A 3-D MICROFLUIDIC COMBINATORIAL CELL CULTURE ARRAYM. C. Liu and Y C. Tai

California Institute of Technology, Pasadena, CA, USA

ABSTRACTWe present the development of a three-dimensional

(3-D), on-glass combinatorial cell culture array chipfeatured with integrated three-input, eight-outputcombinatorial mixers and cell culture chambers. Thedevice is designed to simultaneously screen for the effectsof multiple compounds and concentrations on culturedcells. Experimentally, we first developed a precise way tocharacterize the combined compound concentrationprofile at each chamber with a fluorescence method. Wethen successfully demonstrated the functionality of thecell assay by culturing neuron cells on this device andscreening the ability of three chemicals to attenuate celldeath caused by cytotoxic hydrogen peroxide. Based onthe same technology, the number of inputs and outputs ofthe combinatorial mixer can be scaled-up to construct lab-on-chip devices for performing high-throughput cell-based assay and highly parallel and combinatorialchemical or biochemical reactions with reduced labors,reagents, and time.

INTRODUCTIONMicrofluidic devices have the potential to become

inexpensive platforms for high-throughput cell-basedscreenings, and recently, many devices with cell culturingand assaying abilities have been developed [1,2].However, most of the cell culturing devices can onlyscreen for a single compound at once [3,4]. Amicrofluidic device that can screen for the combinatorialeffect of multiple compounds on cells is valuable becausecells are sustained in complex environments and cell fatesare dictated by the integration of numerous extracellularsignals. For example, combinatorial effects are evident incellular processes such as gene regulation [5]. Also,certain combinations of anti-tumor compounds caninteract in a synergistic manner on cancer cells [6]. In ourprevious works, we have demonstrated the method tomonolithically fabricate three-dimensional (3-D)microfluidic networks on silicon substrate, and based onthis fabrication technology, we have constructed a cellculture device with an integrated combinatorial mixer [7].In this work, we extended such fabrication method toconstruct 3-D microfluidic networks on glass substrateand demonstrated a cytotoxic assay using this chip.Although the technology can be extended for morecompounds, this work demonstrated a device for threecompounds as a feasibility study.

DESIGNFigure 1 shows the layout of the 1.7 cm by 1.7 cm

chip. Compounds A, B, and C are flown into eightchambers with different combinations and concentrationsdepending on the flow resistance of the channels. Theoverpass structure allows one microfluidic stream tocrossover another, and a control channel that receivesnone of the three inputs is included. The concentration ofeach compound inside the chamber is determined by thefluidic resistance of the channels. Assuming that thechannel width is much larger than the channel height, the

resistance can be modeled using the simplified formulafor rectangular channel [8]:

R12 uLwh3

R is the resistance, Ft is the fluid viscosity, and L, w, and hare the length, width and height of the channel,respectively. Chambers receiving multiple compoundsare designed to have equal dilution of the inputcompounds.

Figure 1. Design Layout ofDevice

FABRICATIONThe fabrication process for constructing 3-D

microfluidic networks is shown in Figure 2. Briefly, a firstthin adhesion layer of parylene C was deposited (3 rim) onsoda lime glass wafer. The first sacrificial photoresistlayer was spin-coated (14 rim) and patterned to define thefirst-level channels. A second layer of parylene C (10rim) was then deposited to cover the sacrificialphotoresist, forming the first-level channels. Parylene Cwas patterned using oxygen plasma so the areas where theoverpass structures would be joined were exposed. Thisparylene patterning also opened the area where the mixerand the culture chamber would be connected. A secondsacrificial photoresist was spin-coated (25 rim) andpatterned to define the overpass structure and the culturechamber. This second sacrificial photoresist covered theareas that were etched open, and the overpasses spannedseveral of the first-level microfluidic channels. A thirdlayer of parylene C (2 rim) was deposited. The wholechip was planarized with thick SU8 (90Ftm) and paryleneC was patterned to define the access holes. Finally, thechips were soaked in acetone to dissolve the sacrificialphotoresist.

978-1-4244-2978-3/09/$25.00 ©2009 IEEE

overpass 0

+3A+33%CB *Outlet

Inlets f SOX

ControlO%C

Combinatorial mixer cell culture chambers

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(a) ls' parylene C deposition on soda lime glass wafer (3 pm)lst sacrificial photoresist coating and patterning (14 pm)

(b) 2nd parylene C deposition and patterning (10 pm)

(c) 2nd sacrificial photoresist coating and pattering (25 pm)3rd parylene C deposition (2 pm)

during brain ischemia and reperfusion injury and variousneuropathological conditions. The device operation forthe cell assay experiment is shown in Figure 5.Suspension of rat neuronal cell (B35 rat neuroblastomacells) was injected, and cells were efficiently collected byand grown on the membrane. Maintaining cells on themembrane and separating them from the fluid deliverychannel also mimics the in-vivo tissue environment.Following three hours of incubation after cell seeding,each compound was mixed in media containing 1.5 mMhydrogen peroxide and injected at 5 FL/min for 30minutes, followed by incubation with normal media forthree hours. Live/dead stains (Calcein AM/propidiumiodide) were injected, and cytotoxicity was determined bythe ratio of dead cells to total cells.

(d) SU8 coating and pattering (90 pm)Patterning Parylene CSacrifirial nhntnrsiststrinnina in f

glass = Parylene C - Photoresist =SU8Figure 2. Fabrication process flow. The overpass allowsone fluid stream (-4 ) to crossover another ( 0 ), asshown in (d).

EXPERIMENTALFigure 3 shows the packaging method. A transparent

polyester membrane with 0.4 Ftm pores was cut out fromCorning® Transwell® cell culture insert, and a rectangularpiece of the membrane was placed on top of the chambers.A piece of PDMS (Polydimethylsiloxane) with 100 Ftmheight microfluidic networks was fabricated by replicatemolding from a SU8 mold. The PDMS was aligned ontothe chip and the assembly was clamped together byacrylics. Figure 4 shows the layout of the PDMS piece,which has punched holes that match the locations of theinlet and outlet holes on the parylene microfluidic chip.The PDMS piece also has a cell-seeding inlet and a one-to-eight manifold that distributes cells into eightchambers, which align to the location of the culturechambers of the parylene microfluidic chip. With themembrane, the culture chamber turns into a two-levelconfiguration, and the cell culture level is separated fromthe fluid delivery level. Syringes were connected withTeflon tubes, which were plugged into the holes of thepiece of PDMS.

To characterize the concentration of the three inputcompounds inside each chamber, we first injected a redfluorescence solution (Sulforhodamine 101) into input A,while injecting DI water into input B, input C and control,and the fluorescence image of each chamber wascaptured. Then, the fluorescence intensity was measuredusing ImageJ. Fluorescence solution was then switchedto input B, while injecting DI water into other inlets. Thesame procedure was repeated for input C. The injectionwas controlled by syringe pump and was set at 5 UL/minfor duration of 20 minutes.

This microfluidic device was used to assay for theability of 1,5-dihydroxyisoquinoline (ISO), deferoxamine(DFO), and 3-aminobenzoic acid (ABA) to alleviatecytotoxicity of hydrogen peroxide, which is generated

Figure 3. Device packaging. (a) Device packagingschematic. (b) Packaged device with various foodcoloring solutions injected.

Figure 4. Layout of the PDMS piece.

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(a) Combinatorial eliminated the need for coating the channels with cellattachment factors such as collagen or fibronectin. Thedevice was used to assay for effect of three compounds onreducing the cytotoxicity of hydrogen peroxide. Figure 7shows the result of the cytotoxicity experiment, andFigure 8 shows the arrays of cells after the cells werestained by live/dead stains. 1,5-dihydroxyisoquinolineand deferoxamine showed cytoprotective effect againsthydrogen peroxide. 1,5-dihydroxyisoquinoline reducescell death by inhibiting Poly(ADP-Ribose) polymerase,whose over-production in response to hydrogen peroxidecan cause cell death [9]. Deferoxamine reduces cell deathby decreasing the conversion of hydrogen peroxide tohydroxyl radicals that damage DNA. 3-aminobenzoicacid, structurally similar to 1,5-dihydroxyisoquinoline,showed minimal effect on hydrogen peroxide cytotoxicity.In general, this device successfully demonstrated that,even with just three inputs, eight distinct cytotoxicexperiments were done with different combinatorialcompounds using a single chip.

(b)

Figure 5. Device operation for cell-based assay. (a)Cell seeding by injecting cell suspension into the cell-seeding inlet. The one-to-eight manifold on the PDMSsplits the cell suspension and cells flow into thechamber area where they are trapped by the membrane.(b) Injecting various solutions through thecombinatorial mixer inlets for cell assay.

RESULTS AND DISCUSSIONFigure 6 shows the concentration of the three input

compounds at each chamber. The combinatorial mixerwas able to generate the correct combinations of thecompound solutions. The concentration of thecompounds can deviate from the design value because ofdimensional inaccuracy in processing.

100 A

ii 80-

60 1

0

ABC ABC ABC ABC ABC ABC ABC ABC

1 2 3 4 5 6 7 8Chamber number

Figure 6. Concentration of the three input compounds ateach chamber.

Integrating a porous membrane into the devicepackaging greatly facilitated the on-chip cell assayexperiment. Cell seeding was done without high cell lossas in other microfluidic culture devices that load cells byflowing them through channels. This setup also

90

80

70

>60.

.0 50x0._40

40

o 30.

20

10

Iso * + + +

DFO - + + + - + -

ABA- - - + + + +

Figure 7. Cytotoxicity as a result of each treatment. Theeffects of three compounds, 1,5-dihydroxyisoquinoline(ISO), deferoxamine (DFO), and 3-aminobenzoic acid(ABA) on alleviating cell death caused by hydrogenperoxide were tested. Each compound was mixed inmedia containing 1.5 mM hydrogen peroxide.

CONCLUSIONIn this work, we demonstrated the fabrication of a cell

culture chip on glass with integrated combinatorial mixer,and showed a cell-based screening of differentcombinations of compounds. The combinatorial mixercorrectly delivered the distinct combinations of inputcompounds into the chambers and the concentration ofeach input compound at the chamber was measured. Aporous polyester membrane was incorporated into theculture chamber, and a cytotoxicity experiment was doneusing this chip. By scaling-up this technology, chip withhigh-density cell array and integrated high-inputcombinatorial mixer can be constructed. Such chip willenable inexpensive high-throughput cell-based assay, andsignificantly benefits research in a spectrum of fieldsincluding drug screening and systems biology.

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-300 pm

Figure 8. Cells in each chamber after live(green)/dead (red) staining. Some early apoptotic cellsare stained by both dyes.

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