Microchannel acoustophoresis does not impact survival or function of microglia, leukocytes or tumor cells

Mic ochannel Acous opho esis does no Impac Su i al o Func ion o Mic oglia, Leukocy es o Tumo Cells Miguel A. Bu guillos 1,5. , Cecilia Magnusson 6. , Ma ia No din 2. , And eas Lensho 2 , Pe Augus sson 2 , Magnus J. Hansson 7 , Eskil Elme ´ 7 , Hans Lilja 6,8,9,10 , Pa ik B undin 1 , Thomas Lau ell 2,4 , Tomas Deie bo g 3 * 1Neu onal Su i al Uni , Depa men o Expe imen al Medical Science, Wallenbe g Neu oscience Cen e , Lund Uni e si y, Lund, Sweden, 2Depa men o Measu emen Technology and Indus ial Elec ical Enginee ing, Lund Uni e si y, Lund, Sweden, 3Expe imen al Neu oin lamma ion Labo a o y, Depa men o Expe imen al Medical Science, Wallenbe g Neu oscience Cen e , Lund Uni e si y, Lund, Sweden, 4Depa men o Biomedical Enginee ing, Dongguk Uni e si y, Seoul, Sou h Ko ea, 5Depa men o Oncology-Pa hology, Cance Cen um Ka olinska, Ka olinska Ins i u e , S ockholm, Sweden, 6Depa men o Labo a o y Medicine, Lund Uni e si y, Ska ˚ne Uni e si y Hospi al, Malmo ¨, Sweden, 7Mi ochond ial Pa hophysiology Uni , Depa men o Clinical Sciences, Lund Uni e si y, Lund, Sweden, 8Depa men s o Su ge y (U ology) and Labo a o y Medicine, Memo ial Sloan-Ke e ing Cance Cen e , New Yo k, Uni ed S a es o Ame ica, 9Nu ield Depa men o Su gical Sciences, Uni e si y o Ox o d, Ox o d, Uni ed Kingdom, 10 Ins i u e o Biomedical Technology, Uni e si y o Tampe e, Tampe e, Finland Abs ac Backg ound: The use o acous ic o ces o manipula e pa icles o cells a he mic o luidic scale (i.e. acous opho esis), enables non-con ac , label- ee sepa a ion based on in insic cell p ope ies such as size, densi y and comp essibili y. Acous opho esis holds g ea p omise as a cell sepa a ion echnique in se e al esea ch and clinical a eas. Howe e , i has been sugges ed ha he o ce ac ing upon cells unde going acous opho esis may impac cell iabili y, p oli e a ion o cell unc ion ia sub le pheno ypic changes. I his we e he case, i would sugges ha he acous opho esis me hod would be a less use ul ool o many cell analysis applica ions as well as o cell he apy. Me hods: We in es iga e, o he i s ime, se e al key aspec s o cellula changes ollowing acous opho e ic p ocessing. We used wo se ings o ul asonic ac ua ion, one ha is used o cell so ing (10 V pp ope a ing ol age) and one ha is close o he maximum o wha he sys em can gene a e (20 V pp ). We used mic oglial cells and assessed cell iabili y and p oli e a ion, as well as he in lamma o y esponse ha is indica i e o mo e sub le changes in cellula pheno ype. Fu he mo e, we adap ed a simila me hodology o moni o he esponse o human p os a e cance cells o acous opho e ic p ocessing. Las ly, we analyzed he espi a o y p ope ies o human leukocy es and h ombocy es o explo e i acous opho e ic p ocessing has ad e se e ec s. Resul s: BV2 mic oglia we e unal e ed a e acous opho e ic p ocessing as measu ed by apop osis and cell u no e assays as well as in lamma o y cy okine esponse up o 48 h ollowing acous opho esis. Simila ly, we ound ha acous opho e ic p ocessing nei he a ec ed he cell iabili y o p os a e cance cells no al e ed hei p os a e-speci ic an igen sec e ion ollowing and ogen ecep o ac i a ion. Finally, human h ombocy es and leukocy es displayed unal e ed mi ochond ial espi a o y unc ion and in eg i y a e acous opho e ic p ocessing. Conclusion: We conclude ha mic ochannel acous opho esis can be used o e ec i e con inuous low-based cell sepa a ion wi hou a ec ing cell iabili y, p oli e a ion, mi ochond ial espi a ion o in lamma o y s a us. Ci a ion: Bu guillos MA, Magnusson C, No din M, Lensho A, Augus sson P, e al. (2013) Mic ochannel Acous opho esis does no Impac Su i al o Func ion o Mic oglia, Leukocy es o Tumo Cells. PLoS ONE 8(5): e64233. doi:10.1371/jou nal.pone.0064233 Edi o : Sal a o e V. Pizzo, Duke Uni e si y Medical Cen e , Uni ed S a es o Ame ica Recei ed Decembe 3, 2012; Accep ed Ap il 12, 2013; Published May 27, 2013 Copy igh : ß2013 Bu guillos e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This wo k was suppo ed by g an s om he Swedish go e nmen al agency o inno a ion sys ems, VINNOVA, CellCARE, g an No. 2009-00236, he Swedish Resea ch Council g an no. 2012-2229 and 2010-4389, by he Gyllens ie nska K appe up, The Royal Physiog aphic Socie y, C a oo d, A.E. Be ge , Wibe g, Be g all, G&J Kock, Ca l T ygge ounda ions, he SSF S a egic Resea ch Cen e (C ea e Heal h), Swedish Cance Socie y [11-0624], Finnish Funding Agency o Technology and Inno a ion (TEKES), Na ional Cance Ins i u e [R33 CA127768-03, R01CA160816 and P50-CA92629], Sidney Kimmel Cen e o P os a e and U ologic Cance s, and Da id H. Koch h ough he P os a e Cance Founda ion, Na ional Ins i u e o Heal h Resea ch (NIHR) Ox o d Biomedical Resea ch Cen e P og am. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: Lau ell is a boa d membe , ounde and sha eholde o AcouSo AB ha de elops acous opho esis echnology. Lau ell and Lensho a e in en o s o acous opho esis pa en s owned by E ysa e AB o which nei he has any engagemen . Lau ell holds esea ch g an s in he ield o acous opho esis om se e al Swedish unding Agencies; Swedish Science Council, FORMAS, Vinno a, Knu and Alice Wallenbe g Founda ion. Lilja and B undin a e ounde s and sha eholde s o AcouSo AB, which de elops acous opho esis echnology. Lau ell, Augus sson, Magnusson a e in en o s o a pa en applica ion in he acous opho esis ield, owned by Acouso AB. D . Lilja holds pa en s o licensed and comme cialized ee PSA, in ac PSA, and hK2 assays. The au ho s he eby con i m ha he decla ed pa en along wi h any o he ele an decla a ions ela ing o employmen , consul ancy, pa en s, p oduc s in de elopmen o modi ied p oduc s do no al e hei adhe ence o all he PLOS ONE policies on sha ing da a and ma e ials. * E-mail: omas.deie bo [email protected] .These au ho s con ibu ed equally o his wo k. PLOS ONE | www.plosone.o g 1 May 2013 | Volume 8 | Issue 5 | e64233 In oduc ion The use o acous ic o ces o handle pa icles and cells in mic o luidic sys ems (i.e. mic ochannel acous opho esis) is gaining inc eased a en ion [1]. The applica ion in which he acous o- pho esis me hod can be used include pa icle manipula ion [2,3], deple ion [4], washing [5,6,7], ac iona ion [8], a e e en so ing [9,10], concen a ion [11] and cell cycle synch oniza ion [12]. This no el cell manipula ion echnique is label- ee and enables sepa a ion by unique cell p ope ies, e.g. comp essibili y. In iew o i s high ep oducibili y, eliabili y and he ac ha his echnology can be applied o mos cell ypes, acous opho esis holds g ea p omise as a cell manipula ion echnique in se e al esea ch and clinical se ings [13]. While acous opho esis is eme ging as a new echnology in se e al esea ch a eas, he e a e doub s o whe he he induced acous ic o ces and luid handling a e ha m ul o he cells. Ques ions ha a e ele an o his echnology i acous opho e ic applica ions a e o be used wi h clinical se ing. Ea lie s udies on he impac o acous ic esonan sys ems on cells ha e been ecen ly e iewed by Wiklund (2012) [14]. Mo eo e , Ryll and coau ho s s udied Chinese hams e o a y cells in a pe used mac oscale acous ic cell e en ion de ice o 50 days and concluded ha no ha m was obse ed o his cell ype [15]. In ano he s udy, Wang and collabo a o s s udied mouse hyb idoma cells, which we e acous ically apped in a high po osi y polyes e mesh wi h a low in ensi y, esonan acous ic ield [16], concluded ha he acous ic ield p oduced a negligible e ec on cell iabili y in a sho - e m exposu e. Simila ly, Hul s o¨m and colleagues [17] as well as E ande e al. [18], using cos-7 cells om e al monkey kidney and a neu al s em cells espec i ely, s udied cell iabili y in an acous ic ap. Howe e , bo h s udies concluded ha cell iabili y was no a ec ed. Mo eo e , E ande e al. success ully g ew yeas cells wi hin he ap o demons a e ha cell p oli e a ion was no a ec ed [18]. Al hough acous opho e ic echnology shows g ea p omise, acous opho e ic manipula ion o cells in a clinical se ing mus be s udied in de ail. Bazou and colleagues s udied human li e ca cinoma cells (HepG2) in an acous ic ap and de e mined ha cell iabili y and p oli e a ion we e no a ec ed [19]. Using a con inuous low sys em, Jo¨nsson and coau ho s sepa a ed e y h- ocy es om lipid pa icles and concluded ha he e had been no inc ease o hemolysis o e y h ocy es a e passing h ough an acous opho e ic de ice [20]. Recen ly Dykes e al. emo ed pla ele s om pe iphe al blood p ogeni o cell p oduc s by acous opho esis and cell iabili y and colony- o ming abili ies o he p ogeni o cells was s udied. Fu he mo e, mo phological s udies as well as pla ele ac i a ion assays concluded ha he cells we e no ha med by he acous opho e ic ea men [21]. Howe e , he li e a u e s ill lacks a ho ough examina ion on he e ec o mic ochannel acous opho esis using sho - e m acous ic exposu e imes wi h long- e m iabili y and pheno ypic cha ac e iza ion. Especially cha ac e iza ions o impo an long- e m unc ional biological pa ame e s such as in lamma o y esponse, cell ac i a- ion esponse and espi a ion ha e no been s udied in de ail. I he acous opho esis echnology is used in he clinical se ing, he impac on cell su i al and he sub le pheno ypic changes ha may be induced mus be in es iga ed in de ail. Hence, in his s udy we examine se e al key cellula changes ollowing acous opho esis and use mic oglial cells, a cell ype known o eadily eac o en i onmen al cues o es cell iabili y and p oli e a ion. We also moni o mo e sub le changes in cellula pheno ype by analyzing in lamma o y s a us a e acous opho e ic p ocessing. Finally, we use simila me hodology o examine he e ec on human p os a e cance cells and s udy he di e en p ope ies o cell espi a ion using human leukocy es and h ombocy es. Ou s udies indica e ha mic ochannel acous opho esis can be uned o e ec i e cell handling wi hou ha ing any e ec o cell iabili y, p oli e a ion, espi a ion o in lamma o y s a us. Ma e ials and Me hods E hics S a emen The s udy was app o ed by he egional e hical e iew boa d o Lund, Sweden (pe mi numbe 113/2008). Blood samples we e collec ed om heal hy blood dono s a he blood dono cen al, Ska˚ne Uni e si y Hospi al, Lund. Samples we e ob ained a e w i en in o med consen was acqui ed. Acous opho esis De ice and Se up The acous opho esis chip design is illus a ed in igu e 1. The acous opho esis chip was ab ica ed in ,100.-silicon h ough double sided pho oli hog aphy and aniso opic we e ching using KOH. A bo o loa glass lid o seal he low channel was anodically bonded o he chip. The chip s uc u e is comp ised o i u ca ion inle ollowed by a 20 mm long acous ic ocusing channel (squa e c oss-sec ion; 375 mm6150 mm) co esponding o a e en ion ime o abou 1 second in he acous ic ield o a low a e o 100 mL min 21 and a i u ca ion ou le egion. Fo he iabili y expe imen s only one inle and ou le was used. The cen e ou le low a e was con olled by a sy inge pump (WPI sp210iwz, Wo ld P ecision Ins umen s Inc., Sa aso a, FL, USA) and se o 100 mL min 21 and he cell suspension was d awn in o he cen e inle om an Eppendo ube a he same speed. To a oid sedimen a ion in he ube du ing he cou se o he expe imen s he cells we e mixed gen ly by pipe ing. Samples we e collec ed ei he by a 6-po 2- way sample loop (V-451, Upchu ch Scien i ic, Oak Ha bo , WA, USA) wi h a olume o 100 mL connec ed in se ies wi h he ou le o di ec ly in he sy inge ( o he human blood cell espi a ion s udies). To ac ua e he chip a piezoce amic ansduce (PZ26, esonan a 2 MHz, Fe ope m, K is gaa d, Denma k) was used. The piezoce amic was d i en by a wa e o m gene a o (Agilen , 33250A) se a a equency o 1.94 MHz, ampli ied by an in- house-buil ampli ie . The ol age o e he ansduce was measu ed using an oscilloscope (TDS 1002, Tek onix UK L d., B acknell, UK). The empe a u e o he chip was con olled using a Pel ie elemen egula ed by a Pel ie egula o (TC2812-RS232, CoolT onic GmbH, Beinwill am See, Swi ze land) and se o 37uC o limi e ec s emana ing om ele a ed empe a u e exposu e caused by powe dissipa ion in he piezo ce amic a ele a ed ope a ing ol age ha could a ec he iabili y o he cells. The cells we e p ocessed h ough he chip and exposed o ul asound a ei he 10 o 20 V pp ope a ing ol age o un h ough he chip wi hou ul asound exposu e o e eal any in luence o he mic o luidic sys em. Unp ocessed cells we e used as a con ol. The acous ic ene gies he cells we e exposed o we e highe han would no mally be needed o ocus cells in his chip o he pa icula se ings s a ed abo e. In his se up, h ombocy es we e less p one o ocus in he middle o he mic ochannel due o hei small sizes. Es ima ion o he Acous ic Ene gy Densi y To es ima e he acous ic ene gy densi y (E ac ) inside he acous opho e ic mic ochannel polys y ene mic opa icles (,0.02% w ,Ø7mm) (Sigma-Ald ich. Co., S . Louis, MO, USA) suspended in T i on X-100 (0.01%) in PBS we e p ocessed in he de ice. To es ima e E ac he mic opa icles we e d awn in o he chip om he side inle s a low a e o 50 mL min 21 and a clean Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 2 May 2013 | Volume 8 | Issue 5 | e64233 bu e was pumped in o he cen e inle a a low a e o 450 mL min 21 . The beads we e hen ocused in he cen e o he channel and collec ed om he cen e ou le a a low a e o 250 mL min 21 using wo 6-po 2-way sample loops (V-451, Upchu ch Scien i ic, Oak Ha bo , WA, USA), each wi h a olume o 100 mL. Excess luid was disca ded om he side ou le s also a a low a e o 250 mL min 21 . Ma e ial. The luo escen ma ke o DNA 7-Aminoac ino- mycin D (7-AAD), he syn he ic and ogen agonis me hyl ieno- lone (R1881), 2,3-Bis(2-me hoxy-4-ni o-5-sul ophenyl)-2H- e a- zolium-5-ca boxanilide(XTT), Giemsa s ain, 29-(4- hyd oxyphenyl)-5-(4-me hyl-1-pipe azinyl)-2,59-bi-1H-benzimid- azole ihyd ochlo ide hyd a e, bisBenzimide (Hoechs ), and Lipopolysacha ide (LPS om Esche ichia coli, se o ype 026:B6) we e pu chased om Sigma Ald ich (S ockholm, Sweden). Te ame hyl hodamine, e hyl es e , pe chlo a e (TMRE) was pu chased om In i ogen (S ockholm, Sweden). Cy okines we e de ec ed using a Mul iplex ki om Meso Scale Disco e y (Gai he sbu g, USA). Cell cul u e. The mu ine mic oglial BV2 cells we e a kind gi o D . Sand a Cecca elli and we e es ablished and i s cha ac e ized 1992 by Bocchini e al. [22]. All Cell lines we e cul u e in DMEM (GIBCO) and supplemen ed wi h 10% e al bo ine se um (FBS) (Sigma-Ald ich), 55 IU ml 21 penicillin and 55 mgml 21 s ep omycin (Sigma-Ald ich). Fo he expe imen s pe o med wi h he BV2 cells and LPS, he media was supplemen ed wi h 5% FBS. All cells we e cul u ed a 37uCina humidi ied a mosphe e con aining 5% CO 2 . The human p os a e cance cell lines DU145, LNCaP, PC3 and VCaP we e all ob ained om he Ame ican Type Cul u e Collec ion (ATCC) and g own acco ding o ATCC ecommenda ions. Cell iabili y in BV2 cells. The BV2 mic oglial cells we e suspended (4 million cells mL 21 ) in cul u e medium supplemen ed wi h 2% se um o acili a e pheno ypic/in lamma o y changes o he cells. A e he acous opho e ic p ocessing possible induc ion o acu e cell dea h was examined by dye exclusion using ypan blue. XTT assay. 5000 BV2 cells pe well we e seeded in 96-well pla es a e mic ochannel acous opho e ic p ocessing and cul u ed o 24 o 48 h. The XTT assay was pe o med ollowing he manu ac u e’s ecommenda ion and he da a a e p esen ed as pe cen age in espec o he cells exposed o mic o luidic handling wi hou ul asound. TMRE s aining. 20,000 BV2 cells we e seeded in 12-well pla es o measu e hei Mi ochond ial po en ial (DY) a e acous opho e ic p ocessing. A inal concen a ion o 25 nM o TMRE s aining was added o he medium and he luo escence was measu ed using low cy ome y (FACS Can oII, FACSDi a so wa e, BD Biosciences, San Jose, USA). Cell mig a ion. BV2 cells we e seeded in 6-well pla es a e acous opho e ic p ocessing. Twen y- ou hou s a e seeding, a yellow 100-ml-pipe e ip was used o d aw a line h ough he con luen cul u e dish. A e addi ional 24 h o cul u e, he wid h o he line, i.e. how much cells had mig a ed, was analyzed. Hoechs s aining. BV2 cells we e seeded in 12-well pla es and ixed in 4% PFA du ing 20 minu es a 37uC. The pla es we e hen washed wice wi h PBS and Hoechs was added a a concen a ion o 1 mgmL 21 in PBS o 10 min a oom empe a u e. A e wo mo e washes wi h PBS, moun ing media was added o he wells and he numbe o cells ha p esen ed condensed nuclei was quan i ied using an in e ed mic oscope. Fo each well a leas 300 cells we e coun ed. Clonogenic assay. 1000 BV2 cells we e seeded in duplica es in pe i dishes and 10 mL o medium was added o each dish. A e h ee days, 5 mL o medium was exchanged wi h esh media. A e se en days he media was ca e ully emo ed and he cells we e ixed wi h 70% e hanol du ing 10 min a oom empe a u e. A e he ixa ion he cells we e s ained wi h 10% ( / ) GIEMSA s aining o 15 min a oom empe a u e. The pla es we e hen washed wi h MilliQ wa e and d ied a e which hey we e scanned and analyzed using ImageJ 1.43 so wa e. Immunoblo . A e acous opho e ic p ocessing, BV2 cells we e seeded in 6-well pla es and a e one day exposed o LPS o 24 h. The cells we e hen washed wice in PBS and lysed on ice using loading bu e . Then, he cells we e sonica ed 3 imes du ing 5 seconds each ime while kep on ice. All samples we e boiled o 4 min, esol ed on 10% SDS polyac ylamide gels a 100 V and ansblo ed on o ni o-cellulose memb anes o 2 h a 30 V. The memb anes we e blocked o a leas 1 h in a bu e (5% non- a milk powde and 0.1% NaN3 in PBS, pH 7.4) and p obed wi h p ima y an ibodies (dilu ed in 5% bo ine se um albumin and 0.1% NaN3 in PBS, pH 7.4) agains iNOS (San a C uz, sc-650; 1:1000) and b-ac in (Sigma, A5316 1:4000) o e nigh . The memb anes we e hen washed in PBS con aining 0.1% Tween- 20 and incuba ed wi h he seconda y an ibody (dilu ed 1:5000 in 2.5% milk in PBS) o 1 h and washed 3 imes in PBS. The p o ein bands we e isualized by ECL (Ame sham Biosciences) acco ding o he manu ac u e ’s ins uc ions. Cy okine analysis. A e acous opho e ic p ocessing, BV2 cells we e seeded in 96-well pla es. A e 24 h, cells we e exposed Figu e 1. Pic u e and illus a ion o he se up. (a) Pho o o he acous opho esis mic o luidic sys em i s p esen ed by Augus sson e al. [27]. (b) C oss sec ional iew o cell dis ibu ion in he mic ochannel wi hou ul asound (le ) and wi h ul asound o ming an ul asound s anding wa e ( igh ). (c) Illus a ion o acouscous opho - esis chip. Fo he p esen s udy, only one o he channel segmen s was used allowing cells o be exposed o ul asound. doi:10.1371/jou nal.pone.0064233.g001 Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 3 May 2013 | Volume 8 | Issue 5 | e64233 o LPS o 24 h and he supe na an s we e collec ed. The cy okine p oduc ion was measu e using he Meso Scale Disco e y elec ochemiluminescence ki (Th1/Th2 cy okines, Ul a-Sensi i e ki , K15013C-1) using he manu ac u e ’s ins uc ions. Cell iabili y in p os a e cance cell lines. DU145, PC3, LNCaP and VCaP cells we e de ached by ypsin/EDTA and washed wice in PBS and e-suspended in comple e cell g ow h medium. Immedia ely a e acous opho e ic p ocessing (0 h) ypan blue exclusion was used o es ima e he pe cen age o iable cells. The cells we e coun ed in a Bu¨ ke chambe and a e wa d seeded in 6-well pla es, (200,000 cells/well) and g own o 24 o 48 h. Cells we e ha es ed wi h ypsin/EDTA, washed wi h PBS and subsequen ly s ained wi h 7-AAD o 20 minu es. Cell iabili y was es ima ed o 10,000 cells by low cy ome y (FACS Can oII, FACSDi a so wa e, BD Biosciences), 7-AAD- nega i e cells wi h high o wa d sca e s we e selec ed as iable. PSA sec e ion. Cell lines (LNCaP and VCaP) exp essing he and ogen ecep o (AR) we e ha es ed and subjec ed o he acous opho e ic p ocessing. Unp ocessed cells we e used as a con ol g oup. Cells we e seeded in 96-well pla es (10,000 cells/ well), and g own in comple e g ow h medium o 24 h in he p esence o ei he R1881 (1 nM), o ehicle (PBS/DMSO). 80 mL g ow h medium was emo ed om each well o PSA analysis. The samples we e s o ed a 220uC un il analysis. PSA concen- a ions we e measu ed using he DELFIA P os a us F ee/To al PSA assay om Pe kinElme , Tu ku, Finland [23]. Human blood cell espi a ion. Th ombocy es and leuko- cy es om h ee heal hy blood dono s we e subjec ed o acous opho e ic p ocessing and mi ochond ial unc ion was analyzed by high- esolu ion espi ome y as desc ibed in Sjo¨ all e al. [24]. S a is ics. We used analysis o a iance (ANOVA) o s a is ical compa isons be ween expe imen al g oups. Expe imen s we e pe o med in iplica es o mo e. Da a a e exp essed as mean 6SD and P,0.05 was conside ed as signi ican ly di e en . Resul s Acous ic Ene gy Es ima ions To es ima e he acous ic ene gy (E ac ) a he di e en d i ing ol ages, we used 7 mm diame e mic opa icles. Mic opa icles we e used ins ead o cells do o hei homogenous size dis ibu ion, which allows o a be e measu emen . The d i ing ol age squa ed (U pp ) 2 has a linea ela ion o he E ac in he ollowing exp ession; U2 pp E U2 ~Eac~ p2 ako 4 whe e U is he ansi ion ol age applied when he ansi ion om pa icles exi ing h ough he side ou le s o exi ing om he cen e ou le akes place, E is he co esponding acous ic ansi ion ene gy, p a is he acous ic p essu e ampli ude and k o is he comp essibili y o he suspending liquid. Fo his pa icula chip, using he 7 mm polys y ene mic opa icles and a o al low a e o 500 mL min 21 ,U 2 was measu ed o be 147 V pp 2 and E was hen es ima ed om simula ed pa icles ajec o ies o be 69 J/m 3 . Th ough his exp ession E ac o he ol ages o 10 V pp and 20 V pp was calcula ed o be 47 J/m 3 and 188 J/m 3 , which co espond o acous ic p essu e ampli udes o 0.639 MPa and 1.28 MPa, espec i ely. These ene gy le els a e compa able o o he acous opho esis mic ochip p ocessing de ices, which commonly ope a e a acous ic ene gy densi y le els o 10–100 J/m 3 [25,26]. See Augus sson e al. 2012 o a mo e de ailed desc ip ion o he acous ic ene gy densi y es ima ion [27]. Acous ic Cell P ocessing Mic ochannel acous opho e ically p ocessed cells may be damaged due o hyd odynamic shea , ul asonic exposu e o high empe a u es. To a oid cell damage due o high empe a u es, we e alua ed he e ec o acous opho esis on cell iabili y in a con olled empe a u e en i onmen (37uC). Hyd odynamic shea damage was also excluded by using cells p ocessed h ough he chip wi hou ul asound exposu e as con ols. In he acous opho esis mic ochannel, cells we e ocused in he cen e when ope a ed a a hal wa eleng h esonance a 2 MHz. A ampli ude o 10 V pp , all cells we e ocused in he cen e o he mic ochannel. Ampli ude o 20 V pp was also employed o gene a e a s onge acous ic ield han needed o ocus cells, and he eby enable he s udy o possible ha m ul e ec s on he cells when exposed o ele a ed acous ic adia ion o ces. The se up allowed maximum 25 V pp , howe e using his high ol age gene a ed empe a u e luc ua ions abo e he empe a u e c i e ia o 37 ˚C. We es ed he e ec 25 V pp on immedia e cell dea h ( ypan blue exclusion) using he BV2 mic oglial cells wi hou de ec ing any ad e se e ec (da a no shown). As a con ol, cells we e p ocessed h ough he chip wi hou an ac i e ul asound ( unc ion gene a o se o 0 V pp ). In o de o s udy he e ec o acous opho esis on cell su i al and elucida e possible ad e se e ec s when using acous opho esis o cell so ing, a s anda d acous opho esis chip was used ( ig. 1, desc ibed in Ma e ial and Me hods). Cells we e passed h ough he chip a a low a e o 100 mL min 21 co esponding o a e en ion ime o abou 1 second in he acous ic ield, a e en ion ime ele an o mos acous opho e ic p ocessing. Acous ic Cell P ocessing in a Mic ochannel does no Al e Cell Dea h and Viabili y o Mic oglial Cells Viabili y and cell dea h analysis. We se ou o in es iga e possible dele e ious e ec s o acous opho esis by using he mic oglia cell line BV2, an immo alized mouse mic oglial cell used widely and success ully in medical esea ch [28]. Using ypan blue exclusion, no ad e se e ec s on he acu e cell dea h induced by he acous opho esis handling could be de ec ed (C l, cells no en e ing he acous opho esis chip 9.765.7% cell dea h; 0 V pp , 11.468.8% cell dea h; 10 V pp , 8.967.0% cell dea h; 20 V pp , 11.666.9% cell dea h). Nex , we s udied possible long- e m cellula al e a ions beyond he acu e e ec o acous opho esis. A e going h ough he acous opho esis chip, BV2 cells we e seeded again o 24 h o 48 h o in es iga e possible long- e m e ec s. Fi s , we used XTT assay (mi ochond ial dehyd ogenase ac i i y) o s udy a possible al e a ion in he iabili y/p oli e a ion o acous opho esis. How- e e , no di e ences in iabili y o he cells exposed o he di e en condi ions could be de ec ed in he XTT assay a ei he 10 o 20 V pp o acous ic ocusing a any ime poin , 24 o 48 h, a e seeding ( ig. 2a). To u he elucida e he e ec o acous opho esis, BV2 cells we e PFA- ixed hen Hoechs s ained a 24 and 48 h and he numbe o cells unde going cell dea h, con aining apop o ic bodies, we e quan i ied. Despi e a p og essi e cell dea h om 24 o 48 h, we did no ind any changes in numbe o cells unde going apop osis due o he acous opho e ic p ocessing ( ig. 2b). Fu he mo e, we did no de ec any changes in he mi ochond ial po en ial (indi ec ly measu emen o cells a isk o unde going apop osis) by TMRE due o acous opho esis ( ig. 2c). Simila ly, he cell mig a ion speed as desc ibed p e iously [29], was unal e ed a e acous opho e ic p ocessing a 24 o 48 h a e Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 4 May 2013 | Volume 8 | Issue 5 | e64233 cul u ing (da a no shown). The cellula changes o e a longe ime pe iod (7 days) was also assessed o s udy a possible al e a ion in cell u no e using a clonogenic assay howe e , no ad e se e ec we e de ec ed ( ig. 2d) simila o ou ea lie ime poin s (24 o 48 h). In lamma o y esponse. Al hough we could no de ec any acous opho esis-induced al e a ions on cell dea h o cell iabili y, mino cellula al e a ions and pheno ypic changes canno be uled ou . In his aspec , in lamma o y mic oglial cells a e sui able cells o de ec sub le di e ences in hei in lamma o y esponse due o he exposu e o acous ic o ces. We he e o e explo ed he possibili y ha acous opho e ic p ocessing could al e he in lam- ma o y esponse igge ed by a p oin lamma o y esponse. Lipopolyssacha ide (LPS) is a lipoglycan ound in he ou e memb ane o G am-nega i e bac e ia and a e y po en Toll-like ecep o 4 (TLR4) ligand and induce o in lamma ion. LPS- Figu e 2. Unal e ed iabili y o BV2 mic oglial cell line ollowing acous opho e ic p ocessing (10V pp and 20V pp ). BV2 cells we e passed h ough he acous opho esis chip wi h he unc ion gene a o se a 0, 10 and 20 V pp . A e going h ough he chip, he BV2 cells we e seeded again o 24 and 48 h. Cell iabili y was measu ed by XTT (a), apop o ic nuclei appea ance (b) and dec ease o mi ochond ial po en ial -Ym- (c), showing no di e ence be ween expe imen al g oups. Simila , no di e ence was de ec ed by clonogenic assay (d) used o s udy su i al and p oli e a ion a 7 days ollowing acous opho e ic p ocessing. The g aphs show he esul s om a leas h ee sepa a e expe imen s and he da a a e shown as means 6SD. Signi icance alue P,0.05, ns deno es non-signi ican . doi:10.1371/jou nal.pone.0064233.g002 Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 5 May 2013 | Volume 8 | Issue 5 | e64233 challenged BV2 cells we e analyzed o he p oduc ion o inducible ni ic oxide syn hase, iNOS, ( ig. 3a,b) ha is highly induced unde in lamma o y s imulus (such as LPS). The medium was also collec ed and used o measu e he mic oglial elease o p oin lamma o y cy okines (IL-1b, IL-12 and TNF-a; ig. 3c,d,e) and an i-in lamma o y molecules (IL-10; ig. 3 ). We ound ha , bo h he p o ein exp ession o p oin lamma o y iNOS and he elease o p o- and an i-in lamma o y cy okines upon LPS- challenge we e unal e ed a e exposu e o acous opho e ic p ocessing ( ig. 3). In luence o Acous opho esis on Viabili y o Sec e o y Func ion o P os a e Cance Cells Acous opho esis-based mic o luidic de ices a e cu en ly being explo ed as a mean o en ich and disc imina e iable om non- iable umo cells in pe iphe al blood [27]. The e o e, we ini ia ed a de ailed cha ac e iza ion o he iabili y and sec e o y unc ion o umo cells subsequen o subjec ing hem o acous opho e ic p ocessing. We i s cha ac e ized he p os a e cance cell lines DU145, PC3, LNCaP in e e ence o cell iabili y/p oli e a ion using he same XTT assay ha was used o he iabili y es o he BV2 mic oglial cells. We did no ind any changes in cell iabili y a ei he 24 o 48 h a e acous opho e ic p ocessing (da a no shown). Subsequen ly, we de e mined he ex en o cell dea h using ypan blue exclusion di ec ly ollowing acous opho e ic p ocessing wi hou obse ing any change in cell dea h o he di e en p os a e cance cell lines, DU145, PC3, LNCaP o VCaP. Finally, we examined cell dea h a 24 and 48 h using lo escence ac i a ed cell so ing (FACS) and ga ed cells o 7- AAD-up ake. Again, simila o ou esul s abo e, no ad e se e ec ollowing acous opho e ic p ocessing when compa ed o unp o- cessed cells was iden i ied ( ig. 4a–d). We wen on o de e mine whe he acous opho esis al e s he unc ional p ope ies o iable cells p ocessed h ough he mic ochip. PSA sec e ion is a sensi i e indica o o and ogen ecep o (AR) signaling ac i i y in AR-dependen umo cells. We used he p os a e cance cell lines LNCaP and VCaP and s udied bo h endogenous PSA sec e ion as wells as sec e ion ollowing adminis a ion o he syn he ic and ogen R1881. We did no ind any signi ican di e ences in PSA sec e ion in umo cells subjec ed o acous opho e ic p ocessing compa ed o unp ocessed cells, nei he a e adminis a ion o R1881 no o uns imula ed cells. The e we e no measu able di e ences in e e ence o cells p ocessed h ough he mic ochip in absence o ul asound o when hey we e exposed o ul asound a inc easing ol age ( ig. 5a–b). Mi ochond ial Respi a o y Func ion o Human Blood Cells is no Al e ed Following Acous opho esis Acous opho e ic p ocessing o human blood is an eme ging esea ch ield o acous ic cell manipula ion in mic ochannels. To u he elucida e possible cellula al e a ion due o acous opho - esis, we exposed h ombocy es and leukocy es isola ed om human blood o he same acous ic ea men used o he BV2 mic oglial cells and p os a e cance cells desc ibed abo e. High- esolu ion espi ome y was pe o med o e alua e mi ochond ial espi a o y unc ion and in eg i y. Fu he mo e, endogenous oxygen consump ion was e alua ed in in ac cells (da a no shown), and espi a ion du ing maximal oxida i e phospho yla ion (Oxphos) and maximal lux h ough he elec on anspo sys em was assessed using a mul iple subs a e/inhibi o i a ion p o ocol in digi onin-pe meabilized cells. We s udied he maximal espi a ion du ing Oxphos using bo h complex I- and complex II-linked subs a es o h ombocy es ( ig. 6a) and leukocy es ( ig. 6b) and ound no e ec ollowing acous opho e ic p ocessing. We u he in es iga ed he emaining espi a o y ac i i y ollowing inhibi ion o ATP syn hase wi h oligomycin, so called LEAK o s a e 4 espi a ion, and ound no di e ences in espi a o y ac i i y ollowing passage h ough he acous opho esis mic ochip. We also checked o di e ences in o he espi a o y s a es, wi hou de ec ing any al e a ions due o acous opho e ic p ocessing (da a no shown). Any p ocess in e - e ing wi h me abolic pa hways leading o espi a o y inhibi ion would be eadily de ec ed a maximal Oxphos espi a ion. Simila ly, any p ocess in e e ing wi h inne memb ane in eg i y o inc eased u iliza ion o p o on mo i e o ce o o he pu poses han ADP phospho yla ion would be eadily de ec ed a LEAK/ s a e 4 espi a ion. Thus, he e is no indica ion o any nega i e mi ochond ial e ec o an inc ease in ene gy demand in he cells by acous opho e ic cell so ing. Discussion The acous opho e ic echnology allows non-con ac cell han- dling based on in insic cell p ope ies ha includes size, densi y and comp essibili y which enables he de elopmen o no el label- ee cell so ing s a egies [27]. Howe e , ex ensi e analysis on he e ec s o so ing me hodology a e necessa y no only o a echnique o be es ablished in clinical se ings bu also o assu e ha esea ch esul s do no ge ain ed due o a ha sh so ing echnology. Fo a cell handling o so ing echnologies o be alid o clinical o esea ch pu poses, hey need be non-pe u bing o he cells p ocessed. Hence, in his s udy we ha e pe o med a comp ehensi e s udy wi h di e en cells using a ious echniques aimed a de e mining any cellula al e a ions ha acous opho e ic p ocessing could induce. Ou conclusions in his s udy a e ele an o acous opho e ic so ing o cells in mic o luidic sys ems a low a e in he ange up o 100 ml/min (channel c oss-sec ions 375*150 mm) and wi h e en ion imes in he acous ic s anding wa e o abou 1 second, a p essu es up o 1.28 MPa, a an ac ua ion equency o 2 MHz. The e ec o acous opho e ic p ocessing on mic oglial cells was i s elucida ed. These cells a e he in lamma o y su eillance cells o he b ain simila o mac ophages in o he issues [30]. Mic oglia espond easily o sub le changes in he mic oen i onmen , including exposu e o adia ion [31], nanopa icles [32] and mino empe a u e changes [33]. The mu ine mic oglial cell line BV2 is an in lamma o y cell line widely used in medical esea ch [28]. We i s used his cell line and ound no changes in su i al/ apop osis p oli e a ion o mig a ion a 24 h, 48 h o 7 days a e acous opho e ic p ocessing using bo h manual cy ological analysis and low cy ome y (TMRE). A e demons a ing ha acous o- pho esis did no al e aspec s o iabili y, we u he de e mined i acous opho esis could induce any changes in he in lamma o y s a e o he cells. Mic oglia exposed o acous opho e ic p ocessing we e also s imula ed wi h LPS ac i a ing he pa e n ecogni ion ecep o TLR4. This ecep o is known o ac i a e he MyD88 o TRAM/TRIF pa hways. S imula ion o hese pa hways will igge in acellula pa hways ac i a ing in lamma o y ansc ip- ion ac o s (IRF/NFkB/CREB/AP-1) ha will lead o iNOS ac i a ion and cy okine elease. Ou da a show no al e a ion in iNOS and cy okine esponse ollowing acous opho e ic p ocessing sugges ing ha hese in lamma o y pa hways a e no a ec ed by acous opho esis. In iew o he suscep ible ac i a ion o mic oglial cells by a ious s imulus, ou da a sugges ha acous opho esis is a mild cell-handling echnology. P os a e cance is he mos commonly diagnosed cance in men and has he second highes dea h incidence [34]. Howe e , he Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 6 May 2013 | Volume 8 | Issue 5 | e64233 Figu e 3. In lamma o y esponse o BV2 cells upon LPS challenge ollowing acous opho esis is no changed. A e acous opho e ic p ocessing, BV2 cells we e seeded and s imula ed he nex day wi h LPS o 24 h. We obse ed no al e a ion due o acous opho e ic p ocessing in he exp ession o iNOS (inducible ni ic oxide syn hase)(a,b), he elease o p oin lamma o y cy okines IL-1b(x), IL-12 (d), TNF-a(e) o he an i- Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 7 May 2013 | Volume 8 | Issue 5 | e64233 molecula backg ound o p os a e umo p og ession is cu en ly no comple ely unde s ood. Mos p os a e cance cells equi e and ogen s imula ion o g ow h and p oli e a ion and and ogen dep i a ion he apy is he i s line o ea men o mos p os a e cance pa ien s wi h dissemina ing disease. Al hough e ec i e in educing umo g ow h and size, he ea men selec s o and ogen independen cance cells, esul ing in umo s esis an o and ogen he apy and p og ession in o cas a ion- esis an p os a e cance [35]. Molecula in e oga ion o isola ed ci cula - ing umo cells would p o ide aluable in o ma ion on umo p og ession and me as asis. I is c ucial o u u e cell e alua ion, ha he sepa a ion echnology’s in insic p ope ies do no al e in lamma o y cy okine IL-10 ( ). The g aphs show he esul s om a leas h ee sepa a e expe imen s and he da a a e shown as means 6SD. Signi icance alue P,0.05, ns deno es non-signi ican . doi:10.1371/jou nal.pone.0064233.g003 Figu e 4. P os a e cance cell iabili y is no a ec ed by acous opho esis. Cell iabili y was de e mined a e acous opho esis (0, 10 and 20 V pp ), o ou p os a e cance cell lines: DU 145 (a), PC3 (b), LNCaP (c) and VCaP (d). Le panels show he pe cen age o cell dea h di ec ly a e acous opho esis measu ed by ypan blue exclusion. Righ panels show he cell dea h quan i ied a 24 and 48 h a e acous opho esis by low cy ome y. Cells nega i e o he DNA binding luo och ome 7-Aminoac inmycin (7-AAD) we e de ined as iable. A leas 10,000 cells we e coun ed and he pe cen age o dead cells was de e mined. Cells no subjec ed o acous opho esis we e used as con ol cells. The g aphs show he esul s om a leas h ee sepa a e expe imen s and he da a a e shown as means 6SD. Signi icance alue P,0.05, ns deno es non-signi ican . doi:10.1371/jou nal.pone.0064233.g004 Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 8 May 2013 | Volume 8 | Issue 5 | e64233 Figu e 5. Acous ic cell sepa a ion in a mic ochannel does no al e PSA sec e ion by p os a e cance cells. The and ogen ecep o (AR) exp essing cell lines LNCaP and VCaP we e used o e alua e he impac o acous opho esis on PSA sec e ion. A e acous opho esis un a 0, 10 and 20 V pp , he sec e ion o PSA was measu ed in he absence o p esence o he AR ligand R1881 (1 nM o 24 h) in he LNCaP cell line (a) and in he VCaP cell line (b). Cells no p ocessed h ough he chip we e used as con ol cells. The g aphs show he esul s om h ee sepa a e expe imen s and he da a a e shown as means 6SD. Signi icance alue P,0.05, ns deno es non-signi ican . doi:10.1371/jou nal.pone.0064233.g005 Figu e 6. Mi ochond ial espi a o y unc ion in human leukocy es and h ombocy es a e no al e ed ollowing acous opho esis. Leukocy es and h ombocy es we e passed h ough he acous opho esis chip un a 0, 10 and 20 V pp . Maximal espi a ion du ing using bo h complex I- and complex II-linked subs a es o h ombocy es (a) and leuko cy es (b) was unal e ed a e acous opho esis, suppo ing ha acous opho esis does no a ec me abolic pa hways impo an o espi a ion. The emaining espi a o y ac i i y ollowing inhibi ion o ATP syn hase wi h oligomycin, so called Leak o s a e 4 espi a ion, was also una ec ed by acous opho esis, in h ombocy es (c) and leukocy es (d). This da a con i m no e ec o acous opho esis on inne mi ochond ial memb ane in eg i y o changed u iliza ion o p o on mo i e o ce o o he pu poses han ADP phospho yla ion. Unp ocessed cells we e used as con ol cells. The g aphs show he esul s om h ee sepa a e expe imen s and he da a a e shown as means 6SD. Signi icance alue P,0.05, ns deno es non-signi ican . doi:10.1371/jou nal.pone.0064233.g006 Impac o Acous opho esis on Cells PLOS ONE | www.plosone.o g 9 May 2013 | Volume 8 | Issue 5 | e64233