilarultrastructuralcharacteristics.Itissuggestedthattheabilitytogrowinvitroisrelatedtothenumberandqualityofcellswithunlimitedproliferativecapacity present intheoriginalexplant. Unlimitedproliferativecapacityisprobablyconferredbymalignanttransformationorantigenicstimulationofthelymphocyticelements.INTRODUCTIONIn1964,Woodliff(53)indicatedthathumanlymphocyticcellshadnotyetbeengrownincontinuousculturesforsignificantperiods.Sincethattime,ahostofhumancelllinesderivedfromlymphopoietic organs,bonemarrow,andperipheralbloodhavebeenestablished(9,17,18,25,35,36,41,47,50). Thevastmajorityoftheselineswereobtainedfrompatientswithvariousdiseases,mostlymalignantlymphomasandleukemias,whereasnormalcellsgrewverypoorlyornotatall(33). Althoughtheprecisecellularoriginofthecelllinescouldnotbeestablishedwithabsolutecertainty,itwasgenerallyacceptedthattheyoriginatedfrommalignantcells(8). Instudyingtheproliferationkinetics,Rosenfeldetal. (42)categorizedlymphoidculturesaccordingtosurvivalas(a)populationsthatdierapidly,(b)populationswithalimitedlife-span,and(c)establishedlines.Lymphoidcellshaveprecisenutritionalrequirementsforgrowthinvitro(19)whicharehighlypopulationdependent(20). Someculturesoflymphoreticularcellscanbemaintainedforprolongedperiodsbytheadditiontothenutritional1SupportedbyUSPHSResearchGrantCA06939fromtheNationalCancerInstitute,USPHS,andbySpecialResearchCareerDevelopmentAward1FO3CA42078(toB.D.)fromtheNationalCancerInstitute,USPHS.ReceivedOctober5,1971ÂiacceptedFebruary16,1972.medium ofcertainstimulatory agentsthatproducecharacteristicmorphologicalandbiochemicalchangesinthelymphoidcells,aneventcalled"blastictransformation. "Suchtransformationcanbeinducedbyavarietyofagentssuchasphytohemagglutinin,pokeweedmitogen,pyogenicendotoxins,histocompatibilityantigens,etc.Inothercultures,lymphocytesmayundergo"spontaneous"blastictransformation; thisissometimesmoreeasilyaccomplishedbysupplyingthemediumwithheterologousrather than homologous serum (5). Spontaneoustransformationcanalsooccurfromcocultivationoflymphoidcellsobtainedfrom2healthydonors(2,32),duetoantigenicstimulationbyhistocompatibility antigens.However,inthevastmajorityofculturesoflymphoreticularoriginthatgrowforprolongedperiods,thecauseofsuchaspontaneousblastictransformationcannotbededuced.Thepresentstudyencompassesa5-yearsystematicexplorationbyourtissueculturelaboratoryofthecapacityofcellsofthelymphoreticularsystemtogrowinvitrowithouttheadditionofblastogenicinducerstothenutrientmedium.MATERIALSANDMETHODSBiopsies. Freshtissuebiopsieswereutilizedfortheprimaryexpiants.Thematerialwasobtainedfromlymphnodes(45cases)andspleen(7cases). Hemopoietictissuecamefrombonemarrow(5cases)andperipheralblood(1case). Materialfrompretreatedpatientswasrejected,asweresamplesthatcouldnotbeseededimmediatelyaftertheywereobtained.Thetissuewasfinelyminced,andsmallexpiantsweresetupinT-30flasksunderaperforatedcellophanemembrane,aspreviouslydescribed(49). Insomeinstances,loosecellsweresuspendedinmediumandculturedbythespinnerflaskmethod(34). ThecellsweremaintainedinHam'sF-10mediumsupplementedby20%fetalcalfserum,vitamins,glutamine,andpenicillin(100units/ml). Feedingwasperformedinitiallywhenneededandafterwardsregularlyonceortwiceaweek.Serialpropagationwascarriedoutbypreviouslydescribedmethods(14). BoneMarrow.One-halfto1mlofheparinizedbonemarrowmaterialwasplacedinaPetridishcontainingafewmloffreshmedium.ParticleswereselectedwithaPasteurpipetandplacedinT-30flasksunderaperforatedcellophanemembrane.Themediumwaschangedfrequentlytoremoveredcellcontaminants. PeripheralBlood.Leukocyte-containingplasmafrom2pintsofblood,eachfrom2healthydonors(groups0andA)wasMAY1972 1057