Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of 15 N-enriched ions from metabolically labeled 15 N-sphingolipids in the plasma membrane, using highresolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids-both in living cells and during fixation of living cellsexclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous 15 N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and longrange organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.SIMS | stable isotope

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Background & AimsThe 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.MethodsA 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.ResultsChemical 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.ConclusionsThe in vitro human colon crypt array accurately mimicked the architecture, luminal accessibility, tissue polarity, cell migration, and cellular responses of in vivo intestinal crypts.

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The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field. Current Approaches to Modeling Intestinal Physiology The small and large intestine, located after the stomach, comprise the lower human gastrointestinal tract and play crucial roles in nutrient absorption and in housing much of the human microbiome (see Glossary) (Figure 1, Key Figure). In the past decade, model systems have attempted to recapitulate the complex, in vivo intestinal physiology using cell lines derived from intestinal tumors such as Caco-2 cells in place of primary epithelial cells. Advanced organ-on-achip systems were created by culturing Caco-2 cells on the geometrically or mechanically engineered platforms to properly mimic the structural and mechanical properties of the human intestine [1-6]. To mimic the mucosal architecture, porous scaffolds were micromolded to villus-like projections on which Caco-2 cells could be cultured [1,6]. To recapitulate the mechanically dynamic environment, microfluidic systems were developed with fluid flowing both above and below a Caco-2 cell layer growing on a rhythmically stretched flexible surface [2-5]. These systems were designed to mimic the shear forces and contractile motions occurring in the small intestine. Caco-2 cells, as well as other tumor cell lines, have also been used as surrogate intestinal epithelial cells to probe the interactions between multiple tissue types [7]. These organ-on-a-chip models incorporating tumor cells offer new abilities to emulate the structure, function, and physiology of the living human intestine that are not possible with conventional tissue-cultured monolayers. However, as our understanding of these organs progresses, it is clear that these prior tumor model systems fall short in their ability to accurately reflect in vivo physiology because the models do not possess all of the intestinal epithelial subtypes and either lack receptors, transporters, drug-metabolizing enzymes, or express these proteins at levels different from in vivo. Thus, in vitro replicas of the intestines that more accurately replicate intestinal physiology are required and will need to utilize primary cells. Accordingly, a suite of platforms employing primary cells in a variety of assay formats, including organoid [8,9], monolayer, and shape...

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.

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