To improve the robustness of ﬂuidic interfaces and operation of the airway-on-chipover long (≥10 day) culture durations, two approaches were explored and ultimatelyincluded. These consist of: (1) mechanical reinforcement of the reversible PDMS-to-PET-membrane bond through a rigid plastic clamp and (2) ﬂuidic seal enhancement via dispens-ing of uncured PDMS around ﬂuidic interfaces.
The airway-on-chip model features cells cultured on a PET membrane that is sand-wiched between two PDMS microﬂuidic channels. If the PDMS is not bonded well and thereis a gap between the PDMS and the membrane, cells can grow into the thin gap. This canlead to unpredictable culture sizes and cell numbers, which may undermine reproducibilityfrom chip to chip. Mechanical reinforcement of the reversible PDMS-to-PET-membranebond to restrict cell-growth to the microﬂuidic channel was found to be necessary andachieved through an acrylic clamp. Quarter-inch cast acrylic (McMaster-Carr, Elmhurst, IL,USA) was purchased in 12”-by-12” sheets and cut with a VLS 2.30 (Universal Laser Systems,Scottsdale, AZ, USA) CO2laser cutter; outer dimensions of the clamp measured 73 mm-by-48 mm, 20 mm larger than the chip along both the length and width. A square cutout(4 mm-by-4 mm) was centered over each ﬂuidic inlet/outlet port to enable access, and twocircular cut-outs sized for 8–32 threaded rods (McMaster-Carr) were made collinear withthe top channel. Compression was achieved utilizing 8–32 thumb nuts (McMaster-Carr)coupled with washers for improved uniformity of force distribution. Following assembly ofthe chip-clamp unit, 22-gauge stainless steel needles (McMaster-Carr) with a 1” 90-degreebend were inserted into the ﬂuidic access ports with the other end of the needle coupled toa 10 cm length of 0.02” ID microbore Tygon tubing (VWR). The other end of this tubinglength was coupled to a 0.5” 22GA straight dispensing tip with a luer-lock connection forease of ﬂuidic interfacing and handling. A strip of tape was placed on the clamp, overtopof the ﬂuidic lines for added stability during handling. Liquid PDMS was, then, dispensedvia pipette (with the tip scissor-cut to increase the opening size, due to the ﬂuid’s viscosity)into the clamp’s cut-outs to augment the compression ﬁt seal between the needles and thePDMS, and cured at room temperature for 48 h. The thickness of the clamp as well as thePDMS of the top chip layer is sized such that the bent stainless steel needle rests on theacrylic when the bent portion penetrates approximately 3.5 mm into the 5.25 mm thick (with the channel depth subtracted from the overall layer thickness) PDMS (not too deep soas to risk piercing the PDMS of the bottom layer while retaining a stable pressure-ﬁt seal).
2.3. Microﬂuidic Cell Culture
2.3.1. BBB Chip
For the BBB chip, human brain microvascular endothelial cells (HBMECs) (Lonza,Basel, Switzerland, lot# 376.01.03.01.2F) were thawed from passage 6 and used betweenpassage 6–9. HBMECs were cultured onto ﬁbronectin-coated plates and passaged at 80%conﬂuency. Cells were lifted using 0.05% trypsin and cultured with EGM-2 bullet kitcontaining 2% v/v FBS (Lonza, cat. no. CC-3162).
A 1 mL syringe with a 22G dispensing tip was inserted into chip tubing to performall liquid exchanges. For the BBB chip, a 10 min 70% ethanol rinse, followed by two PBSwashes, and an overnight equilibration using EGM-2 media was used to prepare the chipsfor ECM-coating. For the airway-on-chip, a 20 min ethanol incubation preceded coating.
For the BBB chip, a coating of 0.4 mg/mL collagen IV (Sigma Aldrich, St. Louis,MO, USA, cat. no. c5533), 0.1 mg/mL ﬁbronectin (Sigma Aldrich, cat. no. F1141), and0.1 mg/mL laminin (Sigma Aldrich, cat. no. L2020) was applied the day after equilibrationand left overnight in the fridge. Lastly, the chip was washed twice with PBS. Then, HBMECswere seeded into the chip at a density of 5 million cells/mL and then attached to a pumpfor at least 5 days to allow for perfusion through the endothelial channel. A syringe pumpwas used to provide media perfusion resulting in shear stress values of ~0.001 dyne/cm2within the channel. The top channel media was exchanged every two days. The proﬁle ofthe ﬂow rate delivered by the syringe pump was measured using microﬂuidic ﬂow sensors,as described in the Supplementary Materials.
Three vials of Calu-3 human lung adenocarcinoma airway epithelial cell line wereobtained from ATCC and expanded and maintained in T-75 ﬂasks using Eagle’s MinimumEssential Medium (Corning, Corning, NY, USA) supplemented with fetal bovine serum(MilliporeSigma, St. Louis, MO, USA, Cat: F1051, Lot: 19D019), and Antibiotic-Antimycotic(Thermo, Waltham, MA, USA, Cat: 15240-062, Lot: 2441423). Cells were passaged at 80%conﬂuence and used for experiments between Passages 20–25.
Microﬂuidic chips were ﬁlled with a 100µg/mL rat-tail-collagen (Corning, Cat: 354236,Lot: 1049001) solution in PBS and incubated in a biosafety cabinet overnight to completemembrane coating. Following ﬂushing and equilibration with complete culture medium,chips were seeded via syringe-infusion with a suspension containing six-million cells permilliliter. After the suspension was loaded, luer caps (McMaster-Carr) were coupled toeach ﬂuidic line and the chips were transferred to an incubator to allow cells to attach forﬁve hours.
Following the ﬁve-hour attachment period, luer caps were removed from the basalchannel inlet and outlet lines. They were, then, connected to a recirculating ﬂow sys-tem, wherein a peristaltic pump was positioned to pull culture medium through thebasal channel and into a 2 mL medium reservoir containing the same medium used forculture maintenance. The recirculating ﬂow regime enables the application of physiolog-ically relevant shear stresses while minimizing reagent consumption. Reservoir culturemedium was contained in ﬁve milliliter screw-cap MacroTubes™(FroggaBio, Toronto, ON,Canada); their caps were punctured with 16-gauge needles (Becton, Dickinson and Com-pany, Franklin Lakes, NJ, USA), creating an opening just large enough to accommodatethe insertion of the microﬂuidic tubing employed. The tubing expands in the warmerenvironment of the CO2cell culture incubator, creating a tighter seal for the duration ofthe experiment. The following day, the apical channel was washed with fresh completemedium, and maintained in a submerged state for the duration of the experiment.
The reservoir volume was chosen based on manufacturer’s recommendations forTranswell®24-well culture inserts; this recommendation stipulates a culture volume of 600µL to support a culture area of 0.33 square centimeters, equivalent to the airway-on-chip. Exchange of medium in a Transwell plate every two days is a common culture regimethat has been demonstrated to produce functional cultures [38,39]. Direct communicationwith ATCC yielded an upper bound on culture medium stability in a cell-culture incubatorof four days, and this exchange interval has been employed in previously reported organ-on-chip cultures; as such, the 600µL value was doubled to 1.2 mL and a factor of safetywas added to arrive at the 2 mL reservoir volume, with reservoir exchanges and apicalchannel washes taking place every three to four days . The initial ﬂow rate employedwas 300µL/min, which was ramped up following the second media reservoir exchangeand apical channel wash to 540µL/min, corresponding to a wall shear stress between 0.6and 0.7 dyne per square centimeter exerted basally at the higher ﬂow rate—small airwayepithelia experience a range of 0.5–3 dynes per square centimeter at rest due to airﬂowin vivo [10,41].
Cultures were maintained for eleven days, at which point Calu-3 epithelia cultured onTranswell inserts reach peak barrier integrity (measured based on apparent permeability toﬂuorescein isothiocyanate-conjugated dextrans) .
2.4. Cell Culture Analysis
2.4.1. BBB Chip
HBMECs were stained to visualize f-actin and nuclei. All staining was performed in-chip. A 1 mL syringe with a 22G dispensing tip was inserted into the chip tubing to performall liquid exchanges. For f-actin staining, samples were ﬁxed, with 4% paraformaldehydein PBS and left at room temperature for 15 min. Samples were washed twice with PBS for10 min. After washing the ﬁxative 2×5 min with PBS, samples were incubated with the66µM dimethyl sulfoxide (DMSO) Alexa-Fluor-488 Phalloidin stock solution at a 1:400dilution in PBS for 1 h at room temperature. Samples were left in a dark, covered containerto prevent photobleaching and evaporation while staining. Then, samples are washed2×5 min with PBS (1×). For nuclei staining, cells were either stained live or ﬁxed,with Hoechst (Thermoﬁsher Scientiﬁc, Waltham, MA, USA, cat. no. H3570) at a 1:1000dilution in EGM-2 or PBS. Live cells were washed with EGM-2 and ﬁxed cells were washed2×5 min with PBS (1×).
BBB chips were disassembled using an Exacto knife and pliers, and the membranewas removed and placed onto a glass slide with tweezers. The membrane was placedwith the cell side facing upwards. ProLong®gold Anti-Fade containing 40,6-diamidino-2-phenylindole (DAPI) was used to mount the membrane and a cover slip was placed ontothe membrane; because the cells were stained with Hoechst, the DAPI was not necessarybut was included in the commercial formulation. The edges of the coverslip were sealedusing clear nail polish to prevent evaporation. The mounted samples were stored at 4 ◦C.
On Day 11, the airways-on-chip were ﬁxed in situ for preservation and staining. Allsteps involving manual syringe-infusion of chips are conducted separately for each channel,while the other is occluded; for example, when washing with PBS, the apical channel iswashed ﬁrst while the basal channel is closed with luer-caps. Infusion of a channel isconﬁrmed both visually in the channel, for example by tracking small bubbles that may beintroduced during injection, as well as via conﬁrmation of three droplets forming at theoutlet of the channel being infused (chosen conservatively, based on the internal volumeof the channel). The single-channel infusion with the second channel blocked minimizedthe chance of crossﬂow, which is especially important earlier in the experiment when cellshave not adhered or formed a barrier tighter than the porous membrane itself.
Chips were washed with PBS three times; following this, a 4% methanol-free formalde-hyde solution in PBS (Thermoﬁsher, Pierce) was infused to ﬁll each channel. This solutionwas incubated for 15 min at room temperature, following which an additional three PBSwashes were performed. Cells were then permeabilized by injection of a 0.02% Tween-20 solution in PBS, which was incubated for 15 min followed by an additional three PBSwashes. Chips were then blocked with a 1% BSA solution for 90 min before loading theworking concentration of Alexa-Fluor 488 Phalloidin (Thermoﬁsher) stain (165 nM in PBScontaining 1% BSA), which was incubated for 45 min. Chips were, then, washed a ﬁnalthree times before ﬂuidic and clamp disassembly.
Four incisions along each edge of the membrane, through the PDMS, were madeto extract the cell-laden membrane from the chip. Extracted membranes were promptlylaid onto a microscope slide with two drops of ProLong Gold antifade reagent with DAPI(Thermoﬁsher); a coverslip was carefully placed atop the membrane and the mountingmedia was cured under a coverslip at room temperature in the dark overnight. Thefollowing morning, slides were transferred to a slide-box for storage at 4 ◦C.
Images of HBMECs were taken on an inverted microscope (Zeiss, Observer.Z1,Oberkochen, Germany). Images of Calu-3s were taken on an inverted microscope withepiﬂuorescent capabilities and intermediate magniﬁcation switching from 1.0×to 1.5×(Nikon Eclipse Ti2-E (Nikon, Tokyo, Japan)); 10×/NA 0.3, dry (Nikon: MRH10105) and40×/NA 0.6, dry (Nikon: MRH48430) objectives were used in conjunction with 432 nm(Semrock, Rochester, NY, USA, 36 nm bandwidth, cat: FF01-432/36) and 515 nm (Sem-rock, 30 nm bandwidth, cat: FF01-515/30) single bandpass emission ﬁlters for DAPI andAlexa-Fluor-488-Phalloidin microscopy, respectively).
2.6. PDMS Thickness Uniformity Characterisation
In microﬂuidic devices employing pressure clamps to aid in sealing (such as thatdepicted in Figure 4), the thickness uniformity of the PDMS critically impacts both thequality of the seal as well as channel integrity. Thickness nonuniformity can lead to unevencompression across the device, in turn leading to leakage in regions of low compressionor channel deformation in regions of higher compression. We introduced the use of trans-parency ﬁlms during curing to improve thickness uniformity and thus device performance.In order to characterize the impact of employing transparency ﬁlms during PDMS curing, aseparate PDMS microﬂuidic device with two, smaller, microﬂuidic channels was fabricatedthat would better highlight channel integrity under compression. Master molds werecreated on the same printers as for the organ-on-chip devices and fabrication methodswere identical to the organs-on-chips. Three devices were fabricated without the use of atransparency ﬁlm to quantify its effects on the ﬂatness of a cured device, and three deviceswere fabricated with a transparency ﬁlm for comparison.