Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction.

Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood.

Microphysiological System Design: Simplicity Is Elegance.

Design parameters for microphysiological systems (MPS) are driven by the need for new tools to answer questions focusing on human physiology in a robust and reliable manner. Within this perspective, engineering benchmarks and principles are identified to guide the construction of new devices in the MPS field, with emphasis placed on the design principles common to all tissues, as well as those unique to a subset of tissues. Leading organ replica technologies that recapitulate various functions of the brain, heart, intestine, and lung are highlighted as examples that meet the identified benchmarks and standards, with current barriers for large scale production and commercialization discussed.

Automated platform for cell selection and separation based on four-dimensional motility and matrix degradation.

Motility and invasion are key steps in the metastatic cascade, enabling cells to move through normal tissue borders into the surrounding stroma. Most available in vitro assays track cell motility or cell invasion but lack the ability to measure both simultaneously and then separate single cells with unique behaviors. In this work, we developed a cell-separation platform capable of tracking cell movement (chemokinesis) and invasion through an extracellular matrix in space and time.

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.

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 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.

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).

Generation of Mouse Colon Crypts.

Organoid culture has had a significant impact on studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found 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 , 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 crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface.

Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.

Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity.

Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system.

The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems. We have developed a microraft array methodology that automatically measures the ability of individual T cells to kill a population of target cells and viably sorts specific cells into a 96-well plate for expansion. A human T cell culture was generated against the influenza M1p antigen.

In Vitro Polarization of Colonoids to Create an Intestinal Stem Cell Compartment.

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.

Array-Based Platform To Select, Release, and Capture Epstein-Barr Virus-Infected Cells Based on Intercellular Adhesion.

Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis.

A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis.

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.

Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma.

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets.

Isolation and in vitro culture of rare cancer stem cells from patient-derived xenografts of pancreatic ductal adenocarcinoma.

Described is the construction of a large array of releasable microstructures (micropallets) along with screening and isolation protocols for sorting rare, approximately 1 in 10,000, cancer stem cells (CSCs) from a heterogeneous cell population. A 10.1 × 7.