Creation of a spatially complex mucus bilayer on an in vitro colon model.

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X-Y-Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air-liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 µm thick) possessing an inner (149 ± 50 µm) and outer (435 ± 111 µm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8-10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X-Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease.

Mucus-coated, magnetically-propelled fecal surrogate to mimic fecal shear forces on colonic epithelium.

The relationship between the mechanical forces associated with bowel movement and colonic mucosal physiology is understudied. This is partly due to the limited availability of physiologically relevant fecal models that can exert these mechanical stimuli in in vitro colon models in a simple-to-implement manner. In this report, we created a mucus-coated fecal surrogate that was magnetically propelled to produce a controllable sweeping mechanical stimulation on primary intestinal epithelial cell monolayers. The mucus layer was derived from purified porcine stomach mucins, which were first modified with reactive vinyl sulfone (VS) groups followed by reaction with a thiol crosslinker (PEG-4SH) via a Michael addition click reaction. Formation of mucus hydrogel network was achieved at the optimal mixing ratio at 2.5 % w/v mucin-VS and 0.5 % w/v PEG-4SH. The artificial mucus layer possessed similar properties as the native mucus in terms of its storage modulus (66 Pa) and barrier function (resistance to penetration by 1-µm microbeads). This soft, but mechanically resilient mucus layer was covalently linked to a stiff fecal hydrogel surrogate (based on agarose and magnetic particles, with a storage modulus of 4600 Pa). The covalent bonding between the mucus and agarose ensured its stability in the subsequent fecal sliding movement when tested at travel distances as long as 203 m. The mucus layer served as a lubricant and protected epithelial cells from the moving fecal surrogate over a 1 h time without cell damage. To demonstrate its utility, this mucus-coated fecal surrogate was used to mechanically stimulate a fully differentiated, in vitro primary colon epithelium, and the physiological stimulated response of mucin-2 (MUC2), interleukin-8 (IL-8) and serotonin (5HT) secretion was quantified. Compared with a static control, mechanical stimulation caused a significant increase in MUC2 secretion into luminal compartment (6.4 × ), a small but significant increase in IL-8 secretion (2.5 × and 3.5 × , at both luminal and basal compartments, respectively), and no detectable alteration in 5HT secretion. This mucus-coated fecal surrogate is expected to be useful in in vitro colon organ-on-chips and microphysiological systems to facilitate the investigation of feces-induced mechanical stimulation on intestinal physiology and pathology.

In vitro co-culture of Clostridium scindens with primary human colonic epithelium protects the epithelium against Staphylococcus aureus.

A complex and dynamic network of interactions exists between human gastrointestinal epithelium and intestinal microbiota. Therefore, comprehending intestinal microbe-epithelial cell interactions is critical for the understanding and treatment of intestinal diseases. Primary human colonic epithelial cells derived from a healthy human donor were co-cultured with Clostridium scindens (C. scindens), a probiotic obligate anaerobe; Staphylococcus aureus (S. aureus), a facultative anaerobe and intestinal pathogen; or both bacterial species in tandem. The co-culture hanging basket platform used for these experiments possessed walls of controlled oxygen (O2) permeability to support the formation of an O2 gradient across the intestinal epithelium using cellular O2 consumption, resulting in an anaerobic luminal and aerobic basal compartment. Both the colonic epithelial cells and C. scindens remained viable over 48 h during co-culture. In contrast, co-culture with S. aureus elicited significant damage to colonic epithelial cells within 24 h. To explore the influence of the intestinal pathogen on the epithelium in the presence of the probiotic bacteria, colonic epithelial cells were inoculated sequentially with the two bacterial species. Under these conditions, C. scindens was capable of repressing the production of S. aureus enterotoxin. Surprisingly, although C. scindens converted cholic acid to secondary bile acids in the luminal medium, the growth of S. aureus was not significantly inhibited. Nevertheless, this combination of probiotic and pathogenic bacteria was found to benefit the survival of the colonic epithelial cells compared with co-culture of the epithelial cells with S. aureus alone. This platform thus provides an easy-to-use and low-cost tool to study the interaction between intestinal bacteria and colonic cells in vitro to better understand the interplay of intestinal microbiota with human colonic epithelium.

Monolithic Silica Microbands Enable Thin-Layer Chromatography Analysis of Single Cells.

A picoliter thin-layer chromatography (pTLC) platform was developed for analyzing extremely miniature specimens, such as assay of the contents of a single cell of 1 picoliter volume. The pTLC chip consisted of an array of microscale bands made from highly porous monolithic silica designed to accept picoliter-scale volume samples. pTLC bands were fabricated by combining sol-gel chemistry and microfabrication technology. The width (60-80 µm) and depth (13 µm) of each band is comparable to the size of single cells and acted to reduce the lateral diffusion and confine the movement of compounds along the microbands. Ultrasmall volumes (tens of pL) of model fluorescent compounds were spotted onto the microband by a piezoelectric microdispenser and successfully separated by pTLC. The separation resolution and analyte migration were dependent on the macropore size (ranging from 0.3 to 2.3 µm), which was adjustable by changing the porogen concentration during the sol-gel process. For a 0.3 µm macropore size, attomoles of analyte were detectable by fluorescence using standard microscopy methods. The separation resolution, theoretical plate number, and separation times ranged from 1.3 to 2.1, 4 to 357, and 2 to 8 min, respectively, for the chosen model biological lipids. To demonstrate the capability of pTLC for separating analytes from single mammalian cells, cells loaded with fluorescent lipophilic dyes or sphingosine kinase reporter were spotted on microbands, and the single-cell contents separated by pTLC were detected from their fluorescence. These results demonstrate the potential of pTLC for applications in many areas where miniature specimens and high-throughput parallel analyses are needed.

Development of a Primary Human Intestinal Epithelium Enriched in L-Cells for Assay of GLP-1 Secretion.

Type 2 diabetes mellitus is a chronic disease associated with obesity and dysregulated human feeding behavior. The hormone glucagon-like peptide 1 (GLP-1), a critical regulator of body weight, food intake, and blood glucose levels, is secreted by enteroendocrine L-cells. The paucity of L-cells in primary intestinal cell cultures including organoids and monolayers has made assays of GLP-1 secretion from primary human cells challenging. In the current paper, an analytical assay pipeline consisting of an optimized human intestinal tissue construct enriched in L-cells paired with standard antibody-based GLP-1 assays was developed to screen compounds for the development of pharmaceuticals to modulate L-cell signaling. The addition of the serotonin receptor agonist Bimu 8, optimization of R-spondin and Noggin concentrations, and utilization of vasoactive intestinal peptide (VIP) increased the density of L-cells in a primary human colonic epithelial monolayer. Additionally, the incorporation of an air-liquid interface culture format increased the L-cell number so that the signal-to-noise ratio of conventional enzyme-linked immunoassays could be used to monitor GLP-1 secretion in compound screens. To demonstrate the utility of the optimized analytical method, 21 types of beverage sweeteners were screened for their ability to stimulate GLP-1 secretion. Stevioside and cyclamate were found to be the most potent inducers of GLP-1 secretion. This platform enables the quantification of GLP-1 secretion from human primary L-cells and will have broad application in understanding L-cell formation and physiology and will improve the identification of modulators of human feeding behavior.

Suspended Collagen Hydrogels to Replicate Human Colonic Epithelial Cell Interactions with Immune Cells.

The human colon plays a critical role in fluid and salt absorption and harbors the largest immune compartment. There is a widespread need for in vitro models of human colon physiology with its innate immune system. A method is described to produce a cassette with a network of struts supporting a suspended, non-chemically cross-linked collagen hydrogel scaffold compatible with the co-culture of primary gastrointestinal epithelium and migratory inflammatory cells. The epithelial monolayer cultured on the suspended collagen possesses a population of polarized and differentiated cells similar to that present in vivo. This epithelial layer displays proper barrier function with a transepithelial electrical resistance (TEER) ≥ 1,500 Ω cm2 and an apparent permeability ≤10-5 cm2 s-1 . Immune cells plated on the basal face of the scaffold transmigrated over a period of 24 h to the epithelial layer in response to epithelial production of IL-8 induced by luminal stimulation of Clostridium difficile Toxin A. These studies demonstrate that this in vitro platform possesses a functional primary colonic epithelial layer with an immune cell compartment capable of recruitment in response to pro-inflammatory cues coming from the epithelium.

Human 2D Crypt Model for Assaying Intestinal Stem Cell Proliferation and Differentiation.

Intestine is a common site of adverse drug effects in clinical trials; thus, improved in vitro models for preclinical screening of pharmaceutical compounds are sought. A planar, self-renewing human intestinal monolayer platform based on primary adult gastrointestinal stem cells, termed the 2D crypt model, has been developed to screen for the effects of various compounds on the intestinal epithelium. The 2D crypt platform is based on a standard 12-well plate format and consists of cell culture inserts with a collagen film overlaying an impermeable film patterned with an array of micron-scale holes. This two-chamber format enables a gradient of growth factors to be applied such that the tissue self-organizes into spatially segregated stem and differentiated cell compartments. The patterned monolayer mimics a gut epithelium in possessing a stem cell niche, migrating proliferative and differentiated cells. Once established, the 2D crypts replicate many aspects of in vivo physiology, including cell migration, maturation, and apoptotic cell death. The planar geometry of the system simplifies dosing, sampling, and imaging during assay. An immunofluorescence-based assay was established to quantitatively assess cell density, proliferation, migration, viability, and the abundance and localization of postmitotic lineages as a function of time. The model was used to perform a small-scale screen of compounds, including signaling molecules, endogenous hormones/cytokines, and microbial metabolites, on tissue homeostasis. Hit compounds that significantly impacted proliferation and/or differentiation were readily identified. The 2D crypt platform represents a significant innovation in the development of microphysiological systems for emulating the gut epithelium for compound screens.

A Platform for Co-Culture of Primary Human Colonic Epithelium With Anaerobic Probiotic Bacteria.

An in vitro platform was designed and optimized for the co-culture of probiotic anaerobic bacteria with a primary human colonic epithelium having a goal of assessing the anti-inflammatory impact of the probiotic bacteria. The device maintained a luminal O2 concentration at <1% while also supporting an oxygenated basal compartment at 10% for at least 72 h. Measurement of the transepithelial resistance of a confluent colonic epithelium showed high monolayer integrity while fluorescence assays demonstrated that the monolayer was comprised primarily of goblet cells and colonocytes, the two major differentiated cell subtypes of the colonic epithelium. High monolayer barrier function and viability were maintained during co-culture of the epithelium with the probiotic obligate anaerobe Anaerobutyricum hallii (A. hallii). Importantly the device supported a static co-culture of microbes and colonic epithelium mimicking the largely static or low flow conditions within the colonic lumen. A model inflamed colonic epithelium was generated by the addition of tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) to the basal and luminal epithelium sides, respectively. Co-culture of A. hallii with the LPS/TNF-α treated intestine diminished IL-8 secretion by ≥40% which could be mimicked by co-culture with the A. hallii metabolite butyrate. In contrast, co-culture of the inflamed epithelium with two strains of lactic acid-producing bacteria, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium adolescentis (B. adolescentis), did not diminish epithelial IL-8 secretion. Co-culture with colonic epithelial cells from different donors demonstrated a consistent anti-inflammatory effect by A. hallii, but distinct responses to co-culture with LGG and B. adolescentis. The demonstrated system offers a simple and easily adopted platform for examining the physiologic impact of alterations in the intestinal epithelium that occur in the presence of probiotic bacteria and their metabolites.

A technology of a different sort: microraft arrays.

A common procedure performed throughout biomedical research is the selection and isolation of biological entities such as organelles, cells and organoids from a mixed population. In this review, we describe the development and application of microraft arrays, an analysis and isolation platform which enables a vast range of criteria and strategies to be used when separating biological entities. The microraft arrays are comprised of elastomeric microwells with detachable polymer bases (microrafts) that act as capture and culture sites as well as supporting carriers during cell isolation. The technology is elegant in its simplicity and can be implemented for samples possessing tens to millions of objects yielding a flexible platform for applications such as single-cell RNA sequencing, subcellular organelle capture and assay, high-throughput screening and development of CRISPR gene-edited cell lines, and organoid manipulation and selection. The transparent arrays are compatible with a multitude of imaging modalities enabling selection based on 2D or 3D spatial phenotypes or temporal properties. Each microraft can be individually isolated on demand with retention of high viability due to the near zero hydrodynamic stress imposed upon the cells during microraft release, capture and deposition. The platform has been utilized as a simple manual add-on to a standard microscope or incorporated into fully automated instruments that implement state-of-the-art imaging algorithms and machine learning. The vast array of selection criteria enables separations not possible with conventional sorting methods, thus garnering widespread interest in the biological and pharmaceutical sciences.

Image-Based Live Cell Sorting.

Technologies capable of cell separation based on cell images provide powerful tools enabling cell selection criteria that rely on spatially or temporally varying properties. Image-based cell sorting (IBCS) systems utilize microfluidic or microarray platforms, each having unique characteristics and applications. The advent of IBCS marks a new paradigm in which cell phenotype and behavior can be explored with high resolution and tied to cellular physiological and omics data, providing a deeper understanding of single-cell physiology and the creation of cell lines with unique properties. Cell sorting guided by high-content image information has far-reaching implications in biomedical research, clinical medicine, and pharmaceutical development.

Enterochromaffin Cell-Enriched Monolayer Platform for Assaying Serotonin Release from Human Primary Intestinal Cells.

Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.

An in vitro intestinal platform with a self-sustaining oxygen gradient to study the human gut/microbiome interface.

An oxygen gradient formed along the length of colonic crypts supports stem-cell proliferation at the normoxic crypt base while supporting obligate anaerobe growth in the anoxic colonic lumen. Primary human colonic epithelial cells derived from human gastrointestinal stem cells were cultured within a device possessing materials of tailored oxygen permeability to produce an oxygen-depleted luminal (0.8% ± 0.1% O2) and oxygen-rich basal (11.1% ± 0.5% O2) compartment. This oxygen difference created a stable oxygen gradient across the colonic epithelial cells which remained viable and properly polarized. Facultative and obligate anaerobes Lactobacillus rhamnosus, Bifidobacterium adolescentis, and Clostridium difficile grew readily within the luminal compartment. When formed along the length of an in vitro crypt, the oxygen gradient facilitated cell compartmentalization within the crypt by enhancing confinement of the proliferative cells to the crypt base. This platform provides a simple system to create a physiological oxygen gradient across an intestinal mimic while simultaneously supporting anaerobe co-culture.

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking.

BACKGROUND: The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. RESULTS: The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. CONCLUSIONS: The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function.

A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies.

Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability ( Papp) of <5 × 10-7 cm s-1, while propranolol demonstrated a Papp of 9.69 × 10-6 cm s-1. Rh123 was transported in a luminal direction ( Papp,efflux/ Papp,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo.

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

Formation of arrays of planar, murine, intestinal crypts possessing a stem/proliferative cell compartment and differentiated cell zone.

A simple, in vitro intestinal model recapitulating key aspects of crypt architecture and physiology would facilitate our understanding the impact of drugs, foods and microbial metabolites on the intestine. To address the limitations of previously reported intestinal in vitro platforms, we developed a planar crypt array that replicated the spatial segregation and physiologic responses of primary mouse intestinal epithelial cells in the large intestine. Collagen was coated across an impermeable film possessing an array of microholes creating two regions of distinct stiffness and porosity (above and outside the microholes). Primary mouse colon epithelial cells formed a continuous monolayer across the array with a proliferative cell zone above the microholes and a nonproliferative or differentiated cell region distant from the microholes. Formation of a chemical gradient of growth factors across the array yielded a more complete or in vivo-like cell segregation of proliferative and differentiated cells with cell migration outward from the proliferative cell zone into the differentiated zone to replace apoptotic dying cells much as occurs in vivo. Short chain fatty acids (microbial metabolites) applied to the luminal surface of the crypt array significantly impacted the proliferation and differentiation of the cells replicating the known in vivo effects of these fatty acids. Importantly this planar crypt array was readily fabricated and maintained, easily imaged with properties quantified by microscopy, and compatible with reagent addition to either the luminal or basal fluid reservoirs. The ability to observe simultaneously stem/proliferative and differentiated cell behavior and movement between these two compartments in response to drugs, toxins, inflammatory mediators or microbial metabolites will be of widespread utility.

Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer.

BACKGROUND & AIMS: The successful culture of intestinal organoids has greatly enhanced our understanding of intestinal stem cell physiology and enabled the generation of novel intestinal disease models. Although of tremendous value, intestinal organoid culture systems have not yet fully recapitulated the anatomy or physiology of the in vivo intestinal epithelium. The aim of this work was to re-create an intestinal epithelium with a high density of polarized crypts that respond in a physiologic manner to addition of growth factors, metabolites, or cytokines to the basal or luminal tissue surface as occurs in vivo. METHODS: A self-renewing monolayer of human intestinal epithelium was cultured on a collagen scaffold microfabricated with an array of crypt-like invaginations. Placement of chemical factors in either the fluid reservoir below or above the cell-covered scaffolding created a gradient of that chemical across the growing epithelial tissue possessing the in vitro crypt structures. Crypt polarization (size of the stem/proliferative and differentiated cell zones) was assessed in response to gradients of growth factors, cytokines, and bacterial metabolites. RESULTS: Chemical gradients applied to the shaped human epithelium re-created the stem/proliferative and differentiated cell zones of the in vivo intestine. Short-chain fatty acids applied as a gradient from the luminal side confirmed long-standing hypotheses that butyrate diminished stem/progenitor cell proliferation and promoted differentiation into absorptive colonocytes. A gradient of interferon-γ and tumor necrosis factor-α significantly suppressed the stem/progenitor cell proliferation, altering crypt formation. CONCLUSIONS: The in vitro human colon crypt array accurately mimicked the architecture, luminal accessibility, tissue polarity, cell migration, and cellular responses of in vivo intestinal crypts.

Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins.

Enterotoxins increase intestinal fluid secretion through modulation of ion channels as well as activation of the enteric nervous and immune systems. Colonic organoids, also known as colonoids, are functionally and phenotypically similar to in vivo colonic epithelium and have been used to study intestinal ion transport and subsequent water flux in physiology and disease models. In conventional cultures, organoids exist as spheroids embedded within a hydrogel patty of extracellular matrix, and they form at multiple depths, impairing efficient imaging necessary to capture data from statistically relevant sample sizes. To overcome these limitations, an analytical platform with colonic organoids localized to the planar surface of a hydrogel layer was developed. The arrays of densely packed colonoids (140 µm average diameter, 4 colonoids/mm2) were generated in a 96-well plate, enabling assay of the response of hundreds of organoids so that organoid subpopulations with distinct behaviors were identifiable. Organoid cell types, monolayer polarity, and growth were similar to those embedded in hydrogel. An automated imaging and analysis platform efficiently tracked over time swelling due to forskolin and fluid movement across the cell monolayer stimulated by cholera toxin. The platform was used to screen compounds associated with the enteric nervous and immune systems for their effect on fluid movement across epithelial cells. Prostaglandin E2 promoted increased water flux in a subset of organoids that resulted in organoid swelling, confirming a role for this inflammatory mediator in diarrheal conditions but also illustrating organoid differences in response to an identical stimulus. By allowing sampling of a large number of organoids, the arrayed organoid platform permits identification of organoid subpopulations intermixed within a larger group of nonresponding organoids. This technique will enable automated, large-scale screening of the impact of drugs, toxins, and other compounds on colonic physiology.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells.

BACKGROUND & AIMS: Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. METHODS: The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. RESULTS: The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. CONCLUSIONS: This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.

Chemical fixation to arrest phospholipid signaling for chemical cytometry.

Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24±2% and 139±12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24h yielded recoverable lipid of 61±3% (C6-PIP2) and 55±5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.