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Human gut morphogenesis establishes crypt-villus features of 3D epithelial microarchitecture and spatial organization.This unique structure is required to maintain gut homeostasis by protecting the stem cell niche in the basal crypt from exogenous microbial antigens and their metabolites.In addition, intestinal villi and secreting mucus present functionally differentiated epithelial cells with a protective barrier on the intestinal mucosal surface.Therefore, recreating 3D epithelial structures is crucial for the construction of in vitro gut models.Notably, the organic mimetic gut-on-a-chip can induce spontaneous 3D morphogenesis of the intestinal epithelium with enhanced physiological functions and biomechanics.Here, we provide a reproducible protocol to robustly induce intestinal morphogenesis in the gut on a microfluidic chip as well as in a Transwell embedded hybrid chip.We describe detailed methods for device fabrication, culturing of Caco-2 or intestinal organoid epithelial cells in conventional settings as well as on a microfluidic platform, induction of 3D morphogenesis, and characterization of established 3D epithelia using multiple imaging modalities .This protocol achieves regeneration of functional gut microarchitecture by controlling basolateral fluid flow for 5 d.Our in vitro morphogenesis method employs physiologically relevant shear stress and mechanical motion and does not require complex cell engineering or manipulation, which may outperform other existing techniques.We envision that our proposed protocol could have broad implications for the biomedical research community, providing a method to regenerate 3D intestinal epithelial layers in vitro for biomedical, clinical, and pharmaceutical applications.
Experiments demonstrate that intestinal epithelial Caco-2 cells cultured in gut-on-a-chip1,2,3,4,5 or bilayer microfluidic devices6,7 can undergo spontaneous 3D morphogenesis in vitro without a clear understanding of the underlying mechanism.In our recent study, we found that removal of basolaterally secreted morphogen antagonists from culture devices is necessary and sufficient to induce 3D epithelial morphogenesis in vitro, which has been demonstrated by Caco-2 and patient-derived intestinal organoids. Epithelial cells were validated.In this study, we specifically focused on the cell production and concentration distribution of a potent Wnt antagonist, Dickkopf-1 (DKK-1), in gut-on-a-chip and modified microfluidic devices containing Transwell inserts, termed ” Hybrid Chip”.We demonstrate that addition of exogenous Wnt antagonists (such as DKK-1, Wnt repressor 1, secreted frizzled-related protein 1, or Soggy-1) to the on-chip gut inhibits morphogenesis or disrupts the prestructured 3D epithelial layer , suggesting that antagonistic stress during culture is responsible for intestinal morphogenesis in vitro.Therefore, a practical approach to achieve robust morphogenesis at the epithelial interface is to remove or minimally maintain the levels of Wnt antagonists in the basolateral compartment by active flushing (eg, in gut-on-a-chip or hybrid-on-a-chip platforms) or diffusion .Basolateral media (eg, from Transwell inserts into large basolateral reservoirs in wells).
In this protocol, we provide a detailed method for fabricating gut-on-a-chip microdevices and Transwell-insertable hybrid chips (steps 1-5) to culture intestinal epithelial cells on polydimethylsiloxane (PDMS)-based porous membranes (steps 6A, 7A, 8, 9) or polyester membranes of Transwell inserts (steps 6B, 7B, 8, 9) and induced 3D morphogenesis in vitro (step 10).We also identified cellular and molecular features indicative of tissue-specific histogenesis and lineage-dependent cellular differentiation by applying multiple imaging modalities (steps 11-24).We induce morphogenesis using human intestinal epithelial cells, such as Caco-2 or intestinal organoids, in two culture formats with technical details including surface modification of porous membranes, creation of 2D monolayers, and intestinal biochemical and Reproduction of the biomechanical microenvironment.in vitro.To induce 3D morphogenesis from 2D epithelial monolayers, we removed morphogen antagonists in both cultured forms by flowing the medium into the basolateral compartment of the culture.Finally, we provide a representation of the utility of a regenerable 3D epithelial layer that can be used to model morphogen-dependent epithelial growth, longitudinal host-microbiome co-cultures, pathogen infection, inflammatory injury, epithelial barrier dysfunction, and probiotic-based therapies Example.influences.
Our protocol may be useful to a broad range of scientists in basic (eg, intestinal mucosal biology, stem cell biology, and developmental biology) and applied research (eg, preclinical drug testing, disease modeling, tissue engineering, and gastroenterology) broad impact.Because of the reproducibility and robustness of our protocol to induce 3D morphogenesis of the intestinal epithelium in vitro, we envision that our technical strategy can be disseminated to audiences studying the dynamics of cell signaling during intestinal development, regeneration or homeostasis .In addition, our protocol is useful for interrogating infection under various infectious agents such as Norovirus 8, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clostridium difficile, Salmonella Typhimurium 9 or Vibrio cholerae. Audiences of disease pathology and pathogenesis are also useful.The use of an on-chip gut microphysiology system may allow longitudinal co-culture 10 and subsequent assessment of host defense, immune responses and pathogen-related injury repair in the gastrointestinal (GI) tract 11 .Other GI disorders associated with leaky gut syndrome, celiac disease, Crohn’s disease, ulcerative colitis, pouchitis, or irritable bowel syndrome can be simulated when 3D intestinal epithelial layers are prepared using the patient’s 3D intestinal epithelial layers , these diseases include villous atrophy, crypt shortening, mucosal damage, or impaired epithelial barrier.Biopsy or stem cell-derived intestinal organoids12,13.To better model the higher complexity of the disease environment, readers may consider adding disease-relevant cell types, such as patient peripheral blood mononuclear cells (PBMCs), to models containing 3D intestinal villus-crypt microarchitectures. tissue-specific immune cells, 5.
Since 3D epithelial microstructure can be fixed and visualized without the sectioning process, viewers working on spatial transcriptomics and high-resolution or super-resolution imaging may be interested in our mapping of the spatiotemporal dynamics of genes and proteins on epithelial niches. Interested in technology.Response to microbial or immune stimuli.Furthermore, longitudinal host-microbiome crosstalk 10, 14 that coordinate gut homeostasis can be established in the 3D intestinal mucosal layer by co-culturing various microbial species, microbial communities or fecal microbiota, especially in the gut-on-a-chip. in the platform.This approach is particularly attractive to audiences studying mucosal immunology, gastroenterology, human microbiome, culturomics and clinical microbiology seeking to cultivate previously uncultured gut microbiota in the laboratory.If our in vitro morphogenesis protocol can be adapted to scalable culture formats, such as multiwell inserts in 24, 96 or 384 well plates that continuously replenish basolateral compartments, the protocol can also be disseminated to those developing pharmaceutical, biomedical Or high-throughput screening or validation platforms for the food industry.As a proof-of-principle, we recently demonstrated the feasibility of a multiplex high-throughput morphogenesis system scalable to a 24-well plate format.In addition, multiple organ-on-a-chip products have been commercialized16,17,18.Therefore, the validation of our in vitro morphogenesis method can be accelerated and potentially adopted by many research laboratories, industry or government and regulatory agencies to understand cellular reprogramming of in vitro gut morphogenesis at the transcriptomic level to test drugs or biotherapeutics The absorption and transport of drug candidates was assessed using 3D gut surrogates or using custom or commercial organ-on-a-chip models to assess the reproducibility of the gut morphogenesis process.
A limited number of human-relevant experimental models have been used to study intestinal epithelial morphogenesis, mainly due to the lack of implementable protocols to induce 3D morphogenesis in vitro.In fact, much of the current knowledge about gut morphogenesis is based on animal studies (eg, zebrafish20, mice21 or chickens22).However, they are labor- and cost-intensive, can be ethically questionable, and most importantly, do not precisely determine human developmental processes.These models are also very limited in their ability to be tested in a multi-way scalable manner.Therefore, our protocol for regenerating 3D tissue structures in vitro outperforms in vivo animal models as well as other traditional static 2D cell culture models.As previously described, utilizing 3D epithelial structures allowed us to examine the spatial localization of differentiated cells in the crypt-villus axis in response to various mucosal or immune stimuli.3D epithelial layers can provide a space to study how microbial cells compete to form spatial niches and ecological evolution in response to host factors (eg, inner versus outer mucus layers, secretion of IgA and antimicrobial peptides).Furthermore, 3D epithelial morphology can allow us to understand how the gut microbiota structures its communities and synergistically produces microbial metabolites (eg, short-chain fatty acids) that shape cellular organization and stem cell niches in the basal crypts.These features can only be demonstrated when 3D epithelial layers are established in vitro.
In addition to our method of creating 3D intestinal epithelial structures, there are several in vitro methods.Intestinal organoid culture is a state-of-the-art tissue engineering technique based on the cultivation of intestinal stem cells under specific morphogen conditions23,24,25.However, the use of 3D organoid models for transport analysis or host-microbiome co-cultures is often challenging because the intestinal lumen is enclosed within the organoid and, therefore, the introduction of luminal components such as microbial cells or exogenous antigens is limited. Access to organoid lumens can be improved using a microinjector,26,27 but this method is invasive and labor-intensive and requires specialized knowledge to perform.Furthermore, traditional organoid cultures maintained in hydrogel scaffolds under static conditions do not accurately reflect active in vivo biomechanics.
Other approaches employed by several research groups utilize prestructured 3D hydrogel scaffolds to mimic the gut epithelial structure by culturing isolated human intestinal cells on the gel surface.Fabricate hydrogel scaffolds using 3D-printed, micro-milled, or lithographically fabricated molds.This method shows the self-organized arrangement of isolated epithelial cells in vitro with physiologically relevant morphogen gradients, establishing a high aspect ratio epithelial structure and stroma-epithelial crosstalk by including stromal cells in the scaffold.However, the nature of prestructured scaffolds may prevent the display of the spontaneous morphogenetic process itself.These models also do not provide dynamic luminal or interstitial flow, lacking the fluid shear stress that intestinal cells need to undergo morphogenesis and gain physiological function.Another recent study used hydrogel scaffolds in a microfluidic platform and patterned intestinal epithelial structures using laser-etching techniques.Mouse intestinal organoids follow etched patterns to form intestinal tubular structures, and intraluminal fluid flow can be recapitulated using a microfluidics module.However, this model neither exhibits spontaneous morphogenetic processes nor includes gut mechanobiological movements.3D printing techniques from the same group were able to create miniature gut tubes with spontaneous morphogenetic processes.Despite the complex fabrication of different gut segments within the tube, this model also lacks luminal fluid flow and mechanical deformation.Additionally, model operability may be limited, especially after the bioprinting process is complete, perturbing experimental conditions or cell-to-cell interactions.Instead, our proposed protocol provides spontaneous gut morphogenesis, physiologically relevant shear stress, biomechanics that mimic gut motility, accessibility of independent apical and basolateral compartments, and re-creation of complex biological microenvironments of modularity.Therefore, our in vitro 3D morphogenesis protocol may provide a complementary approach to overcome the challenges of existing methods.
Our protocol is entirely focused on 3D epithelial morphogenesis, with only epithelial cells in culture and no other types of surrounding cells such as mesenchymal cells, endothelial cells, and immune cells.As previously described, the core of our protocol is the induction of epithelial morphogenesis by removing morphogen inhibitors secreted at the basolateral side of the introduced medium.While the robust modularity of our gut-on-a-chip and hybrid-on-a-chip allows us to recreate the undulating 3D epithelial layer, additional biological complexities such as epithelial-mesenchymal interactions33,34, extracellular Matrix (ECM) deposition 35 and, in our model, crypt-villus features that convey stem cell niches in basal crypts remain to be further considered.Stromal cells (eg, fibroblasts) in the mesenchyme play a key role in the production of ECM proteins and regulation of intestinal morphogenesis in vivo35,37,38.The addition of mesenchymal cells to our model enhanced the morphogenetic process and cell attachment efficiency.The endothelial layer (ie, capillaries or lymphatics) plays an important role in regulating molecular transport39 and immune cell recruitment40 in the gut microenvironment.Furthermore, vasculature components that can be connected between tissue models are a prerequisite when tissue models are designed to demonstrate multi-organ interactions.Therefore, endothelial cells may need to be included to model more accurate physiological features with organ-level resolution.Patient-derived immune cells are also essential for displaying innate immune responses, antigen presentation, innate adaptive immune crosstalk, and tissue-specific immunity in the context of mimicking intestinal disease.
The use of hybrid chips is more straightforward than gut-on-a-chip because the device setup is simpler and the use of Transwell inserts allows for scalable culture of gut epithelium.However, commercially available Transwell inserts with polyester membranes are not elastic and cannot simulate peristaltic-like movements.Furthermore, the apical compartment of the Transwell insert placed in the hybrid chip remained stationary with no shear stress on the apical side.Clearly, the static properties in the apical compartment rarely enable long-term bacterial co-culture in hybrid chips.While we can robustly induce 3D morphogenesis in Transwell inserts when using hybrid chips, the shortage of physiologically relevant biomechanics and apical fluid flow may limit the feasibility of hybrid chip platforms for potential applications.
Full-scale reconstructions of the human crypt-villus axis in gut-on-a-chip and hybrid-on-a-chip cultures have not been fully established.Since morphogenesis begins from an epithelial monolayer, 3D microarchitectures do not necessarily provide morphological similarity to crypts in vivo.Although we characterized the proliferating cell population near the basal crypt domain in the microengineered 3D epithelium, the crypt and villous regions were not clearly demarcated.Although higher upper channels on the chip lead to increased height of the microengineered epithelium, the maximum height is still limited to ~300–400 µm.The actual depth of human intestinal crypts in the small and large intestines is ~135 µm and ~400 µm, respectively, and the height of the small intestinal villi is ~600 µm41.
From an imaging standpoint, in situ super-resolution imaging of 3D microarchitectures may be limited to the gut on a chip, since the required working distance from the objective lens to the epithelial layer is on the order of a few millimeters.To overcome this problem, a distant objective may be required.Furthermore, making thin sections for imaging specimen preparation is challenging due to the high elasticity of PDMS.Furthermore, since the layer-by-layer microfabrication of the gut on a chip involves permanent adhesion between each layer, it is extremely challenging to open or remove the upper layer to examine the surface structure of the epithelial layer.For example, by using a scanning electron microscope (SEM).
The hydrophobicity of PDMS has been a limiting factor in microfluidic-based studies dealing with hydrophobic small molecules, since PDMS can nonspecifically adsorb such hydrophobic molecules.Alternatives to PDMS may be considered with other polymeric materials.Alternatively, surface modification of PDMS (eg, coating with lipophilic materials 42 or poly(ethylene glycol) 43 ) can be considered to minimize adsorption of hydrophobic molecules.
Finally, our method has not been well characterized in terms of providing a high-throughput screening or “one-size-fits-all” user-friendly experimental platform.The current protocol requires a syringe pump per microdevice, which takes up space in a CO2 incubator and prevents large-scale experiments.This limitation can be significantly improved by the scalability of innovative culture formats (eg, 24-well, 96-well, or 384-well porous inserts that allow continuous replenishment and removal of basolateral media).
To induce 3D morphogenesis of human intestinal epithelium in vitro, we used a microfluidic chip intestinal device containing two parallel microchannels and an elastic porous membrane in between to create a lumen-capillary interface.We also demonstrate the use of a single-channel microfluidic device (a hybrid chip) that provides continuous basolateral flow beneath polarized epithelial layers grown on Transwell inserts.In both platforms, morphogenesis of various human intestinal epithelial cells can be demonstrated by applying directional manipulation of flow to remove morphogen antagonists from the basolateral compartment.The entire experimental procedure (Figure 1) consists of five parts: (i) microfabrication of the gut chip or Transwell insertable hybrid chip (steps 1-5; Box 1), (ii) preparation of intestinal epithelial cells (Caco-2 cells) or human intestinal organoids; boxes 2-5), (iii) culture of intestinal epithelial cells on intestinal chips or hybrid chips (steps 6-9), (iv) induction of 3D morphogenesis in vitro (step 10) and (v) ) to characterize the 3D epithelial microstructure (steps 11-24).Finally, an appropriate control group (discussed further below) was designed to validate the effectiveness of in vitro morphogenesis by comparing epithelial morphogenesis to spatial, temporal, conditional, or procedural controls.
We used two different culture platforms: gut-on-a-chip with straight channels or nonlinear convoluted channels, or hybrid chips containing Transwell (TW) inserts in a microfluidic device, fabricated as described in Box 1, and step 1 -5.”Device Fabrication” shows the main steps in making a single chip or a hybrid chip.”Culture of Human Intestinal Epithelial Cells” explains the cell source (Caco-2 or human intestinal organoids) and culture procedure used in this protocol.”In vitro morphogenesis” shows the overall steps in which Caco-2 or organoid-derived epithelial cells are cultured on an intestinal chip or on Transwell inserts of a hybrid chip, followed by induction of 3D morphogenesis and the formation of a characterized epithelial structure.The program step number or box number is displayed below each arrow.The application provides examples of how established intestinal epithelial layers can be used, for example, in cell differentiation characterization, gut physiology studies, establishment of host-microbiome ecosystems, and disease modeling.Immunofluorescence images in “Cell Differentiation” showing nuclei, F-actin and MUC2 expressed in the 3D Caco-2 epithelial layer generated on the gut chip.MUC2 signaling is present in goblet cells and mucus secreted from mucosal surfaces.Fluorescent images in Gut Physiology show mucus produced by staining for sialic acid and N-acetylglucosamine residues using fluorescent wheat germ agglutinin.The two overlapping images in “Host-Microbe Co-Cultures” show representative host-microbiome co-cultures in the gut on a chip.The left panel shows the co-culture of E. coli expressing green fluorescent protein (GFP) with microengineered 3D Caco-2 epithelial cells.The right panel shows the localization of GFP E. coli co-cultured with 3D Caco-2 epithelial cells, followed by immunofluorescence staining with F-actin (red) and nuclei (blue).Disease modeling illustrates healthy versus leaky gut in gut inflammation chips under physiological challenge with bacterial antigens (eg, lipopolysaccharide, LPS) and immune cells (eg, PBMC; green).Caco-2 cells were cultured to establish a 3D epithelial layer.Scale bar, 50 µm.Images in bottom row: “Differentiation of cells” adapted with permission from reference.2. Oxford University Press; Reproduced with permission from Ref.5. NAS; “Host-Microbe Co-Culture” adapted with permission from ref.3. NAS; “Disease Modeling” adapted with permission from reference.5. NAS.
Both gut-on-chip and hybrid chips were fabricated using PDMS replicas that were demolded from silicon molds by soft lithography1,44 and patterned with SU-8.The design of the microchannels in each chip is determined by considering hydrodynamics such as shear stress and hydrodynamic pressure1,4,12.The original gut-on-a-chip design (Extended Data Fig. 1a), which consisted of two juxtaposed parallel straight microchannels, has evolved into a complex gut-on-a-chip (Extended Data Fig. 1b) that includes a pair of curved microchannels to induce Increased fluid residence time, nonlinear flow patterns, and multiaxial deformation of cultured cells (Fig. 2a–f) 12.When more complex gut biomechanics need to be recreated, complex gut-on-a-chips can be chosen.We have demonstrated that the convoluted Gut-Chip also strongly induces 3D morphogenesis in a similar time frame with a similar degree of epithelial growth compared to the original Gut-Chip, regardless of the cultured cell type.Therefore, to induce 3D morphogenesis, linear and complex on-chip gut designs are interchangeable.PDMS replicas cured on silicon molds with SU-8 patterns provided negative features after demolding (Fig. 2a).To fabricate the gut on a chip, the prepared upper PDMS layer was sequentially bonded to a porous PDMS film and then aligned with the lower PDMS layer by irreversible bonding using a corona treater (Fig. 2b–f).To fabricate hybrid chips, cured PDMS replicas were bonded to glass slides to create single-channel microfluidic devices that could accommodate Transwell inserts (Fig. 2h and Extended Data Fig. 2).The bonding process is performed by treating the surfaces of the PDMS replica and glass with oxygen plasma or corona treatment.After sterilization of the microfabricated device attached to the silicone tube, the device setup was ready to perform 3D morphogenesis of the intestinal epithelium (Figure 2g).
a, Schematic illustration of the preparation of PDMS parts from SU-8 patterned silicon molds.The uncured PDMS solution was poured onto a silicon mold (left), cured at 60 °C (middle) and demolded (right).The demolded PDMS was cut into pieces and cleaned for further use.b, Photograph of the silicon mold used to prepare the PDMS upper layer.c, Photograph of the silicon mold used to fabricate the PDMS porous membrane.d, A series of photographs of the upper and lower PDMS components and the assembled on-chip intestinal device.e, Schematic of the alignment of the upper, membrane, and lower PDMS components.Each layer is irreversibly bonded by plasma or corona treatment.f, Schematic of the fabricated gut-on-a-chip device with superimposed convoluted microchannels and vacuum chambers.g, Setup of gut-on-a-chip for microfluidic cell culture.The fabricated gut on a chip assembled with a silicone tube and syringe was placed on a coverslip.The chip device was placed on the lid of a 150 mm Petri dish for processing.The binder is used to close the silicone tube.h, Visual snapshots of hybrid chip fabrication and 3D morphogenesis using hybrid chips.Transwell inserts prepared independently to culture 2D monolayers of intestinal epithelial cells were inserted into the hybrid chip to induce intestinal 3D morphogenesis.The medium is perfused through microchannels beneath the cell layer established on the Transwell insert.Scale bar, 1 cm.h Reprinted with permission from reference.4. Elsevier.
In this protocol, the Caco-2 cell line and intestinal organoids were used as epithelial sources (Fig. 3a).Both types of cells were cultured independently (Box 2 and Box 5) and used to seed the ECM-coated microchannels of on-chip gut or Transwell inserts.When cells are confluent (>95% coverage in flasks), routinely cultured Caco-2 cells (between passages 10 and 50) in T-flasks are harvested to prepare dissociated cell suspensions by trypsinization fluid (box 2).Human intestinal organoids from intestinal biopsies or surgical resections were cultured in Matrigel scaffold domes in 24-well plates to support the structural microenvironment.Medium containing essential morphogens (such as Wnt, R-spondin, and Noggin) and growth factors prepared as described in Box 3 was supplemented every other day until the organoids grew to ~500 µm in diameter.Fully grown organoids are harvested and dissociated into single cells for seeding onto gut or Transwell inserts on a chip (Box 5).As we have previously reported, it can be differentiated according to disease type12,13 (eg ulcerative colitis, Crohn’s disease, colorectal cancer, or normal donor), lesion site (eg, lesion versus non-lesioned area) and gastrointestinal location in the tract (eg, duodenum, jejunum, ileum, cecum, colon, or rectum).We provide an optimized protocol in Box 5 for culturing colonic organoids (coloids) that typically require higher concentrations of morphogens than small intestinal organoids.
a, Workflow for the induction of gut morphogenesis in the gut chip.Caco-2 human intestinal epithelium and intestinal organoids are used in this protocol to demonstrate 3D morphogenesis.The isolated epithelial cells were seeded in the prepared gut-on-a-chip device (chip preparation).Once cells are seeded (seeded) and attached (attached) to the PDMS porous membrane on day 0 (D0), apical (AP) flow is initiated and maintained for the first 2 days (flow, AP, D0-D2).Basolateral (BL) flow is also initiated along with cyclic stretching motions (stretch, flow, AP and BL) when a complete 2D monolayer is formed.Intestinal 3D morphogenesis occurred spontaneously after 5 days of microfluidic culture (morphogenesis, D5).Phase contrast images show representative morphology of Caco-2 cells at each experimental step or time point (bar graph, 100 µm).Four schematic diagrams illustrating the corresponding cascades of gut morphogenesis (top right).The dashed arrows in the schematic represent the direction of fluid flow.b, SEM image showing the surface topology of the established 3D Caco-2 epithelium (left).The inset highlighting the magnified area (white dashed box) shows the regenerated microvilli on the 3D Caco-2 layer (right).c, Horizontal frontal view of established Caco-2 3D, claudin (ZO-1, red) and continuous brush border membranes labeled F-actin (green) and nuclei (blue) Immunofluorescence confocal visualization of epithelial cells on intestinal chips.Arrows pointing to the middle schematic indicate the location of the focal plane for each confocal view.d, Time course of morphological changes in organoids cultured on a chip obtained by phase contrast microscopy on days 3, 7, 9, 11, and 13.The inset (top right) shows the high magnification of the provided image.e, DIC photomicrograph of organoid 3D epithelium established in the gut on slice taken on day 7.f, Overlaid immunofluorescence images showing markers for stem cells (LGR5; magenta), goblet cells (MUC2; green), F-actin (grey) and nuclei (cyan) grown on gut chips for 3 days, respectively (Left) and 13-day (middle) organoids on the epithelial layer.See also Extended Data Figure 3, which highlights LGR5 signaling without MUC2 signaling.Fluorescence images showing the epithelial microstructure (right) of 3D organoid epithelium established in the gut on a chip by staining the plasma membrane with CellMask dye (right) on day 13 of culture.Scale bar is 50 μm unless otherwise stated.b Reprinted with permission from reference.2. Oxford University Press; c Adapted with permission from Reference.2. Oxford University Press; e and f adapted with permission by reference.12 Under Creative Commons License CC BY 4.0.
In the gut on a chip, it is necessary to modify the hydrophobic surface of the PDMS porous membrane for successful ECM coating.In this protocol, we apply two different methods to modify the hydrophobicity of PDMS membranes.For culturing Caco-2 cells, surface activation by UV/ozone treatment alone was sufficient to reduce the hydrophobicity of the PDMS surface, coat the ECM and attach Caco-2 cells to the PDMS membrane.However, microfluidic culture of organoid epithelium requires chemical-based surface functionalization to achieve efficient deposition of ECM proteins by sequentially applying polyethyleneimine (PEI) and glutaraldehyde to PDMS microchannels.After surface modification, ECM proteins were deposited to cover the functionalized PDMS surface and then introduced into the isolated organoid epithelium.After the cells are attached, the microfluidic cell culture starts by perfusing only the medium into the upper microchannel until the cells form a complete monolayer, while the lower microchannel maintains static conditions.This optimized method for surface activation and ECM coating enables the attachment of organoid epithelium to induce 3D morphogenesis on the PDMS surface.
Transwell cultures also require ECM coating prior to cell seeding; however, Transwell cultures do not require complex pretreatment steps to activate the surface of porous inserts.For growing Caco-2 cells on Transwell inserts, ECM coating on porous inserts accelerates the attachment of dissociated Caco-2 cells (<1 hour) and tight junction barrier formation (<1-2 days).To culture organoids on Transwell inserts, isolated organoids are seeded on ECM-coated inserts, attached to the membrane surface (<3 h) and maintained until the organoids form a complete monolayer with barrier integrity .Transwell cultures are performed in 24-well plates without the use of hybrid chips.
In vitro 3D morphogenesis can be initiated by applying fluid flow to the basolateral aspect of an established epithelial layer.In the gut on a chip, epithelial morphogenesis began when the medium was perfused into the upper and lower microchannels (Fig. 3a).As previously described, it is critical to introduce fluid flow in the inferior (basolateral) compartment for continuous removal of directional secreted morphogen inhibitors.To provide sufficient nutrients and serum to cells bound on porous membranes and generate luminal shear stress, we typically apply dual flow in the gut on a chip.In hybrid chips, Transwell inserts containing epithelial monolayers were inserted into the hybrid chips.Then, the medium was applied under the basolateral side of the porous Transwell insert through the microchannel.Intestinal morphogenesis occurred 3-5 days after initiation of basolateral flow in both culture platforms.
The morphological features of microengineered 3D epithelial layers can be analyzed by applying various imaging modalities, including phase contrast microscopy, differential interference contrast (DIC) microscopy, SEM, or immunofluorescence confocal microscopy (Figures 3 and 4).Phase contrast or DIC imaging can be easily performed at any time during culture to monitor the shape and protrusion of 3D epithelial layers.Due to the optical transparency of PDMS and polyester films, both the gut-on-a-chip and hybrid chip platforms can provide real-time in situ imaging without the need for sectioning or disassembly of the device.When performing immunofluorescence imaging (Figures 1, 3c, f and 4b, c), cells are typically fixed with 4% (wt/vol) paraformaldehyde (PFA), followed by Triton X-100 and 2% (wt/vol) ) bovine serum albumin (BSA), in order.Depending on the cell type, different fixatives, permeabilizers, and blocking agents can be used.Primary antibodies targeting lineage-dependent cell or region markers are used to highlight cells immobilized in situ on the chip, followed by secondary antibodies along with a counterstain dye targeting either nucleus (e.g., 4′,6-diamidino-2-phenylene) indole, DAPI) or F-actin (eg, fluorescently labeled phalloidin).Fluorescence-based live imaging can also be performed in situ to detect mucus production (Fig. 1, “Cell differentiation” and “Gut physiology”), random colonization of microbial cells (Fig. 1, “Host-microbe co-culture”) , the recruitment of immune cells (Fig. 1, ‘Disease Modeling’) or the contours of 3D epithelial morphology (Fig. 3c,f and 4b,c).When modifying the gut on the chip to separate the upper layer from the lower microchannel layer, as described in ref.As shown in Fig. 2, the 3D epithelial morphology as well as the microvilli on the apical brush border can be visualized by SEM (Fig. 3b).The expression of differentiation markers can be assessed by performing quantitative PCR5 or single-cell RNA sequencing.In this case, 3D layers of epithelial cells grown in gut chips or hybrid chips are harvested by trypsinization and then used for molecular or genetic analysis.
a, Workflow for the induction of intestinal morphogenesis in a hybrid chip.Caco-2 and intestinal organoids are used in this protocol to demonstrate 3D morphogenesis in a hybrid chip platform.Dissociated epithelial cells were seeded in prepared Transwell inserts (TW prep; see figure below).Once cells were seeded (seeded) and attached to polyester membranes in Transwell inserts, all cells were cultured under static conditions (TW culture).After 7 days, a single Transwell insert containing a 2D monolayer of epithelial cells was integrated into a hybrid chip to introduce a basolateral flow (Flow, BL), which ultimately led to the generation of a 3D epithelial layer (morphogenesis).Phase contrast micrographs showing morphological features of human organ epithelial cells derived from normal donor (C103 line) ascending colon at each experimental step or time point.The schematics in the upper layers illustrate the experimental configuration for each step.b, Hybrid chips (left schematic) can lead to 3D morphogenesis of organoid epithelial cells with top-down confocal microscopy views taken at different Z positions (upper, middle, and lower; see right schematic and corresponding dotted lines). showed obvious morphological characteristics.F-actin (cyan), nucleus (grey).c, Fluorescence confocal micrographs (3D angled view) of organoid-derived epithelial cells cultured in static Transwell (TW; inset within white dashed box) versus hybrid chip (largest full shot) comparing 2D versus 3D morphology, respectively.A pair of 2D vertical cross-cut views (inset in the upper right corner; “XZ”) also show 2D and 3D features.Scale bar, 100 µm.c Reprinted with permission from reference.4. Elsevier.
Controls can be prepared by culturing the same cells (Caco-2 or intestinal organoid epithelial cells) into two-dimensional monolayers under conventional static culture conditions.Notably, nutrient depletion may result due to the limited volume capacity of the microchannels (i.e. ~4 µL in the top channel on the original gut-chip design).Therefore, epithelial morphology before and after application of basolateral flow can also be compared.
The soft lithography process should be performed in a clean room.For each layer on the chip (upper and lower layers and membranes) and hybrid chips, different photomasks were used and fabricated on separate silicon wafers because the heights of the microchannels were different.The target heights of the upper and lower microchannels of the gut on the chip are 500 µm and 200 µm, respectively.The channel target height of the hybrid chip is 200 µm.
Place a 3-inch silicon wafer in a dish with acetone.Gently swirl the plate for 30 seconds, then air dry the wafer.Transfer the wafer to a plate with IPA, then spin the plate for 30 s to clean.
A piranha solution (mixture of hydrogen peroxide and concentrated sulfuric acid, 1:3 (vol/vol)) can optionally be used to maximize the removal of organic residues from the silicon wafer surface.
Piranha solution is extremely corrosive and generates heat.Additional safety precautions are necessary.For waste disposal, allow the solution to cool and transfer to a clean, dry waste container.Use secondary containers and properly label waste containers.Please follow the facility’s safety guidelines for more detailed procedures.
Dehydrate the wafers by placing them on a 200 °C hot plate for 10 min.After dehydration, the wafer was shaken five times in air to cool.
Pour ~10 g of photoresist SU-8 2100 onto the center of the cleaned silicon wafer.Use tweezers to spread the photoresist evenly on the wafer.Occasionally place the wafer on a 65°C hot plate to make the photoresist less sticky and easier to spread.Do not place the wafer directly on the hot plate.
SU-8 was evenly distributed on the wafer by running spin coating.Program an incoming rotation of the SU-8 for 5–10 s to propagate at 500 rpm at an acceleration of 100 rpm/s.Set the main spin for 200 µm thickness patterning at 1,500 rpm, or achieve a 250 µm thickness (making a 500 µm height for the upper layer of the gut on the chip; see “Critical steps” below) set at an acceleration of 300 rpm/s 30 seconds at 1,200 rpm.
The main spin speed can be adjusted according to the target thickness of the SU-8 pattern on the silicon wafer.
To fabricate SU-8 patterns of 500 µm height for the upper layer of the gut on the chip, the spin coating and soft bake steps of this Box (steps 7 and 8) were sequentially repeated (see step 9) to produce two layers of 250 µm A thick layer of SU-8, which can be layered and joined by UV exposure in step 12 of this box, making a layer 500 µm high.
Soft bake the SU-8 coated wafers by carefully placing the wafers on a hot plate at 65 °C for 5 min, then switch the setting to 95 °C and incubate for an additional 40 min.
To achieve a 500 μm height of the SU-8 pattern in the upper microchannel, repeat steps 7 and 8 to generate two 250 μm thick SU-8 layers.
Using the UV Mask Aligner, perform a lamp test according to the manufacturer’s instructions to calculate the exposure time of the wafer.(exposure time, ms) = (exposure dose, mJ/cm2)/(lamp power, mW/cm2).
After determining the exposure time, place the photomask on the mask holder of the UV mask aligner and place the photomask on the SU-8 coated wafer.
Place the printed surface of the photomask directly on the SU-8 coated side of the silicon wafer to minimize UV dispersion.
Expose the SU-8 coated wafer and photomask vertically to 260 mJ/cm2 of UV light for the predetermined exposure time (see step 10 of this box).
After UV exposure, SU-8-coated silicon wafers were baked at 65°C for 5 min and 95°C for 15 min on each hot plate to fabricate patterns with a height of 200 μm.Extend the post-bake time at 95 °C to 30 min to fabricate patterns with a height of 500 µm.
The developer is poured into a glass dish, and the baked wafer is placed in the dish.The volume of SU-8 developer may vary depending on the size of the glass plate.Make sure to use enough SU-8 developer to completely remove unexposed SU-8.For example, when using a 150 mm diameter glass dish with a 1 L capacity, use ~300 mL of SU-8 developer.Develop the mold for 25 minutes with occasional gentle rotation.
Rinse the developed mold with ~10 mL of fresh developer followed by IPA by spraying the solution using a pipette.
Place the wafer in a plasma cleaner and expose to oxygen plasma (atmospheric gas, target pressure 1 × 10−5 Torr, power 125 W) for 1.5 min.
Place the wafer in a vacuum desiccator with a glass slide inside.Wafers and slides can be placed side by side.If the vacuum desiccator is divided into several layers by a plate, place the slides in the lower chamber and the wafers in the upper chamber.Drop 100 μL of trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane solution on a glass slide and apply vacuum for silanization.
Thaw a vial of frozen Caco-2 cells in a 37°C water bath, then transfer the thawed cells to a T75 flask containing 15 mL of 37°C prewarmed Caco-2 medium.
To pass Caco-2 cells at ~90% confluency, first warm Caco-2 medium, PBS, and 0.25% trypsin/1 mM EDTA in a 37°C water bath.
Aspirate the medium by vacuum aspiration.Wash cells twice with 5 mL of warm PBS by repeating vacuum aspiration and adding fresh PBS.