SING L E CELL SUSPE N SION C U LTUR E USING P OLYH E …1% penici cells were ob y of Michiga n 10%...
Transcript of SING L E CELL SUSPE N SION C U LTUR E USING P OLYH E …1% penici cells were ob y of Michiga n 10%...
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
r-
g of d e
or l-er
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