SINGL

1D

ABSTRACT

In this wocell resolutioncell loadings to achieve a sof skov3 cellstraced for mo

KEYWORD

INTRODUC Cell adhesitachment leadare required tand the abilitythousands of ever, cell aggthe media excwe propose a ture over time In the procmany sub-popThe ability toconventional sphere-formatextensive stud Microfluidbeen limited tcrochambers quality of impolyHEMA. Cand cultured fto integrate po

Figure(a) the

LE CELL FOR

YDept. of Electr

2Dep

T ork, we reportn. The proposto an exact po

single cell suss and sphere fre than ten da

DS: Suspension

CTION ion to the extrds to anoikis, to detach fromy to evade apocells were loa

gregation, whichange, some single cell an

e for viability cess of metastapulations in co form a sphemammosphe

tion platform dy of differentic technologieto adherent cuin our previou

mage. In this wCells can be lfor more thanolyHEMA in

e 1. Schematie entire device

SUSPENR ANOIKI

Yu-Chih Chrical Engineerpt. of Biomedi

t a single cell sed chip uses aosition in the spension cultuformation of Says.

n Culture, Ano

racellular matwhich trigger

m the ECM anoptosis in suspaded in the suich inevitablycells can be

noikis chip thatest without inasis, detachedcancer cells, iere in suspensre assays, whcan easily tra

t sub-populaties allow singleulture. We deus work [8]; h

work, we repoloaded in eachn ten days in c

microfluidics

ic diagram ofe and (b) an e

NSION CUIS ASSAY

hen1, Patrickring and Compical Engineeri

suspension cha hydrodynamarray of micr

ure in each miSUM159 and

oikis Assay, S

trix (ECM) is rs the cell deatnd be viable ipension markspension cultu

y happens, maeasily lost inat can exactly nterruption of

d cells should t is believed sion environmhich load a face each singlion behaviors.e cell analysemonstrated su

however, the port a chip withh microchambontinuous me and the first m

f a single cellenlarged micro

ULTUREY AND SPk Ingram2, X

mputer Scienceing, University

hip which canmic capturing srochambers. Picrochamber. MCF-7 cells;

Sphere Culture

essential in mth by apoptosin circulations the malignanure dish and c

ay skew survivpipetting opeload single ce

f cell positionibe able to forthat cancer st

ment (mammofew thousand le cell and mo. s for heterogeuspension cultpatterned surfah the surface ber at single ceedia perfusionmicrofluidic p

l suspension co-chamber.

E USING PPHERE FXia Lou1 ande, University ofy of Michigan

n perform anoscheme based

PolyHEMA coWe performe

; and the beha

e, Single Cell

maintaining cesis [1]. Howev

[2]. Anoikis ncy of cancer

cell viability wval rates in theration; as a reell in a chambing. rm a new colotem-like cellsosphere assay)

cells in a suonitor its sphe

eneous populature by integrace requires emodified usinell resolution . To the best oplatform for si

culture chip:

POLYHEFORMATd Euisik Yoof Michigan, An, Ann Arbor, M

ikis assay andd on differenceoating is integed two sets ofavior of each

, polyHEMA

ellular homeosver, in the metserves as a nacells [3]. In c

was monitoredhe conventionaesult the lengtber and provid

ony from met (CSC) are cr) indicates steuspension dishere formation

ation cell grourating hydrophxpensive DRIng Poly(2-hydin the same w

of our knowleingle cell anoi

Figure 2. Fsteps.

EMA COATION on1,2

Ann Arbor, MIMI, USA

d sphere forme in flow resisgrated with mif experiments:single cell wa

coating

stasis. Disrupttastasis procesatural barrier conventional ad after a few dal dish assaysth of assay is de continuous

astatic cancerritical in tumoemness [6-7].h, the proposprocess; thus

ups, but most phobic surface IE tools and ddroxyethyl meway reported pedge, this is thikis assays.

Fabrication p

ATING

I, USA

mation at singlestance to guideicrofabrication: anoikis assayas successfully

tion of cell atss, tumor cellfor metastasi

anoikis assaysdays [4]. Hows. Also, duringlimited. Hereperfusion cul

r cells. Amongorigenesis [5] Compared to

sed single-cels, allowing the

platforms haveinside the mi

deteriorates theethacrylate) opreviously [8]he first attemp

process

e e n y y

t-s s

s, w-

g e, l-

g ]. o ll e

e i-e

or ], pt

16th International Conference on Miniaturized Systems for Chemistry and Life Sciences

October 28 - November 1, 2012, Okinawa, Japan978-0-9798064-5-2/μTAS 2012/$20©12CBMS-0001 106

DESIGN AN Figure 1 shoptimal ratio suspension cu60mg/mL of psecond), and cured PDMS overnight to m EXPERIME Skov3 cellLab (Universin RPMI lin/streptomytained from DMI, USA) an1% penicillinused in anoiwere grown mented with and other supcell loading, cells in solutiinjected to ththe inlet and thus cells canminutes, the fresh media fo

RESULTS AND DICUSSION Figure shows cells ctured in the fricated devwith a singcell capture rover 90%. Tcapture mechnism outpforms the othprevious trifor miniaturizanoikis assa[10]. Two typof cells loaded to repsent heterogneous cgroups. As a is believed toand adherent apoptosis. Thconfirming th Figure 6 shSUM159 celling the 64-mi3.14% of MCperiments usiIn addition to

ND FABRICAhows the scheof flow rates

ulture, the popolyHEMA inthen bonded tis used as a

minimize any

NTAL s were provid

sity of Michigwith 10% cin. SUM159

Dr. Wicha’s Lnd cultured in n/streptomycinikis assays. Fin serum-freeB27, 20 ng/m

pplements suggtrypsin/EDTA

ion, which is e inlet. Liquioutlet can g

n be captured cell solution

for the assay.

IS-

3 ap-

fab-vice gle-rate The ha-

per-her ials zed ays pes are

pre-ge-cell proof of conc

o enhance the culture. The

he live/dead (ghe enhanced suhows the sphls in the devicircowell devicCF-7 cells in ting the 64-miro the quantitat

Figurecultureday 1 aand (d)

ATION ematic diagram in two micro

olyHEMA, whn 95% ethanoltogether. Sincglue to fastenresidue stress

ded from Dr. gan, MI, USA

FBS and 9 and MCF-7 Lab (Universit

DMEM withn. Same cultFor sphere foe DMEM-F12

ml bFGF, 20 ngested by Dr. A is used to diluted to 10

id height diffegenerate a gra

hydrodynamiin the inlet

cept, skov3 (ovcell survival attached skovgreen/red) staiurvival rate is here formationes formed sph

ces. MCF-7 (bthe devices forcowell devicive analysis o

e 4. Skov3 celle: (a, b) adheand day 4, (c)

d) suspended d

m of a single cofluidic paths hich can inhibl. Both the PDce polyHEMAn the device. Ts as illustrated

BuckanovichA) and culture

1% penicicells were ob

ty of Michiganh 10% FBS anture media arormation, cel2 (1:1) suppleng/ml EGF, 2%

Wicha. Beforre-suspend th

06 cells/mL anerence betweeavity flow, anically. After 1is replaced b

varian cancerin suspension

v3 cells prolifeining is used tobserved whe

n of SUM159heres after tenbreast adenocarmed sphere aes. The prelim

of sphere form

ls in adherenterent cells in ) suspended li

dead cell on da

cell suspensiofor hydro-dy

bit cell adhesiDMS layer andA absorbs watThe PDMS g

d in Figure 2.

’s ed il-b-n,

nd re ls e-% re he nd en nd 10 by

) cells are usen culture [11]. ferated during to monitor ceen cells are ex9 (breast carcn days. The vaarcinoma) cellafter ten daysminary result

mation rate, qu

t and suspensa micro-well ive cell on dayay 4.

on culture chipynamic captureion, is coatedd substrate areer and swells,

glue is then cu

ed for anoikis Figure 4 shothe four daysll viability. Fi

xposed to 50ncinoma) cellsariation was 6ls were also st, and the varimatches well

ualitative obse

ion on

ay 4

Figure for 6 dafor susp

p. Flow channe of single ce

d on substratee treated by ox, bonding streured at a lowe

assays. Hepaws the compas culture, whiigure 5 summg/mL HGF. from a singl

.84% from foutudied in the sation was 2.4with that in t

ervation can b

5. Single cell ays in polyHEpension cultur

nels are designells by gravitye by slow evaxygen plasma ength may deger temperature

atocyte growtharison of susple the suspend

marizes anoikis

e cell in ten ur repeated exsuspension cul6% from eighthe suspensione also perform

Figure 3skov3 cellsSUM159 (whole deand (b) schamber p

anoikis assayEMA treated mre.

ned to give any flow [9]. Foaporation from

(300 Watt, 60grade. The une (65 degrees

h factor (HGFpension cultureded cells wens experiments

days. 72% oxperiments uslture platformht repeated exn dish culture

med by contin

3. Capturedls (green) and(red) cells: (a)evice picturesingle micro

picture.

y of skov3 cellsmicrochambers

n or m 0

n-s)

) e

nt s,

of s-

m. x-e. n-

d d

a) e -

ls rs

107

uously monitother studied b

CONCLUSIO We successpolyHEMA cSUM159 andfrom the captstudy of canc

ACKNOWL This work Chemical Genliance AwardCenter at the

REFERENC[1] P. Mehl[2] N. Sethi[3] D. Hana[4] N. T. W[5] P. B. Gu[6] G. Dont[7] J. Visva[8] P. Ingram[9] J. Chung[10] A. P. Ra[11] S. Watan CONTACT *Y.C. Chen, t

oring the procby labeling the

Figpenday

ON sfully develop

coating inside d MCF-7 cellstured single ceer metastasis

LEDGEMENTwas supporte

nomics at the d from KAUST

University of

CES en and A. Puii and Y. Kangahan, and R. A

Woods, H. Yamupta, C. L. Chtu, W. M. Abdader & G. Lindm, M. Im, S. Mg, Y.-J. Kim aago et al., Cytonabe, T. Kish

tel: +1-734-56

cess of spheree cells after lo

gure 6. The dension culture y 0, (b) day 2,

ped a microfluthe microwel

s using the faells for more tin multiple sta

TS ed in part by Life Sciences

T. The cells wf Michigan.

isieux, Natureg, Nature Rev. A. Weinberg, Cmaguchi, F. Y.haffer and R. Adallah, J. M. Fdeman, NatureMcDermott, Mand E. Yoon, Aotechnology, imoto, and O.

65-9976; yuch

formation. Thading and con

evelopment of inside a poly(c) day 4, (d)

uidic platformll array. We d

abricated chipthan ten daysages.

the Thermo Fs Institute at thwere provided

e Rev. Cancer,Cancer, 11, 7

Cell, 144, 646. Lee, et al, CaA. Weinberg, NFoley and M. Se Reviews CanM. Wicha, andAppl. Phys. Le56, 81-90 (20. Yokosuka, P

hchen@umich

he behavior dntinuously trac

f a SUM159 spyHEMA surfaday 6, (e) day

m for single cedemonstrated a. The behavio. The propose

Fisher Scientihe University

d by Dr. Buck

, 6, 449-458 (2735-748 (20116–674 (2011).ancer Res., 67Nat. Med., 15S. Wicha, Genncer, 8, 755–7d E. Yoon, Miett, 98(12), 3708).

Pancreas, 40(4

h.edu

difference betwcing them.

phere from a ace-coated miy 8 and (f) day

ll anoikis assaanoikis assaysor of heterogeed single-cell

fic Screeningof Michigan,

kanovich and D

2006). 1).

7 (22), 10744–(9), 1010–101

nes Dev., 17, 1768 (2008). icroTAS, 1539701 (2011).

4), 608-614 (2

ween different

single cell in icro-chamber:y 10.

ay and sphere s for skov3 ceeneous cells csuspension-cu

Technology , and in part bDr. Wicha at

–10752 (2007)12 (2009). 1253–1270 (2

9-1541 (2011)

2011).

t sub-populati

sus-: (a)

formation byells and spherecould be succeulture chip can

Grant under by Academic E

the Compreh

).

003).

).

ion can be fur

y incorporatinge formation oessfully tracedn facilitate the

the Center foExcellence Alhensive Cance

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108