Colonic mucus forms a critical barrier to intestinal contents, providing the protection necessary for intestinal and organismal health. The mucus is composed of gel-forming mucin secreted by goblet cells residing in the epithelial layer lining the colon; yet, our knowledge of many of the attributes and functions of mucus and the goblet cells remains limited. A planar array of colonic cryptlike structures with a thick covering of goblet cell-generated mucus was developed to mimic the differentiated colonic epithelium and provide an easily accessible physiologic mucus layer for the evaluation of mucus barrier function in response to intestinal microbiota and toxins. The human microphysiological system (MPS) was created using an impermeable thin film patterned with a geometrical array of a 10 μm × 10 μm scale through holes overlaid with collagen and primary colonic stem cells. The array dimensions, collagen thickness, and growth factor concentration were optimized to assess the cell density, proliferation, migration, differentiation, and mucus thickness. A 175 μm center-to-center distance between the through holes or stem cell niches and a collagen thickness of 10 μm were found to be optimal to enable long-term culture (≥23 days) with a discrete stem/proliferative cell region and a differentiated cell zone enriched in goblet cells and supporting a 250 μm-thick adherent mucus layer. The mucus layer acted as an effective barrier to block the access of the Staphylococcus aureus α-hemolysin toxin to the epithelial cells as well as to protect the cell layer from both Staphylococcus aureus and Lactobacillus rhamnosus. The intestinal mucus MPS will be a useful tool for emulating the intestinal epithelium to study the interplay of stem cell renewal, goblet cell differentiation, mucus dynamics, and microbiota-mucus-host interactions.

Abstract The ability to separate and detect lipid metabolites in single cells has been achieved using a miniaturized thin-layer chromatography (TLC) platform termed picoTLC (pTLC). However, continued innovations in the matrix fabrication, lipid reporter development, and single-cell workflow will broaden the applicability of pTLC to single-cell assays. In this study, we report three advances: a simple post-fabrication process [hydrothermal treatment (HT)] to modify the pTLC silica matrix, a novel clickable lipid reporter to assay metabolism of alkylglycerol (an ether lipid), and a fix-click strategy to prepare cells for the pTLC assay of a metabolized lipid reporter. HT was performed by heating pTLC chips in a silica-saturated aqueous solution with cetyltrimethylammonium bromide (CTAB) followed by slow cooling to generate a co-precipitate of silica/CTAB on the silica matrix. After extraction of CTAB, a mesoporous silica deposition was present with a 3-fold increase in surface area of the silica matrix on the HT processed pTLC chip. The separation performance on pTLC was significantly improved by HT, supporting the separation of five model fluorescent lipids with a resolution >1 for all adjacent lipids that was not achievable without HT. Given the growing importance of ether lipids such as alkylglycerol and its relatives in health and disease, a novel clickable lipid (S)-3-(hexadec-15-yn-1-yloxy)propane-1,2-diol (Reporter 1) was synthesized as a reporter for measuring alkylglycerol metabolism in addition to a companion nonmetabolizable lipid as a control (R)-4-((hexadec-15-yn-1-yloxy)methyl)-2,2-dimethyl-1,3-dioxolane (Reporter-2). A fix-click strategy was adapted to provide a simple, easy-to-use workflow for measurement of the reporter lipid metabolism in single cells by pTLC. These combined advances enabled the identification of metabolites formed from the novel lipid reporters in single leukemic cells and will further broaden the application of pTLC to report lipid metabolism at the single-cell level.

Cell-to-cell heterogeneity in lipid signaling underlies variations in response and recurrence for many cancers, including leukemias. A highly parallel, miniaturized thin-layer chromatographic platform capable of assaying single cells was developed. Ultrasmall volumes (50 pL) of standard fluorescent lipids were separated with excellent repeatability, reproducibility, and limits of detection. Sphingosine-cyanine 5 (Sph-Cy5) was loaded into cells, and the single-cell contents were separated to identify Sph-Cy5 and two metabolites, Sph-1-phosphate-Cy5 (S1P-Cy5) and hexadecanoic acid Cy5 (HA-Cy5). In leukemic cells, the CD34+ blast cells demonstrated significantly greater conversion of Sph-Cy5 to its phosphorylated form compared to that of the CD34- cells. After treatment with a sphingosine kinase (SphK) inhibitor, the level of formation of S1P-Cy5 remained significantly greater for the inhibited CD34+ cells relative to that of the inhibited CD34- cells. Over 1200 single cells were rapidly assayed using 8 chips within 4 h. Sphingosine kinase activity in the CD34+ blast cells of 3 patients with acute myeloid leukemia was assayed with and without inhibitors. The patient cells displayed intertumor and intratumor heterogeneity, and subsets of cells with distinct enzymatic activities and products, highlighting the diversity of the cells within a clinical sample and between patients.

Aqueous two-phase extraction (ATPE) is an effective and scalable liquid-phase processing method for purifying single species of single-wall carbon nanotubes (SWCNTs) from multiple species mixtures. Recent metrological developments have led to advances in the speed of identifying solution parameters leading to more efficient ATPE separations with greater fidelities. In this feature article, we review these developments and discuss their vast potential to further advance SWCNT separations science towards the optimization of production scale processes and the full realization of SWCNT-enabled technologies.

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X-Y-Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air-liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 +/- 180 mu m thick) possessing an inner (149 +/- 50 mu m) and outer (435 +/- 111 mu m) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8-10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X-Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease.

A combination of analytical ultracentrifugation (AUC) and fluorescence spectroscopy are utilized to orthogonally probe compositions of adsorbed surfactant layers on the surface of (7,5) species single-wall carbon nanotubes (SWCNTs) under conditions known to achieve differential partitioning in aqueous two-phase extraction (ATPE) separations. Fluorescence emission intensity and AUC anhydrous particle density measurements independently probe and can discriminate between adsorbed surfactant layers on a (7,5) nanotube comprised of either of two common nanotube dispersants, the anionic surfactants sodium deoxycholate and sodium dodecyl sulfate. Measurements on dispersions containing mixtures of both surfactants indicate near total direct exchange of the dominant surfactant species adsorbed to the carbon nanotube at a critical concentration ratio consistent with the ratio leading to partitioning change in the ATPE separation. By conducting these orthogonal measurements in a complex environment reflective of an ATPE separation, including multiple surfactant and polymer solution components, the results provide direct evidence for the hypothesis that it is the nature of the adsorbed surfactant layer that primarily controls partitioning behavior in selective ATPE separations of SWCNTs.

The relationship between the mechanical forces associated with bowel movement and colonic mucosal physiology is understudied. This is partly due to the limited availability of physiologically relevant fecal models that can exert these mechanical stimuli in in vitro colon models in a simple-to-implement manner. In this report, we created a mucus-coated fecal surrogate that was magnetically propelled to produce a controllable sweeping mechanical stimulation on primary intestinal epithelial cell monolayers. The mucus layer was derived from purified porcine stomach mucins, which were first modified with reactive vinyl sulfone (VS) groups followed by reaction with a thiol crosslinker (PEG-4SH) via a Michael addition click reaction. Formation of mucus hydrogel network was achieved at the optimal mixing ratio at 2.5 % w/v mucin-VS and 0.5 % w/v PEG-4SH. The artificial mucus layer possessed similar properties as the native mucus in terms of its storage modulus (66 Pa) and barrier function (resistance to penetration by 1-μm microbeads). This soft, but mechanically resilient mucus layer was covalently linked to a stiff fecal hydrogel surrogate (based on agarose and magnetic particles, with a storage modulus of 4600 Pa). The covalent bonding between the mucus and agarose ensured its stability in the subsequent fecal sliding movement when tested at travel distances as long as 203 m. The mucus layer served as a lubricant and protected epithelial cells from the moving fecal surrogate over a 1 h time without cell damage. To demonstrate its utility, this mucus-coated fecal surrogate was used to mechanically stimulate a fully differentiated, in vitro primary colon epithelium, and the physiological stimulated response of mucin-2 (MUC2), interleukin-8 (IL-8) and serotonin (5HT) secretion was quantified. Compared with a static control, mechanical stimulation caused a significant increase in MUC2 secretion into luminal compartment (6.4 × ), a small but significant increase in IL-8 secretion (2.5 × and 3.5 × , at both luminal and basal compartments, respectively), and no detectable alteration in 5HT secretion. This mucus-coated fecal surrogate is expected to be useful in in vitro colon organ-on-chips and microphysiological systems to facilitate the investigation of feces-induced mechanical stimulation on intestinal physiology and pathology.

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity. Therapeutic safety and efficacy depend on the correct encapsidation of the DNA in individual virus particles, which is often characterized by the single scalar value of the ratio of full capsids with complete viral genomes to the total viral capsid number [the full-to-total (FTT) ratio]. This study reports on the interlaboratory and intertechnique variations of measurement methods for FTT among a cohort of organizations. The analytical methods used were sedimentation velocity analytical ultracentrifugation (SV-AUC) with UV/Vis and/or Rayleigh interference optics, size exclusion chromatography (SEC) with multi-angle light scattering (MALS), and tandem UV/Vis and/or refractive index, cryogenic electron microscopy, dual-wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR, or ddPCR). FTT measurements for both AAV5 and AAV8 serotypes were similar, except for PCR-ELISA. The optical techniques (UV spectroscopy/SEC-MALS) showed <10% SD between laboratories, likely from the uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in both PCR and ELISA protocols and instrumentation. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle critical quality attributes such as FTT.

A complex and dynamic network of interactions exists between human gastrointestinal epithelium and intestinal microbiota. Therefore, comprehending intestinal microbe-epithelial cell interactions is critical for the understanding and treatment of intestinal diseases. Primary human colonic epithelial cells derived from a healthy human donor were co-cultured with Clostridium scindens (C. scindens), a probiotic obligate anaerobe; Staphylococcus aureus (S. aureus), a facultative anaerobe and intestinal pathogen; or both bacterial species in tandem. The co-culture hanging basket platform used for these experiments possessed walls of controlled oxygen (O2) permeability to support the formation of an O2 gradient across the intestinal epithelium using cellular O2 consumption, resulting in an anaerobic luminal and aerobic basal compartment. Both the colonic epithelial cells and C. scindens remained viable over 48 h during co-culture. In contrast, co-culture with S. aureus elicited significant damage to colonic epithelial cells within 24 h. To explore the influence of the intestinal pathogen on the epithelium in the presence of the probiotic bacteria, colonic epithelial cells were inoculated sequentially with the two bacterial species. Under these conditions, C. scindens was capable of repressing the production of S. aureus enterotoxin. Surprisingly, although C. scindens converted cholic acid to secondary bile acids in the luminal medium, the growth of S. aureus was not significantly inhibited. Nevertheless, this combination of probiotic and pathogenic bacteria was found to benefit the survival of the colonic epithelial cells compared with co-culture of the epithelial cells with S. aureus alone. This platform thus provides an easy-to-use and low-cost tool to study the interaction between intestinal bacteria and colonic cells in vitro to better understand the interplay of intestinal microbiota with human colonic epithelium.

Sorting single-chirality enantiomers is the ultimate goal for single-wall carbon nanotube (SWCNT) separation. In this work, aqueous two-phase extraction (ATPE) is used to obtain highly purified (>80%) left- and right-handed (6,5) SWCNTs with limited experimental steps. The optimized surfactant conditions are presented, and analytical ultracentrifugation (AUC) and near-infrared photoluminescence (PL) measurements are used to examine the surfactant wrapping and partition coefficient change conditions (PCCCs) required for separation. Our study provides valuable insights into the underlying mechanisms behind surfactant-assisted SWCNT separations and facilitates the use of SWCNT enantiomers in fundamental studies and applications in the future.

Surfactant-controlled aqueous two-polymer phase extraction is a sensitive method for separating specific (n,m) structures of single-walled carbon nanotubes (SWCNTs). Using empirically determined values for the overall surfactant compositions that enable differential extraction, it is possible to isolate many (n,m) species across a broad diameter range from < 0.8 nm to > 1.5 nm, however, researching new methods requires extensive new separation experiments. To overcome this barrier, we recently advanced a fluorescence-based methodology for determining extraction conditions for specific (n,m) species SWCNTs and their enantiomers that does not rely on the performance of separations, and can be performed at well controlled environmental conditions with orders of magnitude less nanotube sample. This method is based on the finding that the change in the nature of the extraction is directly correlated with a dramatic change in fluorescence intensity of the particular (n,m) structure. Using analytical ultracentrifugation, we have now orthogonally determined the density of the bound surfactant on specific nanotube (n,m)s below, near, and above the surfactant concentrations yielding the extraction change, as well as for the two co-surfactants separately. These results enable evaluation of the hypothesis that the fluorescence and extraction conditions changes are the result of a change in the composition of the dominant adsorbed surfactant at a specific composition ratio.

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.

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.

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.

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.

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.

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. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1β, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

In this special publication, we report on a modified synthetic process that results in a stable suspension of Au-PEI nanoparticles, Au-PEI@NIST. Synthesized materials were characterized using an orthogonal approach that included dynamic light scattering (DLS), UV-Vis absorbance (UV-Vis), transmission electron microscopy (TEM), atomic force microscope (AFM), and thermogravimetric analysis (TGA). Very importantly, the end-product in this study demonstrated excellent colloidal stability in multiple aqueous environments including over a wide pH and temperature range of physiological relevance. The product also exhibited long-term storage stability under ambient conditions. Furthermore, the cytotoxicity of Au-PEI@NIST was investigated using the CHO K1 cell line and exhibited dose proportional toxicity, which suggested potential for use as a positive control in nanotoxicology studies. The results and stringent testing regimen point toward an effective and sustainable approach that can serve as a clarified starting point for the development of more complex conjugates for nanomedicine area.

In this talk I will present a methodology for rapid and quantitative determination of surfactant concentrations that result in top or bottom-phase partitioning of individual (n,m) SWCNTs species in an aqueous two-polymer phase extraction using near-infrared fluorescence measurements. This methodology enables quantitative evaluation of parameters affecting ATPE such as temperature, polymer molecular weight and composition, and surfactant choice without conducting an actual separation. Additional advantages of the technique include a very low sample concentration for measurement, rapid measurement, and no mass transfer derived limitations in interpreting results. Using the technique, we have investigated different combinations of surfactants to explore the effects of surfactant chemistry. In most cases, two transitions in the NIR fluorescence are observed with co-surfactant concentration for each (n,m) species, which we identify as differential separation of left and right-handed SWCNT enantiomers. Specific trends with (n,m) diameter and with variation of surfactant chemistry will be discussed.

Technologies capable of cell separation based on cell images provide powerful tools enabling cell selection criteria that rely on spatially or temporally varying properties. Image-based cell sorting (IBCS) systems utilize microfluidic or microarray platforms, each having unique characteristics and applications. The advent of IBCS marks a new paradigm in which cell phenotype and behavior can be explored with high resolution and tied to cellular physiological and omics data, providing a deeper understanding of single-cell physiology and the creation of cell lines with unique properties. Cell sorting guided by high-content image information has far-reaching implications in biomedical research, clinical medicine, and pharmaceutical development.