bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Highly scalable and standardized organ-on-chip platform with
TEER for biological barrier modeling
Hoang-Tuan Nguyen1,2*, Siiri-Liisa Rissanen1, Mimosa Peltokangas1, Tino Laakkonen1, Jere Kettunen1,
Lara Barthod1, Ragul Sivakumar1, Anniina Palojärvi1, Pauliina Junttila1, Jussi Talvitie1, Michele Bassis3,
Sarah L. Nickels3, Jens C. Schwamborn3, Sebastien Mosser1, Prateek Singh 1
1

Finnadvance Ltd., Aapistie 5, 90220 Oulu, Finland

2

Faculty of Biochemistry and Molecular Medicine, and Biocenter Oulu, University of Oulu, Finland

3

Developmental and Cellular Biology, Luxembourg Centre for Systems Biomedicine (LCSB), Universityof Luxembourg, 7, avenue des Hauts-Fourneaux, 4362 Esch-sur-Alzette, Luxembourg* Correspondence author

Abstract
The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro
models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and holdgreat promise for tissue and disease modeling. However, the non-standardized design of these chips
and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS).
Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is
applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts
ensure compatibility with existing laboratory workflows and high-throughput data collection andanalysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers,
primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The
integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeatedmonitoring of barrier integrity over long time periods.
Together with automated confocal imaging and compound permeability testing analyses, wedemonstrate the flexibility of the AKITA platform for establishing human-relevant models forpreclinical drug and precision medicine's efficacy, toxicity, and permeability under physiologicalconditions.

Keywordsorgan-on-chip; high throughput screening; drug discovery; microfluidics; TEER; blood-brain barrier;shear stress; vascular barriers; Lung; skinbioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1. Introduction
Drug development is a lengthy and expensive process with a high failure rate, as only 10% of drugs
entering clinical trials make it to the market 1. One of the main reasons for this failure is the poor
predictivity of current preclinical models 1–4. To solve this major issue, a new generation of in vitro
models has been developed, called organ on chip (OOC). Those allow the modeling of relevant humanphysiological features 5–9. By emulating perfusion, mechanical stimulation, and other physiologicalparameters essential to tissue and organ physiology, the OOC devices are expected to mimic
physiological conditions better than conventional cell culture models.
OOC technology has advanced significantly in recent years, with numerous innovations that have
improved these models' functionality, versatility, and reliability. OOCs are microfluidic devices that
aim to replicate the structure and function of human organs in a controlled environment, enabling
researchers to study physiological processes and drug responses in vitro 7,10. One of the majoradvantages of OOCs is their ability to mimic the complex intercellular interactions that occur withinorgans, enabling researchers to study disease mechanisms and drug responses by targeting yet
untested in vitro physiological parameters. For instance, a lung OOC may be used to assess
chemotherapy response, or how air pollution affects lung function 11. Similarly, blood-brain barrier(BBB) OOCs can be used for PK/PD tests to assess the capacity of drug compounds to reach the brainor study the inflammation of brain vasculature and how it affects the onset of neurodegenerative
diseases.
Current in vitro models partially recapitulate the in vivo microenvironment, but there is a strong bodyof literature indicating the physiologically relevant cues that guide tissue function. For instance, theflow has a significant impact on cells and tissue 12. It provides perfusion at lower flow rates, whichhelps to deliver nutrients and oxygen to cells, and removes waste products 13–18. At higher flow rates,
flow induces wall shear stress (WSS) on cells and tissue, which can influence their function andbehavior 19–22. Shear stress is involved in various physiological processes such as blood vessel dilation23, inflammation 24, and wound healing 25. Additionally, WSS has been shown to affect celldifferentiation 26, proliferation 27, and gene expression, which can have implications for tissue
engineering and disease modeling. Furthermore, maintaining near-physiological concentrations ofautocrine and paracrine signals requires a low medium-to-cell ratio within in vitro culture systems.
OOC technology has the potential to aid the development of new therapies, however, theirdevelopment has been carried out at the expense of throughput 28,29. Indeed they require more
specialized and labor-intensive procedures to fabricate and maintain. Furthermore, OOCs are rarelybioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

compatible with the industry-standard workflow and infrastructure because they require different
equipment, protocols, and expertise compared to traditional cell culture models. Those adoption
barriers decrease the likelihood for pharmaceutical companies to adopt and integrate OOC technology
widely into their drug development pipelines.
To address those limitations, we present a scalable - from high to low throughput – automationcompatible OOC platform for applied kinetic in vitro assays (AKITA). It is suited for the modeling ofendothelial-epithelial barriers, and the culture of organoid or cell migration. Its format is compatible
with ANSI/SLAS standard 96-well (24 units of OOC) and 384-well (128 units of OOC) plates, compatible
with common laboratory workflows. For high ease of manipulation, the flow is induced by a tubelesssystem - a rocker - whose angle can be set up to precisely control the flow rates. Furthermore, fullplate impedance spectroscopy allows the real-time recording of barrier integrity via trans-epithelial
electrical measurements (TEER).
As an example, we show the generation of skin-, BBB-, and lung-on-chip were generated and validated
by a fluorescent permeability assay, TEER measurements, and automatized confocal microscopy. This
work aims at providing seamless integration of OOC technology into the current disease modeling anddrug study pipeline of both academic research and the pharmaceutical industry.

2. Materials and methods
2.1. Microfluidic plate design
The AKITA plates comply with the ANSI/SLAS cell culture plate standard, having the form anddimensions of the 96 or 384-well plate. The plate incorporated 24 (96-well plate) or 128 (384-wellplate) microfluidic units (one unit corresponds to one test or experimental condition). Each unit
regroups three wells to form the complex structures underneath. In this paper, we mainly explain thedesign and manufacturing of the AKITA Plate 96. For each unit (three linked wells) the middle well
plays as an open-top chamber; the other two wells are linked together through a microchannel on thebottom. At the area where the microchannel is crossing the bottom of the middle well, a semipermeable polyethylene terephthalate (PET) membrane is integrated to separate the twocompartments. The width and height of the microchannel were designed to gradually ramp up from
0.3 mm to 6 mm and from 0.15 mm to 0.3 mm from the 2 inlets toward the middle area, to ensure
the smooth liquid laminar flow and avoid any turbulence. The membrane was 9 μm thick with a porebioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

size of 3 μm, acting as an interfacing layer between the microchannel and the open-top chamber. The
design of the AKITA Plate allows a bilateral flow through the microchannel using a rocker platform topass the liquid from one well to the other while keeping the open-top chamber static with no liquid
circulation. This combination of both flow (microchannel) and static (middle well) conditions acting on
both sides of the membrane give a versatile platform for multiple in vitro co-culture models where
one cell type can experience the flow-induced shear stress while the other cell type remains static or
exposed to very low inertial flow.
An additive manufacturing-assisted soft-lithography procedure was developed to fabricate thecomplex structure of the plate. With the membrane clamped in between, the 3D printed 2-part
negative mold was cast with a 10:1 mixture of the Polydimethylsiloxane (PDMS) prepolymer andcuring agent. Finally, the demolded PDMS structure was bonded onto a 100 μm thick PDMS film usinga plasma cleaner (Harrick Plasma Inc., US). The process yields a full plate with multiple layers while
reducing the need for many bonding processes between layers.

2.2. Label-free TEER assessment of barrier integrity
To monitor the development of the tissue barriers formed on the AKITA plate 96, we used the AKITA
Lid device developed by Finnadvance, to enable high throughput, label-free, non-invasive TEER
measurements of 24 channels simultaneously. The AKITA Lid consisted of an analog front-end for an
impedance analyzer with a frequency sweeping spectrum from 100 Hz to 200 kHz, a digital back-end
for main function control, and a detachable electrode array. The electrode array circuit facilitated 144
gold-plated chopstick-like electrodes. The tetrapolar configuration was used for the sensing, there are
one pair of excitation electrodes and one pair of measurement electrodes. Theoretically, theelectrodes of each pair are placed on the opposite side of each other, i.e., on both sides of themembrane, via the inlet/outlet reservoirs and open-top chamber. The impedance values aremeasured and transmitted to the computer software in three ways, depending on the laboratory’s
accessibility: Wi-Fi, serial port, and micro-sd card. The measurements were performed before themedium change, at 37 °C, to reduce the variations in temperature. For the cells under air-liquidinterface (ALI), the culture medium was added to the open-top chamber just for measurement andremoved after it. TEER values were calculated by fitting the recorded impedance data to an electricalequivalent circuit of tissue barriers described elsewhere 30. In short, the circuit includes a serial
combination of a resistor representing culture medium resistance, a resistor in parallel with acapacitor representing the cell layer, and a constant phase element CPE (electrode-medium interface).

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2.3. Computational studies
1) Gravity-driven fluid dynamic simulation
Flow rate, shear stress, and flow streams were simulated using finite element analysis (COMSOLMultiphysics). A 3D computer-aided design (CAD) model of the chip was created from Solidworks(Dassault Systèmes) and imported into COMSOL. Free and porous medium flow physics was selected
for modeling the fluid flow which is governed by Navier Stokes and Brinkman equations 31. The
incompressible flow was used with Phosphate buffer solution (dynamic viscosity = 0.001 Pa.s, Density= 1005.6 Kg/m3) as the fluid material. PDMS was selected as the boundary walls and a no-slip
condition was applied to the walls. Porous material was included, and the membrane was selected asa porous medium. The gravitational force was included as a volumetric force which induceshydrostatic pressure for driving fluid inside the channel.
The moving mesh was applied for simulating the rocker motion where the hydrostatic pressurechanges with respect to the tilting angles. A normal mesh was used, and a time-dependent study was
implemented for 150 seconds to study the velocity, flow rate, and shear stress from 10o to 30o tilting
angles. Shear stress was calculated by multiplying the shear rate and fluid viscosity whereas the flow
rates were determined by taking the surface integral of fluid velocity in the microchannel under themembrane.
2) Electrical simulation for TEER sensitivity
Sensitivity field distribution on the membrane where the cells grow was simulated using COMSOLMultiphysics. The 3D geometry of the chip with submerged electrodes was designed in Solidworks andimported into COMSOL. Electric field physics was used for studying sensitivity on the cell layer at
different electrode setups (tetrapolar configuration with 4 or 6 electrodes). The electrodes are 0.4 mm
in diameter, and 10 mm in length, with gold as the material. Alternative current (AC) of 10 µA wasinjected through the Current Carrying (CC) electrodes and Voltage Sensing (VS) electrodes were set as
floating potential. The model was simulated at TEER values ranging from 101 to 103 Ω.cm2, for covering
the TEER range of the most popular in vitro tissue barrier models. A wide-range frequency domain(10E1 – 10E6 Hz) study was used. Parametric sweeps at different TEER values were simulated andcompared with the help of sensitivity distribution fields (at different electrode diameters). Fine mesh
was used in solving all the models.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2.4. Cell culture
Primary mouse Brain Microvascular Endothelial Cells (mPBMECs, CellBiologics) were cultured in acomplete Endothelial Cell Medium (CellBiologics) in petri-dish pre-coated with 0,2% gelatin (Merck
Life Science). Immortalized human cerebral microvascular endothelial cell line (hCMEC/D3 Merck Life
Science) were cultured on 250µg/ml collagen-I (Advanced Biomatrix) coated petri dish in EndoGROMV complete culture medium (Merck Life Science) supplemented with 1% PenStrep (Gibco) and1ng/ml basic fibroblast growth factor (Merck Life Science). Adenocarcinoma human alveolar basal
epithelial A549 cells (Sigma) were cultured in Ham's F-12 K medium (Fisher Scientific) supplemented
with 10 % Fetal bovine serum (FBS) (Serana), 1 % penicillin/streptomycin (Gibco), and 2 mM LGlutamine (Sigma). Human umbilical vein endothelial cells (HUVECs) were kindly provided by prof.
Eklund from Oulu University. HUVECs were cultured on an attachment factor (Gibco) coated petri dishin Endothelial cell growth medium 2 (ECGM2) with a supplement kit (Promocell), 10% FBS, and 1%
PenStrep. Normal human dermal adult fibroblasts (NHDFa, Lonza) were grown in DMEM (Gibco)
supplemented with 10% FBS and 1% PenStrep. Normal human epidermal neonatal keratinocytes(NHEKneo, Lonza) were cultured on KGM Gold keratinocyte growth medium (Lonza). In all theexperiments, cells were cultured at 37 °C with a 5 % CO2 concentration.

2.5. Blood-brain barrier-on-chip
Mouse blood-brain barrier (BBB) model was established with a tri-culture of mouse brain
microvascular endothelial cells cultured inside the microchannel in a complete endothelial cell
medium, and a mixture of primary mouse brain astrocytes (mAs, ScienCell) and mouse primary brain
pericytes (mPe, iXCells) cultured on the open-top chamber in 1:1 mixture of Astrocyte Medium-animal(ScienCell) and pericyte growth medium (iXCells). Whereas the human BBB model was establishedwith hCMEC/D3 cultured inside the microchannel with EndoGRO-MV complete culture medium. The
triculture model of the human BBB was established with a mixture of human primary cerebellar
astrocytes (hAS, ScienCell) and human primary brain vascular pericytes (hPe, ScienCell) cultured on
the open-top chambers in 1:1 mixture of complete astrocyte medium (ScienCell) and complete
pericyte medium (ScienCell).
AKITA Plate microchannels and open-top chambers were coated with 70µL and 50µL respectively, of
100µg/ml human fibronectin (Corning) diluted in phosphate-buffered saline (PBS, Gibco). The coating
solution was incubated for 4 hours at +37 oC, 5% CO2, following an overnight incubation at +4 oC. On
the day of the cell loading the coating solution was removed and replaced with the suited culture
medium. The endothelial cells in both human and mouse models were seeded to microchannels inbioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2E6 cells/ml cell concentration. The plate was flipped upside down and incubated upside down for 4
hours at +37 oC, 5% CO2, allowing the cells to attach to the top side of the microchannel. Astrocytes
and pericytes in both models were seeded to the open-top chambers in 1E6 cells/ml concentration toform a triculture model. All cells for both human and mouse BBB were seeded to the AKITA Platestraight after thawing from liquid nitrogen.
The plates were either cultured in static conditions or on the AKITA Wave rocker with a 20-degree
tilting angle, 1 RPM speed, and 40-second hold time at extreme angles. The medium was replenishedevery day in the microchannels and open-top chambers. The cells were grown on AKITA® Plate for 57 days prior to permeability assay or fixing.

2.6. Blood-Brain barrier and midbrain organoid co-culture
The neurovascular unit was established on the AKITA Plate 96 with a coculture of hCMEC/D3 cells
inside the microchannel and a midbrain organoid (OrganoTherapeutics) in the open-top chamber.
Briefly, the endothelial barrier with monoculture of hCMEC/D3 cells was established as described athuman BBB formation above, only using hCMEC/D3 cells. The human BBB was cultured for sevendays under flow conditions allowing a barrier formation. On day seven of the experiment themedium on the open-top chamber was changed to midbrain organoid culture medium(OrganoTherapeutics) and the midbrain organoids were loaded to the open-top chambers one byone. To assess if the viability of the organoids or the simple BBB remains stable during the coculture
period, midbrain organoids and BBB were also monocultured on the AKITA Plate as a control. The
formed neurovascular unit was further cultured for 48h.

2.7. Skin-on-chip
The skin-on-chip model was established by culturing human dermal fibroblast in a collagen mixture
on top of the porous membrane, following human epidermal keratinocyte culture on top of the dermal
layer. The AKITA plate units were coated with 100 µg/ml collagen-I solution diluted in PBS as explainedin the blood-brain barrier model. On the day of cell seeding, the fibroblasts were diluted to a final
concentration of 1E6 cells/ml in a mixture with 100 µg/ml of collagen-1 and culture medium andloaded into the open-top chambers. After 3 days of culture, keratinocytes were added on top of the
fibroblast layer in a concentration of 1E6 cells/ml. The cells were left to attach in static conditions in
the incubator overnight. Monoculture units were also cultured as a control.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

After attachment, the plates were either cultured in static conditions or on the AKITA Wave rocker(20° tilting angle, 1 RPM speed, and 40-second hold time) to achieve dynamic flow. After 3 days ofseeding keratinocytes, the units were exposed to the air, and an air-liquid interface (ALI) was created.
During the coculture, the mixture of 1:1 DMEM and KGM medium was used for the model, and the
medium was changed daily. The model was cultured for 11 days under ALI, following the permeability
assay, and fixing.

2.8. Lung-on-chip
The lung-on-chip model was established with a coculture of HUVECs cultured inside the microchanneland A549 cultured on the open-top chambers. The AKITA plate’s units were coated with 100 µg/ml
collagen-I diluted in PBS as explained in the blood-brain barrier model. Then A549 cells were seeded
to the open-top chambers in 0,25E6 cells/ml concentration. The plate was incubated statically for 4
hours at +37 oC, 5% CO2, allowing the cells to attach. Next, HUVECs were seeded to microchannels in
2E6 cells/ml cell concentration, and the plate was flipped upside down and incubated overnight at +37oC, 5% CO2. On the next day, the plate was flipped back right side up.

As with the BBB and skin models, the plates were either cultured on static conditions or on the AKITA
Wave rocker with the 20° tilting angle, 1 RPM speed, and 40-second hold time at extreme angles. The
medium was replenished every day in the microchannels and open-top chambers. After culturing cells
for 5 days under the submerged environment with culture medium on both sides of the membrane
the medium was removed from open-top chambers, and A549 epithelial cells were exposed to the air.
As a comparison, control units were cultured under a submerged environment during the wholeexperiment. The culturing was continued for an additional 4 days for full barrier maturation.

2.9. Permeability assay
Fluorescent-labeled molecules, 0,4 kDa sodium fluorescein (Sigma), 70 kDa TRITC-dextran (TdB Labs),
and 4 kDa FITC-dextran (TdB Labs) were used to test the barrier permeability of all the organ models.
Briefly, the medium of the open-top chamber was first changed to 70 µl of fresh medium, and medium
background samples were collected. Next, reservoirs of the microchannel were emptied and 100 µl of
tracer solution in culture medium (0,5 mg/ml for dextran, 0,05 mg/ml for sodium salt) was added to
the reservoirs. The AKITA Plate was placed on the rocker and incubated for either 60 or 120 min. Afterincubation, samples from both the open-top chamber and microchannel reservoir were collected to a
microtiter plate.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The fluorescent intensity of the samples was measured with a plate reader (TECAN infinite M1000Pro).
For 4 kDa and 0,4 kDa tracers, 498 nm excitation wavelength, and 518 nm emission wavelength wereused. For the 70 kDa tracer 554 nm excitation wavelength and 578 nm emission wavelength wereused. The permeability coefficient at desired time point was determined as previously reported 32.
Briefly, apparent permeability (Papp) was calculated using the following equation (1):𝑉

𝑃𝑎𝑝𝑝 = 𝐴∙[𝐶]𝑖𝑛 ∙𝑑[𝐶]𝑝𝑎𝑠𝑠
𝑑𝑡(1)

Where V is chamber volume of the passed tracer (cm3), A is membrane surface area (cm2), [C]in is
initial tracer concentration (ng/ml), d[C]pass is passed tracer concentration (ng/ml), and dt is the timeof the experiment (s).

2.10. Reversible barrier opening
For reversible barrier opening assay, a BBB model was established as described in mouse blood-brain
barrier-on-chip formation with few exceptions, instead of fibronectin, a 250µg/ml collagen-I was used
as coating solution and the barrier was established without pericytes. On day seven of the experiment,
first the initial permeability of the established BBB was assessed with a permeability assay with a
70kDa dextran as described above. After the permeability assay, the normal culture medium waschanged to a hypertonic culture medium (Finnadvance) and incubated for 1 h. The opened barrier
permeability was measured with 70kDa dextran solution supplemented with a hypertonic culture
medium. After this, the culture medium was changed yet again to the normal isotonic medium toallow the cells to recover for 1 hour from the opening of the barrier, to ensure the recovery thepermeability assay was once again performed with the 70kDa dextran.

2.11. Live staining and imaging
During the experiments, cell confluency and viability were observed with live staining and imaging.
For this purpose, Calcein-AM (Fisher Scientific) was used. Calcein-AM was diluted into a fresh culture
medium with a final concentration of 2 µM and the culture medium on both microchannels and opentop chambers was replaced with a staining solution, following 30 min incubation. Next, the fresh
culture medium was changed, and the units were imaged with a fluorescence microscope.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

2.12. Immunofluorescence staining
At the end of the experiments, the organ-on-chip units were fixed with 4% PFA and continued to theimmunofluorescence staining. To minimize nonspecific binding the samples were blocked for 4 h atroom temperature with a blocking buffer containing 1% of Tween20 (Fisher Scientific) and 3 % of BSA(VWR) in PBS. For skin-on-chip, permeabilization with 0.1% Triton X100 (Fisher scientific) in PBS was
performed for 10 minutes prior to blocking.
Primary antibodies

Dilution

Secondaryantibodies

Dilution

Phalloidin CF660R(BioNordika)

1:200

-

-

MUC5AC, antihuman, MousemAb(Fisher
Scientific)

1:100

CF488A Goat Anti
Mouse IgG (H+L),
Highly CrossAdsorbed (min X Rat)
(Biotium)

1:2000--DAPI (Biotium)

1:1000

Phalloidin CF594(Biotium)

1:200

-

-

CD31 ((PECAM-1),
anti-mouse, GoatpAb (Bio-Techne)

10µg/ml

CF488A Goat AntiMouse IgG (H+L),
Highly CrossAdsorbed (min X Rat)
(Biotium)

1:2000--DAPI (Biotium)

1:1000

CF594

1:200

-

-

Collagen IV, antihuman, Rabbit pAb(Abcam)

1:200

CF680 Donkey AntiRabbit, Highly CrossAdsorbed (Biotium)

1:2000Loricrin,antihuman/anti-mouse,
Rabbit pAb (Abcam)

1:200

CF680 Donkey AntiRabbit, Highly CrossAdsorbed (Biotium)

1:2000-DAPI (Biotium)

1:1000Lung

Blood-brain barrier

Phalloidin(Biotium)

Skin-

Table 1: immunofluorescence staining reagents.
Primary antibodies used, listed in Table 1, were diluted into a blocking buffer, and incubated overnightat +4 °C. After incubation, the cells were washed three times with PBST (1% Tween20 in PBS). Similarly,
secondary antibody dilutions were prepared in a blocking buffer and incubated overnight at +4 °C,

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

stained cells were washed 3 times with PBST. Membranes of BBB and lung models with stained cells
were cut out of the AKITA Plate and mounted with Immu-Mount (Fisher Scientific) between
microscopy glass slides for imaging. Skin-on-chip units were imaged directly on the Plate.

2.13. Microscopy and image analysis
For live imaging, either Olympus CellSense time-lapse imaging system with an Olympus XM10 CCD
camera and 4x objective with a GFP filter, or Zeiss Axio Zoom.V16 fluorescence stereo microscopewith Hamamatsu Orca Fusion BT (monochrome) and Zeiss Axiocam 305 (color) cameras with PlanApo
Z 0.5x/0.125 objective were used. For fixed BBB and lung samples, imaging was performed using a
Leica TCS SP8 confocal with a DMI8 microscope using LAS X 3.5.2 acquisition software. The used
objective was HC PL APO 20x/0.75 CS2 DRY with excitation of 405 nm, 489 nm, and 661 nm solid-statelasers. Fixed skin-on-chip units were imaged with Leica STELLARIS 8 DIVE multiphoton confocal
microscope using a Spectral fluorescence detector (3 HyD S). The used objective was HC PL APO
20x/0.75 CS2 (Air) with excitation of 405 nm, 561 nm, and 638 nm lasers.
Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) samples wereprepared and imaged in collaboration with the electron microscopy core facility of Biocenter Oulu.
Cells were fixed with fixative (1 % glutaraldehyde, 4 % formaldehyde in 0.1M phosphate buffer,provided by Biocentre) and stored at +4 °C before sample processing. For SEM imaging, Sigma HD VP
FE-SEM was used with 5 kV electron high tension and a pixel size of 44,66 nm. For TEM imaging, Tecnai
G2 Spirit 120 kV TEM with Veleta and Quemesa CCD cameras were used.
Image analysis was made with ImageJ (Rasband., 2018) and Bitplane Imaris 3D software (Imaris., 2022)
for fluorescence and confocal microscopy images, respectively. All the imaging was performed withthe collaboration of Biocenter from the University of Oulu. Construction of the schemes was
performed using Biorender software.

2.14. Statistical analysis
All data are presented as mean ± standard deviation. For all experiments, “N” represents theexperimental repetitions. The homoscedasticity and homogeneity of variances were tested with the
Shapiro-Wilk test and Bartlett test from all the collected data. Two-way analysis of variance (ANOVA)
and Tukey’s multiple comparisons test were used to test differences between conditions. For
inhomogeneous data, the Kruskal-Wallis test and the Dunn test with Bonferroni correction were used.
The confidence levels of p<0,05 (*), p<0,01 (**), and p<0,001 (***) were used as significant differencesbioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

in all statistical tests. R 4.2.3 software (R Core Team, 2023) was used for data analysis. The exact
number of repeats performed for each experiment is indicated in the corresponding figure legends.

3. Results and Discussion
3.1. Microfluidic plate design and manufacturing
The AKITA Plate 96 comprises three main layers, the top reservoirs, the track-etched PET membrane,
and the bottom microchannel (Fig. 1a-c). Due to the complexity of multiple layers, non-planar
structures, and material characteristic differences, the fabrication of the plate was facing many
challenges. However, we have optimized the production process to create a monolithic plate with an
integrated membrane, without the existence of layer gaps, and eliminate the bonding steps between
layers. As a result, a standardized well plate format OOC platform was designed and manufactured toenable a wide range of physiological barrier models in vitro (Fig. 1d).
Figures 1a & 1b show the 3D rendered full plate designs of the AKITA Plate 96 (24 individual
microfluidic units) and AKITA Plate 384 (128 individual microfluidic units). Within the context of thispaper, we only discuss the results from the AKITA plate 96. Figure 1c illustrates how multiple layers ofcells/tissues are interfacing with each other through the thin semi-porous membrane while coupling
both the dynamic flow and static conditions for adapting to different cell types. With 24 (co)culture
units on one plate, the experiment throughput was scaled up significantly compared to normal
microscope slide-sized OOC devices.
The easy-access design with inlet/outlet reservoirs, and open-top chambers that are compatible withmultichannel pipette or robotic liquid handling systems made the AKITA Plate 96 a suitable platform
for high throughput screening applications. Microscopy imaging is an essential readout method forevery cell-based model, Figure 1e shows an array of 24 live images of the cells’ Calcein AM signal at
4X objective taken from the AKITA Plate. While current 3D organ-on-chip models are difficult to be
imaged due to their complex, non-standard 3D structures, our plate design enables high-resolution
fluorescent or confocal fluorescent imaging thanks to the consistent cell culture constructs on themembrane across the units on the same plate and between multiple plates. Furthermore, the short
focal distance (400 μm) from the plate substrate to the membrane where the cells are growing also
plays an important role in the adaptation to most of the long working distance objectives of existing
microscopy systems.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1. AKITA microfluidic plate design. (a) 3D rendered AKITA Plate 96 with 24 culture units. (b) 3D
rendered AKITA Plate 384 with 128 culture units. (c) Side view of a culture unit on the AKITA Plate. The
open-top chamber provides static culture conditions while the microchannel enables dynamic culture
with the medium recirculated between the two inlet/outlet reservoirs. The semi-permeable PET
membrane allows the cells on the open-top chamber and the microchannels to interact and form
physiological barriers. (d) The manufactured AKITA Plate 96 is loaded with red dye in the microchannels
and blue dye in the open-top chambers. (e) Top view of the AKITA Plate 96 matching with 24 live CAM
staining images. The CAM signal shows the cells living in the membrane regions.

3.2.

Pumpless flow optimization

Flow control is an important factor to establish the cell culture on-chip. Here we aim to eliminate theuse of pumps for our high throughput platform as pump-based systems require a complex setup
involving tubing, connectors, and valves, which can be challenging to assemble, maintain and scale
up. With the AKITA Plates, we used a simpler approach to induce the flow through the microfluidic
channels of the plate through the periodic tilting of the AKITA Wave rocker. Due to the hydrostaticpressure differences between the two reservoirs during the tilting periods, the liquid flows from one
inlet to the other, bi-directionally. We performed the flow optimization by both in silico simulationand practical experiment (Fig. 2h) to ensure the precise flow profiles for specific tissue models. Figure
2a illustrates the consistent flow streams at the free-standing membrane region where the barriers

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

are formed. As a result, a uniform shear stress distribution was observed, thus ensuring the same
responses from the cells across the membrane area (Fig. 2b).

Figure 2. Bilateral flow simulations. (a) Arrow volume represents the velocity field of fluid inside themicrochannel at the 20° rotation angle. (b) Surface plot showing the shear stress at the ceiling surfaceof the microchannel at the 20° tilting angle. Shear stress under the membrane area (zoomed-in picture)is uniform but smaller compared to the shear stress at the narrow regions of the microchannel. (c)Tilting motions of the plate due to the rocker’s movements. (d) The AKITA Wave rocker with 4 AKITA
Plates stacking for dynamic cell culture. The rocker’s trays are designed to be compatible with
ANSI/SLAS microtiter plates and robotic gripping arms. (e) Calculated values of the average shear
stress at the membrane region and flow rates at different tilting angles. (f) Flow rate and average
shear stress on the membrane over time during the rocker’s working cycles (tilting angle = 20°, rotation
speed = 1 RPM, holding time on the left and right angles = 40 seconds). (g) The volume of liquid turnoverat different tilting angles. (h) Comparison between simulation and experimental values of the
microchannel’s flow rate.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

As shown in Figure 2c, the plate is tilted from left to right periodically, yielding the changes in theheight of the medium reservoir, increasing the volume of liquid passing through the channel over time
(Fig. 2g). Furthermore, as the tilting angle is raised, the shear stress is linearly increased. Figures 2e &2f show the linearity between the flow rate and shear stress, at different tilting angles and over time.
Especially, figure 2f illustrates the steady flow rate periods when the rocker is held at a certain angle
for 40 seconds. Based on those computed values, with different tissue types, the tilting angles, tilting
time, and duty cycles can be optimized to give suitable shear stress and flow rate for the cells to grow.
For instance, with our chip design, a rocker profile consisting of a tilting angle of 20o, 1 RPM speed,
and 40 seconds holding time at the extreme left and right positions would yield 70 uL of medium
passing through the microchannel in each period, and an average shear stress of 0.06 Pa across themembrane area.

3.3. Optimization of the electrode setups for TEER
measurements and comparison with standard
equipment
To implement the TEER measurements for the AKITA Plate 96 we used the AKITA Lid device, developed
by Finnadvance Ltd. The AKITA Lid consists of 144 submerged electrodes addressing the 24 units ofthe plate sequentially through a multiplexing circuit for full spectrum (100 Hz to 200 kHz) sweeping
and impedance measuring of the whole plate within 3 minutes. The AKITA Lid uses the tetrapolar
configuration for impedance measurement, two or three pairs of electrodes were dipped into 3
reservoirs of each unit containing medium, acting as the current injection and voltage measurement
electrodes. Figures 3a & 3d illustrate the two setups of electrodes addressing both sides of themembrane for TEER measurements. It is noted that the electrode characteristics play an importantrole in the signal quality. Hence, we performed several tests to ensure that the AKITA Lid can give
reliable results. The tests include measurement sensitivity due to electrode placements and electrodedimensions and comparison with commercially available TEER measurement equipment.
The electrode’s geometry, arrangement, and microfluidic design have a great influence on the electricfield during the TEER measurements 33,34. Over the area of interest, if there is a non-uniform electricfield, only some portions of the cell layer contribute to the inferred TEER values, which do not
represent the value of the whole cell layer. Planar sensitivity distribution on the cell layers on the
AKITA Plate’s membrane was simulated with COMSOL using the reciprocal theorem 35. The sensitivity
distribution was simulated at TEER values ranging from 101 to 103 Ω.cm2, covering the TEER range ofbioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

most of the in vitro organ models 36,37. Figure 3b shows the normalized planar sensitivity (%) withheight expression on the cell layer at 10 kHz frequency for a 4-electrode setup (Fig. 3a), with one pairof electrodes submerged in the open-top chamber, and the other pair inserted in the reservoir on theright side. Because of the lack of electrodes on the left reservoirs, electric current flow between thetwo electrode pairs tends to concentrate more on the right side (Fig. 3c), resulting in the lowest
sensitivity observed on the left area of the membrane. Hence, we added two extension electrodes
into the left reservoirs, merely a simple duplication of the other two electrodes in the right reservoir,
forming a 6-electrode setup (Fig. 3d) to distribute the electric field across the membrane more evenly(Fig. 3f).

Figure 3. AKITA Lid TEER device. (a) A single cell culture unit with submerged chopstick-like electrodes.
(b) Normalized planar sensitivity distribution (%) at 10 kHz of the membrane surface at different TEER
values for 0.4 mm diameter gold electrodes. In both electrode setups, sensitivity peaks are observedon the edges of the cell surface and the sensitivity valleys are present at the center of the cell surfaces,
but a uniform sensitivity is found over the barrier surface in 6-electrode setup at low TEER values (10Ω.cm2). Note the logarithmic scale is different between the two electrode setups. (c) Current density
streamline plot for the normal current (blue color) and the reciprocal current (red color). (d) The AKITA
Lid is in operation with the AKITA Plate 96. (e) Impedance measurement made by the AKITA LID and
the EVOM1 for solutions of varying conductivity: 1X PBS versus 0.01, 0.1, or 1M NaCl. Impedance ismeasured at 12.5 Hz for the EVOM and is the average of the impedance values between 500Hz and
30kHz for the AKITA Lid. N=4

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

With 6 electrodes in total for the tetrapolar configuration, the simulation results showed asymmetrical distribution (Fig. 3e) of the sensitivity on the membrane and lower down the electricalresistance caused by the microfluidic channel. Furthermore, the 4-electrode TEER simulation results
by Helm et al. have shown that the measurement sensitivity varies for different TEER values atdifferent sweeping frequencies. For low-frequency sweeping, a uniform sensitivity is noticed on thecell layer with high TEER values. In contrast, at high-frequency sweeping, a more uniform sensitivity isobserved on the cell layer at lower TEER values. Sensitivity is directly proportional to the TEER at higher
frequencies whereas, at very low frequencies, sensitivity is inversely proportional to TEER 38.
Importantly, as observed in our simulation results, the non-uniformity still occurs even with the 6electrode setup, and it was also mentioned elsewhere that the chopstick-like electrodes could notform a homogeneous electrical field along the cell layer 39. Both Zoio et al. and Yeste et al. simulated
the sensitivity of the chopstick STX2 electrodes (World Precision Instruments) with 6.5mm and 12mmTranswell inserts, and both had non-uniformity of the sensitivity with “sensitivity valleys” 33,40. WithAKITA Lid, two mitigation steps can be performed in the future to resolve the problem. First is toincorporate the electrodes into the AKITA Plate 96, to reduce the distance from the electrodes to themembrane and the distance between electrodes, providing a more uniform electric field distribution.
The second is to widen the sweeping frequency range, to cope with the tissue barriers at low and high
TEER values. Further simulation studies are required for optimizing the parameters such as electrodearrangement (distance between the electrodes), electrode shape, and electrode positioning (distance
between the electrode tip and cell layer) for attaining a more uniform sensitivity at the cell layer.
Despite the drawbacks, the AKITA Lid illustrates a significant improvement compared to theconventional TEER measurement systems using manual single chopstick-like electrodes with low
throughput and high variations 33. Furthermore, with a wide frequency range, AKITA Lid can performmeasurements at much higher TEER values compared to conventional fixed-frequency TEER
measurement systems.
To validate our AKITA Lid, we performed TEER measurements on the AKITA Plate 96 filled with buffers
at different ion concentrations and compared it to the same measurements by EVOM1 (World
Precision Instruments). To ensure measurement consistency, both devices should have the same
electrode geometry 41. We thus customized an electrode connector to interface the EVOM1 with ourelectrode array, to fit with the AKITA Plate 96. Figure 3h shows the impedance measured for the
0.01M, 0.1M, and 1M NaCl, as well as 1X PBS. As expected, as the ions concentration increases, theimpedance decreases. The AKITA LID measured an impedance more than four times higher for the
0.01M NaCl buffer compared to the 0.1M NaCl, and an impedance about 30 times higher for the 0.01M

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

NaCl buffer compared to the 1M NaCl buffer. The EVOM and our AKITA LID have consistent impedance
measurement results, except for the least ion-concentrated buffer: the EVOM was saturated at 20
kOhms impedance due to its fixed frequency AC current (12 Hz). Both devices show very low standard
deviation as our electrode unit ensured a fixed positioning in the well.

3.4. Blood-brain barrier-on-chip
The application versatility of the AKITA Plate allows multiple BBB modeling opportunities. For proofof-concept, we established both mouse and human BBB models for high molecular weight compound
screening and reversible barrier opening assay for enhanced compound delivery to the brain. BBB-onchip models were established with endothelial cells in the microchannel co-cultured with speciesspecific astrocytes and pericytes (Fig. 4a).
The endothelial cells were exposed to bilateral flow to ensure tight barrier formation. On day 5 of theexperimentation, we assessed the permeability of the 70kDa dextran tracer through the triculture
models of the human BBB (hBBB, Fig. 4b) and the mouse BBB (mBBB, Fig. 4c). As previously reported,inducing the flow in the microchannel improved the barrier tightness significantly when compared tohBBB or mBBB culture in a static environment 42. We also observed by electron microscopy that theendothelial cells are forming monolayers inside the microchannel (Fig. 4d). Additionally, the confocal
microscopy analysis performed directly in the Akita plate showed the segregation of both cell
populations at each side of the membrane. Indeed, the expression of the CD31 endothelial marker
was specifically expressed at the bottom side of the membrane, while phalloidin stained the actin of
both cell populations (Fig. 4f).
To enhance the delivery of drug compounds into the brain, transient opening of the BBB via theinfusion of hypertonic solution can be performed both clinically 43 and in rodents 44. We aimed at
reproducing this clinical protocol in our barrier model and optimized a reversible barrier opening assay
using the hyperosmolar solution for BBB disruption. For this purpose, we established an mBBB with acoculture of mouse primary endothelial cells and mouse primary astrocytes. On day 7 of theexperiment, the initial tightness of the barrier was verified to be close to values observed in vivo byusing 70kDa dextran as a tracer (Fig. 4e) 45. After exposure to a hyperosmolar culture medium, weobserved the transient opening of the barrier followed by recovery of the permeability scores after 1
hour back to the initial state (Fig. 4e).
Previously reported models have also shown successful cultures of BBB and midbrain organoids ontranswell. The AKITA platform is compatible for organoid culture, hence we established a BBBmidbrain organoid co-culture model in a scalable throughput, with enhanced physiological flow
conditions. First, we established a simple hBBB model with the monoculture of hCMEC/D3 cells inbioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

the microchannel and cultured the cells for seven days under dynamic conditions to allow barrier
maturation. On the 7th day of the experiment, we added a midbrain organoid to the open-top
chamber and monitored the viability of the coculture for an additional two days with calcein AM
staining and live cell imaging (Fig. 4g). The viability of the organoid and the hBBB remained stable
over the 48h co-culture, which provided a sufficient time window for the treatment with additional
compounds.

Figure 4. Blood-brain barrier-on-chip on AKITA Plate. (a) A scheme representing the BBB-on-chipmodel on AKITA® Plate with astrocytes and pericytes co-cultured with brain microvascular endothelial
cells on opposing sides of the porous membrane. Both human (hBBB) and mouse (mBBB) models havetricultures of species-specific cells. (b) Apparent permeability (Papp) of 70kDa dextran tracer for hBBB

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

model cultured on AKITA Plate with and without dynamic flow. (c) Papp of 70kDa dextran tracer for
mBBB model cultured on AKITA Plate with and without dynamic flow. (d) Transmission electron
microscopy image from tight junction structure of the endothelial cells of the mBBB cultured on AKITA
Plate. Tight junctions between the endothelial cells are marked with a black arrow (e) Papp of 70kDa
dextran during the reversible barrier opening assay for mBBB model. (f) Confocal image of the mBBB
cultured on AKITA® Plate showing co-culture monolayers on opposing sides of the porous membrane.
Tight junction expression can be seen only in the endothelium side of the mBBB model. Blue DAPI,
green CD31, red phalloidin. (g) Live imaging of BBB and organoid co-culture on AKITA® Plate,brightfield and Calcein AM signals shown. Data were analyzed using Two-way ANOVA and Tukey’s
multiple comparison tests, **p<0,01, ***p<0,001, N=3

3.5. Skin-on-chip
The structure of our full-thickness skin-on-chip model is displayed in Fig. 5a with a basal layer of dermal
fibroblast covered by keratinocytes. Because high amounts of calcium have been shown to increase
the maturation of keratinocytes, both high and low concentrations have been tested in our skin-onchip model 46.
The dermal fibroblasts were cultivated for three days prior to the addition of keratinocytes. During
the 17 days of tissue maturation, barrier integrity was monitored by TEER and showed a progressive
tightness increase over time (Fig. 5b). After 14 days of co-culture required for keratinocyte maturation
47,48(experiment day 17), barrier permeability was compared between TEER (Fig. 5c) and the diffusionof 0.4 kDa low molecular weight dextran from top to bottom (Fig. 5d). We noticed a high correlation
between both TEER and dextran measurements (Fig. 5b-c). While both datasets showed a low impact
of calcium concentrations on barrier permeability, an increased permeability under flow condition
was observed (Fig 5. b-c). Those results indeed correlate with the poor physiological relevance of
having flow in skin tissue; the addition of an endothelial cell layer may be required to elicit improved
flow - and physiological relevance - outcomes.
Finally, the stratification of the skin tissue was observed by confocal microscopy in the Akita plate.
Keratinocyte cornification was monitored with Loricrin 49 while Collagen IV labels the collagen basal
membrane 50 (Fig. 5e). Both stains show higher tissue stratification in the low calcium concentration
which contrasts with previous studies 46. In our system, the air-liquid interface only allows the deliveryof culture media to the basal fibroblasts, which may impair the latter. Such observation confirms that

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

our skin model may be used to study impairment in skin stratification, barrier integrity, and compound
absorption.

Figure 5. Skin-on-chip on AKITA Plate. (a) A scheme representing the skin-on-chip model in AKITA®
Plate. Fibroblasts and keratinocytes are seeded on top of each other and cultured under the air-liquidinterface. (b) TEER values for units measured during the model maturation up to 17 days, day 0
corresponds to fibroblast seeding. The units are cultured with or without dynamic flow, as well as withor without extra Ca2+ in the culture medium (High/Low). As a control fibroblast alone cultivated in static
condition and low Ca2+ are displayed here (Fib.). (c) The TEER values acquired at tissue maturation, daybioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

17 are plotted for statistical analysis. The no-cell control (blank) is available for comparison. (d)Apparent permeability values for the same skin-on-chip units measured with 0,4 kDa dextran. (e) Crosssectional confocal images from stained skin-on-chip models cultured either with high or low Ca2+
medium. Stained with DAPI (blue), phalloidin (green), and collagen IV (left panel, red) or loricrin (rightpanel, red). N=3 for monocultures and N =12 for cocultures and no-cell controls (blanks) except N =6
for coculture units with low Ca2+ medium with 4 kDa permeability. Data were analyzed using two-way
ANOVA tests and Tukey’s multiple comparison tests for dextran permeability and Kruskal-Wallis test
together with the Dunn test for panel (c).

3.6. Lung-on-chip
The application capacity of a high-throughput platform for lung modeling was optimized with the coculture of Adenocarcinoma human alveolar basal epithelial A549 cells and human umbilical vascularendothelial cells (HUVEC) as depicted in Figure 6a. Confocal microscopy analysis of a day 10 epithelium
cultivated in an air-liquid interface shows the expression of the maturation marker mucus protein
MUC5AC on the apical side of the epithelium (Fig. 6b).
Additionally, we show already after 2 days of air-liquid interface culture that the combination of bothflow and air-liquid interface improve barrier tightness observed with the 70 kDa dextran tracer (Fig.
6c). Surprisingly, for the 4 kDa lower molecular size tracer, both flow and air-liquid interface increased
the tightness of the model even alone (Fig. 6d). Overall, dynamic flow seems to have a higher effecton barrier tightness, as reported previously 51. With the 4kDa dextran tracer, our model appears toachieve higher tightness compared to previous studies which obtained 8,44E-6 cm/s 52, 1.70E-5 cm/s
53

. While with the 70kDa tracer our models show comparable permeability compared to literature

3,8E-6 cm/s 53 and 6,98E-8 cm/s 54. In contrast with Chang et al. 55, the air-liquid interface had a limited
impact on the tightness of our system, likely due to the use of different cell lines in the studies.
The A549 are type II cells that exhibited the formation of microvilli on their apical side observed byelectron microscopy (Fig. 6e) 56,57. Together with the apical expression of the mucus protein MUC5AC,(Fig. 6f), this further confirmed the polarization of the lung epithelium and demonstrated its
functionality. As can be seen from Figure 6f, dynamic flow also increased mucus production comparedto the static culture. This model may be further used for the high throughput assessment of drug
compound absorption, the test of anti-inflammatory compounds, or the effect of pro-inflammatory
stimuli and pollutants on lung-on-chip barrier integrity.

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 6. Lung-on-chip on AKITA Plate. (a) Scheme representing the lung-on-chip model in AKITA®
Plate. Lung epithelial cells (A549) and endothelial cells (HUVEC) are seeded on the other side of theporous membrane. (b) Confocal image of the A549 on the lung-on-chip barrier. Blue DAPI, green
MUC5AC, red phalloidin. (c) Apparent permeability values (Papp) for lung-on-chip units cultured withor without dynamic flow, and with or without an air-liquid interface (ALI) measured with 70 kDa
dextran tracer. (d) Apparent permeability values (Papp) for lung-on-chip units cultured with or without
dynamic flow, and with or without an air-liquid interface (ALI) measured with 4 kDa dextran tracer. (e)SEM image from microvilli structure on top of the epithelial layer cultured under the air-liquid interface
in AKITA Plate. (f) Cross-sectional confocal images of lung-on-chip barriers cultured either in dynamic

bioRxiv preprint doi: https://doi.org/10.1101/2023.08.02.551612; this version posted August 5, 2023. The copyright holder for this preprint(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

flow or static environment. Stained with DAPI (blue) and MUC5AC (green). Data were analyzed using
Two-way ANOVA tests and Tukey’s multiple comparison tests. N=6.

4. Conclusions
We present a versatile and scalable OOC platform, providing a low to high-throughput reproducible
solution for in vitro tissue barrier modeling with pumpless flow control. It may be used to study thepharmacodynamics of drug compounds crossing biological barriers, and barrier integrity via rapid
label-free TEER measurements, of high-content screening with confocal and standard fluorescent
microscopy. The current laboratory equipment such as microscopes, automated liquid handlingrobots, high-content screening systems, etc. can adopt the AKITA Plates into their workflows
seamlessly thanks to the standard design and simplicity of the plate handling.

Declaration of Interest statement
The authors have filed patents for technologies presented in this paper. The authors declare no
conflict of interest.

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