Clostridioides difficile-mucus interactions encompass shifts in gene expression, metabolism, and biofilm formation.

In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides diffiicile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight a flexibility in metabolism that may influence pathogenesis.

Bifidobacterium infantis associates with T cell immunity in human infants and is sufficient to enhance antigen-specific T cells in mice.

Bacille Calmette-Guerin (BCG) vaccine can elicit good TH1 responses in neonates. We hypothesized that the pioneer gut microbiota affects vaccine T cell responses. Infants who are HIV exposed but uninfected (iHEU) display an altered immunity to vaccination. BCG-specific immune responses were analyzed at 7 weeks of age in iHEU, and responses were categorized as high or low. Bifidobacterium longum subsp. infantis was enriched in the stools of high responders, while Bacteroides thetaiotaomicron was enriched in low responders at time of BCG vaccination. Neonatal germ-free or SPF mice orally gavaged with live B. infantis exhibited significantly higher BCG-specific T cells compared with pups gavaged with B. thetaiotaomicron. B. infantis and B. thetaiotaomicron differentially affected stool metabolome and colonic transcriptome. Human colonic epithelial cells stimulated with B. infantis induced a unique gene expression profile versus B. thetaiotaomicron. We thus identified a causal role of B. infantis in early-life antigen-specific immunity.

Automated microarray platform for single-cell sorting and collection of lymphocytes following HIV reactivation.

A promising strategy to cure HIV-infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single-cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.

Analytical Techniques for Single-Cell Biochemical Assays of Lipids.

Lipids are essential cellular components forming membranes, serving as energy reserves, and acting as chemical messengers. Dysfunction in lipid metabolism and signaling is associated with a wide range of diseases including cancer and autoimmunity. Heterogeneity in cell behavior including lipid signaling is increasingly recognized as a driver of disease and drug resistance. This diversity in cellular responses as well as the roles of lipids in health and disease drive the need to quantify lipids within single cells. Single-cell lipid assays are challenging due to the small size of cells (∼1 pL) and the large numbers of lipid species present at concentrations spanning orders of magnitude. A growing number of methodologies enable assay of large numbers of lipid analytes, perform high-resolution spatial measurements, or permit highly sensitive lipid assays in single cells. Covered in this review are mass spectrometry, Raman imaging, and fluorescence-based assays including microscopy and microseparations.

Automated microarray for single-cell sorting and collection of lymphocytes following HIV reactivation.

A promising strategy to cure HIV infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T-lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of various LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5 %, 11.2 %, and 12.1 % percentage of cells respectively, reactivated similar to that observed in other experimental systems. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T-lymphocytes that were either fast or slow reactivators were sorted, and single-cell RNA-sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found. These results advance our conceptual understanding of HIV reactivation dynamics at the single-cell level toward a cure for HIV.

Microraft arrays for serial-killer CD19 chimeric antigen receptor T cells and single cell isolation.

Chimeric antigen receptor T (CAR-T) cell immunotherapies have seen success in treating hematological malignancies in recent years; however, the results can be highly variable. Single cell heterogeneity plays a key role in the variable efficacy of CAR-T cell treatments yet is largely unexplored. A major challenge is to understand the killing behavior and phenotype of individual CAR-T cells, which are able to serially kill targets. Thus, a platform capable of measuring time-dependent CAR-T cell mediated killing and then isolating single cells for downstream assays would be invaluable in characterizing CAR-T cells. An automated microraft array platform was designed to track CD19 CAR-T cell killing of CD19+ target cells and CAR-T cell motility over time followed by CAR-T cell collection based on killing behavior. The platform demonstrated automated CAR-T cell counting with up to 98% specificity and 96% sensitivity, and single cells were isolated with 89% efficiency. On average, 2.3% of single CAR-T cells were shown to participate in serial-killing of target cells, killing a maximum of three target cells in a 6 h period. The cytotoxicity and motility of >7000 individual CAR-T cells was tracked across four microraft arrays. The automated microraft array platform measured temporal cell-mediated cytotoxicity, CAR-T cell motility, CAR-T cell death, and CAR-T cell to target cell distances, followed by the capability to sort any desired CAR-T cell. The pipeline has the potential to further our understanding of T cell-based cancer immunotherapies and improve cell-therapy products for better patient outcomes.

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.

Scalable Additive Construction of Arrayed Microstructures with Encoded Properties for Bioimaging.

Microarrays are essential components of analytical instruments. The elements of microarrays may be imbued with additional functionalities and encodings using composite materials and structures, but traditional microfabrication methods present substantial barriers to fabrication, design, and scalability. In this work, a tool-free technique was reported to additively batch-construct micromolded, composite, and arrayed microstructures. The method required only a compatible carrier fluid to deposit a material onto a substrate with some topography. Permutations of this basic fabrication approach were leveraged to gain control over the volumes and positions of deposited materials within the microstructures. As a proof of concept, cell micro-carrier arrays were constructed to demonstrate a range of designs, compositions, functionalities, and applications for composite microstructures. This approach is envisioned to enable the fabrication of complex composite biological and synthetic microelements for biosensing, cellular analysis, and biochemical screening.

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.

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.

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.

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.

"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.

Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a "fix and click" method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The "fix and click" strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using "fix and click" paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.

G protein-coupled receptor kinase 3 modulates mesenchymal stem cell proliferation and differentiation through sphingosine-1-phosphate receptor regulation.

BACKGROUND: The bone marrow niche supports hematopoietic cell development through intimate contact with multipotent stromal mesenchymal stem cells; however, the intracellular signaling, function, and regulation of such supportive niche cells are still being defined. Our study was designed to understand how G protein receptor kinase 3 (GRK3) affects bone marrow mesenchymal stem cell function by examining primary cells from GRK3-deficient mice, which we have previously published to have a hypercellular bone marrow and leukocytosis through negative regulation of CXCL12/CXCR4 signaling. METHODS: Murine GRK3-deficient bone marrow mesenchymal stromal cells were harvested and cultured to differentiate into three lineages (adipocyte, chondrocyte, and osteoblast) to confirm multipotency and compared to wild type cells. Immunoblotting, modified-TANGO experiments, and flow cytometry were used to further examine the effects of GRK3 deficiency on bone marrow mesenchymal stromal cell receptor signaling. Microcomputed tomography was used to determine trabecular and cortical bone composition of GRK3-deficient mice and standard ELISA to quantitate CXCL12 production from cellular cultures. RESULTS: GRK3-deficient, bone marrow-derived mesenchymal stem cells exhibit enhanced and earlier osteogenic differentiation in vitro. The addition of a sphingosine kinase inhibitor abrogated the osteogenic proliferation and differentiation, suggesting that sphingosine-1-phosphate receptor signaling was a putative G protein-coupled receptor regulated by GRK3. Immunoblotting showed prolonged ERK1/2 signaling after stimulation with sphingosine-1-phosphate in GRK3-deficient cells, and modified-TANGO assays suggested the involvement of ß-arrestin-2 in sphingosine-1-phosphate receptor internalization. CONCLUSIONS: Our work suggests that GRK3 regulates sphingosine-1-phosphate receptor signaling on bone marrow mesenchymal stem cells by recruiting ß-arrestin to the occupied GPCR to promote internalization, and lack of such regulation affects mesenchymal stem cell functionality.

Photoactivatable Reporter to Perform Multiplexed and Temporally Controlled Measurements of Kinase and Protease Activity in Single Cells.

Peptide bioreporters were developed to perform multiplexed measurements of the activation of epidermal growth factor receptor kinase (EGFR), Akt kinase (Akt/protein kinase B), and proteases/peptidases in single cells. The performance characteristics of the three reporters were assessed by measuring the reporter's proteolytic stability, kinetic constants for EGFR and Akt, and dephosphorylation rate. The reporter displaying optimal performance was composed of 6-carboxyfluorescein (6-FAM) on the peptide N-terminus, an Akt substrate sequence employing a threonine phosphorylation site for Akt, followed by a tri-D arginine linker, and finally an EGFR substrate sequence bearing a phosphatase-resistant 7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-htc) residue as the EGFR phosphorylation site. Importantly, use of a single electrophoretic condition separated the mono- and diphosphorylated products as well as proteolytic forms permitting the quantitation of multiple enzyme activities simultaneously using a single reporter. Because the Akt and EGFR substrates were linked, a known ratio (EGFR/Akt) of the reporter was loaded into cells. A photoactivatable version of the reporter was synthesized by adding two 4,5-dimethoxy-2-nitrobenzyl (DMNB) moieties to mask the EGFR and Akt phosphorylation sites. The DMNB moieties were readily photocleaved following exposure to 360 nm light, unmasking the phosphorylation sites on the reporter. The new photoactivatable reporter permitted multiplexed measurements of kinase signaling and proteolytic degradation in single cells in a temporally controlled manner. This work will facilitate the development of a new generation of multiplexed activity-based reporters capable of light-initiated measurement of enzymatic activity in single cells.

Passive Control of Silane Diffusion for Gradient Application of Surface Properties.

Liquid lithography represents a robust technique for fabricating three-dimensional (3D) microstructures on a two-dimensional template. Silanization of a surface is often a key step in the liquid lithography process and is used to alter the surface energy of the substrate and, consequently, the shape of the 3D microfeatures produced. In this work, we present a passive technique that allows for the generation of silane gradients along the length of a substrate. The technique relies on a secondary diffusion chamber with a single opening, leading to a directional introduction of silane to the substrate via passive diffusion. The secondary chamber geometry influences the deposited gradient, which is shown to be well captured by Monte Carlo simulations that incorporate the passive diffusion and grafting processes. The technique ultimately allows the user to generate a range of substrate wettabilities on a single chip, enhancing throughput for organ-on-a-chip applications by mimicking the spatial variability of tissue topographies present in vivo.

Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus.

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

Stem/Proliferative and Differentiated Cells within Primary Murine Colonic Epithelium Display Distinct Intracellular Free Ca2+ Signal Codes.

The second messenger, intracellular free calcium (Ca2+ ), acts to transduce mitogenic and differentiation signals incoming to the colonic epithelium. A self-renewing monolayer of primary murine colonic epithelial cells is formed over a soft, transparent hydrogel matrix for the scalable analysis of intracellular Ca2+ transients. Cultures that are enriched for stem/proliferative cells exhibit repetitive, high frequency (≈25 peaks h-1 ), and short pulse width (≈25 s) Ca2+ transients. Upon cell differentiation the transient frequency declines by 50% and pulse width widens by 200%. Metabolites and growth factors that are known to modulate stem cell proliferation and differentiation through Wnt and Notch signaling pathways, including CHIR-99021, N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), bone morphogenetic proteins (BMPs), and butyrate, also modulate Ca2+ oscillation patterns in a consistent manner. Increasing the stiffness of the supportive matrix from 200 Pa to 3 GPa shifts Ca2+ transient patterns toward those resembling differentiated cells. The ability to monitor Ca2+ oscillations with the spatial and temporal resolution offered by this platform, combined with its amenability to high-content screens, provides a powerful tool for investigating real-time communication within a wide range of primary tissues in addition to the colonic epithelium.

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.

Magnetically-propelled fecal surrogates for modeling the impact of solid-induced shear forces on primary colonic epithelial cells.

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 decreased 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.