Abstract

​

Regeneration of intestinal epithelium is fueled by a heterogeneous population of rapidly proliferating stem cells (ISCs) found in the base of the small intestine and colonic crypts. ISCs populations can be enriched by fluorescence-activated cell sorting (FACS) based on expression of combinatorial cell surface markers, and fluorescent transgenes. Conventional ISC culture is performed by embedding single ISCs or whole crypt units in a matrix and culturing in conditions that stimulate or repress key pathways to recapitulate ISC niche signaling. Cultured ISCs form organoid, which are spherical, epithelial monolayers that are self-renewing, self-patterning, and demonstrate the full complement of intestinal epithelial cell lineages. However, this conventional "bulk" approach to studying ISC biology is often semiquantitative, low throughput, and masks clonal effects and ISC phenotypic heterogeneity. Our group has recently reported the construction, long-term biocompatibility, and use of microfabricated cell raft arrays (CRA) for high-throughput analysis of single ISCs and organoids. CRAs are composed of thousands of indexed and independently retrievable microwells, which in combination with time-lapse microscopy and/or gene-expression analyses are a powerful tool for studying clonal ISC dynamics and micro-niches. In this protocol, we describe how CRAs are used as an adaptable experimental platform to study the effect of exogenous factors on clonal stem cell behavior.

Abstract

​

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.

Abstract

​

BACKGROUND & AIMS: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. METHODS: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. RESULTS: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. CONCLUSIONS: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.

Abstract

​

Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.

Abstract

​

The relationship between intestinal stem cells (ISCs) and the surrounding niche environment is complex and dynamic. Key factors localized at the base of the crypt are necessary to promote ISC self-renewal and proliferation, to ultimately provide a constant stream of differentiated cells to maintain the epithelial barrier. These factors diminish as epithelial cells divide, migrate away from the crypt base, differentiate into the postmitotic lineages, and end their life span in approximately 7 days when they are sloughed into the intestinal lumen. To facilitate the rapid and complex physiology of ISC-driven epithelial renewal, in vivo gradients of growth factors, extracellular matrix, bacterial products, gases, and stiffness are formed along the crypt-villus axis. New bioengineered tools and platforms are available to recapitulate various gradients and support the stereotypical cellular responses associated with these gradients. Many of these technologies have been paired with primary small intestinal and colonic epithelial cells to re-create select aspects of normal physiology or disease states. These biomimetic platforms are becoming increasingly sophisticated with the rapid discovery of new niche factors and gradients. These advancements are contributing to the development of high-fidelity tissue constructs for basic science applications, drug screening, and personalized medicine applications. Here, we discuss the direct and indirect evidence for many of the important gradients found in vivo and their successful application to date in bioengineered in vitro models, including organ-on-chip and microfluidic culture devices.

Abstract

​

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.

Abstract

​

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.

Abstract

​

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo has not been recapitulated in the organoid systems. We have used a microengineered platform with suitable extracellular matrix contacts and stiffness to generate a self-renewing mouse colonic epithelium that replicates key architectural and physiological functions found in vivo, including a surface lined with polarized crypts. Chemical gradients applied to the basal-luminal axis compartmentalized the stem/progenitor cells and promoted appropriate lineage differentiation along the in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model.

Abstract

​

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.

Abstract

​

The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.

Abstract

​

Stem cells reside in ‘niches’, where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell–niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a ‘microraft array’ (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell–niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

Abstract

​

BACKGROUND: New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising efficiency and reproducibility. Here murine crypt isolation yield and quality are optimized, and colonoid culture efficiency measured in microfabricated culture devices. RESULTS: An optimal incubation time of 60 min in a chelating buffer released 280,000 +/- 28,000 crypts from the stroma of a single colon with 79.3% remaining intact. Mechanical agitation using an average acceleration of 1.5 x g liberated the highest quality crypts with 86% possessing well-defined lumens. Culture in 50% Matrigel resulted in the highest colonoid formation efficiency of 33 +/- 5%. Immunostaining demonstrated that colonoids isolated under these conditions possessed stem/progenitor cells and differentiated cell lineages. Microfabrication substrates (glass, polystyrene, PDMS, and epoxy photoresists: SU-8 and 1002-F) were tested for compatibility with colonoid culture. PDMS promoted formation of 3-D colonoids containing stem/progenitor cells, while other substrates promoted outgrowth of a 2-D epithelial monolayer composed of differentiated cells. CONCLUSION: Improved crypt isolation and 3-D colonoid culture, along with an understanding of colonic epithelial cell behavior in the presence of microfabrication substrates will support development of 'organ-on-a-chip' approaches for studies using primary colonic epithelium.

Abstract

​

The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm, depth = 150 μm) followed by addition of Matrigel that formed micropockets locking the crypts within the microwells. Embedded crypts first underwent 3D expansion inside the wells. After the cells filled the microwells, they migrated onto the surrounding surface forming a 2D monolayer in the array regions without Matrigel. This unique 2D/3D hybrid culture generated a continuous, millimeter-scale colonic epithelial tissue in vitro, which resembled the polarized architecture (i.e. distinct proliferative and non-proliferative zones) and geometry of the colonic epithelium in vivo. This work initiates the construction of a "colon-on-a-chip" using primary cells/tissues with the ultimate goal of producing the physiologic structure and organ-level function of the colon.

Abstract

​

The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm, depth = 150 μm) followed by addition of Matrigel that formed micropockets locking the crypts within the microwells. Embedded crypts first underwent 3D expansion inside the wells. After the cells filled the microwells, they migrated onto the surrounding surface forming a 2D monolayer in the array regions without Matrigel. This unique 2D/3D hybrid culture generated a continuous, millimeter-scale colonic epithelial tissue in vitro, which resembled the polarized architecture (i.e. distinct proliferative and non-proliferative zones) and geometry of the colonic epithelium in vivo. This work initiates the construction of a "colon-on-a-chip" using primary cells/tissues with the ultimate goal of producing the physiologic structure and organ-level function of the colon.