1374 OPTICS LETTERS / Vol. 35, No. 9 / May 1, 2010

Investigation of portable in situ fluorescenceoptical detection for microfluidic 3D

cell culture assays

Jong-ryul Choi,1 Jong Hwan Sung,2 Michael Louis Shuler,2,3 and Donghyun Kim1,*1School of Electrical and Electronic Engineering, Yonsei University, 120-749 Seoul, Korea

2Department of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA3Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853, USA

*Corresponding author: kimd@yonsei.ac.kr

Received January 11, 2010; revised March 9, 2010; accepted March 15, 2010;posted March 25, 2010 (Doc. ID 122463); published April 23, 2010

A portable fluorescence optical detection system was developed to demonstrate real-time in situ analysis ofcells that are three-dimensionally cultured in an extracellular matrix under microfluidic environment. Thesystem was designed to provide a large field of view in the lateral plane to average out cellular processes inan axial layer and simultaneously diffraction-limited axial resolution. In this proof-of-concept study, the de-tection system was applied to quantitative analyses of short-term measurements of cell staining and cellcytotoxicity and long-term monitoring of a cell-invasion assay. For assays, colon cancer cells were cultured ina Matrigel or alginate matrix. The measured data were largely consistent with predicted results and re-vealed quantitatively cell dynamics specific to 3D cell cultures. The detection system has a potential as asingle package to investigate 3D cultures in a microfluidic system. © 2010 Optical Society of America

OCIS codes: 120.3890, 170.1530, 280.4788.

Microfluidic cell culture systems have drawn tremen-dous interests for cell-based studies, as they suggestthe potential to mimic animal physiology in vitro andcan be used for testing cytotoxicity of biochemicalsprior to animal tests in the drug discovery process.Microfluidic cell cultures can provide an environmentthat is closer to in vivo dynamics compared to con-ventional cell cultures, since many culture param-eters can be adjusted to study specific issues rangingfrom cell-to-cell interactions to co-culture responsesto drug chemicals. Most of these microfluidic cell cul-ture systems are based on two-dimensional (2D) cul-tures in a monolayer formation of cells. However, cellbehavior in a 2D culture can differ significantly fromthat of cells in tissue matrix. For this reason, a recenttrend has been to develop three-dimensionally cul-tures in microfluidic cell culture devices to bettersimulate an in vivo environment [1–4]. For example,the hepatoma cell line (HepG2/C3A), human hepato-cellular carcinoma, lacks authentic enzymatic activ-ity when cultured in conventional 2D culture buthigher enzymatic activity of HepG2/C3A wasreported in a 3D Matrigel matrix in a microfluidicdevice [5].

One of the key issues to be addressed in cell-basedstudies using 3D cultures is a need for a noninvasiveanalytical method to determine precisely the dynam-ics in the culture. Among various approaches, opticalmethods, especially fluorescence detection, have beengenerally employed for the analysis of cellularchanges in various cell-based studies including 2Dmicrofluidic cell culture systems [6,7]. For 3D culturesystems, different strategies have been employed.For instance, aggregation of 3D cultured mammaliancells in a rotating bioreactor was measured by detect-ing the light transmitted after being partially

absorbed by aggregated cells [8]. Optical coherence

0146-9592/10/091374-3/$15.00 ©

tomography (OCT) has been used to study cell dy-namics in 3D tissue models [9]. However, OCT suf-fers from low resolution typically on the order of afew micrometers and, more importantly, does not pro-vide functional parameters.

In this Letter, we take a more direct approach toinvestigate 3D cell culture dynamics by developing aportable 3D in situ fluorescence optical detection sys-tem (3D-ISFODS). The 3D-ISFODS measures thefluorescence signal directly from 3D cultured cellsthat are stained by fluorescence indicators and em-bedded within an extracellular matrix (ECM) forquantitative analysis. There are several advantagesof the system over traditional optical sectioningmethods, for example, standard confocal microscopes.First of all, we are mostly interested in the changesof dynamics in the axial direction of a 3D culture. Inthe lateral plane, only average cell behavior is of in-terest. This requires an extremely short depth of fo-cus axially and yet a focus large enough to observemany cells in a lateral field of view. Also, 3D-ISFODSis compact and robust to allow continuous real-timemeasurements in an incubation environment. Thereal-time detection by the 3D-ISFODS offers a greatadvantage over conventional analytical instrumentssuch as a fluorescence plate reader or a microscope,which can only take end-point measurements. On theother hand, a commercial microincubator may beused in combination with confocal microscopy. In thiscase, however, compatibility with custom-made mi-crofluidic cell culture systems is limited. For in-stance, management of tubes that interconnect to ex-ternal pumps can be cumbersome and interrupt thewhole fluidic system by affecting cell viability and in-creasing the chances of contamination. One may in-tegrate a MEMS-driven 3D scanning confocal lens di-

rectly onto cell-based assays [10], which suffers from

2010 Optical Society of America

May 1, 2010 / Vol. 35, No. 9 / OPTICS LETTERS 1375

a short scan distance that can be axially scanned aswell as the difficulty associated with fabrication andmultiple uses.

We address these concerns by implementing the3D-ISFODS that scans a 3D culture only axially in astandard incubator, while cells are cultured in a 3Dconstruct maintained under a regulated microfluidicenvironment. The 3D-ISFODS takes advantage ofthe compact and portable nature for real-time moni-toring in situ. Furthermore, design issues and re-quirements for optical detection have been identifiedand optimized to investigate microfluidic 3D cell cul-tures. The feasibility is demonstrated with cell stain-ing, cell cytotoxicity, and cell-invasion assays. In par-ticular, cell invasion represents a process by whichcancer cells actively enter into a tissue, often by se-creting enzymes to digest away matrix that other-wise prevents the entry of cancer cells. Cell invasioncombined with staining and cytotoxicity is an idealsubject of study, as the invasion of cancer cells in 3Dculture environments has only recently begun to beinvestigated.

The schematic of the 3D-ISFODS is shown in Fig.1. The system employs a fiber coupled laser diodewith 532 nm peak wavelength (Shanghai LaserTech., China) and a photomultiplier tube (PMT, Oriel,Stratford, Conn., USA). For the elimination of out-of-focus signal, a pinhole aperture ��=500 �m� wasplaced in front of the PMT. No pinhole was used onthe excitation side to produce a large field of view inthe lateral plane. The excitation light is focused byan objective lens (NA=0.55, 50X, working distance=13 mm, 378–805–3, M PLAN APO, Mitutoyo Corp.,Japan) with minimal field curvature. The axial scan-ning was motorized in a step of 10 �m. The field ofview in the lateral plane and diffraction-limited axialdepth of field produced by the system were theoreti-cally estimated as 480 and 1.8 �m, respectively. Ini-tially, the 3D-ISFODS was calibrated with a stan-dard confocal microscope (Bio-Rad MRC-600, Bio-Rad Laboratories, Hercules, CA, USA) for 1-mm-thick alginate matrix embedded with fluorescentbeads (�=10 �m, F-8836, Invitrogen) and stainedcells (data not shown).

HCT-116, a colon cancer cell line, was cultured inT-flasks in McCoy’s 5a medium with 10% FBS with

Fig. 1. (Color online) (a) Optical setup for the used 3D mi-crofluidic assay (OB, objective; P, pinhole; F, filter; and DM,dichroic mirror). Also shown are the schematics of the 3Dcell culture assays for studies of (b) cell staining, (c) cell cy-

totoxicity, and (d) cell invasion (not scaled).

37°C temperature and 5% CO2. For cell staining, cellcytotoxicity, and cell-invasion assays, a simple cellculture device was constructed by attaching a single,circular chamber to a glass slide and a 1-mm-thicksilicone gasket, as shown in Figs. 1(b)–1(d). In thecell staining assay, cell staining solution, 1 �M Cell-tracker Orange CMRA (Invitrogen, Carlsbad, Calif.)was applied on 3D alginate matrix. In the cell cyto-toxicity assay, 5% Triton X-100 solution in DPBS wasapplied on the 3D matrix 20 min after cell stainingsolution. For cell invasion, a Boyden chamber assaywas used to investigate cell migration through 3D hy-drogel matrix.

Figure 2 presents the measured axial distributionof fluorescence intensity when cell-staining solutiondiffuses down through 3D alginate-cell matrix. Cellsin the top layers of the 3D matrix are exposed soonerto the staining solution than those in the bottom lay-ers. The diffusion of the staining solution makes thefluorescence intensity increase over time, and thefluorescent intensity is almost always higher in anupper layer until the solution is sufficiently diffused.Note that the diffusion characteristics look differentdepending on the axial location of cells. In the upperlayers (z=0.35 and 0.43 mm: z is the depth from sur-face), fluorescent intensity increases quickly, whereasit increases slowly in the lower (z=0.62 and 0.75mm). This is well explained with a transport modelas shown in Fig. 2. Here, the solid curves representtheoretical increase in the fluorescence, which is ingood agreement with measured fluorescence, exceptnear the bottom of the 3D matrix �z=0.75 mm�. Thedisparity in the case of z=0.75 mm or larger is due toa time delay associated with the cellular uptake offluorescence not considered in the model and cell den-sity variation. The inset shows the relationship be-tween axial location and staining time when half ofthe cells in a layer become stained. The relationshipis almost linear �R=0.94335� and provides an aver-age axial staining speed as 57 �m/min.

Figure 3 shows the axial distribution of fluores-cence intensity measured over 150 min for cell cyto-toxicity assay. An initial increase in fluorescence in-tensity due to the cell staining solution is followed bya decrease as a result of Triton X-100 solution after

Fig. 2. (Color online) Fluorescent intensity measured bythe 3D-ISFODS for cell-stained solution (celltracker or-ange) applied on the 3D alginate-cell matrix at t=0 min.Solid curves represent calculated trends based on a trans-

−10 2

port model with diffusivity D=2.4�10 m /s.

1376 OPTICS LETTERS / Vol. 35, No. 9 / May 1, 2010

20 min. The effect of Triton X-100 solution kicks insooner in the middle so that peak fluorescence isachieved approximately at 28th minute, while it islater at 46th minute in the lower layer �z=0.82 mm�.During this period, the effect of the cytotoxic solutionis mixed with the diffusion of staining solution. Thestaining effect is stronger in the middle layer so thatoverall fluorescence intensity can increase slightly,which appears as a kink. Soon, the effect of cytotoxicsolution dominates and cellular fluorescence de-creases exponentially. The cytotoxicity model agreeswell with the experimental data: R=0.9058 for z=0.68 mm and R=0.9790 for z=0.82 mm.

Using a long-term cell-based assay, we comparedthe cell invasion in Matrigel and alginate hydrogel.For quantitative evaluation, relative axial movement��z� was defined as �z= ��zfz /�fz�t− ��zfz /�fz�t=0,where summations are performed from z=z0 to zt. z0and zt denote an initial and a final axial point respec-tively. fz represents the fluorescence intensity mea-sured at the axial points. The time when an experi-ment is over is t. Overall, �z represents an averageaxial distance over which cells have traveled overtime t. Figure 4 shows �z measured with HCT-116cells in the cell-invasion assay. In a Matrigel-basedcell-invasion assay, cells moved to chemo-attractant(McCoy’s 5 media with 10% FBS) by 150 �m during aspan of 24 h. Cell invasion was found to follow expo-nential axial movement with R=0.9953. Cell inva-sion due to chemo-attraction was initially linear atthe velocity of 0.83 �m/min, which is consistent withthe literature [9]. The invasion rate decreased mono-tonically as chemo-attractors became homogeneouslydistributed [11], and cells eventually stopped inva-sion. In contrast, �z=15 �m in the direction awayfrom chemo-attractor for alginate-based assays, pos-sibly due to expansion of alginate matrix. The dras-tically different behavior of the cells in Matrigel andalginate stems mainly from the fact that Matrigel isa naturally derived basement membrane matrix. Incontrast, alginate does not contain components that

Fig. 3. (Color online) Fluorescent intensity measured withcell cytotoxicity solution applied at t=20 min. Diffusivitywas assumed as D=2.4�10−10 m2/s.

can be digested by cellular proteases and, thus, much

more resistant to cell-invasion process than Matrigel.In summary, we describe the development of a com-

pact and portable in situ confocal fluorescence detec-tion system that allows real-time monitoring of cellu-lar changes in 3D microfluidic cell cultures. Forproof-of-concept, the system was applied to short-term assays of cell staining and cell cytotoxicity andalso a long-term measurement using a cell-invasionassay. This novel system should be advantageous inmonitoring cellular process in 3D gels enhancingin vitro studies on cell physiology.

The authors thank the support by theKorea Science and Engineering Foundation throughNational Core Research Center for NanomedicalTechnology (grants R15-2004-024-00000-0,M10755020003-08N5502-00310, and R01-2007-000-20821-0). This work was also sponsored by the“System IC 2010” project of Ministry of KnowledgeEconomy and partly by National Science Foundation(USA) and Army Corps of Engineers (CERL,W9132T-07).

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