DOI: 10.6084/m9.figshare.14036855
title: Magnetically-Propelled Fecal Surrogates for Modeling the Impact of Solid-Induced Shear Forces on Primary Colonic Epithelial Cells

description: Images (converted to 8-bit TIFF files), CellProfiler pipeline, design schematics, and scripts supporting the research article, "Magnetically-Propelled Fecal Surrogates for Modeling the Impact of Solid-Induced Shear Forces on Primary Colonic Epithelial Cells."<br><b>Motor controller files:</b> The ".ino" file includes relevant Arduino scripts for stepper motor operation. The ".cpp" file is an updated library for the Adafruit motor shield that may be required for microstepping.<br><br><b>Acrylic frame designs:</b> The ".ai" files were directly utilized for laser cutting 0.25 in thick acrylic sheets on a Universal Laser Systems ILS9.75 to produce the acrylic frame that supports the stepper motors, neodymium magnets, and 6-well cell culture plate.<br><b>Hoechst 33342 / EdU / MUC2 / ALP images:</b> Flattened into median intensity projections from <i>z</i>-series optical sections and analyzed for differentiated lineage allocation using empirical thresholding in Fiji. <i>Naming structure:</i> "[fluorescent stains]_[C or MM][replicate number]_Median_Z.TIF," where "C" indicates control sample and "MM" indicates magnetic agarose-exposed sample.<br><b>Hoechst 33342 / F-actin images: </b>Analyzed for changes in cellular height using the line measure tool in Fiji. <i>Naming structure:</i> "[fluorescent stains]_[C or MM][replicate number].TIF," where "C" indicates control sample and "MM" indicates magnetic agarose-exposed sample.<br><br><b>Hoechst 33342 / ZO-1 flattened images:</b> Flattened into maximum intensity projections from <i>z</i>-series optical sections and analyzed for cell and nuclear morphology using the included CellProfiler pipeline ("MM Morphology Analysis.cpproj") and R scripts. <i>Naming structure:</i> "[fluorescent stains]_[C or MM][replicate number]_Z2_[sub-replicate number]_5ADVMLE_Maximum_Z.TIF," where "C" indicates control sample and "MM" indicates magnetic agarose-exposed sample.<br><b>Abstract:</b> The colonic epithelium is continuously exposed to an array of biological and mechanical stimuli as its luminal contents are guided over the epithelial surface through regulated smooth muscle contraction. In this report, the propulsion of solid fecal contents over the colonic epithelium is recapitulated through noninvasive actuation of magnetic agarose hydrogels over primary intestinal epithelial cultures, in contrast to the vast majority of platforms that apply shear forces through liquid microflow. Software-controlled magnetic stepper motors enable experimental control over the frequency and velocity of these events to match in vivo propulsive contractions, while the integration of standardized well plate spacing facilitates rapid integration into existing assay pipelines. The application of these solid-induced shear forces did not deleteriously affect cell monolayer surface coverage, viability, or transepithelial electrical resistance unless the device parameters were raised to a 50× greater contraction frequency and 4× greater fecal velocity than those observed in healthy humans. At a frequency and velocity that is consistent with average human colonic motility, differentiation of the epithelial cells into absorptive and goblet cell phenotypes was not affected. Protein secretion was modulated with a two-fold increase in luminal mucin-2 secretion and a significant reduction in basal interleukin-8 secretion. F-actin, zonula occludens-1, and E-cadherin were each present in their proper basolateral locations, similar to those of static control cultures. While cellular height was unaffected by magnetic agarose propulsion, several alterations in lateral morphology were observed including decreases in circularity and compactness, and an increase in major axis length, which align with surface epithelial cell morphologies observed in vivo and may represent early markers of luminal exfoliation. This platform will be of widespread utility for the investigation of fecal propulsive forces on intestinal physiology, shedding light on how the colonic epithelium responds to mechanical cues.