Development of a Microfluidic Device for Blood Vessel

Realization and Characterization

Barbara Sofia Fialho Simoes

Instituto Superior Tecnico, Lisboa, Portugal

November 2016

Abstract

The circulatory system is responsible for the flow of blood, nutrients, biochemicals, oxygen andother gases, to and from the cells. The circulatory system works as a powerful mean to fight diseases.However, through angiogenesis, it can also be related to cancer development. Currently, microfluidicsystems and tissue engineering have merged towards the study of blood vessels in vitro. Here, the studyof a microfluidic system for blood vessel realization and characterization is presented. In particular,with different designs and different approaches. Viscous finger patterning and passive pumping wereused to create lumens through collagen type I and human umbilical vein endothelial cells (HUVECs)were seeded in the lumen. Successfully formed lumens were characterized in terms of dimensions.Keywords: Microfluidic Systems, Passive Pumping, Viscous Finger Patterning, Lumen, HUVECs

1. Introduction

The study of microfluidic systems that allow thegeneration of lumen structures through extracellu-lar matrix (ECM) hydrogels is an important steptowards the development of functional blood ves-sel models. Blood vessel models have been devel-oped to generate in vitro assays with physiologi-cally relevant three-dimensional (3D) lumen struc-tures for drug screening and the study of angiogen-esis. To date, several methods have been proposedto produce 3D vessel models, including templating3D microvessels models, 3D bioprinting and self-organization models [6]. However the perfect mimicof a native blood vessel is yet to be achieved.

The endothelium comprises approximately 60trillion endothelial cells (ECs), showing heterogene-ity in structure, function and mechanisms, accord-ing to the vascular beds in which the ECs are lo-cated [1] [2] [12]. When ECs are removed from theirnative microenvironment, they undergo phenotypicchanges [1]. Therefore, in vitro conditions require acontrolled microenvironment including: the appro-priate ECM, co-culture with non-ECs allowing cell-cell contact, incubation with plasma/serum of hu-man origin, dynamic conditions to implement flow,and a 3D structure. Different in vitro models ofblood vessels have been realized with human um-bilical vein endothelial cells (HUVECs). HUVECsunder static conditions remain polygonal with cob-blestone appearance, their intracellular actin fibersare randomly oriented and the tight junction VE

cadherin is detected at contact regions [15]. HU-VECs alignment can be either due to shear inducedby flow, or to strains induced by surface stretching[24].

The ECM provides structural support to the vas-cular endothelium, and consist of an essential scaf-fold for ECs proliferation, migration, morphogen-esis, survival and blood stabilization [10]. Colla-gen comprises one-third of the total protein amountin humans, it accounts for three quarters of thedry weight of the skin, and it is the most preva-lent component of the ECM [23]. These propertiesmake collagen ideal as a natural hydrogel biomate-rial. However, because of its structure, the physicalproperties of collagen are influenced by the gela-tion conditions: temperature, pH, ionic strengthand gelation rate [25]. In microsystems, like mi-crofluidic systems, the condition-dependent way ofcollagen gelation is even more pronounced due tothe larger surface-to-volume ratio, working volume,and material [25].

To achieve luminal structures, templating mod-els either require cylindrical rods which limitsthe lumen formation to simple linear structures[6] [7] [21], or biocompatible sacrificial layers thathave to be rigid enough for handling and with melt-ing point higher than the gelation temperature ofthe chosen ECM hydrogel [13]. Additionally, litho-graphic methods for sacrificial layers stamping con-fer rigid corners which lead to approximately cylin-drical vessels [8] [13] [26] [30]. 3D bioprinting can

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be used to template sacrificial layers or direct bio-printing of cells embedded in the sacrificial layer.Bioprinting methods have the capacity to producecomplex vascular networks but are time consum-ing, not always biocompatible and vessels dimen-sions depend on the printed droplet size [19]. Self-organization methods explore angiogenesis (vascu-larization from pre-existing vessels) and tubulogen-esis for perfusable microvessel formation, which re-quires weeks until a perfusable system is achieved[6] [19].

Here, the method of viscous finger patterning(VPF) through passive pumping (PP) is presentedin order to create lumens [4] [5]. The method isbased on the displacement of a more viscous fluid(collagen type I, in partial gelation) by a less vis-cous fluid (culture media). Passive pumping is usedto flow the media through the collagen type I and itdiffers from active pumping by the fact that it pas-sively pumps the liquid by mean of droplets [27]. Amicrofluidic system fabricated by soft lithographyusing polydimethylsiloxane (PDMS) is presentedwith different designs. The hydrophobicity pro-vided by the PDMS surface helps perform passivepumping [3] [14] [22] and consequently VFP. HU-VECs were studied under different collagen typeI conditions and their proliferation was comparedto the proliferation inside lumens. The study pre-sented here is a step towards optimization, and theformation of blood vessels in a microfluidic chip.

2. Materials and Methods2.1. Device Design

The device design was drawn with CleWin5 soft-ware. Straight and y-shaped channels were de-signed with different widths and lengths. A straightchannel with dimensions W: 500 µm x H: 500 µmx L: 8200 µm (S500) and a straight channel withdimensions W: 1000 µm x H: 500 µm x L: 8200 µm,a y-shaped channel with W: 500 µm x H: 500 µm xL: 9600 µm (Y500) and a y-shaped channel with W:1000 µm x H: 500 µm x L: 9600 µm (Y1000) bothwith 50 ◦ between the arms and a main arm with4180 µm. A wider channel with two outlets and in-lets was also designed, with dimensions W: 1500 µmx H: 500 µm x L: 9600 µm (P2). The resulting maskwas fabricated at the NanoLab cleanroom facilitiesof the MESA+ Institute for Nanotechnology, on Cron glass. This mask was used for photolitographyprocess.

2.2. Device Fabrication

The master mold was fabricated with photolithog-raphy and devices were fabricated with soft lithog-raphy. Two layers of SU8-100 were spun, ex-posed successively, and developed to generate amaster mold for 500 µm height fluidic channels.Polydimethylsiloxane (PDMS) base and cross-linker

were mixed at a 10:1 weight ratio. The PDMS waspoured over the SU-8 master and degassed undervacuum at room temperature for 20-30 min. Next,PDMS was cured at 60 ◦C for 3 h and removed fromthe master. PDMS was cut and punched for 1 mmand 2 mm of diameter for inlet and outlet, respec-tively. The resulting piece of PDMS was bonded af-ter plasma activation (Plasma cleaner Harrick, RF18 W, 45 s) to either PDMS coated, or non coatedmicroscope cover slips.

2.3. Preparation of ECM hydrogel and cell culture

Collagen type I solutions were prepared with fi-nal concentrations between 6 mg/mL and 7 mg/mLby neutralization of an acidic collagen solution (10mg/mL; rat tail collagen type I in acetic acid; Ibidi).In order to avoid concentration variability due toCollagen type I viscosity, every volume of Collagentype I 10 mg/mL was weighted and corrected withthe density obtained at RT of 6 mg/µL. The stockcollagen type I solution was diluted with acetic acid17.5 mM to collagen type I solution 8mg/mL. Thefurther dilutions and neutralizations used the di-luted collagen I at 8 mg/mL. First, 10 × phosphate-buffered saline (PBS) was added to a volume of col-lagen (one-tenth of the final total volume) to make1 × PBS after neutralization. Then, a basic solu-tion (1 N NaOH) was calculated to perform a vol-ume of 2.3% of the collagen type I and neutralizeit. Volume of deonized water (ddH2O) was addedto adjust collagen type I final concentration. Fi-nally NaOH was added adjusting its volume untilthe collagen type I solution was neutral. The colla-gen type I was kept in ice for specific times to applya longer nucleation phase, when molecular assemblyoccurs [20] before channel loading.

Human umbilical vein endothelial cells (HU-VECs) were cultured in endothelial growth media(EGMTM -2) supplemented with SingleQuotsR kit.All cultures were maintained at 37 ◦C in a humidi-fied atmosphere containing 5% CO2. The HUVECsseeded in the lumens were used between passages 3and 8, at a cell density of 1500 cell/µL.

2.4. Viscous Finger Patterning and HUVECs Inser-tion

To pattern lumens through collagen type I, the col-lagen was first loaded into a microchannel with thehelp of a micropippet. Prior to loading, the mi-crochannels were coated with fibronectin (FN) 0.1mg/mL for 20-30 min in order to improve collagentype I adhesion to PDMS [9] [29] [31]. After load-ing, the channels were incubated at 37 ◦C for 0 to4 min, to that gelation partially occurs and to in-crease of viscosity of the solution. Passive pumpingwas used to pattern a lumen through the collagentype I. A droplet of 10 µL of media was placed atthe outlet and a droplet of 1 to 2 µL of media was

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placed at the inlet. Through viscous finger pattern-ing (VFP) the media displaced the collagen type I atthe center of the microchannel, patterning a lumenalong the length of the microchannel. Incubationat 37 ◦C was done between 30 s and 3 min. After-wards, HUVECs were inserted by passive pumpingand allowed to seed at the bottom of the lumen for10 min. In order to attach cells on the other innerwalls of the lumen, the process was repeated withthree rotations of 90 ◦.

2.5. Viability, live/dead and proliferation assays

The metabolic activity of HUVECs was quantifiedusing 10% (v/v) PrestoBLue (Thermo Fisher) inEGMTM -2 at days 1, 2, 5 and 7. The sampleswere washed after media removal at room temper-ature, the PrestoBlue solution was added, and thesamples were incubated at 37 ◦C for 2h. Then 200µL of supernatant was collected allowing 2 readingsof each sample for fluorescence measurement withplate reader (Victor). Analysis were performed intriplicates. The conditions tested were: differentcollagen type I concentrations (3, 5.5, 6, 6.5 and7 mg/ml) all with a neutral gelation pH; differentgelation pHs (6, 7, 8 and 9) all at collagen type I6 mg/mL; different passage numbers for HUVECs(6 and 11) and different HUVECs initial densities(2000 cells/well or 4000 cells/well) (these last con-ditions were measured until day 5). All the sam-ples were tested in triplicates. Statistical analysisof data was performed with GraphPad Prism.

The live/dead stains (Invitrogen) were preparedin PBS, using calcein (0.25µL/mL) and enthid-ium homodimer-1 (3µL/mL). 100µL of the result-ing staining solution was added to each well, aftermedia removal and PBS washing and incubated for45 min at RT. The assay was performed at day 8using samples with either different collagen concen-trations (3, 5.5, 6, 6.5 and 7 mg/ml) at neutral gela-tion pH or different gelation pHs (6, 7, 8 and 9) allat collagen type I 6 mg/mL.

The proliferation assay was performed to HU-VECs on collagen type I in 96-well samples andon collagen type I that formed a lumen in a mi-crofluidic system. Cell tracker (red CMTPX) wasused with a dilution of 1:1000 in PBS. 100µL of thestaining solution was added to each well, after me-dia removal and PBS washing, and incubated for 30min at 37 ◦C. The assay was performed at days 1,2, and 5 for conditions with different passage num-bers for HUVECs (6 and 11) and different HUVECsinitial densities (2000 cells/well or 4000 cells/well).This assay was performed to lumens during 6 days.

2.6. HUVECs staining

HUVECs inserted in lumens were stained for theirnucleus with either propidium iodide (PI) 1mg/mLdiluted 1:500 in PBS, 15 min (5%, CO2, 37 ◦C) or

Hoechst 33342 (Thermo Fisher) 10mg/ml diluted1:2000 in PBS, 10 min at RT, or stained for theircytoskeleton with Actin green (Thermo Fisher), 2drops/mL of PBS, 10 min (5%, CO2, 37 ◦C).

2.7. HUVECs immunostaining

HUVECs were fixed with 10% formaldehyde for15 min at RT, permeabilized with 0.1% PBS Tri-ton X-100 for 5 min at RT and blocked for non-specific protein-protein interactions with 1% BSAfor 1 h in the incubator (5%, CO2, 37 ◦C). Then,HUVECs were incubated with anti-VE cadherin(Thermo Fisher, PA5-19612, 1µg/mL) overnightat 4 ◦C. Next, goat anti-rabbit IgG H&L (AlexaFluor 488 preadsorbed, Thermo Fisher) was addedat 1:1000 dilution in PBS for 1h at RT.

2.8. Imaging

To visualize lumens through collagen type I hydro-gels, the seeded HUVECs were stained as previouslydescribed. Samples were imaged with confocal mi-croscopy (Nikon confocal A1). An excitation sourcetuned to 405 nm, 488 nm, or 561 nm was used. Zstacks were acquired and volume-rendered imageswere generated using NIS Elements Analysis Soft-ware.

2.9. Characterization

Lumens dimensions were characterized under dif-ferent conditions (microchannels, collagen type Iconcentration and neutralization and incubationtimes). In total, 9 lumens in collagen type I6mg/mL (7 S500 and 2 S1000), 3 lumens in col-lagen type I 6.5 mg/mL (1 Y1000 and 2 S1000) and1 lumen in collagen type I 7mg/ml (1 S500) werecharacterized. The measurements were performedin two different locations per lumen, three timeseach, with ImageJ software.

3. Results and Discussion

3.1. Passive Pumping and Viscous Finger Pattern-ing

Passive pumping was used to pattern lumens in col-lagen type I hydrogels through viscous finger pat-terning. Thirteen lumens were confirmed and char-acterized. Figure 1 and Figure 2 show representa-tive images of lumens formed through collagen typeI hydrogel in microchannels S500 and S1000, respec-tively. For these experiments, collagen type I withconcentrations of 5, 6, 6.5 and 7 mg/ml was usedwith different incubation times.

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(a) Cross-section

(b) Top View

Figure 1: Confocal microscopy imaging of exemplarlumens: S500 channel (W: 500 µm x H: 500 µmx L: 8200 µm). HUVEC nuclei were stained withHoechst 33342. Scale bars: 500 µm.

(a) Cross-section

(b) Oblique projection

Figure 2: Confocal microscopy imaging of exemplarlumens: S1000 channel (W: 1000 µm x H: 500 µmx L: 8200 µm). HUVEC cytoskeleton was stainedwith actin green. Scale bars: 500 µm.

Incubation times were optimized in order to per-ceive which times allow the formation of a lumenand how the lumen diameter varies. Table 1 sum-marizes different times tested and the respectivecases of study (I-N). Time 0 represents the timelapsed after collagen type I neutralization, time 1represents the first incubation time, after collagentype I loading in the microchannels and before VFP,and lastly time 2 represents the second incubationtime, after VFP.

Table 1: Different incubation times and differentstudy cases: Time 0 - time lapsed after hydro-gel neutralization; Time 1 - 1 ◦ incubation timeafter hydrogel insertion in the channel; Time 2 -2 ◦ incubation time, after VFP. Lumen successfullyachieved (+) or failed (-).

Time Case I J K L M N0 1:2 2:2 10:0 18:3 1:0 21:11 1:0 2:0 2:0 2:0 2:0 3:02 2:0 1:0 1:0 2:0 3:0 1:0Lumen (-) (+) (+) (-) (-) (+)

Cases J, K and N allowed the realization of lu-mens, while cases I, L and M did not. Not achievinga lumen could be due to fast gelation, preventingVFP. Gelation of collagen has two phases: a nucle-ation phase where molecular assembly occurs and arapid growth phase where cross-linking takes place[20]. Consequently, the combination of times 0 and1 has a maximum time until gelation occurs. Time 0marks the beginning of the first phase of gelation ofcollagen type I and time 1 must allow partial gela-tion. Table 1 indicates that lumens were createdwith a time 0 of either 2 or 21 minutes and a time1 of either 2 or 3 minutes. From these experiments,the variance in time 0 indicates that it was not de-cisive on lumen achievement because it allowed hy-drogels in partial gelation for VFP in any exper-iment. Meanwhile time 1 was decisive for lumenachievement and it must be longer than 1 minute.Concerning time 2, only 1 minute of incubation af-ter VFP allowed the creation of a lumen. However,time 2 was also not decisive for lumen formationbecause it represented the time after VFP, when apotential lumen was already achieved. Time 2 as-sured enough stability after VFP being 1 minutelong, which means, longer times would also assureenough stability. Table 2 describes the concentra-tions of collagen type I in which the lumens wereachieved according to the respective time case. Thefact that lumens were not created in the other cases(I, L and M) could be caused by lumen collapse, in-complete VFP, or incomplete inner coverage withHUVECs.

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Table 2: Confirmed lumens with different condi-tions: different collagen type I conditions and timecases. All lumens were confirmed with confocal mi-croscopy with exception of 1, which was confirmedwith a trapped bubble (Y1000).

Collagen I(pH: 7-7.5)

TimeCase

Channel Lumens

6 mg/mL KS500 7S1000 2

6.5 mg/mLN Y1000 1J S1000 2

7 mg/mL K S500 1

(a) FLuorescence image of the bot-tom of a lumen: Anti-VE cadherinimmunostaining, with goat anti-rabbitIgG H&L Alexa Fluor 488 (green). Nu-cleus, PI (red). Scale bar: 250µm.

(b) Confocal imaging, top projection ofa lumen: Anti-VE cadherin immunos-taining, with goat anti-rabbit IgG H&LAlexa Fluor 488 (green). Nucleus, PI(red). Scale bar: 400µm.

Figure 3: Focal plane and confocal top 3D projec-tion of a lumen and the same collapsed lumen.

Figures 3(a) and 3(b) confirm the collapse of a lu-men in a wider microchannel (P2) W: 1500 µm x H:500 µm x L: 9600 µm. Figure 3(a) depicts the bot-tom of a lumen totally covered, while Figure 3(b)represents a confocal top projection. The cell den-sity was measured for both cases: Figure 3(a) hada cell density of 3.9 x 102 cell/cm2 and Figure 3(b)

a cell density of 6.5 x 102 cell/cm2 which confirms alumen collapse. These microchannels ( W: 1500 µmx H: 500 µm x L: 9600 µm) did not have a suitableaspect ratio for lumen formation.

3.2. Lumens: CharacterizationTable 3 summarizes the lumen inner dimensions foreach of the thirteen confirmed lumens. The di-mensions were measured by considering their cross-section as elliptical. The values of the majorand minor axes are shown in terms of percentageof channel dimension, channels S1000/Y1000 andS500 having widths of 1000 and 500 µm, respec-tively, and a height of 500 µm. The measurementswere carried out with ImageJ software, in two dif-ferent locations per lumen, three times each.

Table 3: Lumen dimensions: different collagen typeI conditions and channels. The results are in termsof channels percentage: major axis in percentage ofchannel width (varied between 500 and 1000 µm)and minor axis in percentage of channel height (500µm).

Collagen I(pH: 7-7.5)

LayoutMajoraxis (%)

Minoraxis (%)

6 mg/mLS500 50±12 42±11S1000 45±1 51±2

6.5 mg/mLY1000 37±0 -S1000 35±6 35±5

7 mg/mL S500 63±1 38±4

Table 3 shows that wider and taller lumens werecreated in lower a collagen type I concentration of6 mg/mL compared with the lumen size in a highercollagen type I concentration (7 mg/ml). In addi-tion, the neutralization and first incubation times(times 0 and 1) were five times longer for the widerand taller lumens. It has been reported that highercollagen type I concentrations led to a smaller lu-men width [4]. Moreover, the time between neu-tralization and first incubation time, before VFP,influences the nucleation phase. Longer nucleationphases lead to thicker collagen fibers, which in turn,leads to a more flexible hydrogel (where fibers aresparsely distributed) [25] [28] and larger lumen di-ameter.

As seen in Table 3 the microchannels with asquare cross-section yielded a lumen with, on av-erage, an ellipsoidal cross-section instead of a circu-lar cross-section. This indicates a different distribu-tion of forces when the media displaced the collagentype I hydrogel. This difference could be due to theuneven heat transfer, from the microchannel lim-its, which in turn leads to uneven gelation. Whenthe collagen was introduced to the microchannels, itwas in contact with PDMS along three walls, except

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the bottom surface which consisted of a microscopecover slip. The material with the higher thermalconductivity (the cover slip), contributed to a fastergelation of the collagen that was in direct contactwith it. Some microscope cover slips were coatedwith PDMS and compared as shown in Table 4.

Table 4: Difference between major and minor axes.Results for lumens prepared under different condi-tions: channel width and time case.

Collagen I(pH: 7-7.5)

LayoutPDMScoating

Axis differ-ence (%)

6 mg/mLS500

Yes 8±167 mg/mL No 25±4

Past studies showed that the interfacesPDMS/PDMS have significant lower thermalconductivity compared with PDMS/glass slideinterfaces [11]. Table 4 show a comparisonbetween two lumens that were formed by use ofthe same timings (case K) and microchannels withthe same dimensions. The difference consisted inthe collagen type I concentration and the use ofcover slips coated or not with PDMS. The lowerdifference between the major and the lower axisindicates a higher proximity to a circular shape.The difference in axis was shown to be lower withthe lumen formed in microchannels that werebonded to a PDMS coated cover slip. However,considering the standard deviation, both cases inTable 4 show a similar difference. The standarddeviation of both Tables 3 and 4 resulted fromnon-uniform diameters along the lumens’ length.Regardless, the overall lumen characterization wassuccessful.

3.3. Cells: Viability assayThe viability tests were performed in order to un-derstand the behavior of the HUVECs under differ-ent proportion conditions of collagen type I: usingdifferent concentrations at neutral gelation pH anddifferent gelation pHs at a concentration 6 mg/mL.This assay was perfomed in micro-wells.

Figure 4 shows an increase in metabolic activityover time for all the samples, except for the sam-ple with collagen type I 5.5 mg/mL that showed nochange in activity between the fifth and the sev-enth day. At day 7, Fig.4 shows that the collagentype I at the highest concentration (7 mg/mL) hadmore activity than other concentrations. In paral-lel to these conditions, cells in hydrogel-free wellsare shown as a control and present substantiallylower metabolic activity at all time points exceptday 1. The higher activity in the first day in thecontrol may reveal a higher stress from the cells thatwere not seeded in collagen. The different collagen

type I concentrations have different fiber density,and consequently present different cell-fiber contactpoints. The metabolic activity showed by HUVECson different collagen type I concentrations couldbe related to the cell-fiber contact points. Hence,higher metabolic activities were obtained with ei-ther higher, or lower collagen type I concentrations(higher or lower number of cell-fiber contact points).In order to study the higher metabolic activity dueto the number of cell-fiber contact points, furtheranalysis should be done on the interaction betweenHUVECs and collagen fibers.

Figure 4: HUVEC metabolic activity on Collagen Iwith different concentrations, and neutral gelation.Initial cell density: 1500 cells/well. The controlswere collagen-free samples. Fluorescence is relativeto the fluorescence measured at cell-free collagen Isamples at the conditions tested. Activity was mea-sured at days 1, 2, 5 and 7. Significant differencesare noted as **** (p ≤ 0.0001) and non-significantas ns (p >0.05).

Table 5: Live HUVECs were quantified fromLIVE/DEAD assay performed at day 8, aftermetabolic assay to collagen type I at different con-centrations (3, 5.5, 6, 6.5, 7 mg/mL, with gelationat pH neutral). The percentages correspond to thecell densities measured at day 8, relative to the ini-tial seeding density.

Collagen I (mg/ml) 3 5.5 6 6.5 7% of growth 105 73 68 158 127

Figure 5 shows that all the samples exhibit an in-crease in metabolic activity over time. Additionally,the hydrogel sample that gave the highest metabolicactivity after 7 days was the hydrogel prepared withpH 9. This assay was conducted with hydrogel-free controls, which confirms that in all the casescells have higher metabolic activity and possiblyproliferate better with collagen type I. Literature

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has shown that gelation at higher pH is faster andresults in more rigid collagen type I with thinnerfibers [28]. Additionally, it has been reported that,generally ECs show a stiffness-dependent spreadingbehavior. When on soft gels, ECs do not spreadfast or over very far compared to stiffer gels. Thesebehavioral could also apply to HUVECs.In the end, different cell behaviors can be related todifferent metabolic activity, which were expressedon both figures 4 and 5.Table 5 and Table 6 summarize the percentage ofcell densities at day 8, relative to the initial seedingdensity. Three random areas from triplicates werequantified with ImageJ software.

Figure 5: HUVECs metabolic activity on CollagenI with different gelation pHs, at 6mg/mL. Initialcell density: 1500 cells/well. The controls werecollagen-free samples. Fluorescence is relative tothe fluorescence measured at cell-free collagen Isamples at the conditions tested. Activity was mea-sured at day 1, 2, 5 and 7. Significant differencesare noted as **** (p ≤ 0.0001) and non-significantas ns (p >0.05).

Table 6: Live HUVECs were quantified fromLIVE/DEAD assay performed at day 8, aftermetabolic assay to collagen type I at 6 mg/mL, withdifferent gelation pHs (6,7, 8 and 9) The percent-ages correspond to the measured cell densities atday 8, relative to the initial seeding density.

Collagen I (gelation pH) 6 7 8 9% of growth 94 68 177 227

The recommended passage number to use HU-VECs is up to passage 7 due to phenotypic changesthat occur over time. Figure 6 depicts the metabolicactivity of HUVECs with different passage num-bers, time points and initial cell densities. The col-lagen type I hydrogels were prepared with 6 mg/mLat pH 7.5. The control samples were hydrogel-free

samples. Almost all samples grown in hydrogelshow an increase in metabolic activity over time.The hydrogel-free control at passage 11 (initial den-sity of 2000 cells/well) and the hydrogel sample atpassage 6, (initial density of 4000 cells/well) had adecrease in metabolic activity between days 1 and2. As the passage number varies, the cell lineagephenotype will also vary. A higher activity was ex-pected from samples with a higher initial seedingdensity, and from cells with a lower passage. Cellswith a lower passage number are expected to bestronger and proliferate faster than cells with higherpassage number [18]. The shown results met theseexpectations.

Figure 6: HUVECs metabolic activity on CollagenI, seeded with different initial densities and pas-sage number 6 (P6) or 11 (P11). The controlswere collagen-free samples. Fluorescence is relativeto the fluorescence measured at cell-free collagen Isamples at the conditions tested. Activity was mea-sured at days 1, 2, 5 and 7. Significant differencesare noted as **** (p ≤ 0.0001) and non-significantas ns (p >0.05)

3.4. Cells: Cell tracker assay

The proliferation assay was performed in parallel tothe viability assay while testing different HUVECpassage numbers and different initial cell densities,at days 1, 2 and 5. Figure 7 shows that cells prolif-erated over time in all the samples.

Figure 7 shows a decrease between the second andfifth day for the sample with HUVECs at passage 6,with an initial cell density of 2000 cells/well. How-ever, the error associated with the second day issubstantially high. Errors associated with this as-say are relative to the non uniform distribution ofHUVECs on the hydrogel surface. In terms of mor-phology, pictures at the fifth day did not show dif-ferent particularities to distinguish cells used withdifferent passage numbers (data not shown).

Overall, HUVECs clearly proliferated on the sur-face of 2D collagen type I.

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Figure 7: HUVECs cell densities on collagen typeI hydrogels with different passage numbers and ini-tial cell seeding densities. Full symbols: HUVECsat passage 6 (P6) or 11 (P11), with initial cell den-sities of 2000 or 4000 cells/well. Empty symbolscorrespond to the respective controls. All the sam-ples were prepared with collagen type I at 6 mg/mLand using a gelation pH of 7.5, except for the controlsamples that were hydrogel-free. Measurements atdays 1, 2 and 5. Error bars represents the standarderror of triplicates.

3.5. Lumens: Cell tracker assayThe aim of this assay was to understand differencesbetween proliferation on top of a 2D hydrogel (onmicro wells) and in a 3D hydrogel structure (inside alumen). Figures 8(a) and 8(b) show representativepictures of the cells inside lumens of collagen typeI, 6mg/mL with gelation at neutral pH. This assaywas performed using 8 different channels, along 6days (data not shown). The analysis of all channelsshowed no evident proliferation behavior over time.

(a) day 4 (b) day 6

Figure 8: Cell tracker assay performed in a Y500channel - fluorescence imaging. Cells were trackedover 6 days. Scale bars: 400 µm.

In what concerns the method used to track cellproliferation, some improvements could be imple-mented. Using cell tracker and a fluorescence mi-croscope to analyze a 3D structure requires the visu-alization of the same focal planes over time, whichmakes it difficult to compare lateral proliferation.For this reason, the measurement of cell densitiesover time was not performed.

Comparing these results with the results of theviability assay, the differences are clear: HUVECs,

either at passage 6 or 11, with different initial den-sities, proliferate on top of the collagen type I pre-pared using the same conditions. However, thesame proliferation was not visible inside the lumens.This leads to conclude that there is a differencein behavior between HUVECs grown on a 2D sur-face and a curved 3D structure. To enhance pro-liferation of HUVECs, co-cultures with mural cellslike smooth muscle cells, or pericytes or fibroblastscould be used [16]. The co-culture with mesenchy-mal stem cells also showed to improve HUVECsproliferation [17], especially bone marrow-derivedhuman mesenchymal stem cell showed phenotypictransition toward mural cells [16].

4. Conclusions

Viscous finger patterning through passive pumping,applied to collagen type I is presented as a simpleand easy method to create lumen in an ECM ma-trix. From an estimated number of 720 microchan-nels tested, only thirteen lumens were confirmedand characterized. According to the analysis on theapplied conditions, the reason why these few chan-nels were confirmed and characterized could be dueto lumen collapse, incomplete viscous finger pat-terning (back flow), and lumen incomplete cover-age with HUVECs. Future work should focus oncanceling each variable that provoke the listed rea-sons (for instance, collagen could be stained insteadof HUVECs required for lumen confirmation) andfurther optimization.

The analysis of HUVECs either on top of 2Dor 3D collagen type I hydrogels (lumens), underdifferent concentrations and gelation pHs condi-tions, showed that HUVECs survived over time andshowed proliferation on the surface of hydrogels dis-posed on micro wells, but not inside lumens. Fur-ther work could test dynamic conditions by addingflow to confirmed lumens and co-culture.

In conclusion, this study is a step in the right di-rection for the formation of vessels in a microfluidicchip. These conditions will need to be further op-timized to create a viable and working model thatcan revolutionize blood vessel testing and its appli-cations in drug delivery.

Acknowledgements

The author would like to thank Dr. Severine Le Gacand Jean Baptiste for all the support and valuablediscussions in the Netherlands, and everyone whohelped in any way from DBE, NanoLab and BIOSdepartments. From Portugal, Dr. Frederico Fer-reira for all his availability. The author would alsolike to thank family and friends.

This project was performed at University ofTwente, in The Netherlands, and defended at In-stituto superior Tecnico, in Portugal.

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