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\begin{document}

\title{A Generic Pump-free Organ-on-a-Chip Platform for Assessment of Drug
Bioavailability}



\author[1]{Yaqiong Guo}%
\author[1]{Yingying Xie}%
\author[1]{Jianhua Qin}%
\affil[1]{Dalian Institute of Chemical Physics Department of Biotechnology}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Organ-on-a-chip technology has shown great potential in disease modeling
and drug evaluation. However, traditional organ-on-a-chip devices are
mostly pump-dependent with low throughput, which makes it difficult to
leverage their advantages. In this study, we have developed a generic,
pump-free organ-on-a-chip platform consisting of a 32-unit chip and an
adjustable rocker, facilitating high-throughput dynamic cell culture
with straightforward operation. By utilizing the rocker to induce
periodic fluid forces, we can achieve fluidic conditions similar to
those obtained with traditional pump-based systems. Through constructing
a gut-on-a-chip model, we observed remarkable enhancements in the
expression of barrier-associated proteins and the spatial distribution
of differentiated intestinal cells compared to static culture.
Furthermore, RNA sequencing analysis unveiled enriched pathways
associated with cell proliferation, lipid transport and drug metabolism,
indicating the ability of the platform to mimic critical physiological
processes. Additionally, we tested seven drugs which represent a range
of high, medium, and low in vivo permeability using this model and found
a strong correlation between their Papp values and human Fa, indicating
reliable and predictive simulation outcomes for drug absorption. Our
findings highlight the potential of this pump-free organ-on-a-chip
platform as a valuable tool for advancing drug development and enabling
personalized medicine.%
\end{abstract}%



\sloppy


\textbf{AGeneric Pump-free Organ-on-a-Chip Platform}

\textbf{for Assessment of Drug Bioavailability}

\textbf{Yaqiong Guo\textsuperscript{1*}, Yingying
Xie\textsuperscript{1,5*},Jianhua Qin\textsuperscript{1,2,3,4,5}}

\textsuperscript{1}Division of Biotechnology, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian, China

\textsuperscript{2}Beijing Institute for Stem Cell and Regenerative
Medicine, Chinese Academy of Sciences, Beijing, China.

\textsuperscript{3}University of Science and Technology of China, Hefei,
China

\textsuperscript{4}Suzhou Institute for Advanced Research, University of
Science and Technology of China, Suzhou, China

\textsuperscript{5}University of Chinese Academy of Sciences, Beijing,
China.

\textsuperscript{*} These authors equally contributed to this work.

Corresponding author: Jianhua Qin.

E-mail address: \emph{jhqin@dicp.ac.cn}

\textbf{Key words} : organ-on-a-chip, in-vitro model, high-throughput,
drug testing

\textbf{Abbreviations used in the paper:}

CCK-8: Cell counting kit-8 assay

Fa: fraction absorbed

FBS: fetal bovine serum

FITC: fluorescein isothiocyanate

GO: gene ontology

H\&E: Hematoxylin and eosin

HBSS: Hank's Balanced Salt Solution

KEGG: Kyoto Encyclopedia of Genes and Genomes

P\textsubscript{app}: apparent permeability coefficient

PBS: phosphate-buffered saline

PC: polycarbonate

PET: polyethylene terephthalate

PFA: paraformaldehyde

PS: penicillin/streptomycin

RNA-Seq: RNA sequencing

SEM: scanning electron microscopy

TEER: transepithelial electrical resistance

\par\null

\textbf{Abstract:}

Organ-on-a-chip technology has shown great potential in disease modeling
and drug evaluation. However, traditional organ-on-a-chip devices are
mostly pump-dependent with low throughput, which makes it difficult to
leverage their advantages. In this study, we have developed a generic,
pump-free organ-on-a-chip platform consisting of a 32-unit chip and an
adjustable rocker, facilitating high-throughput dynamic cell culture
with straightforward operation. By utilizing the rocker to induce
periodic fluid forces, we can achieve fluidic conditions similar to
those obtained with traditional pump-based systems. Through constructing
a gut-on-a-chip model, we observed remarkable enhancements in the
expression of barrier-associated proteins and the spatial distribution
of differentiated intestinal cells compared to static culture.
Furthermore, RNA sequencing analysis unveiled enriched pathways
associated with cell proliferation, lipid transport and drug metabolism,
indicating the ability of the platform to mimic critical physiological
processes. Additionally, we tested seven drugs which represent a range
of high, medium, and low \emph{in vivo} permeability using this model
and found a strong correlation between their P\textsubscript{app} values
and human Fa, indicating reliable and predictive simulation outcomes for
drug absorption. Our findings highlight the potential of this pump-free
organ-on-a-chip platform as a valuable tool for advancing drug
development and enabling personalized medicine.

\textbf{Introduction}

In the recent decade, researchers have continuously developed many in
vitro models, from 2D to 3D, and particularly, organ chips have evolved
from conceptual to powerful tools that hold the potential to partially
replace traditional in vitro models and animal
models.\textsuperscript{{[}1-3{]}} Nevertheless, the widespread adoption
of organ chip systems currently faces certain challenges, including the
reliance on labor-intensive external pumps and limited
throughput.\textsuperscript{{[}4-6{]}} In addition, although
microfluidic systems reduce the use of cells and drugs, they also
increase the difficulty of characterization and detection. For example,
more precise manual operations or complex sensors are often required
when measuring TEER, which can be challenging to achieve in small
chips.\textsuperscript{{[}7, 8{]}}Overcoming these challenges
necessitates addressing both biological and engineering obstacles. This
endeavor entails developing innovative microfluidic designs or platforms
capable of precisely controlling the flow of fluids, as well as
establishing reproducible and physiologically relevant cell culture
microenvironment.\textsuperscript{{[}9-11{]}}

Currently, a variety of organ chips, such as
intestine-on-a-chip,\textsuperscript{{[}12{]}}liver-on-a-chip,\textsuperscript{{[}13{]}}brain-on-a-chip,\textsuperscript{{[}14{]}}
have been developed. Of the various organ-on-chip devices, the
intestine-on-a-chip are one of the most widely used models. A
traditional intestine-on-a-chip is typically composed of two channels
separated by a porous membrane lined with intestinal epithelial
cells.\textsuperscript{{[}15-17{]}} To replicate the dynamic in vivo
microenvironment, the medium is commonly circulated through the channels
using either syringes or peristaltic pumps. Intestine-on-a-chip systems
allow for the analysis of drug absorption, toxicity, and efficacy, as
well as the simulation of intricate host-microbe
interactions.\textsuperscript{{[}18-21{]}}

As we all know, the Caco-2 monolayer cultured on a transwell insert is
widely recognized as one of the most commonly utilized models for
studying human intestinal barrier absorption due to its simplicity and
cost-effectiveness.\textsuperscript{{[}22{]}}However, despite the
widespread use of the transwell model in research, the cell culture
process involved a prolonged time period, and the two-dimensional growth
of cells in static culture does not entirely replicate the in vivo
environment.\textsuperscript{{[}23, 24{]}} Due to the effectiveness of
organ chips in advancing the development of novel drugs and therapies,
it offers a more physiologically relevant and reproducible alternative
to traditional in vitro models.\textsuperscript{{[}25{]}} On the other
hand, existing chips used for drug evaluation often require
high-precision instruments for detection due to low drug volume, which
potentially increases the cost of application. Therefore, developing a
high-throughput organ chip system that eliminates the need for complex
pumps and valves has the potential to revolutionize drug discovery and
toxicity testing.

\par\null

In this study, we present a generic, pump-free and high-throughput 3D
organ-on-a-chip platform that enables dynamic cell culture in vitro
similar to traditional chip system. A rocker was utilized to generate
periodic fluid force in our system which replaces the pump and
intertwined pipelines. Using our platform, we established a
gut-on-a-chip system that simulates human intestine. 3D stereoscopic
structure and enhanced expression of intestinal barrier-associated
proteins (ZO-1) was observed in our platform, which was deficient in
static cultures. We also observed the spatial distribution of
differentiated (Villin, MUC-2) and proliferative cells (Ki67) markers,
suggesting that cell differentiation on the chip occurs similarly to
that in vivo. RNA sequencing analysis demonstrated that cells showed
significantly enriched pathways and up-regulated genes associated with
drug metabolism function compared to those in static cultures. Based on
these findings, we evaluated the drug bioavailability using our platform
and achieved reliable and predictive simulation results, thus providing
a promising tool for predicting the intestinal drug absorption of new
drugs and a valuable asset for drug development and personalized
medicine.



\textbf{Materials and Methods}

\textbf{Device fabrication}

The upper and lower layers of the chip were designed using SolidWorks
software and sent to Shenzhen Future Factory for mold production and
injection molding. After chip cleaning, the ultra-thin double-sided
adhesive was precisely cut into the defined shape and then being used to
securely encapsulate a polyethylene PET film between the upper and lower
layers of the chip in a controlled dust-free environment. The PET film
had a thickness of 10 \selectlanguage{greek}μ\selectlanguage{english}m and a pore size of 2 \selectlanguage{greek}μ\selectlanguage{english}m. To ensure the sterile
conditions for cell culture, the assembled chips underwent overnight UV
irradiation and additionally treated with a 5\% PS solution for a
minimum of 4 hours before being utilized. The specific chip size can be
obtained in fig. S1.

\textbf{Cell culture}

Caco-2 cells were cultured in high-glucose Dulbecco's modified Eagle's
medium supplemented with 10\% FBS and 1\%PS. HT-29 cells were maintained
in McCoy's 5A medium supplemented with 10\% FBS and 1\% PS. All cells
were cultured in an incubator maintained at 37  with a 5\%
CO\textsubscript{2} atmosphere. All cell culture reagents are purchased
from Gibco.

To seed the cells on the PET membrane, a ratio of 9:1 was used,
resulting in a total cell count of approximately 50,000 cells. The
cell-seeded chips were then placed on a rocker operating at 10 rpm and \selectlanguage{ngerman}±
8°, facilitating gentle fluid perturbation within the middle hole and
ensuring regular fluid flow in the underlying channels. The culture
medium was refreshed every two days to maintain cell viability and
provide essential nutrients for growth. In terms of media
supplementation, 150 \selectlanguage{greek}μ\selectlanguage{english}L was introduced into the middle hole of the chip,
while 600 \selectlanguage{greek}μ\selectlanguage{english}L was added to the lower layer. This allocation aimed to
maintain fluid level equilibrium between the middle hole and the two
side holes, and ensuring adequate nutrient supply and waste removal
throughout the experiment.

\textbf{Finite element simulation}

Simulations were conducted using the ''two-phase laminar flow'' and
''incompressible Navier-Stokes'' application modes within the Comsol
Multiphysics Finite Element Program (COMSOL 3.5, Comsol AB, Burlington)
to visualize the flow characteristics along the centerline of the upper
and lower surfaces of the membrane. The oscillatory motion was applied
with the bottom center point as the axis, employing an angular
displacement of \selectlanguage{ngerman}± 8° and a period of 6 seconds. The results revealed
that the distribution of fluid shear stress on the membrane surface
exhibited intermediate symmetry and temporal variation.

\textbf{Live/Dead staining}

Following the protocol of the LIVE/DEAD® Viability/Cytotoxicity Assay
Kit (Invitrogen, America), a working solution was prepared by combining
20 \selectlanguage{greek}µ\selectlanguage{english}L of EthD-1 stock solution and 5 \selectlanguage{greek}µ\selectlanguage{english}L of calcein AM stock solution in
10 mL of D-PBS (Meilunbio, China). This working solution was applied to
cells that had undergone two washes with D-PBS. After a 30-minute
incubation, fluorescence microscopy was employed to capture images.

\textbf{CCK-8 assay}

On the sixth day of culture, a complete medium containing 10\% CCK-8
(Meilunbio, China) was prepared. The cells were washed twice with PBS
and then treated with the respective FITC-dextran solutions. After one
and two hours of treatment, 100 \selectlanguage{greek}μ\selectlanguage{english}L of medium from the lower layer was
collected to measure the fluorescence intensity using a plate reader
(Infinite\selectlanguage{ngerman}® 200 PRO, Tecan, Switzerland).

\textbf{Immunofluorescence imaging}

The PET membrane with cells was carefully excised, and washed with PBS
twice. Subsequently, the cells were fixed with 4\% PFA for 15 minutes.
Permeabilization was achieved by treating the cells with 0.2\% Triton
X-100 for 7 minutes, followed by blocking with goat serum for 40 minutes
at room temperature. The samples were then incubated overnight at 4 \selectlanguage{english}
with a diluted antibody solution of ZO-1 (Abcam, ab221547, UK), MUC-2
(Abcam, ab11197, UK), Villin (Abcam, ab109516, UK) or Ki67 (Abcam,
ab16667, UK), and subsequently with secondary antibodies at room
temperature for 1 hour, protected from light. Hoechst (Meilunbio, China)
staining was used to visualize the nuclei. For visualization of F-actin,
samples were stained with phalloidin (Solarbio, China). Washing with PBS
was performed three times. Confocal microscopy (FV-1000; Olympus, Tokyo,
Japan) was utilized to capture images. Image processing was conducted
using Image J.

\textbf{H\&E staining imaging}

The PET membrane with cells was cut off carefully, washed with PBS twice
and fixed with 4\% PFA for more than 24 hours and dehydrated in a graded
series of alcohols in a dehydrator followed by being embedded in
paraffin and sliced. Then, the paraffin-embedded sample was dewaxed in
an environmentally friendly dewaxing solution, followed by dehydration
in a series of alcohol solutions and a final water wash. The sections
were stained with Hematoxylin for 3-5 minutes, rinsed with water,
differentiated, rinsed again, and counterstained with Eosin for 5
minutes. Then the sections were dehydrated in a series of alcohol
solutions and cleared with xylene, and mounted with neutral resin.
Finally, the sections were viewed under a microscope, and images were
taken for analysis. The cell nuclei appear blue, while the cytoplasm
appears red.

\textbf{SEM imaging}

To prepare a sample for SEM imaging, PET membrane with fixed cells were
washed with PBS and then fixed with electron microscopy fixative. The
sample was then washed with PBS for 3 times (15 min each) and post-fixed
with OsO\textsubscript{4}. After being dehydrated with a series of
ethanol concentrations (30\%, 50\%, 70\%, 80\%, 90\%, 95\% ethanol for
15 min, two changes of 100\% ethanol for 15 min and isoamyl acetate for
15 min), the sample was dried with a critical point dryer, attached to
metallic stubs, and coated with gold before being observed and imaged
with a scanning electron microscope.

\textbf{TEER measurement}

The TEER measurement was based on commercial EVOM3 instrument (World
Precision Instruments, America) and a simple device compatible with our
chip for measuring TEER was designed \textbf{(Fig. S2)} . Four platinum
wires were fixed on the PC cover after being cleaned several times with
Piranha solution (A mixture of concentrated sulfuric acid and 30\%
hydrogen peroxide (3:1)), acetone, ethanol, and water, directly
inserting into the solution as electrodes after sterilization by wiping
with 70\% ethanol. Washed the cell by PBS buffer once and the
measurement was carried out in the PBS after the electrode was soaked in
PBS for half an hour before use. The blank channel of the chip was
measured in PBS before inoculating cells as blank control.

\textbf{Permeability test}

FITC labeled dextran with molecular weights of 40 kDa and 4 kDa was
prepared at a concentration of 1 mg/ml using complete medium. The cells
were washed twice with PBS and then treated with the respective
FITC-dextran solutions. After one hour and two hours of treatment, 100
\selectlanguage{greek}μ\selectlanguage{english}L of medium from the lower layer was collected to measure the
fluorescence intensity using a plate reader (Infinite\selectlanguage{ngerman}® 200 PRO, Tecan,
Switzerland).

\textbf{RNA} \textbf{sequencing}

Total RNAs were extracted using TRIzol Reagent and DNA digestion was
performed using DNase I. The quality of RNA samples was assessed by
measuring the A260/A280 ratio using the Nanodrop\textsuperscript{TM} One
spectrophotometer (Thermo Fisher Scientific Inc). Furthermore, RNA
integrity was confirmed by conducting 1.5\% agarose gel electrophoresis.
Finally, qualified RNA samples were quantified using the Qubit3.0 with
the Qubit\textsuperscript{TM} RNA Broad Range Assay kit (Life
Technologies, Q10210). For stranded RNA sequencing library preparation,
2 \selectlanguage{greek}μ\selectlanguage{english}g of total RNA was utilized. The
KC-Digital\textsuperscript{TM}Stranded mRNA Library Prep Kit for
Illumina\selectlanguage{ngerman}® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd., China) was
employed, following the manufacturer's instructions. This kit
incorporates unique molecular identifiers consisting of 8 random bases
to label the pre-amplified cDNA molecules. This approach helps to
mitigate duplication bias during PCR and sequencing steps. Library
products within the range of 200-500 bps were enriched, quantified, and
subsequently sequenced on the DNBSEQ-T7 sequencer (MGI Tech Co., Ltd.,
China) using the PE150 model.

\textbf{RNA-Seq data analysis}

The initial raw sequencing data underwent preprocessing steps.
Trimmomatic (version 0.36) was employed to filter out low-quality reads
and remove reads contaminated with adapter sequences. Clean reads were
further processed using in-house scripts to address duplication bias
introduced during library preparation and sequencing.

The de-duplicated sequences were aligned to the reference genome of Homo
sapiens using STAR software (version 2.5.3a) with default parameters.
Reads mapped to the exon regions of each gene were counted using
featureCounts (Subread-1.5.1; Bioconductor), and Reads Per Kilobase per
Million mapped reads values were calculated. Differential gene
expression analysis between groups was conducted using the edgeR package
(version 3.12.1). Statistical significance of gene expression
differences was assessed using a p-value cutoff of 0.05 and a
fold-change cutoff of 2. GO analysis and KEGG enrichment analysis for
differentially expressed genes were performed using KOBAS software
(version: 2.1.1). A p-value cutoff of 0.05 was used to determine
statistically significant enrichment.

\textbf{Drug bioavailability evaluation}

Solutions of seven drugs (Antipyrine, Propranolol hydrochloride,
Hydrochlorothiazide, (±)-Sulpiride, and Ranitidine hydrochloride) were
purchased from Shanghai Aladdin Biochemical Technology. Atenolol and
Furosemide were purchased from Dalian Meilune Biochemical Technology.
The drugs were prepared at a concentration of 2 \selectlanguage{greek}μ\selectlanguage{english}mol/ml in HBSS from
Meilunbio. Prior to drug absorption evaluation, cells were washed twice
with HBSS for 30 minutes. Then, 150 \selectlanguage{greek}μ\selectlanguage{english}L of the drug solution was added to
the middle well, while 600 \selectlanguage{greek}μ\selectlanguage{english}L of HBSS was added to the lower well. After
a 3-hour incubation, 500 \selectlanguage{greek}μ\selectlanguage{english}L of medium from the lower well was collected
and centrifuged at 8000 rpm for 10 minutes. The supernatant was
quantified by liquid chromatography (LC20A, Shimadzu, Japan) with a
chromatographic column size of 250 mm*4.6 mm, 5 \selectlanguage{greek}μ\selectlanguage{english}m, C18. The injection
volume was 10 \selectlanguage{greek}μ\selectlanguage{english}L. Antipyrine was detected at a wavelength of 254 nm,
Propranolol hydrochloride at 223 nm, Verapamil at 278 nm,
Hydrochlorothiazide at 270 nm, Ranitidine hydrochloride at 230 nm,
Atenolol at 240 nm, (\selectlanguage{ngerman}±)-Sulpiride at 230 nm, and Furosemide at 279 nm.

\textbf{Statistical analysis}

Data were expressed as the means ± SEM and analyzed by performing
Student's t-test. Significance levels were denoted as follows: *p
\textless{} 0.05, **p \textless{} 0.01, and ***p \textless{} 0.001. The
sample size is at least three and all experiments have been repeated at
least twice. In addition, the data were handled with Excel, PowerPoint,
and GraphPad Prism.

\textbf{Result}

\textbf{Design and operation of the generic organ-on-a-chip platform}

As shown in \textbf{Fig. 1A} , each chip consists of 32 chip units, with
each chip unit consisting of three horizontally arranged wells. The
holes at both ends are connected by the passageway of the lower channel,
where culture the medium was perfused with a rocker. The middle hole is
designated for cell culture and is separated from the lower layer by a
transparent PET porous membrane. The specifications of the porous
membrane can be chosen according to specific requirements. This design
not only reduces the consumption of culture medium and other materials
but also facilitates pipetting operations and characterizations with
enhanced convenience.

To validate the usability of the chip, we constructed a simple
gut-on-a-chip model. In the specific experiments, Caco-2 and HT-29 cells
were seeded in a 9:1 ratio, totaling about 50,000 cells, in the central
well of the chip unit to form a compact layer of intestinal epithelial
cells on the membrane \textbf{(Fig. 1B)} . Then the chip was placed on a
rocker at a speed of 10 rpm and an angle of ± 8° to induce basal fluid
flow. To further investigate the fluid dynamics within the chip,
computational simulations were performed using COMSOL software. The
results indicate that the fluid shear stress (\textasciitilde{}0.4
dyn/cm\textsuperscript{2}) at the center of the membrane was relatively
lower compared to the surrounding regions (0.8\textasciitilde{}2
dyn/cm\textsuperscript{2}) \textbf{(Fig. 1C)} . Additionally, the fluid
shear stress beneath the membrane remained stable at around 0.4
dyn/cm2\textbf{(Fig. S3)} . These observed shear force values align well
with physiological data.\textsuperscript{{[}26{]}}

It should be noted that although we focus on seeding cells on the upper
side of the membrane in this work, the chip design \textbf{(Fig. 1B)}
allows for easy cell seeding on the opposite side of the membrane by
simply seeding cells in the bottom channel and inverting the chip. This
unique feature enables the co-culture of multiple cell types, thus
highlighting the scalability of our system and its potential for diverse
applications. For instance, a multicellular model of inflammatory bowel
disease can be established on this platform by seeding intestinal
epithelial cells in the central well, inoculating intestinal endothelial
cells on the opposite side of the membrane, and introducing immune cells
into the bottom channel. Additionally, the versatility of the system
extends further with the ability to control the cultivation conditions.
By adjusting the angle and speed of the rocker, different shear forces
can be easily applied to achieve distinct cultivation conditions for
various experimental needs.

\textbf{Characterization of the intestinal epithelium on
theorgan-on-a-chip platform}

Previous studies have elucidated the crucial role of basal-side fluid
flow in promoting the morphogenesis of Caco-2
cells.\textsuperscript{{[}27, 28{]}} This effect is attributed to the
accumulation of Wnt signaling pathway inhibitors, such as DKK-1, beneath
the membrane under static conditions, leading to hindered cell
proliferation and impaired morphogenesis of the Caco-2 cell layer. The
introduce of fluid flow can effectively eliminate the accumulation of
these inhibitory molecules. In our research, a rocker with adjustable
angle and speed was employed to induce reciprocating fluid flow. This
dynamic flow not only dilutes the inhibitory molecules but also
facilitates the occurrence of intestinal-like morphogenesis that closely
mimics in vivo conditions.

In our chip system, a prominent distinction was observed between the
perfused and static conditions. Under perfusion conditions, the
intestinal cell layer cultured on the chip developed 3D villous
structures, while cells without perfusion predominantly remained in a
single-layer state. After three days of culture with fluid, noticeable
disparities were observed under an optical microscope \textbf{(Fig. 2A)}
. The intestinal cells under flow conditions formed convex and stacked
structures, appearing as dark arc curves or circular structures with
well-defined boundaries. Similar to transwell or petri dish culture,
intestinal cells can be cultured on the chip for up to 30 days, but
after the sixth day, there is no significant change in the morphology of
the cells under the optical microscope \textbf{(Fig. S4)} .
Consequently, cells after six days of fluid introduction were taken for
characterization.

Live/dead staining images revealed that most of the cells exhibited
normal growth after six days of perfused culture on the
chip\textbf{(Fig. 2D)} . Additionally, a CCK-8 assay was performed to
assess cell viability under static and perfused conditions,
demonstrating that the viability of cells cultured on the chip was
comparable to or even higher than that of cells cultured in a static
state \textbf{(Fig. 2E)} . Furthermore, differences in cell layer height
and microvillus morphology were observed through H\&E staining
\textbf{(Fig. 2B)} and SEM\textbf{(Fig. 2C)} , respectively. These
results highlight the successful establishment of an intestinal-like
morphogenesis on our chip system under perfusion conditions.

\textbf{Improved functionality of intestinal epithelium under flow
condition}

To assess the integrity of the cell layer, we evaluated the permeability
of the intestinal barrier by measuring the fluorescence intensity of
FITC-dextran with varying molecular weights that permeated the cell
layer within a specific time frame \textbf{(Fig. 2F)} . Notably,
regardless of molecular weight, the P\textsubscript{app} of dextran in
the perfused chip was significantly lower than that observed in the
static culture. In the 1-hour experiment, the P\textsubscript{app} of 4
kDa-dextran was as low as 5 x 10\textsuperscript{-7}cm/s, indicating a
substantial enhancement in the integrity of the intestinal barrier in
the perfusion environment.

To further assess the integrity of the intestinal barrier, we measured
the TEER using a commercially available instrument, EVOM3, which was
facilitated by our chip design \textbf{(Fig. S3)} . Interestingly, there
was minimal disparity between the TEER values of the perfused and static
cultures \textbf{(Fig. 2G)} , deviating from previous studies. Both
groups displayed an upward trend in TEER over the six-day culture
period, eventually reaching an approximate value of 100
\selectlanguage{greek}Ω\selectlanguage{ngerman}·cm\textsuperscript{2}. While this value may seem relatively low
compared to other reports, it is essential to note that co-culturing
Caco-2 and HT-29 cells typically leads to lower TEER
values.\textsuperscript{{[}29, 30{]}} Consequently, the observed TEER
value aligns more closely with physiological conditions in humans.

Immunofluorescence staining of ZO-1 \textbf{(Fig. 3C)} revealed the
presence of well-formed tight junctions and brush border formation in
the perfusion environment, indicating a more robust barrier compared to
static culture. Moreover, the detection of Villin \textbf{(Fig. 3F)} in
apical cells provided additional evidence for the presence of intestinal
villi in the perfusion environment, which was further supported by SEM
images showing well-defined microvilli \textbf{(Fig. 2C)} . Furthermore,
by analyzing the distribution of nuclei in images at different levels,
we could clearly visualize the hollow structure of intestinal
villi\textbf{(Fig. 3A)} . The expression of MUC-2 was also detected. As
shown in \textbf{Fig. 3D} , MUC-2 was evenly distributed on the
intestinal villus, and superposition with the bright field image
provided a clear view of the villi's shape. Side-view confocal 3D images
allowed us to calculate the height of the villi, and the results
indicated that the average villi height in static culture was about 33
\selectlanguage{greek}μ\selectlanguage{english}m \textbf{(Fig. 3E)} , while the average villi height on the chip was
close to 90 \selectlanguage{greek}μ\selectlanguage{english}m. Additionally, the expression of Ki67 \textbf{(Fig. 3G)}
indicated active proliferation of intestinal cells, suggesting the
potential for further growth into higher intestinal villi.

\textbf{Transcriptional analysis of the intestinal cells}

To gain a comprehensive overview of the transcriptional responses to
basal fluid, we performed RNA-seq analysis of intestinal epithelial
cells in the chip. Differentially expressed genes were identified to
elucidate the transcriptomic differences between the presence and
absence of fluid. Based on the biological repeated correlation test of
the samples, a clear distinction could be observed between the perfused
and static groups, while within each group, independent reproducibility
was confirmed \textbf{(Fig. 4B)} . By applying the criteria of an
absolute value of log fold change \textgreater{} 1 and a p-value
\textless{} 0.05, we identified 151 up-regulated genes and 304
down-regulated genes in the perfused group compared to the static
condition \textbf{(Fig. 4A)} .

The biological processes associated with these genes were further
characterized using GO and KEGG pathway enrichment analysis. Using the
GO database, we classified the differentially expressed genes into
biological process, molecular function, and cell component categories.
Among all the enriched terms, the biological process classification was
the most prominent and of primary interest. \textbf{Fig. 4C} presents
the top 20 terms in descending order of p-value. Notably, in the context
of our system, several up-regulated genes of cell under fluid flow were
associated with cellular activities, including ``regulation of
epithelial cell proliferation'', ``negative regulation of endothelial
cell apoptotic process'', and ``cellular response to dexamethasone
stimulus''. Additionally, terms related to material metabolism, such as
''negative regulation of cholesterol storage'', ''linoleic acid
metabolic process'', and ''glutathione derivative biosynthetic
process'', indicated that fluid stimulation impacts the metabolic
function of cells. Interestingly, in the enrichment analysis of
down-regulated genes, we identified the terms ``cellular response to
fluid shear stress'' and ``cellular response to mechanical stimulus''.
Furthermore, terms related to cell adhesion, such as ``cell surface''
and ''anchored component of membrane'', were also present. Whether this
is a negative feedback regulation of cells in the gut-on-a-chip system
we constructed, it still needs a further exploration.

Furthermore, the KEGG enrichment analysis \textbf{(Fig. 4D)} revealed
that the presence of fluid in the culture enhances several pathways
associated with drug metabolism compared to static culture. These
pathways include ``Metabolism of xenobiotics by cytochrome P450'',
``Drug metabolism - other enzymes'', and ``Drug metabolism - cytochrome
P450''. Moreover, the enrichment of up-regulated genes in pathways such
as ``Fat digestion and absorption'' and ``Metabolic pathways'' indicates
an improvement in the metabolic function of cells in the presence of
fluid. These findings underscore the ability of our platform to achieve
better physiological function simply by providing fluid with a rocker,
as compared to regular static cultivation.

\textbf{Assessment of drug bioavailability after exposure to}

\textbf{gut-on-a-chip}

The results of RNA-seq have heightened our focus on the potential
application of the gut-on-a-chip system in the field of in vitro drug
bioavailability evaluation. It is well-established that various orally
administered drugs exhibit different levels of absorption across the
epithelial barrier in vivo. As a result, an absorption test is essential
for each new drug before its clinical application. In this study, we
aimed to utilize the gut-on-a-chip system to measure the permeability
(P\textsubscript{app}) values of seven drugs: Antipyrine, Propranolol
hydrochloride, Hydrochlorothiazide, Ranitidine hydrochloride, Atenolol,
Sulpiride, and Furosemide. These drugs were selected based on the
Biopharmaceutics Classification System Biowaivers Guidance for Industry,
and they represent a range of high, medium, and low in vivo permeability
with well-documented bioavailability.\textsuperscript{{[}31{]}}

After cultivating cells on the chip using the consistent method, we
employed high-performance liquid chromatography to measure the drug
concentration reaching the bottom channel of the chip following a
three-hour drug incubation period \textbf{(Fig. 5A)} . Subsequently, we
established a correlation between the drug permeability coefficient and
its in-vivo bioavailability. The P\textsubscript{app} values were
determined using the following formula: P\textsubscript{app} = (dQ/dt) /
(A \selectlanguage{ngerman}· c\textsubscript{0}). In this equation, dQ represents the amount of
drug passing through the membrane during the experimental duration, dt
denotes the experimental time, A signifies the membrane area, and
c\textsubscript{0} corresponds to the initial concentration of the drug
introduced from the apical side. The experimental results showed a clear
positive correlation between the P\textsubscript{app} values and
bioavailability in both the static and perfused chips \textbf{(Fig. 5B)}
. However, the linear fit of the dynamic results was significantly
superior, with an R\textsuperscript{2} value of 0.7085 for the perfused
chip and only 0.3081 for the static chip. These findings indicate that
the perfused chip holds greater potential as a platform for predicting
drug absorption.



\textbf{Discussion}

In this study, we successfully developed a generic pump-free
organ-on-a-chip platform and used it to mimic the physiological
characteristics of the human intestinal barrier. After determining the
appropriate flow rate through fluid simulation, the intestinal cells
cultured on our platform proliferate and differentiate vigorously,
forming an intestinal barrier and demonstrating its function through the
oscillation of the rocker to provide fluid. Subsequently, we
investigated the impact of fluid dynamics on cells in our system using
RNA-seq analysis, which revealed significant differences in metabolic
pathways. Based on these promising results, we proceeded to evaluate the
bioavailability of drugs using our gut-on-a-chip model, achieving
excellent linear relationship simulations. These data conclusively
confirm the applicability of our platform and its potential as a
substitute for traditional organ chip models.

Looking ahead, our chip platform has the capability to simulate various
human physiological systems, particularly those at the interface in
vivo, such as the blood-brain barrier, alveoli, glomeruli, and more. By
enabling the culture of different types of cells on both sides of the
membrane and providing shear force through a rocker mechanism to
simulate different body fluid conditions, we can further enhance the
versatility of our platform. However, we recognize the need to address
specific limitations and challenges to maximize the potential of our
chip system. This includes enhancing the longevity and stability of
cultured cells, optimizing fluidic dynamics within the chip, and
expanding the range of cell types to better emulate the complexity of
the human body.

In the future, we hope to establish a powerful model that integrate
organ-on-a-chip technology with advanced analytical techniques and
computational models aiming to transcend the limitations of animal
testing, especially in drug development and toxicity studies. By
leveraging these cutting-edge tools, we anticipate achieving a deeper
understanding of drug behavior, leading to the identification and
development of safer and more effective therapeutics, ultimately
benefiting human health and transforming the landscape of pharmaceutical
research.

\par\null

\textbf{Acknowledgements}

This research was supported by the National Key R\&D Program of China
(No. 2022YFA1104700), National Nature Science Foundation of China (No.
31971373, 32101163), the Strategic Priority Research Program of the
Chinese Academy of Sciences, Grant (No. XDB29050301), Yunnan Key
Research and Development Program (No.202003AD150009), Innovation Program
of Science and Research from the DICP, CAS (DICP I202128).

\textbf{AuthorContributions}

Yaqiong Guo: Conceptualization, methodology, project administration,
visualization, and writing--review\&editing; Yingying Xie: Data
curation, investigation, project administration, writing and editing.
Jianhua Qin: Supervision, funding acquisition, writing--review\&editing.

\textbf{Conflict of Interest}

The authors declare no commercial or financial conflict of interest.

\textbf{Availability of data and materials}

We declare that all data generated or analyzed during this study are
included in this article.



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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig 1.} \textbf{Schematic illustration of the platform. (A)} The
schematic of chip and cell culture. The chips are transferred onto a
rocker placed in a cell culture incubator 24 hours after cell
seeding.\textbf{(B)} 3D sectional view of the chip. The central hole
displays the cell culture in the upper layer and the fluid flow in the
interconnected lower layer. \textbf{(C)} Computational simulations
reveal the magnitude of fluid shear stress above the membrane.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig 2.} \textbf{Characterization of the intestinal epithelium on
the organ-on-a-chip platform. (A)} Microscopic bright field image (Scale
bar: 100 um), \textbf{(B)} side HE staining image (Scale bar: 100 um)
and \textbf{(C)} SEM image (Scale bar: 10 um) of chip without (up) /with
(down) fluidic force provided by rocker. \textbf{(D)} Live/Dead staining
in static chip and perfused chip (Scale bar: 100 um). \textbf{(E)} Cell
activity in static chip and perfused chip by CCK-8 kit.
\textbf{(F)}Permeability test by detecting the fluorescence intensity of
FITC-Dextran crossing through the cell layer within a certain
time.\textbf{(G)} The TEER value of static chip (red) and perfused chip
(black).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig 3. Morphologic and differential characterization of the
intestinal epithelium on the gut-on-a-chip platform. (A)} The
longitudinal section of chip and the immunofluorescence images of cells
cultured in gut-on-a-chip with fluidic force, in which expression of
ZO-1 (red) at different height could be observed. Corresponding to the
schematic, I represents apical side and sequentially V represents basal
side. Besides, nuclei are dyed by Hoechst (blue) while F-actin is marked
by phalloidin (green), the same below. (Scale bar: 50 \selectlanguage{greek}μ\selectlanguage{english}m).
\textbf{(B)}Cross-sectional view of expression of ZO-1 in static chip
(top) and perfused chip (below). (Scale bar: 100 \selectlanguage{greek}μ\selectlanguage{english}m). \textbf{(C)} The
integral 3D image of intestinal cells layer in static chip (top) and
perfused chip (below). \textbf{(D)} Expression of MUC-2 in perfused chip
(Scale bar: 50 \selectlanguage{greek}μ\selectlanguage{english}m). \textbf{(E)} The height of villi measured by Image J
according to the side view of immunofluorescence.
\textbf{(F\&G)}Cross-sectional view of expression of Villin and Ki67 in
static chip (F) and perfused chip (G) (Scale bar: 50 \selectlanguage{greek}μ\selectlanguage{english}m).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig 4.} \textbf{Transcriptional analysis of the cells in
perfused or static condition.} \textbf{(A)} Volcano plots of the
dysregulated genes in perfusion culture. \textbf{(B)} Cluster analysis
of differentially expressed genes. \textbf{(C)} GO and \textbf{(D)} KEGG
pathway enrichment analysis of perfused chip contrast static chip
up-regulation (left) /down-regulation (right) differentially expressed
genes.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig 5. Application of gut-on-a-chip in drug bioavailability
assessment. (A)} The schematic of high throughout gut-on-a-chip used in
drug absorption prediction experiment. \textbf{(B)} Comparison of drug
permeation across cell barrier formed on static or perfused chip with
human in vivo bio-availability. Every point in the plot represents a
drug in the table.

\selectlanguage{english}
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