Modeling Gut Neuro-Epithelial Connections in a Novel Micro uidic Device

Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions – to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut’s neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.


Introduction
Epithelia are made up of a diversity of cell types.In the GI tract, all epithelial cells contribute to the barrier [1][2][3][4] , while specialized sensory cells called enteroendocrine cells (EECs) sense chemical [5] and mechanical [6] stimuli in the luminal space.Also common to epithelia-covered organs is the communication between epithelia and sensory neurons via neuro-epithelial connections.Neuro-epithelial connections underlie all of the major senses, including touch, taste, smell, hearing and sight.In the internal organs, the neuro-epithelial connections form the basis of interoception [7,8] .The gut is unique among the interoceptive organs because in addition to extrinsic neuronal innervation, it has an expansive intrinsic nervous system, affectionately known as the "second brain."Both extrinsic and intrinsic (enteric) sensory neurons form neuro-epithelial connections.Intriguingly, the gut's intrinsic primary afferent (sensory) neurons (IPANs) are genetically similar to the extrinsic primary afferent neurons (ExPANs) [9] .
The intrinsic neuro-epithelial sensory system in the gut involves epithelial sensing of luminal stimuli and relaying them to IPANs, which trigger a range of physiologic effects, such as control of vascular and intestinal smooth muscle and epithelial secretion, that are critical for proper digestion.Therefore, sensing luminal signals by the GI epithelium and its communication to enteric neurons are important elements contributing to GI function. [10]euro-epithelial interactions have been challenging to study both in vivo and in vitro.Characterization in vivo is confounded by complex tissue anatomies -innervation complexity and large distances between the soma of innervating neurons and their target epithelia, high turnover and migration of epithelial cells, and active motility.Given the epithelia's rapid turnover and the relative stability of neurons, culture conditions for these two populations differ dramatically.Epithelial cells have been cultured alone or cocultured with neurons in vitro [11][12][13][14] .However, these preparations require the use of culture conditions (media and substrate functionalization) that favor either epithelia or neurons, affecting their functions.The random neuro-epithelial connections in these cultures are challenging to orchestrate or selectively stimulate cellular compartments in a predictable manner.Our goal was to develop a culture system for placing intestinal epithelial and neuronal cells in distinct but proximal compartments to de ne and monitor neuro-epithelial connections (see Fig. 1).
Micro uidic devices have been used extensively for cultivation of mammalian cells.Some of the early examples included hepatocytes alone or together with stromal cells where micro uidic perfusion was used to supply oxygen and nutrients. [16,17] ubsequent work included cultures of intestinal epithelial cells within micro uidic devices providing shear and cyclical strain to stimulate and maintain phenotype of the cells. [15,18,19] Te complexity of micro uidic cultures has progressed from cell lines (e.g.[26][27] Despite the increasing complexity of gut-on-chip cultures, to the best of our knowledge, these devices have not incorporated and studied enteric neurons innervating and establishing contact with intestinal epithelial cells.
Micro uidic devices have also been used extensively for cultivation and studies of connectivity of neurons and glia. [28,29] 4] In this paper, we describe the development of a micro uidic device for co-cultivation of intestinal epithelial cells with enteric neurons.As shown in Fig. 1B, the device contained two micro uidic compartments interconnected by microgrooves that guided neuronal processes into the epithelil compartment.The micro uidic device allowed to co-cultivate intestinal epithelial cells and enteric neurons while observation interactions between the two cellular compartments.

Fabrication of micro uidic devices 2.2.1 Mold fabrication
Two master molds were fabricated by photolithography, one for the ow layer (cell culture compartments, media transport channels and media reservoirs) and one for the valve layer.The design was done using CAD software (AutoCAD 2019, Autodesk Inc.) and exported as single les for each layer.The mold for the ow layer was fabricated on a 4-inch silicon wafer by sequential spin-coating and UV exposure of four SU-8 photoresist layers.1) 50 µm tall alignment marks were fabricated using SU-8 2025.2) 2.5 µm tall microgroove features were fabricated using SU-8 2002.3) 100 µm tall features for neuronal compartment and transport channel were fabricated using SU-8 2050.4) 300 µm tall features for the epithelial compartment and transport channel were fabricated using SU-8 2100.Each of the four steps involved spin-coating of resist, soft exposure bake, UV exposure, post exposure bake, developing and hard bake steps that were performed following the manufacturer's instructions for each type of SU-8 resist.
Exposure of all SU-8 layers was performed using a Micro Pattern Generator (µPG 101, Heidelberg, Germany).The valve mold was fabricated on a separate 4-inch silicon wafer and consisted of a single layer of SU-8 2100 resist with 400 µm tall features.The fabrication of this layer followed a sequence of steps described above and was accomplished with the same UV exposure tool.After both molds were fabricated, they were exposed to chlorotrimethylsilane in a closed chamber for ~ 30 min.The molds were then kept on a Petri dish until use.

Assembly of micro uidic devices
Micro uidic devices were fabricated using multilayer soft lithography.Brie y, the micro uidic device was composed of two layers of PDMS: ow and valve layer.The ow layer (bottom) was composed of two parallel cell culture compartments interconnected by microgrooves.The epithelial compartment contained a semicircle array of pillars and an injection port.The valve PDMS layer (top) was designed to place a valve above the injection port.A 20:1 and 5:1 wt/wt ratio of PDMS-curing agent was poured on the ow and valve mold, respectively; degassed for 30 min, and partially cured at 80°C for 18 min.
Afterwards, the PDMS valve layer was detached, cut, and the 0.5mm inlet of the valve was punched.The valve PDMS slab was aligned on the ow layer and further baked at 80°C for 2 h to promote bonding between the two layers.Next, the assembly was peeled off and inlets of the neuronal chamber were punched using 14-Ga needles.The inlets for the epithelial compartments were created using a 3 mm diameter puncher.Two strips of invisible tape (2 mm x 7 mm) were placed along the injection port and on the surface of a previously cleaned cover glass to protect the region of the valve during oxygen plasma treatment. [35]The PDMS assembly and the cover glass were exposed to oxygen plasma at 30 W for 3 min.The tape strips were removed from the assembly and the coverglass for alignment and bonding.
Two 8 mm (d) × 8 mm (h) Pyrex cloning cylinders were bonded with uncured 10:1 PDMS mix on the neuronal chamber inlets, meanwhile two 10 mm (d) × 10 mm (h) cylinders were secured at the epithelial chamber inlets.The devices were cured at 80°C for 30 min.

Functionalization of micro uidic devices prior to cell seeding
Neuronal and epithelial compartments were functionalized with poly-L-ornithine and collagen I, respectively, using a well-established protocol [36,37] .Brie y, the channels and compartments of a micro uidic device were incubated with 2.5% APTES in 95% ethanol for 20 min followed by a quick wash with 99% ethanol before being dried with nitrogen gas and incubated at 80°C for 1h.This step was designed to remove water and promote formation of an aminosilane layer on the glass.Consequently, the chambers were lled with 10 mM Bs 3 in 1x PBS and incubated for 1h at room temperature (RT).
Afterwards, a micro uidic device was washed with distillated water and dried with nitrogen gas.Subsequently, the epithelial and neuronal compartments were infused with 0.3 mg/ml of collagen type I and 0.5 mg/mL poly-L-ornithine, respectively, and incubated for 1h.Bs 3 is a homobifunctional crosslinker covalently linking amines on glass to the amino groups on proteins or polypeptides.After the functionalization step, the devices were washed with fresh 1x PBS, degassed for 1 h and UV-sterilized for 1 h prior to seeding cells.

Diffusion characterization in the micro uidic device
Media reservoirs of micro uidic devices were lled with equal volumes of 1x PBS and mounted on an inverted uorescence microscope (IX-83, Olympus) using 10× long distance objective for timelapse imaging.Prior imaging, the saline solution in the epithelial compartment was exchanged by FITC-Dextran (MW 4kDa) at a concentration of 100 µM in 1x PBS.After levels of solution equilibrated in reservoirs, uorescence images were acquired from the central region of the device every 10 min for 4.5 h.Fluorescence intensity analysis was performed using ImageJ.
Prior to seeding into the micro uidic device, media was removed from reservoirs feeding epithelial compartments and a house vacuum line was connected to the micro uidic device for valve actuation (opening).30 µL of HC media containing organoid fragments at ~ 3x10 5 cell/ml concentration were gently aspirated using a 25-Ga needle connected via Tygon tubing (06419-05; Cole-Parmer) to 1ml BD Luer-Lok syringe (30 9628, BD).The needle was then introduced into the injection port and 1-2 µL of cell suspension was released in the epithelial chamber.Afterwards, the needle was removed, and the device was disconnected from the vacuum which returned the valve to its normally closed state.The micro uidic device was incubated for ~ 1 h at 37°C with 5% CO 2 to ensure cell attachment.Then, 500 µL of HC media was added into one of the reservoirs feeding epithelial compartment to 1) ush away unattached cells and 2) supply media in a su cient amount for cultivation.Devices with cells were maintained at 37°C with 5% CO 2 with daily media exchanges.At the end of culture, the intestinal epithelial cells were exposed to calcein, ethidium homodimer and Hoechst to assess cell viability.Live/Dead assay was used per manufacturer's instructions.

Neuronal isolation and seeding into a micro uidic device
All animal experiments were performed under the National Institutes of Health (NIH) guidelines for ethical care and use of laboratory animals with the approval of the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic, Rochester, MN.Primary cultures of the myenteric plexus of the mouse small intestines were derived from transgenic Avil-CreERT2::tdTomato mice.This strain was created by breeding Avil-CreERT2 mice (Jax 032027) [41] with B6.Cg-Gt(ROSA)26Sor tm14(CAG−tdTomato )Hze/J mice (Ai14; Jax 007914) [42] to hemizygosity and homozygosity, respectively.Cells of the small intestine myenteric plexus were isolated from dissected external muscle layers of the small intestine, as previously described. [9]Brie y, tissues were incubated rst in a solution of 0. A micro uidic device was rst primed with supplemented Neurobasal A media for 5 min at 37 ºC.Then, the media was aspirated from the reservoirs feeding the neuronal compartments and replaced with 5 µL of neuronal cell suspension (1x10 6 cell/mL).The device was then incubated for ~ 1h at 37°C to ensure cell attachment.Afterwards, the unattached cells were washed away from the cell compartment by adding 300 µL of fresh Neurobasal media into one media reservoir, creating a difference in hydrostatic pressure and driving media into the device.After aspirating media with unattached cells, 500 µL a of Neurobasal media supplemented with 5 µM A83-01 (TGF-β1 inhibitor) were placed into each media reservoir feeding the neuronal compartment.Devices with neuronal cells were cultured at 37°C, 5% CO 2 with daily exchanges of Neurobasal A media supplemented with 5 µM A83-01.

Creating neuro-epithelial co-cultures in a micro uidic device
A micro uidic device was assembled and functionalized with cell-adhesion ligands as described in sections 2.2 and 2.3, respectively.Epithelial cells derived from human colon organoids were seeded into the epithelial compartment as described in section 2.4.During seeding of epithelial cells, the neuronal side of the micro uidic device was lled with Neurobasal media (see supplementary Figure S1).The epithelial cells were allowed to attach inside the device for 1 h, after which excess cells were washed away by lling one media reservoir with 300 µL of media and allowing media to equilibrate between the reservoirs.Neuronal cells were seeded 24 h after introduction of epithelial cells, according to the protocol described in section 2.5.The neuronal cells were incubated for 1 h, after which excess cells were washed away with Neurobasal media and cultured in Neurobasal media supplemented with 5 µM A83-01.Micro uidic cultures were maintained at 37°C, 5% CO 2 with daily media exchanges.

Live-cell imaging in micro uidic devices
Cells were cultured and imaged in the micro uidic device.Imaging was performed on a Zeiss LSM980 confocal laser scanning microscope (Carl Zeiss Microscopy, LLC, White Plains, NY) using a 40X, 1.2 numerical aperture (NA) water-immersion objective lens and equipped with stage-top incubation set to 37°C and 5% CO 2 .Z-stacks were acquired every 2 h for 62 ho (32 frames total).Microscope control, postacquisition image analysis, and 3D projections were done using Zen 3.4 (blue edition, Carl Zeiss Microscopy, LLC, White Plains, NY).

Results and Discussion
We describe the development and characterization of neuro-epithelial co-cultures assembled in a micro uidic device.Intestinal epithelial cells and enteric neurons were shown to maintain phenotype and form connections in this micro uidic device.

Design of the micro uidic device
The design criteria for the micro uidic device were to: 1) culture intestinal epithelial cells and enteric neurons into distinct but neighboring compartments, 2) provide guides for the neuronal projections into the epithelial compartment and 3) enable visualization of the epithelial and neuronal compartments.The resulting micro uidic device is shown in Fig. 2A.It contained two cell culture compartments -larger compartment (4 cm by 1.5 mm and 300 µm in height) to be populated with epithelial cells derived from human colon organoids and a smaller compartment (4 cm by 0.5 mm with 100 µm height) for culturing enteric neurons.The two compartments were connected by microgrooves 150 µm in length and 2.5 µm in height (See Fig. 2B).The dimensions of the compartments were guided by several considerations.The neuronal compartment was made long and narrow to minimize the distance of neuronal soma from the grooves that allowed access to the epithelial compartment, thus increasing the chances of neuronal projections reaching the epithelial cells.The epithelial compartment was made taller to accommodate organoid fragments that ranged from 50 to 200 µm in size.Given that intestinal organoids were used as the source of epithelial cells, we wanted to ensure e cient seeding in the region of the device where neuro-epithelial connections were most likely to occur.To achieve this, we incorporated an injection port that allowed us to insert a needle and transfer organoid fragments directly into the epithelial compartment (see Fig. 2C). [35]This injection port was protected by a normally closed valve.The valve was opened by applying a house vacuum and reverted to its default (closed) state when disconnected from the vacuum.In addition, posts were incorporated in a semicircular con guration around the area of epithelial cell injection (see Fig. 2C).The posts had a pitch of 30 µm and were used to retain organoid fragments within the epithelial seeding area and in proximity to microgrooves.The posts did not, however, interfere with diffusion of nutrients from the media reservoirs to the epithelial compartment.Media was delivered from reservoirs (cylinders) located at the inlet and outlet of each compartment (see Fig. 2A) and was exchanged every 24 h.Devices were designed with simplicity of use in mind.No lines for controlling the injection port/valve and no ow for delivering media were required during culture.

Characterizing diffusion of molecules between epithelial and neuronal compartments
As shown in Fig. 2B, our device contained microgrooves designed for guiding neuronal projections into the epithelial compartment.Such micro-constrictions have been used widely for axonal guidance in micro uidic devices. [28]We wanted to assess experimentally how presence of microgrooves affected the exchange of molecules between the two micro uidic compartments.To accommodate epithelial cell fragments from organoids the epithelial compartment was designed to to be 8.7 times larger than the neuronal compartment (2.26 vs. 0.26 µL).Fluorescent dextran (MW 4 kDa) was used as a tracer molecule, was infused into the epithelial compartment, and its appearance in the neuronal compartment was monitored using uorescence microscopy.As shown in Fig. 3A, after 4.5 h, the neuronal chamber reaches ~ 35% of the intensity compared to the epithelial chamber, suggesting that the timeframe to reach equilibrium is ~ 13 h.Diffusion was expected to also occur from neuronal to epithelial compartment, but was challenging to detect given the volume difference and dilution of signal.Overall, our characterization pointed to the existence of a robust exchange of signals between the two micro uidic compartments.

Intestinal epithelial in the micro uidic device
Dissociated organoids were into the micro uidic device through an injection port described in the preceding section and were evenly distributed within the attachment area de ned by the array of posts (see Fig. 4A).Injecting cells directly into the culture chamber allowed for e cient use of primary intestinal organoids.Organoid fragments attached to the collagen-coated glass surface of the epithelial compartment 2 h after seeding and were observed to organize into small patches 24h after seeding (see Fig. 4A).These patches expanded, with epithelial layer reaching 50 to 80% con uence after 4 days and full con uence after 7 days of culture.Similar dynamics of organoid fragments planarizing into patches and expanding into a con uent layer were observed with the neuronal compartment containing either HC (epithelial) or Neurobasal media (data not shown).
The epithelial compartment of the micro uidic device was characterized by immuno uorescence staining for zonulin (ZO-1) and E-cadherin, markers of well-differentiated interconnected intestinal epithelium.The results, shown in Fig. 4B andC, highlight that the epithelial layer in the micro uidic device had high levels of ZO-1 and e-cadherin expression and that cells exhibited cuboidal morphology typical of columnar epithelium. [43]Intestinal epithelial cells were con ned to the epithelial compartment and did not cross over into the neuronal compartment during 7 days in culture (data not shown).The dynamics of organoid planarization and epithelial layer formation observed in our device were consistent with planarized and cultured colon organoids in gut-on-chip devices with apical and basolateral compartments. [22,44,45] Tviability of cells within the micro uidic epithelial layer was assessed at day 10 of culture.As highlighted by Fig. 4D, the epithelial layer was contiguous and occupied an area that extended beyond the initial seeding area demarcated by posts.Live/dead staining showed that 98 to 99% of cells within the layer were viable (Fig. 4D).The results in Fig. 4 highlight our ability to planarize organoids and form intestinal epithelial layer inside the micro uidic device.

Enteric neuronal cultures in the micro uidic device
As the next step, we set out to assess enteric neuronal cell uidic device.The cells were isolated from myenteric plexus and represented a mixture of non-uorescent cells, including neurons (~ 8%) and glia (~ 75%), with neuronal IPANs (advillin+) that expressed tdTomato.As shown in Fig. 5, IPANs (red uorescence) and other cells (no uorescence) were uniformly distributed in the neuronal compartment shortly after seeding.Based on our previous work, mixed culture was deemed bene cial for maintaining healthy neurons in vitro. [33]However, we observed excessive proliferation of these likely nonneuronal cells in the micro uidic devices in some of the early experiments and supplemented the media with TGF-β inhibitor to remedy this. [46,47] s seen from Fig. 5, the proportion of uorescent IPANs and non-uorescent cells remained constant over the course of 6 days in culture.As may be appreciated from higher magni cation images, after about 4 days, the micro uidic neuronal cultures reassembled into clusters with features resembling a ganglionated plexus.
Neuronal cultures changed phenotype over time.A small number of neurons extended projections into the microgrooves 48h after seeding (see Fig. 5).After 72h of culture, neurons formed ganglion-like structures with a complex network of processes covering most of the neuronal compartment.At this time point, some neuronal projections entered the microgrooves, however, the projections frequently looped back into the neuronal chamber (see Fig. 5).This was independent of what culture media (neuronal or epithelial) was present in the unseeded epithelial compartment.

Creating neuro-epithelial co-cultures in the micro uidic device
To create co-cultures, a micro uidic device was rst populated with colon organoid fragments, which were allowed to acclimate for 24h.At this point, fragments were attached (this process starts within 2h of seeding) and began to form interconnected patches of epithelium (see Fig. 6A).Dissociated myenteric cells were seeded into the neuronal compartment of the device that already contained patches of epithelial cells.In the rst two days of co-culture, we did not observe appreciable differences in the growth of neuronal processes compared to neuronal cells cultured alone.However, by day 3, IPAN neuronal processes were observed to traverse microgrooves, reach into the epithelial compartment, and make connections with epithelial cells.Other myenteric neuronal types (non-uorescent labeled) were observed to extend their processes to the epithelial compartment as IPAN neurons (data not shown).In days 4-6 of culture, numerous neuronal projections were observed in the epithelial compartment making connections with epithelial cells (see supplementary video 1).Interestingly, IPAN projections appeared to seek out epithelial patches as they were planarizing (Fig. 6A, D6), but epithelial cells also seemed to migrate toward neural processes and cause changes in those processes, including neurite pruning and redirection, upon contact (supplementary video 1).
Figure 6A shows a single epithelial cell innervated by multiple neuronal processes at day 6 of culture.In other instances, a single neuronal projection appeared to interact with multiple epithelial cells (see Fig. 6B).Some neuronal processes were observed to reach hundreds of micrometers into the epithelial compartment to make these connections.
We quanti ed the number and length of neuronal processes present in the epithelial compartment in the co-culture vs. mono-culture scenarios.The data, summarized in Fig. 6(D,E), highlight that neuronal projections reached the epithelial compartment faster, in greater numbers and were longer when epithelial cells were present.
We note that in both cases, the epithelial compartment contained epithelial media meaning that factors in the media alone were unlikely to explain the differences in neuronal projections.These differences may be attributed to the presence of yet to be elucidated paracrine cues of epithelial origin that attract and guide neuronal projections.
These results highlight our ability to use a novel micro uidic device for creating neuro-epithelial cocultures where phenotype of an individual cell type was maintained and where enteric neurons innervated the intestinal epithelial layer.

Conclusion
We designed and fabricated a novel micro uidic device for modeling neuro-epithelial interactions in the gut.To the best of our knowledge, this is the rst demonstration of intestinal epithelial cells and enteric neurons co-cultured in a micro uidic device.We demonstrated that colon organoid fragments planarized in the device and formed a contiguous layer that retained markers of epithelium and was viable for at least 10 days.Enteric neuronal cells retained normal morphology and formed in vivo-like ganglion clusters in the micro uidic devices.Importantly, the microgrooves separating neuronal and epithelial compartments limited cells crossing over while allowing for neuronal projections to reach epithelium.Our micro uidic device was mounted on a glass cover slip that allowed for high-resolution time-lapse microscopy of neuro-epithelial interactions.In the future, this micro uidic device will be used to examine the formation, structure, and function of neuro-epithelial connections, and may help to understand disease mechanisms underlying functional gastrointestinal disorders, such as irritable bowel syndrome (IBS).Enteric neurons cultured in the micro uidic device.A series of micrographs showing time-dependent changes in morphology of neuronal cultures.At day 0 (D0) IPANs (red) and non-uorescent cells were injected into the chamber.The epithelial compartment was lled with epithelial HC media.After 48 h (D2), a complex network of neuronal processes formed in the compartment with some processes entering microgrooves.At day 4 (D4) and day 6(D6), neuronal processes exited microgrooves on the epithelial side but did not extend into the compartment.Scale bar: left column 500 µm, right column 100 µm.