Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.
The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure for the in vitro modeling of interferon gamma (IFNγ)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.
The human intestine is a complex and multitasking organ capable of self-regeneration. It is divided into the small and large intestine. The primary function of the small intestine is to further digest food coming from the stomach, absorb all the nutrients, and pass the residue on to the large intestine, which recovers the water and electrolytes. The small intestine is further divided into multiple anatomically distinct regions: the duodenum, jejunum, and ileum, each of which is adapted to perform specific functions. For example, the duodenum helps break down of the chyme (stomach contents) to enable the proper absorption of nutrients involving proteins, carbohydrates, vitamins, and minerals in the jejunum. This proximal part of the small intestine is also the main site of intestinal drug absorption and metabolism, and it is characterized by the higher expression of drug-metabolizing enzymes (e.g., CYP3A4) compared to their expression in the ileum and colon1. In addition to its main role in digesting and absorbing of nutrients, the intestine is also an effective barrier against potentially harmful luminal contents, such as pathogenic microorganisms, microbial metabolites, dietary antigens, and toxins2,3. It is noteworthy that the human colon is inhabited by a large number of microorganisms, far exceeding that of total cells in the human body, which provide many benefits to nutrition, metabolism, and immunity. Therefore, the maintenance of the integrity of the mucosal barrier formed by intestinal epithelial cells is critical for allowing the symbiotic relationship between the gut microbiota and the host cells by physically separating them to avoid unnecessary immune cell activation2. In addition, programmed intestinal cell death plays an essential role as a self-protective mechanism preventing infected cells from persisting or proliferating—thereby disseminating potential pathogens3—while the continuous self-renewal of intestinal epithelium every four to seven days compensates for the cell loss ensuring barrier integrity and tissue homeostasis. Impairments of described intestinal functions, including nutrient absorption, barrier integrity, or imbalance in intestinal cell death and self-renewal, may result in the development of a range of gastrointestinal disorders, including malnutrition and inflammatory bowel disease (IBD)2,3.
Previously, animal models and transformed cancer-derived intestinal cell lines have been used to study the physiological and pathophysiological functions of human intestinal tissue. However, increasingly prominent concerns about the translatability of animal research to humans, caused by the presence of significant disparities between the two species, highlighted a need for human-relevant alternative methods4. The commonly used in vitro intestinal cell lines include T84, Caco-2, and HT29 cells. While they mimic certain aspects of the intestinal barrier function and membrane transport, they are characterized by an altered expression of drug metabolizing enzymes5, surface receptors, and transporters4. In addition, they lack intestinal segment specificity and fail to recapitulate the complexity of intestinal epithelium, with each model containing only one out of the five epithelial cell types present in the intestine6.
Recently, human intestinal organoid cultures established from fresh biopsies of the small intestine and colon7,8 or induced pluripotent stem cells (iPSC)9 were introduced as alternative experimental models with potential to complement, reduce and perhaps replace animal experimentation in the future. While iPSCs can be obtained in a non-invasive manner, establishing organoids from iPSCs requires the use of complex and lengthy protocols (with several experimental steps) and generates cultures resembling human fetal tissue. In contrast, biopsy-derived organoids are highly scalable, as they can leverage the inherent renewal capacity of intestinal tissue and can be indefinitely passaged and propagated in vitro. Importantly, biopsy-derived organoids maintain the disease and intestinal region-specific characteristics of the primary tissue from which they were developed and emulate the cellular diversity of the intestinal epithelium. Organoids can be used as patient-specific avatars in vitro to unravel the biology and pathogenesis of various gastrointestinal disorders and improve their therapeutic management. Although intestinal organoids have achieved an impressive degree of physiological functionality, they still fail to reproduce the complexity of the native organs due to their lack of critical stromal components—including blood vessels, connective tissue, peripheral nerves, and immune cells—as well as mechanical stimulation. Mechanical parameters, such as flow, shear stress, stretch, and pressure, are known to influence tissue morphogenesis and homeostasis in vivo and were previously shown to improve maturation of cells in vitro10,11,12,13. An additional important drawback of organoid systems is the inaccessibility of the lumen and, thus, to the apical side of the epithelium. This presents a challenge for investigating various mechanisms associated with the polarized expression of ion and drug transporters, host-microbiome interactions, and pharmaceutical toxicity testing. Lastly, organoid cultures suffer from considerable variability in size, morphology, and function, owing to the stochastic nature of the in vitro self-organization process and cell fate choices. Therefore, to realize the full potential of intestinal organoids in disease modeling, drug screening, and regenerative medicine, it is necessary to explore new strategies that reduce the variability in organoid development, improve the access to the luminal compartment, and incorporate missing cell-cell interactions.
Organ-on-a-Chip technology has introduced many techniques for the incorporation of mechanical forces and fluid flow to intestinal cell cultures in vitro. However, since most of the initial proof-of-concept studies have used cancer-derived cell lines that did not exhibit sufficient cellular diversity, the relevance of these systems has been questioned. Recently, we have synergistically combined intestinal organoids and organ-on-a-chip technology to incorporate the best features of each approach into one in vitro system14,15,16. The resulting intestine-chip recapitulates the multicellular architecture of intestinal epithelium, the presence of epithelial-endothelial tissue interface, and the mechanical forces of fluid flow and stretch, enabling emulation of organ-level functions in vitro. Additionally, the use of primary-tissue-derived organoids (which can be sampled from different regions of the human intestine) as a starting material increases this model’s versatility, as chips representing human duodenum, jejunum, ileum, and colon can be established following similar seeding and culture procedures. Importantly, Intestine-Chips enable real-time assessment of: the intestinal barrier integrity; activity of the brush border and drug-metabolism enzymes; production of mucins; secretion of cytokines; and interaction of intestinal cells with pathogenic and commensal microorganisms, as demonstrated in the previously published reports. Notably, when intestine chips were established using organoids generated from different individuals’ tissue, these models captured the expected inter-donor variability in the functional responses to various drugs and treatments. Altogether, merging organoids with Organ-on-a-Chip technology opens the door to more advanced, personalized, in vivo-relevant models that could improve the physiological relevance and accuracy of the in vitro findings as well as their extrapolation to humans. Here, a detailed protocol is presented for establishing the intestine chip and its application in the studies of physiological functions of the two intestinal segments: duodenum and colon. Firstly, the methods for the assessing the activity of the drug-metabolizing enzyme CYP3A4 in the duodenum chip, as well as its induction by prototypical compounds such as rifampicin and Vitamin D3, are described. Secondly, the steps required to model “leaky gut” in the colon chip are outlined in the protocol, with the disruption of the epithelial barrier being performed using hallmark cytokines implicated in the pathogenesis of the IBD. Briefly, organoids derived from human biopsies are propagated in vitro, subjected to enzymatic digestion, and introduced in the top channel of the chip. In the presence of continuous perfusion with growth-factor-enriched media they develop into a confluent epithelial monolayer with 3D architecture and a readily accessible apical cell surface. The bottom, "vascular" chip compartment is seeded with microvascular endothelial cells isolated from the small or large intestine. The epithelium and endothelium are separated by a porous stretchable membrane, which facilitates the paracrine interactions between the two tissues and, when subjected to cyclic deformations, emulates peristalsis-like motions of the human gut. The co-culture is maintained under the dynamic flow conditions generated by luminal and vascular perfusion with appropriate cell culture media. Finally, we describe numerous types of assays and endpoint analyses that can be performed directly on-chip or from sampled cell culture effluents.
NOTE: All cell cultures should be handled using a proper aseptic technique.
The human intestinal organoids employed in this study were obtained from Johns Hopkins University and all methods were carried out in accordance with approved guidelines and regulations. All experimental protocols were approved by the Johns Hopkins University Institutional Review Board (IRB #NA 00038329).
1. Preparation of the cell culture reagents
2. Culture of Human Intestinal Microvascular Endothelial Cells (HIMECs)
3. Microfabrication and preparation of the chip
4. Activation and ECM coating of the membrane
5. Seeding of Intestinal Microvascular Endothelial Cells (HIMECs) in the bottom channel of the chip
NOTE: Small intestinal and colonic HIMECs are seeded in the bottom channel of the duodenum and colon chip, respectively.
6. Seeding of organoid fragments in the top channel of the chip
NOTE: Organoids isolated from biopsies of various intestinal regions can be cultured in the intestine chip7. Follow the procedures described in Fujii et al. for the isolation of human intestinal crypts and establishment of organoid cultures22. Here, duodenal and colonic organoids are used to generate the duodenum and colon chips, respectively. Given the high batch-to-batch and donor-to-donor variability in organoid formation and growth, it is suggested to perform a pilot evaluation of cell density in the organoid suspension culture (24-well plate format) to achieve the optimal seeding density of 8 million cells/mL.
7. Dynamic culture of intestine chip – initiation, and maintenance of flow and peristalsis-like motions
8. CYP450 induction using prototypical CYP inducers in the duodenum chip
NOTE: The cytochrome P450 (CYP450) induction assay enables assessing whether the test compound increases the mRNA levels and/or catalytic activity of specific CYP450 enzymes. Here, we describe the protocol for the evaluation of CYP3A4 induction by the industry standard and regulator recommended in vitro CYP inducers, Rifampicin (RIF) and 1,2-dihydroxy vitamin D3 (VD3). The presented method may be used to identify the potential of various test compounds to induce different isoforms of CYP450 in human intestinal tissue. Specific sets of primers and probe substrate will need to be selected for each enzyme isoform to be evaluated.
9. Disruption of the epithelial barrier using proinflammatory cytokines in colon chip
NOTE: This protocol describes disruption of the intestinal epithelial barrier by the cytokine interferon gamma (IFNγ)26,27,28,29. The cytokine is dosed in the bottom channel of the colon chip given the basolateral expression of IFNγ receptor on intestinal epithelial cells. The proinflammatory stimulus is introduced in the chip on day 5 of the culture, as soon as the Papp has been stabilized below 0.5 x 10-6 cm/s. A similar dosing regimen can be used for other proinflammatory cytokines and barrier disruptive agents.
Figure 1D summarizes the timeline of the intestine chip culture and illustrates the intestinal endothelial cells and organoids before and upon seeding on the chip. Moreover, it demonstrates the distinct morphological differences between the duodenum and colon chips, highlighted by the presence of the villi-like formations in the duodenum chip and representative of the small intestinal architecture.
Figure 3A,B demonstrate the fold CYP3A4 induction responses in the duodenum chip from three different organoid donors. The chips were exposed to 20 µM RIF or 100 nM VD3 for 48 h and were used to assess the mRNA levels (A) and/or the fold catalytic activity (B) of the CYP450 enzyme. Increased levels of testosterone metabolite, 6β-hydroxytestosterone (6β-OH-T), consistent with the elevated CYP3A4 mRNA gene expression was observed in the duodenum chip exposed to RIF and VD3 indicating appropriate induction responses across all three donors tested. The means ± SEM of n = 3 biologically independent chips are shown.
Figure 4 depicts the representative results for the modeling of the leaky gut syndrome in the colon chip, including (A,C) changes in epithelial cell morphology and tight junction integrity, (B) decrease in the barrier function, (D) induction of apoptosis and (E) increased cytokine secretion in response to the IFNγ treatment.
Figure 4A shows representative brightfield images of the control and IFNγ-treated (50 ng/mL, 48 h) epithelial monolayer of the colon chip. The chips were removed from the culture module and the epithelial morphology was assessed under the microscope daily. Images were acquired using a brightfield microscope and a 10x objective. Colon chips treated with IFNγ show a compromised cell morphology and loss of the columnar epithelium.
Figure 4B demonstrates a representative result of the apparent permeability assay performed on colon chips cultured under baseline conditions or upon stimulation with IFNγ. 3 kDa Dextran Cascade Blue tracer was added to the top channel reservoir to a final concentration of 100 µg/mL. IFNγ at a final concentration of 50 ng/mL was dosed into the bottom channel on day 5 of the culture. Chips were perfused at 60 mL/h. Next, the media was collected daily (up to day 72 h post-exposure) from inlet and outlet reservoirs of both the top and bottom channels. 50 µL of each sample was collected, processed as described in step 9.2.1 of the protocol, and examined for fluorescence at 375-420 nm using a plate reader. A significant increase of the epithelial paracellular permeability was observed following 48 h of IFNγ stimulation.
Figure 4C shows representative immunofluorescent images of the epithelial tight (Zonula Occludens 1 – ZO-1, Occludin, Claudin-4) and adherens (E-cadherin) junctions in control and IFNγ-treated (50 ng/mL, 48 h) colon chip. The chips were fixed with 4% Paraformaldehyde (PFA) for 15 min at room temperature and further processed as described in step 9.2.2 of the protocol. Images were acquired using a confocal microscope and 20x long-distance objective and processed using Fiji version 2.0 according to standard protocols. Treatment with IFNγ triggers displacement of ZO-1 and Claudin-4 and internalization of Occludin and E-cadherin, as shown by the increased cytoplasmic signal.
Figure 4D represents the time-course of Caspase 3 cleavage in colon chip in response to 50 ng/mL of IFNγ indicative of apoptosis activation. Epithelial cells were lysed on the chips and protein samples were purified according to standard methods. The intracellular content of the total and cleaved Caspase 3 was measured as described in step 9.2.3 of the protocol. IFNγ induces activation of apoptosis, as described previously29, in the colonic epithelial cells cultured on the chip, following 48 h of stimulation.
Figure 4E demonstrates the time-course of the cytokine's secretion from the colon chip upon stimulation with 50 ng/mL IFNγ. 200 µL of effluent media were collected daily from the outlet reservoirs of both top and bottom channels. Effluent samples were stored at -80°C and 24 h before the analysis were thawed overnight at 4°C. The cytokine levels in the chip culture media were assessed using Meso Scale Discovery technology as described in step 9.2.4 of the protocol. IFNγ induced a polarized secretion of proinflammatory molecules in the colon chip, as shown by the basolateral secretion of Vascular Cell Adhesion Protein 1 (VCAM-1) and Interleukin 6 (IL-6) and the apical secretion of Intercellular Adhesion Molecule 1 (ICAM-1) and Serum Amyloid protein A (SAA). Serum levels of the soluble forms of VCAM-1, ICAM-1, IL-6, and SAA are used in clinical laboratories as an indicator of inflammation30,31.
Figure 1: Establishment of the intestine chip15,16. (A) Schematic of the chip activation and coating. Briefly, the chips are placed in a chip cradle, perfused with ER1 solution and activated under UV light. Next, the chips are washed with ER2 and DPBS. Finally, the chips are coated with an ice-cold ECM solution, specific for each cell type, and incubated overnight at 37°C. (B) Schematic depicting the process of introducing organoids in the chip. Organoids are transferred from the 24-well plate into a conical tube and incubated on ice in the presence of the BMM dissociation solution to recover organoids from the solubilized BMM. Organoid suspension is then centrifuged and evaluated for the presence of solubilized BMM residues. If a transparent layer of BMM is still visible above the cell pellet, the process should be repeated. If a well-defined pellet is observed with no visible gel residues, organoids are dissociated enzymatically and introduced in the top channel (blue) of the chip. The bottom channel (magenta) of the chip is seeded with tissue-specific microvascular endothelial cells. (C) Schematic showing the connection of the chips to the culture module, which supports media flow and cyclic peristalsis-like stretch. The priming cycle enables the generation of the cell culture medium droplets over the chip ports and at the end of the portable module resistors. This ensures the creation of the liquid-liquid interface between the chip and the portable module, facilitating the chip sliding into the module and their secure connection. Lastly, the portable modules are placed in trays, and trays are inserted into the culture module. (D) Experimental timeline highlighting the major steps for the establishment of the biopsy-derived intestine chip platform, including duodenum and colon chip. Brightfield images illustrate the endothelial and organoid-derived cells at day 0, before and after seeding in the chip, as well as the formation of confluent and 3D epithelial tissue at day 8 of chip culture. Please click here to view a larger version of this figure.
Figure 2: Collection of effluent media from the intestine chip. (A) Schematic depicting the collection of media effluent from the portable module. Media are collected from the outlet reservoirs of the top and bottom channel and stored at -80 °C until further ELISA or LC-MS / LC-MS/MS analysis. (B) Schematic showing the collection of effluent samples using a multichannel pipette and their transfer into a 96-well black-walled plate for downstream assessment of epithelial apparent permeability (Papp). Please click here to view a larger version of this figure.
Figure 3: CYP3A4 induction in the duodenum chip15. (A) Induction of CYP3A4 mRNA levels in duodenum chip by 48 h exposure to 20 µM RIF and 100 nM VD3. Mean ± SEM, N = 3 chips/condition/donor, Two-way ANOVA, Tukey's post hoc test, ****p < 0.0001 (compared across DMSO controls). (B) Induction of CYP3A4 activity in the duodenum chip exposed to prototypical inducers as assessed by LC-MS/MS quantification of probe substrate metabolite: 6β-hydroxytestosterone. A significant increase in CYP3A4 activity was observed in the duodenum chip treated with 20 µM RIF or 100 nM VD3 for 48 h. Mean ± SEM, N = 3 chips/condition/donor, Two-way ANOVA, Tukey's post hoc test, ****p < 0.0001. Please click here to view a larger version of this figure.
Figure 4: Disruption of the epithelial barrier in the colon chip using IFNγ16. (A) Brightfield images showing the morphology of the epithelial cells under baseline conditions and upon stimulation with 50 ng/mL IFNγ for 48 h. Scale bar: 100 µm. (B) Apparent permeability of the epithelial barrier to 3 kDa Dextran over the course of a 72 h stimulation with 50 ng/mL of IFNγ. Mean ± 95% CI, N = 5-9 chips/condition, Two-way ANOVA, Tukey's post hoc test, ****p < 0.0001. (C) Immunofluorescence images depicting the disruption of the epithelial tight and adherens junctions upon stimulation with 50 ng/mL of IFNγ for 48 h. Zonula Occludens 1 (ZO-1) (red) tight junctions, E-cadherin (red) adherens junctions, Occludin (red) tight junctions, Claudin-4 (red) tight junctions, 4′,6-diamidino-2-phenylindole (DAPI) (blue) nuclei. Scale bar: 50 µm. (D) Induction of Caspase 3 cleavage in colon chip by the treatment with 50 ng/mL of IFNγ. Four different time points were assessed across 72 h of exposure. Mean ± 95% CI, N = 3-6 chips/ condition, Two-way ANOVA, Tukey's post hoc test, ****p < 0.0001. (E) Increased secretion of cytokines: Vascular Cell Adhesion Protein 1 (VCAM-1) and Interleukin 6 (IL-6), in the bottom channel, and Intercellular Adhesion Molecule 1 (ICAM-1) and Serum Amyloid protein A (SAA) in the top channel effluent media, over the course of a 72 h stimulation of the colon chip with 50 ng/mL of IFNγ. Mean ± 95% CI, N = 3 chips/ condition, Two-way ANOVA, Tukey's post hoc test, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****p < 0.0001. Please click here to view a larger version of this figure.
The combination of organ-on-a-chip technology and intestinal organoids holds promise for accurate modeling of human intestinal physiology and pathophysiology. Here, we provide a simple and robust step-by-step protocol (outlined in Figure 1) for establishment of the intestine chip containing biopsy-derived small intestinal or colonic epithelium and intestinal microvascular endothelial cells co-cultured in a microfluidic device. This chip-based simulation of the human intestine incorporates physiological, luminal, and vascular flow and peristalsis-like motions. In addition, we describe procedures for the assessment of critical intestinal functions, such as drug metabolism in the duodenum chip and barrier function in the colon chip.
To recapitulate epithelial-endothelial cellular interactions, which are essential for the maintenance of gut homeostasis and therefore the accurate modeling of intestinal tissue on chip, it is critical to establish functional and intact cell monolayers at both sides of PDMS membrane. The first step toward successful chip seeding is chemical activation of the PDMS surface that allows for development of a stable linkage between the PDMS surface and ECM proteins. Improper activation may hamper the attachment of cells and result in the formation of incomplete cellular monolayers. The activation solution must be prepared fresh as it is light sensitive and prone to degradation. During UV activation and subsequent ECM coating, it is important to ensure an even distribution of reagents within each channel. The specific composition and concentration of solutions used to coat the top and bottom channels have been optimized to suit the needs of the epithelial and endothelial cells, respectively. If changes to cellular composition of the intestine chip are required, the ECM conditions might need to be re-optimized to achieve optimal results.
Following ECM coating, it is important to seed HIMECs at high cell seeding density (8 x 106 cells/mL) to obtain a complete and homogenous monolayer on the bottom side of the PDMS membrane. If full cell confluency is not being achieved, confluency, it is advised to further increase the density of the endothelial cell suspension during the seeding or to delay the seeding of organoid fragments in the top channel of the chip until HIMECs become fully confluent. Use of fragmented organoids, rather than suspension of single cells obtained through enzymatic digestion of organoids, was previously shown to improve the success and reproducibility of the intestinal monolayer formation within the intestine chip14. Therefore, it is strongly advised to closely monitor the organoid digestion process to ensure the optimal time of incubation with the enzyme resulting in organoid fragments composed of around 10-30 cells (40-100 µm in size). Inadequate dissociation can result in reformation of the cystic organoid structure in the microfluidic chips instead of the desired monolayer. In addition, it's critical to avoid excessive organoid dissociation into single cells that could lead to diminished cell survival, recovery, and failure to form confluent monolayer within the chip.
Incorporation of fluid flow (shear stress) and cyclic strain into the intestine chip culture, supported using the culture module, has been shown to enhance the formation of the functional intestinal barrier and to promote the spontaneous development of the 3D architecture of the epithelial tissue13. However, when performing microfluidic experiments with the use of Organs-on-Chips, it is critical to pre-warm and degas the cell culture media prior to use to avoid formation of microbubbles in the channels which could lead to cellular stress or even detachment from the membrane.
Besides the specific applications shown here, the intestine chip model can be used to address a variety of scientific questions, from basic science to discovery and testing of novel drugs or drug delivery technologies. It can facilitate studies of human intestinal development, stem cell maturation, and epithelial cell function; particularly in the context of evaluating the role of mechanics in the growth and homeostasis of the intestinal tissue. Recent discoveries revealed that dynamic equilibrium of the healthy intestinal epithelium relies on its ability to precisely coordinate various forces that define crypt-villi architecture, compartmentalize cell types, direct cell migration, and regulate cell identity, proliferation, and death in space and time32. The intestine chip provides an opportunity to better elucidate the interplay between mechanical forces (e.g., tension or shear stress), cell fate, and function to answer some of many fascinating questions that remain in the emerging field of intestinal mechanobiology. Moreover, it may allow for studies of sensing and intestinal transport processes, thanks to the presence of hormone-secreting enteroendocrine cells and correct localization of various nutrient transporters15,16. The intestine chip model can also be modified to incorporate immune cells as well as commensal or pathogenic microorganisms for studies on gut inflammatory disorders, host-pathogen, and host-microbiome interactions, as well as to precisely predict off-target toxicities of immuno-oncology products, as previously shown17,20,33,34,35.
In addition, the use of clinical biopsy specimens from individuals with specific genotypic and phenotypic characteristics can enable analysis of patient-specific disease mechanisms as well as response to therapies, thereby helping to advance personalized medicine in the future.
The major limitation of this method is that fragments from a large number of organoids (~60-80) are required to form a confluent intestinal epithelium on each chip. This is because organoid fragments require high-density seeding to ensure that an adequate number of intestinal stem cells are present to support the proliferative expansion of the monolayer and the establishment of a 3D tissue architecture. A fully differentiated intestinal epithelium on a chip possesses all the major intestinal epithelial cell types (absorptive enterocytes, Paneth cells, goblet cells and enteroendocrine cells) and transcriptional profile closely resembling the in vivo counterpart. However, the current model still lacks other important components of the living intestine, including intestinal fibroblasts, resident immune cells (e.g., macrophages, intraepithelial lymphocytes, and dendritic cells), and the enteric nervous system.
While these are potential drawbacks, previous studies have shown that the power of the Organ-on-a-Chip technology approach lies in its ability to emulate the structural and functional complexity of human organs by progressively integrating various components of in vivo tissues and their microenvironment one by one36,37. This synthetic biology approach to in vitro tissue engineering provides a way to study the contribution of individual cellular and molecular components to organ-level physiological and pathophysiological responses at varying levels of system complexity. Moreover, it allows for the biochemical, genetic, and microscopic analysis of the interacting tissues to be performed individually and in real-time, gaining insight into how intercellular signals and tissue-tissue interactions contribute to specific organ-level behaviors. Another notable characteristic of intestine chip technology is versatility. Precise control over microenvironmental elements enables fine tuning of media flow rates and strain parameters to reproduce natural forces experienced by cells in the human body, such as the shear stress sensed by the endothelial cells exposed to blood flow and the cyclic peristaltic motions of the intestinal tissue. Furthermore, synergistic engineering of organoids and organs-on-chips enables creation of vascularized Organs-on-Chips representing various regions of intestinal tissues that could be fluidically linked with each other to study physiological interactions between the small and large intestines. Thus, we believe that the intestine chip represents a superior model of the intestinal epithelium and might help to implement the 3Rs (Reduction, Refinement, and Replacement) principle in basic science as well as in the preclinical and regulatory setup.
The authors have nothing to disclose.
We thank Professor Mark Donowitz for providing the intestinal biopsy-derived organoids and Brett Clair for designing the scientific illustrations of the chip, portable and culture module. All the rest of the scientific illustrations were generated using the BioRender.
small intestine Human Intestinal Microvascular Endothelial Cells | AlphaBioRegen | ALHE15 | 0.5 cells M/ml ; cryopreserved |
colon Human Intestinal Microvascular Endothelial Cells | AlphaBioRegen | ALHE16 | 0.5 cells M/ml ; cryopreserved |
Biopsy-derived Human Duodenal Organoids | John Hopkin's University | – | The organoids were provided by Professor Mark Donowitz (Institutional Review Board Number: NA_00038329). |
Biopsy-derived Human Colonic Organoids | John Hopkin's University | – | The organoids were provided by Professor Mark Donowitz (Institutional Review Board Number: NA_00038329). |
Zoë CM-1™ Culture Module | Emulate Inc. | – | Culture module |
Orb-HM1™ Hub Module | Emulate Inc. | – | 5% CO2, vacuum stretch, and power supply |
Chip-S1™ Stretchable Chip | Emulate Inc. | – | Organ-Chip |
Pod™ Portable Modules | Emulate Inc. | – | Portable module |
UV Light Box | Emulate Inc. | – | – |
Chip Cradle | Emulate Inc. | – | 1 per square culture dish |
Steriflip®-HV Filters | EMD Millipore | SE1M003M00 | 0.45 μm PVDF filter |
Square Cell Culture Dish (120 x 120 mm) | VWR | 82051-068 | – |
Handheld vacuum aspirator | Corning | 4930 | - |
Aspirating pipettes | Corning / Falcon | 357558 | 2 mL, polystyrene, individually wrapped |
Aspirating tips | - | Sterile (autoclaved) | |
Serological pipettes | - | 2 mL, 5 mL, 10 mL, and 25 mL low endotoxin, sterile | |
Pipette | P20, P200, P1000 and standard multichannel | ||
Pipette tips | P20, P200, and P1000. | ||
Conical tubes (Protein LoBind® Tubes) | Eppendorf | 0030122216; 0030122240 | 15 mL, 50 mL tubes |
Eppendorf Tubes® lo-bind | Eppendorf | 022431081 | 1.5 mL tubes |
96 wells black walled plate | – | – | for epithelial permeability analysis |
Microscope (with camera) | – | – | For bright-field imaging |
Water bath (or beads) | – | – | Set to 37°C |
Vacuum set-up | - | – | Minimum pressure: -70 kPa |
Cell scrapers | Biotium | 220033 | |
T75 flasks | BD Falcon | 353136 | Cell culture flask |
Emulate Reagent-1 (ER-1) | Emulate Inc. | – | Chip coating solution |
Emulate Reagent-2 (ER-2) | Emulate Inc. | – | Chip coating solution |
Dulbecco’s PBS (DPBS) | Corning | 21-031-CV | 1X |
Cell Culture Grade Water | Corning | MT25055CV | |
Trypan blue | Sigma | 93595 | For cell counting |
TryplE Express | ThermoFisher Scientific | 12604013 | Organoids dissociation and endothelium cells detachment solution |
Advanced DMEM/F12 | ThermoFisher Scientific | 12634028 | Medium |
IntestiCult™ Organoid Growth Medium (Human) | Stem Cell technologies | 06010 | Organoid Growth Medium |
Endothelial Cell Growth Medium MV 2 | Promocell | C-22121 | Endothelial medium |
Fetal bovine serum (FBS) | Sigma | F4135 | Serum |
Primocin™ | InvivoGen | ANT-PM-1 | antimicrobial agent |
Attachment Factor™ | Cell Systems | 4Z0-210 | coating solution for flask |
Matrigel – Growth Factor Reduced | Corning | 356231 | Solubilized basement membrane matrix |
Collagen IV | Sigma | C5533 | ECM component |
Fibronectin | Corning | 356008 | ECM component |
Y-27632 | Stem Cell technologies | 72304 | organoid media supplement |
CHIR99021 | Reprocell | 04-0004-10 | organoid media supplement |
Cell Recovery Solution | Corning | 354253 | Basement mebrane matrix dissociationsolution |
Bovine Serum Albumin (BSA) | Sigma | A9576 | 30%, Sterile |
Cell Culture Grade Water | Corning | MT25055CV | Sterile, Water |
DMSO | Sigma | D2650 | solvent |
3KDa Dextran Cascade Blue | Invitrogen | D7132 | 10 mg powder |
Rifampicin (RIF) | Sigma | Cat# R3501 | CYP inducer |
Testosterone hydrate | Sigma | T1500 | CYP substrate |
1,25-dihyroxy Vitamin D3 (VD3) | Sigma | Cat# D1530 | CYP inducer |
Acetonitrile with 0.1% (v/v) Formic acid | Sigma | 159002 | LCMS stop solution |
IFNγ | Peprotech | 300-02 | |
4% Paraformaldehyde (PFA) | EMS | 157-4 | Fixative |
Triton-X 100 | Sigma | T8787 | |
Normal Donkey Serum (NDS) | Sigma | 566460 | |
anti-Occludin | ThermoFisher Scientific | 33-1500 | tight junctions marker |
anti-Claudin 4 | ThermoFisher Scientific | 36-4800 | tight junctions marker |
anti-E-cadherin | Abcam | ab1416 | epithelial adherens junctions marker |
anti-VE-cadherin | Abcam | ab33168 | endothelial adherent junctions marker |
anti- Zonula Occludens 1 (ZO-1) | Thermo Fischer | 339194 | tight junctions marker |
DAPI | ThermoFisher Scientific | 62248 | nuclear stain |
2-mercaptoethanol | Sigma | M6250 | |
PureLink RNA Mini Kit | Invitrogen | 12183020 | RNA lysis, isolation and purification kit |
SuperScript™ IV VILO™ Master Mix | Invitrogen | 11756050 | reverse transcriptase kit |
TaqMan™ Fast Advanced Master Mix | Applied Biosystems | 4444557 | qPCR reagent |
QuantStudio™ 5 Real-Time PCR System | Applied Biosystems | A28573 | Real-time PCR cycler |
18S primer | ThermoFisher Scientific | Hs99999901_s1 | Eukaryotic 18S rRNA |
CYP3A4 primer | ThermoFisher Scientific | Hs00604506_m1 | Cytochrome family 3 subfamily A member 4 |
Pierce™ Coomassie Plus (Bradford) Assay Kit | ThermoFisher Scientific | 23236 | Protein quantification kit |
MSD Tris lysis buffer | Meso Scale Diagnostics | R60TX-3 | Protein lysis buffer |
Cleaved/Total Caspase-3 Whole Cell Lysate Kit | Meso Scale Diagnostics | K15140D | Caspase 3 detection kit |
V-PLEX Vascular Injury Panel 2 Human Kit | Meso Scale Diagnostics | K15198D | |
V-PLEX Human Proinflammatory Panel II (4-Plex) | Meso Scale Diagnostics | K15053D | |
Zeiss LSM 880 | Zeiss | – | Confocal microscope |
Zeiss LD plan-Neofluar 20x/0.40 Korr M27 | Zeiss | – | 20X long-distance objective lenses |
Zeiss AXIOvert.A1 | Zeiss | – | Brightfield microscope |
Zeiss LD A-Plan 10X/0.25 Ph1 | Zeiss | – | 10X objective lenses |