Development of the protocol
It has been experimentally demonstrated that intestinal epithelial Caco-2 cells cultured in a gut-on-a-chip1,2,3,4,5 or in a bilayered microfluidic device6,7 can undergo spontaneous 3D morphogenesis in vitro without a clear understanding of the underlying mechanism. In our recent study, we identified that the removal of morphogen antagonists that are basolaterally secreted from the culture setup is necessary and sufficient to induce 3D epithelial morphogenesis in vitro, verified with both Caco-2 and patient-derived intestinal organoid epithelia4. In this study, we specifically focused on the cellular production and concentration profile of a potent Wnt antagonist, Dickkopf-1 (DKK-1), in both gut-on-a-chip and a modified microfluidic device that contains a Transwell insert, called a ‘hybrid chip’. We confirmed that exogenous additions of Wnt antagonists (e.g., DKK-1, Wnt inhibitory factor 1, secreted frizzled-related protein 1 or Soggy-1) into a gut-on-a-chip result in the inhibition of morphogenesis or the disruption of prestructured 3D epithelial layers, suggesting that the antagonistic pressure during the culture is responsible for intestinal morphogenesis in vitro. Thus, practical approaches for robust morphogenesis on an epithelial interface are to either remove or minimally maintain the level of Wnt antagonists in the basolateral compartment by actively washing away (e.g., in the gut-on-a-chip or hybrid chip platforms) or diffusing the basolateral culture broth (e.g., from the Transwell insert to a large basolateral reservoir in the well).
In this protocol, we provide detailed methods for fabricating a gut-on-a-chip microdevice and a Transwell-insertable hybrid chip (Steps 1–5), culturing intestinal epithelial cells on either a polydimethylsiloxane (PDMS)-based porous membrane (Steps 6A, 7A, 8, 9) or the polyester membrane of a Transwell insert (Steps 6B, 7B, 8, 9), and inducing 3D morphogenesis in vitro (Step 10). We also identify cellular and molecular characteristics that are indicative of tissue-specific histogenesis and lineage-dependent cytodifferentiation by applying multiple imaging modalities (Steps 11–24). We induce morphogenesis using human intestinal epithelial cells such as Caco-2, or intestinal organoids, in both culture formats with technical details including surface modification of a porous membrane, creation of a 2D monolayer and recapitulation of the biochemical and biomechanical microenvironment of the gut in vitro. To induce 3D morphogenesis from a 2D epithelial monolayer, we remove morphogen antagonists in both culture formats by flowing culture medium into the basolateral compartment of the cultures. Finally, we provide representative examples of the utility of the regenerative 3D epithelial layers that can potentially be used for simulating morphogen-dependent epithelial growth, longitudinal host–microbiome co-cultures, pathogen infection, inflammatory injury, epithelial barrier dysfunctions and probiotic-based therapeutic effect.
Applications of the method
Our protocol can potentially provide a broad impact to a wide spectrum of scientists in both basic (e.g., intestinal mucosal biology, stem cell biology and developmental biology) and applied research (e.g., preclinical drug testing, disease modeling, tissue engineering and gastroenterology). Since our protocol is reproducible and robust to induce 3D morphogenesis of an intestinal epithelium in vitro, we envision that our technical strategy can be disseminated to audiences who study the dynamics of cellular signaling during intestinal development, regeneration or homeostasis. Also, our protocol can be useful for audiences who interrogate mechanisms of disease pathology and pathogenesis under various infectious agents such as norovirus8, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Clostridioides difficile, Salmonella enterica Typhimurium9 or Vibrio cholerae. The use of the gut-on-a-chip microphysiological system can potentially allow a longitudinal co-culture10 and subsequent assessments of host defense, immune response and pathogen-associated damage repair in the gastrointestinal (GI) tract11. Other GI diseases that involve villous atrophy, crypt shortening, mucosal damage or impaired epithelial barrier germane to leaky gut syndrome, celiac disease, Crohn’s disease, ulcerative colitis, pouchitis or irritable bowel syndrome can be emulated when a 3D intestinal epithelial layer is prepared using patient’s biopsy- or stem-cell-derived intestinal organoids12,13. To better mimic a higher complexity of a disease milieu, readers can contemplate adding disease-relevant cell types such as tissue-specific immune cells derived from a patient’s peripheral blood mononuclear cells (PBMCs) in the model that contains 3D intestinal villus–crypt microarchitecture3,5.
Since the 3D epithelial microstructure can be fixed and visualized without a sectioning process, audiences who work on spatial transcriptomics and high- or super-resolution imaging may potentially be interested in our technique to map the spatiotemporal dynamics of genes and proteins on the epithelial niche in response to microbial or immune stimulations. In addition, longitudinal host–microbiome crosstalk that orchestrates intestinal homeostasis can be established in a 3D intestinal mucosal layer10,14, especially in the gut-on-a-chip platform, by co-culturing various microbial species, microbial communities or fecal microbiota. This approach will be particularly compelling to audiences who study mucosal immunology, gastroenterology, human microbiome, culturomics with an effort to culture previously uncultured gut microbiota in laboratories, and clinical microbiology. If our in vitro morphogenesis protocol can be adapted to a scalable culture format such as multiwell porous inserts in a 24-, 96- or 384-well plate that can continuously replenish the basolateral compartment, the protocol can be also disseminated to audiences who develop a high-throughput screening or a validation platform in the pharmaceutical, biomedical or food industries. As a proof-of-principle, we recently demonstrated the feasibility of a multiplex high-throughput morphogenesis system scalable to a 24-well plate format15. In addition, multiple organ-on-a-chip products have been commercialized16,17,18. Hence, the validation of our in vitro morphogenesis method can be accelerated and potentially adopted in many research laboratories, industries or government and regulatory agencies to understand cellular reprogramming of in vitro intestinal morphogenesis at the transcriptome level19, to test absorption and transport of pharmaceutical or biotherapeutic candidates using the 3D gut surrogates or to assess reproducibility of the intestinal morphogenic process using customized or commercialized organ-on-a-chip models.
Comparison with other methods
A limited number of human-relevant experimental models have been utilized to study intestinal epithelial morphogenesis, mainly because of a lack of implementable protocol to induce in vitro 3D morphogenesis. Indeed, a majority of the current knowledge on intestinal morphogenesis is based on animal studies (e.g., zebrafish20, mouse21 or chicken22). However, they are labor and cost intensive, may be ethically questionable and, most importantly, do not accurately dictate the developmental process in humans. These models are also substantially limited for testing in a multiplex scalable way. Thus, our protocol to regenerate a 3D tissue architecture in vitro is advantageous over in vivo animal models as well as other conventional static 2D cell culture models. As previously mentioned, utilizing a 3D epithelial structure allows us to examine the spatial localization of differentiated cells in a crypt–villus axis in response to various mucosal or immune stimulations. The 3D epithelial layer can provide a space to investigate how microbial cells compete to make a spatial niche and ecologically evolve in response to host factors (e.g., inner versus outer mucus layers, secretion of IgA and antimicrobial peptides). Furthermore, 3D epithelial morphology may allow us to understand how the gut microbiota structures their communities and synergistically produces microbial metabolites (e.g., short-chain fatty acids) that shape the cellular organization and stem cell niche in the basal crypt. These features can only be demonstrated when 3D epithelial layers are established in vitro.
There are a couple of in vitro approaches besides our method to create 3D intestinal epithelial structures. The intestinal organoid culture is the most advanced tissue engineering technique based on an intestinal stem cell culture under defined morphogen conditioning23,24,25. However, it is often challenging to use the 3D organoid models for running transport assays or host–microbiome co-cultures because the intestinal lumen is enclosed inside an organoid body, and thus, introduction of luminal components such as microbial cells or exogenous antigens is limited. Using a microinjector can improve the accessibility to the organoid lumen26,27, but this method is invasive and labor intensive and requires expertise to execute. Furthermore, the conventional organoid culture is maintained under a static condition in a hydrogel scaffold, which does not accurately reflect active in vivo biomechanics.
Other approaches that several research groups have adopted leverage a prestructured 3D hydrogel scaffold to mimic the intestinal epithelial architecture by culturing dissociated human intestinal cells on the gel surface28,29,30. Hydrogel scaffolds were fabricated using 3D printed, micromilled or lithographically made molds. This method has shown a self-organized alignment of dissociated epithelial cells with a physiologically relevant morphogen gradient in vitro, establishment of a high-aspect ratio epithelial structure and stromal–epithelial crosstalk by including stromal cells in the scaffold. However, the nature of prestructured scaffolds may preclude demonstrating a spontaneous morphogenesis process per se. These models also do not offer dynamic luminal or interstitial flow, lacking fluid shear stress that intestinal cells need to undergo morphogenesis and gain physiological functions. Another recent study used a hydrogel scaffold in a microfluidic platform and patterned intestinal epithelial structure using a laser etching technology31. Mouse intestinal organoids formed an intestinal tubular structure following the etched pattern, and a luminal fluid flow can be recapitulated using a microfluidic module. However, this model also neither demonstrates a spontaneous morphogenesis process nor includes intestinal mechanobiological motions. The same group’s 3D printing technology enabled an establishment of a mini-intestinal tube with a spontaneous morphogenesis process32. Although this study sophisticatedly fabricated different segments of the gut within a tube, the model also lacks luminal fluid flow and mechanical deformations. Furthermore, the manipulability of the model may be limited, especially to perturb experimental conditions or intercellular interactions once the bioprinting process is completed. On the contrary, our proposed protocol offers spontaneous intestinal morphogenesis, physiologically relevant shear stress, biomechanics emulating intestinal peristalsis, accessibility of apical and basolateral compartments independently, and modularity for re-creating a complex biological microenvironment. Hence, our protocol for in vitro 3D morphogenesis may provide a complementary approach to overcome the challenges of the existing methods.
Limitations
Our protocol focuses entirely on the 3D epithelial morphogenesis, having only the epithelial cells in cultures without other types of surrounding cells such as mesenchymal, endothelial and immune cells. As stated earlier, the core of our protocol is to induce epithelial morphogenesis by removing morphogen inhibitors secreted in the basolateral side in the introduced medium4. While the strong modularity of our gut-on-a-chip and a hybrid chip allows us to re-create undulated 3D epithelial layers, additional biological complexity such as epithelial–mesenchymal interactions33,34, extracellular matrix (ECM) deposition for 3D regeneration35 and crypt–villus characteristics that convey a stem cell niche in the basal crypt36 remains to be further considered in our model. Stromal cells in the mesenchyme (e.g., fibroblasts) play a critical role in producing ECM proteins and regulating intestinal morphogenesis in vivo35,37,38. Inclusion of mesenchymal cells in our model may enhance the morphogenesis process and cell attachment efficiency. Endothelial layers (i.e., capillary vasculatures or lymphatic vessels) play seminal roles in regulating molecular transport39 and immune cell recruitment40 in the intestinal microenvironment. Also, when tissue models aim to demonstrate multiorgan interactions, the vasculature component that can connect between the tissue models is a prerequisite. Therefore, including endothelial cells may be required for simulating more accurate physiological features with organ-level resolutions. In the case of modeling intestinal diseases, patient-derived immune cells are also essential for demonstrating innate immune responses, antigen presentation, innate-adaptive immune crosstalk and tissue-specific immunity.
The use of a hybrid chip is more straightforward than that of a gut-on-a-chip because the device setup is simpler and the use of a Transwell insert allows scalable cultures of intestinal epithelium. However, the commercially available Transwell insert with a polyester membrane is not elastic to emulate intestinal peristalsis-like movements. Furthermore, the apical compartment of the Transwell insert placed in a hybrid chip remains static, lacking shear stress on the apical side. Obviously, a static nature in the apical compartment is seldom capable of long-term bacterial co-cultures in a hybrid chip. Although we can robustly induce 3D morphogenesis in a Transwell insert when using a hybrid chip, a shortage of physiologically relevant biomechanics and apical fluid flow may restrict the implementability of the hybrid chip platform for potential applications.
In both gut-on-a-chip and hybrid chip cultures, the full-size re-creation of the human crypt–villus axis has not been fully established. Since the morphogenesis is initiated from an epithelial monolayer, the 3D microarchitecture does not necessarily provide morphological similarity with an in vivo crypt. Although we characterized the population of proliferative cells near the basal crypt domain in the microengineered 3D epithelium2, the crypt and villus regions are not clearly compartmentalized. Although a taller upper channel of a gut-on-a-chip induces an increased height of a microengineered epithelium4, the maximum height is still limited to ~300–400 µm. The actual depth of human intestinal crypts in vivo in the small and large intestines is ~135 µm and ~400 µm in depth, respectively, and the height of a small intestinal villus is ~600 µm41.
From an imaging standpoint, in situ super-resolution imaging of the 3D microarchitecture may be limited in a gut-on-a-chip because the required working distance from an objective to the epithelial layer is on the order of a couple of millimeters. To overcome this issue, a long-distance objective may be required. Furthermore, because of the high elasticity of PDMS, it is challenging to make a thin section for imaging specimen preparation. In addition, since the layer-by-layer microfabrication of a gut-on-a-chip involves a permanent bonding between each layer, it is extremely challenging to open or remove the upper layer to examine the surface structure of the epithelial layers, for example, by using scanning electron microscopy (SEM).
Hydrophobicity of PDMS has been a limiting factor in microfluidics-based studies dealing with hydrophobic small molecules because PDMS can nonspecifically adsorb such hydrophobic molecules. Other polymeric materials may be considered to substitute PDMS. Alternatively, surface modification of PDMS (e.g., coating with lipophilic material42 or poly(ethylene glycol)43) may be contemplated to minimize the adsorption of hydrophobic molecules.
Finally, our approach has not been fully characterized for providing a high-throughput screening or a ‘one-fit-to-all’-type user-friendly experimental platform. The current protocol requires one syringe pump per each microdevice, which takes up space in a CO2 incubator, preventing large-scale experiments. This limitation may be substantially improved by innovating the scalability of the culture format (e.g., 24-, 96- or 384-well porous inserts that allow the continuous replenishment and removal of basolateral medium).
Experimental design
To induce 3D morphogenesis of human intestinal epithelium in vitro, we use a microfluidic gut-on-a-chip device that contains two parallel microchannels and an elastic porous membrane in the middle to create a lumen–capillary interface. We also demonstrate the use of a single channel microfluidic device, a hybrid chip, that can offer continuous basolateral flow below a polarized epithelial layer grown on a Transwell insert. In both of the platforms, morphogenesis of various human intestinal epithelial cells can be demonstrated by applying a directional manipulation of flow to remove morphogen antagonists from the basolateral compartment. The overall experimental procedure (Fig. 1) is composed of five parts: (i) microfabrication of a gut-on-a-chip or a Transwell-insertable hybrid chip (Steps 1–5; Box 1), (ii) preparation of intestinal epithelial cells (either Caco-2 cells or human intestinal organoids; Boxes 2–5), (iii) culture of intestinal epithelial cells on a gut-on-a-chip or a hybrid chip (Steps 6–9), (iv) induction of in vitro 3D morphogenesis (Step 10) and (v) characterization of 3D epithelial microarchitectures (Steps 11–24). Finally, appropriate control groups (discussed further below) are designed to verify the validity of in vitro morphogenesis by comparing the epithelial morphogenesis with spatial, temporal, conditional or procedural controls.
We use two different culture platforms: a gut-on-a-chip either with a straight channel or a nonlinear convoluted channel, or a hybrid chip that includes a Transwell (TW) insert in a microfluidic device, fabricated as described in Box 1 and Steps 1–5. ‘Device fabrication’ displays the major steps for making either a gut-on-a-chip or a hybrid chip. ‘Culture of human intestinal epithelium’ explains the cell sources used in this protocol (Caco-2 or human intestinal organoids) and the culture process. ‘In vitro morphogenesis’ shows the overall steps of Caco-2 or organoid-derived epithelial cell culture on a gut-on-a-chip or on the Transwell insert of a hybrid chip followed by the induction of 3D morphogenesis and characterization of the formed epithelial structure. Procedure step numbers or box numbers are indicated below each arrow. Applications provides examples that the established intestinal epithelial layers can be utilized for, such as characterization of cytodifferentiation, study of intestinal physiology, establishment of a host–microbiome ecosystem and disease modeling. An immunofluorescence image in ‘Cytodifferentiation’ shows nuclei, F-actin and MUC2 expressed in a 3D Caco-2 epithelial layer produced on a gut-on-a-chip. The MUC2 signal is found in both the goblet-like cells and the secreted mucus on the mucosal surface. A fluorescence image in ‘Intestinal physiology’ displays the production of mucus by staining sialic acid and N-acetylglucosaminyl residues using fluorescent wheat germ agglutinin. Two overlaid images in ‘Host–microbe co-culture’ show the representative host-microbiome co-culture in a gut-on-a-chip. The left image shows the co-culture of green fluorescence protein (GFP)-expressing Escherichia coli with a microengineered 3D Caco-2 epithelium. The right image visualizes the localization of GFP E. coli co-cultured with 3D Caco-2 epithelium followed by the immunofluorescence staining with F-actin (red) and nuclei (blue). Disease modeling illustrates a demonstration of healthy versus leaky gut in a gut inflammation-on-a-chip under physiological challenges by a bacterial antigen (e.g., lipopolysaccharide, LPS) and immune cells (e.g., PBMCs; green). Caco-2 cells were cultured to establish the 3D epithelial layers. Scale bars, 50 µm. Images in bottom row: ‘Cytodifferentiation’ adapted with permission from ref. 2, Oxford University Press; ‘Intestinal physiology’ reproduced with permission from ref. 5, NAS; ‘Host–microbe co-culture’ adapted with permission from ref. 3, NAS; ‘Disease modeling’ adapted with permission from ref. 5, NAS.
Microfabrication of a gut-on-a-chip or a Transwell-insertable hybrid chip (Box 1 and Steps 1–5)
Both the gut-on-a-chip and the hybrid chip are fabricated using PDMS replicas demolded from silicon molds patterned with SU-8 by soft lithography1,44. The design of microchannels in each chip was determined by considering fluid dynamics such as shear stress and hydrodynamic pressure1,4,12. The original design of the gut-on-a-chip (Extended Data Fig. 1a) that contains two parallel straight-lined microchannels apposed to each other has evolved to a convoluted gut-on-a-chip (Extended Data Fig. 1b) that includes a pair of curved microchannels to induce increased fluid residence time, nonlinear flow pattern and the multiaxial deformation of cultured cells (Fig. 2a–f)12. The convoluted gut-on-a-chip may be chosen when more complex intestinal biomechanics need to be reconstituted. We have verified that the convoluted gut-on-a-chip also robustly induces 3D morphogenesis at a similar extent of epithelial growth in a similar time frame regardless of the cultured cell types compared with the original gut-on-a-chip12. For the purpose of inducing 3D morphogenesis, thus, the linear and the convoluted gut-on-a-chip designs are interchangeable. The PDMS replica cured on the silicon molds with SU-8 patterns provides a negative feature once they are demolded (Fig. 2a). To fabricate a gut-on-a-chip, a prepared upper PDMS layer is sequentially bonded to a porous PDMS membrane, then subsequently aligned to the lower PDMS layer by performing irreversible bonding using a corona treater (Fig. 2b–f). To fabricate a hybrid chip, a cured PDMS replica is bonded to a glass slide to establish a single-channel microfluidic device that can hold a Transwell insert (Fig. 2h and Extended Data Fig. 2). The bonding process is performed by treating the surface of the PDMS replica and the glass with oxygen plasma or corona treatment. After sterilization of a microfabricated device connected to silicone tubing, the device setup is ready to use for performing 3D morphogenesis of intestinal epithelium (Fig. 2g).
a, A schematic to prepare a PDMS part from a SU-8-patterned silicon mold. Uncured PDMS solution is poured on a silicon mold (left), cured at 60 °C (middle) and demolded (right). The demolded PDMS is cut into several pieces and cleaned for further use. b, A photograph of a silicon mold for preparing an upper PDMS layer. c, A photograph of a silicon mold for fabricating PDMS porous membranes. d, A series of photos of upper and lower PDMS parts as well as an assembled gut-on-a-chip device. e, A schematic diagram of the alignment of the upper, membrane and lower PDMS parts. Each layer is irreversibly bonded by either plasma or corona treatment. f, A schematic of a fabricated gut-on-a-chip device that has superimposed convoluted microchannels and vacuum chambers. g, Setup of a gut-on-a-chip for microfluidic cell culture. The fabricated gut-on-a-chip assembled with silicone tubing and syringes is placed on a cover slip. The chip setup is placed on a lid of a 150 mm Petri dish for handling. Binder clips are used to close the silicone tubing. h, A visual snapshot of the fabrication of a hybrid chip and the use of a hybrid chip for 3D morphogenesis. A Transwell insert that is independently prepared to culture a 2D monolayer of intestinal epithelial cells is inserted into a hybrid chip to induce intestinal 3D morphogenesis. Culture medium is perfused through the microchannel underneath the cell layer established on the Transwell insert. Scale bars, 1 cm. h reproduced with permission from ref. 4, Elsevier.
Preparation of intestinal epithelial cells (Boxes 2–5 and Step 8)
In this protocol, Caco-2 cell line and intestinal organoids are used as epithelial sources (Fig. 3a). Both types of cells are independently cultured (Boxes 2 and 5) and used for seeding in an ECM-coated microchannel of a gut-on-a-chip or on a Transwell insert. Caco-2 cells (passage number between 10 and 50) routinely cultured in a T flask are harvested when the cells are confluent (>95% coverage in a flask) to prepare a dissociated cell suspension by trypsinization (Box 2). Human intestinal organoids derived from intestinal biopsies or surgical resections are cultured in a dome of Matrigel scaffold plated in a 24-well plate to support the structural microenvironment. Culture medium that contains essential morphogens (e.g., Wnt, R-spondin and Noggin) and growth factors, prepared as described in Box 3, is replenished every other day until the organoids grow up to ~500 µm in diameter. Fully grown organoids are harvested and dissociated into single cells for a seeding into a gut-on-a-chip or on a Transwell insert (Box 5). As we previously reported, diverse intestinal organoid lines can be established and used depending on the disease type12,13 (e.g., ulcerative colitis, Crohn’s disease, colorectal cancer or normal donors), the site of lesion (e.g., diseased versus nondiseased region) and the location in the GI tract (e.g., duodenum, jejunum, ileum, cecum, colon or rectum). We provide an optimized protocol in Box 5 for culturing colonic organoids (colonoids) that typically require a higher concentration of morphogens than small intestinal organoids.
a, A workflow to induce intestinal morphogenesis in a gut-on-a-chip. Both Caco-2 human intestinal epithelium and intestinal organoids are used in this protocol to demonstrate 3D morphogenesis. The dissociated epithelial cells are seeded in a prepared gut-on-a-chip device (Chip prep). Once the cells are seeded (Seeding) and attached (Attachment) on a PDMS porous membrane on day 0 (D0), apical (AP) flow is initiated and maintained for the first 2 d (Flow, AP, D0–D2). When an intact 2D monolayer is formed, basolateral (BL) flow is also initiated along with a cyclic stretching motion (Stretching, Flow, AP and BL). Intestinal 3D morphogenesis spontaneously occurs after 5 d of microfluidic culture (Morphogenesis, D5). Phase-contrast images show representative morphologies of Caco-2 cells at each experimental step or timepoint (Bar, 100 µm). Four schematics illustrate the corresponding cascade of intestinal morphogenesis (right top). Dashed arrows in the schematics indicate the direction of fluid flow. b, An SEM image shows the surface topology of the established 3D Caco-2 epithelium (left). An inset that highlights a zoomed-in area (a white dashed box) shows the microvilli regenerated on the 3D Caco-2 layer (right). c, Immunofluorescence confocal visualization of the tight junction protein (ZO-1, red) and the continuous brush-border membrane labeled for F-actin (green) and nuclei (blue) in a horizontal en face view of an established Caco-2 3D epithelium in a gut-on-a-chip. Arrows directed to the schematic image in the middle indicate the location of the focal plane of each confocal view. d, A time course of morphological changes of an organoid epithelium cultured in a gut-on-a-chip acquired by a phase-contrast microscope on days 3, 7, 9, 11 and 13. The insets (right top) show a high magnification of the provided images. e, A DIC micrograph of an organoid 3D epithelium established in a gut-on-a-chip taken on day 7. f, Overlaid immunofluorescence images showing the markers of stem cells (LGR5; magenta), goblet cells (MUC2; green), F-actin (gray) and nuclei (cyan) on the organoid epithelial layers grown in gut-on-a-chips for 3 (left) and 13 d (middle), respectively. See also Extended Data Fig. 3, which highlights LGR5 signal without MUC2 signal. A fluorescent image shows the epithelial microstructure of a 3D organoid epithelium established in a gut-on-a-chip visualized by staining the plasma membrane using a CellMask dye at day 13 of the culture (right). Scale bars 50 µm, unless otherwise indicated. b reproduced with permission from ref. 2, Oxford University Press; c adapted with permission from ref. 2, Oxford University Press; e and f adapted with permission from ref. 12 under a Creative Commons licence CC BY 4.0.
Culture of intestinal epithelial cells on a gut-on-a-chip or on the Transwell insert of a hybrid chip (Steps 6–9)
In the gut-on-a-chip, it is necessary to modify the hydrophobic surface of the PDMS porous membrane for a successful ECM coating. In this protocol, we apply two different methods to modify the PDMS membrane’s hydrophobicity. For culturing Caco-2 cells, surface activation via UV/ozone treatment alone is enough to reduce hydrophobicity of the PDMS surface, coat the ECM and attach Caco-2 cells on the PDMS membrane. However, the microfluidic culture of organoid epithelium requires chemical-based surface functionalization to achieve efficient deposition of ECM proteins by applying polyethylenimine (PEI) and glutaraldehyde, sequentially, to the PDMS microchannels13. After the surface modification, ECM proteins are deposited to coat the functionalized PDMS surface followed by the introduction of dissociated organoid epithelium. After cell attachment, microfluidic cell culture begins by perfusing culture medium only to the upper microchannel until the cells form an intact monolayer while the lower microchannel maintains static conditions. This optimized approach for surface activation and ECM coating enables the attachment of organoid epithelium to induce 3D morphogenesis on the PDMS surface13.
Transwell cultures also require ECM coating prior to cell seeding; however, Transwell cultures do not require complex pretreatment steps for activating the surface of a porous insert. For growing Caco-2 cells on a Transwell insert, an ECM coating on the porous insert accelerates the attachment of dissociated Caco-2 cells (<1 h) and the formation of tight junction barrier1 (<1–2 d). For culturing organoid cells on a Transwell insert, dissociated organoid cells are seeded on the ECM-coated insert, attached on the membrane surface (<3 h) and maintained until the organoid cells form an intact monolayer with barrier integrity. The Transwell culture is performed in a 24-well plate, and the hybrid chip is not used at this point.
Induction of in vitro 3D morphogenesis (Step 10)
The in vitro 3D morphogenesis can be initiated by applying a fluid flow on the basolateral side of the established epithelial layer. In the gut-on-a-chip, epithelial morphogenesis begins when the culture medium is perfused into both the upper and the lower microchannels (Fig. 3a). As previously described4, it is critical to introduce fluid flow in the lower (basolateral) compartment to continuously remove the directionally secreted morphogen inhibitors. To supply sufficient nutrients and serum to the cells bound on the porous membrane as well as to create a luminal shear stress, we typically apply dual flow in a gut-on-a-chip. In the hybrid chip, a Transwell insert that contains an epithelial monolayer is inserted into a hybrid chip. Then, the culture medium is applied through the microchannel, beneath the basolateral side of the porous Transwell insert. Intestinal morphogenesis occurs in 3–5 d after the basolateral flow is initiated in both culture platforms.
Characterization of 3D epithelial microarchitectures (Steps 11–24)
The morphological characteristics 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 (Figs. 3 and 4). Phase-contrast or DIC imaging can be readily performed at any time during the culture to monitor the shape and the protrusion of 3D epithelial layers. Thanks to the optical transparency of PDMS and the polyester membrane, both the gut-on-a-chip and hybrid chip platforms offer real-time in situ imaging without sectioning or disassembly of the device. When immunofluorescence imaging is performed (Figs. 1, 3c,f and 4b,c), cells are generally fixed with 4% (wt/vol) paraformaldehyde (PFA), then permeabilized and blocked with Triton X-100 and 2% (wt/vol) bovine serum albumin (BSA), sequentially. Depending on the cell type, different fixatives, permeabilizing reagents and blocking reagents may be used. Primary antibodies targeting lineage-dependent cells or regional markers are used for highlighting the cells fixed in situ in the chips, followed by treatment with secondary antibodies as well as counterstaining dyes that target either nucleus (e.g., 4′,6-diamidino-2-phenylindole, DAPI) or F-actin (e.g., fluorescence-labeled phalloidin). Fluorescence-based real-time imaging can be also performed in situ to detect mucus production (Fig. 1, ‘Cytodifferentiation’ and ‘Intestinal physiology’), stochastic colonization of microbial cells (Fig. 1, ‘Host–microbe co-culture’), recruitment of immune cells (Fig. 1, ‘Disease modeling’) or the contours of the 3D epithelial morphology (Figs. 3c,f and 4b,c). When the gut-on-a-chip is modified to separate the upper layer from the lower microchannel layer as described in ref. 2, 3D epithelial morphology as well as the microvilli on the apical brush border can be visualized by SEM (Fig. 3b). Expression of differentiation markers may be assessed by performing quantitative PCR5 or single-cell RNA sequencing19. In this case, epithelial cells of a 3D layer grown in either a gut-on-a-chip or a hybrid chip are harvested by trypsinization, then used for molecular or genetic analyses.
a, A workflow to induce intestinal morphogenesis in a hybrid chip. Both Caco-2 and intestinal organoids are used in this protocol to demonstrate 3D morphogenesis in a hybrid chip platform. The dissociated epithelial cells are seeded in a prepared Transwell insert (TW prep; see the schematic below). Once the cells are seeded (Seeding) and attached on a polyester membrane in a Transwell insert, all the cells are cultured under static conditions (TW culture). After 7 d, an individual Transwell insert that contains a 2D monolayer of epithelial cells is incorporated into a hybrid chip to introduce basolateral flow (Flow, BL) that eventually leads to a generation of a 3D epithelial layer (Morphogenesis). Phase-contrast micrographs show the morphological profile of human organoid epithelial cells derived from the ascending colon of a normal donor (C103 line) at each experimental step or timepoint. Schematics in the upper layer illustrate the experimental configuration at each step. b, The hybrid chip (left schematic) can lead to the 3D morphogenesis of organoid epithelial cells, where the top-down confocal microscopic views taken at different Z-positions (upper, middle and lower; see the right schematic with corresponding dashed lines) show distinct morphological characteristics. F-actin (cyan), nuclei (gray). c, Fluorescence confocal micrographs (3D angled views) of the organoid-derived epithelial cells cultured in a static Transwell (TW; an inset inside a white dashed box) versus a hybrid chip (the largest full shot) comparing the 2D versus 3D morphologies, respectively. A pair of 2D vertical cross-cut views (insets on the right top; ‘XZ’) also display 2D versus 3D characteristics. Scale bars, 100 µm. c reproduced with permission from ref. 4, Elsevier.
Preparation of the control groups
Control groups can be prepared by culturing the same cells (either Caco-2 or intestinal organoid epithelium) into a 2D monolayer in conventional static culture conditions. It is noted that it may be challenging to maintain static conditions in a gut-on-a-chip microdevice for a long time because the limited volume capacity of the microchannels (i.e., ~4 µL in the top channel of the original gut-on-a-chip design) may cause nutrient depletion. Thus, epithelial morphologies before and after applying basolateral flow can be also compared.
