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The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.

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.

Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.

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. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo 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 in vivo, 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 in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model.

Primary patient samples are the gold standard for molecular investigations of tumor biology yet are difficult to acquire, heterogeneous in nature and variable in size. Patient-derived xenografts (PDXs) comprised of primary tumor tissue cultured in host organisms such as nude mice permit the propagation of human tumor samples in an in vivo environment and closely mimic the phenotype and gene expression profile of the primary tumor. Although PDX models reduce the cost and complexity of acquiring sample tissue and permit repeated sampling of the primary tumor, these samples are typically contaminated by immune, blood, and vascular tissues from the host organism while also being limited in size. For very small tissue samples (on the order of 10(3) cells) purification by fluorescence-activated cell sorting (FACS) is not feasible while magnetic activated cell sorting (MACS) of small samples results in very low purity, low yield, and poor viability. We developed a platform for imaging cytometry integrated with micropallet array technology to perform automated cell sorting on very small samples obtained from PDX models of pancreatic and colorectal cancer using antibody staining of EpCAM (CD326) as a selection criteria. These data demonstrate the ability to automate and efficiently separate samples with very low number of cells.

Wnt/β-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/β-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/β-catenin signaling were screened over short (< 6) and long (> 25) generational timescales. To characterize phenotypic divergence these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1,119 clonal colonies in parallel. This screen revealed phenotypic divergence after 25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provide insight into the pathway’s mechanisms of positive feedback and self-inhibition.

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. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.

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. Individual microrafts on a 70 × 70 array were loaded with on average 1 CD8+ cell from the culture and a population of M1p presenting target cells. Target cell killing, measured by fluorescence microscopy, was quantified in each microraft. The rates of target cell death among the individual CD8+ T cells varied greatly; however, individual T cells maintained their rates of cytotoxicity throughout the time course of the experiment enabling rapid identification of highly cytotoxic CD8+ T cells. Microrafts with highly active CD8+ T cells were individually transferred to wells of a 96-well plate, using a needle-release device coupled to the microscope. Three sorted T cells clonally expanded. All of these expressed high-avidity T cell receptors for M1p/HLA*02:01 tetramers, and 2 of the 3 receptors were sequenced. While this study investigated single T cell cytotoxicity rates against simple targets with subsequent cell sorting, future studies will involve measuring T cell mediated cytotoxicity in more complex cellular environments, enlarging the arrays to identify very rare antigen specific T cells, and measuring single cell CD4+ and CD8+ T cell proliferation.

A microfabricated platform was developed for highly parallel and efficient colony picking, splitting, and clone identification. A pallet array provided patterned cell colonies which mated to a second printing array composed of bridging microstructures formed by a supporting base and attached post. The posts enabled mammalian cells from colonies initially cultured on the pallet array to migrate to corresponding sites on the printing array. Separation of the arrays simultaneously split the colonies, creating a patterned replica. Optimization of array elements provided transfer efficiencies greater than 90% using bridging posts of 30 μm diameter and 100 μm length and total colony numbers of 3000. Studies using five mammalian cell lines demonstrated that a variety of adherent cell types could be cultured and effectively split with printing efficiencies of 78-92%. To demonstrate the technique's utility, clonal cell lines with siRNA knockdown of Coronin 1B were generated using the arrays and compared to a traditional FACS/Western Blotting-based approach. Identification of target clones required a destructive assay to identify cells with an absence of Coronin 1B brought about by the successful infection of interfering shRNA construct. By virtue of miniaturization and its parallel format, the platform enabled the identification and generation of 12 target clones from a starting sample of only 3900 cells and required only 5 man hours over 11 days. In contrast, the traditional method required 500,000 cells and generated only 5 target clones with 34 man hours expended over 47 days. These data support the considerable reduction in time, manpower, and reagents using the miniaturized platform for clonal selection by destructive assay versus conventional approaches.

The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration.1–6 These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die.7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis.4,7–9 S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2.6,9–11 In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine.6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies.7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases.10,15–18 Thus, sphingosine signaling is proving to be extremely important in clinical medicine.17–21 Although S1P plays a major role as an extracellular signaling molecule, it is predominately synthesized by SK within cells.6 The precise mechanism and control of S1P cellular export is not clearly understood, although sphingosine and sphingosine derivatives do appear to readily enter and exit living cells.22,23 Recently, the primary reservoir for S1P acting to influence the immune system has been shown to reside in erythrocytes (red blood cells). It remains unclear whether the intracellular pool of S1P is from endogenous kinase activity or from uptake and storage of exogenous S1P.23,24 These studies have been hampered by the inability to quantitatively measure SK activity in cells. The traditional assay for S1P requires a two-step CHCl3 extraction followed by 9-fluorenylmethyl chloroformiate derivatization of the extracts and analysis by HPLC.23 However, these steps reduce the accuracy of quantitative measurements such that only large changes in S1P concentration in the biological sample can be identified.25 Another common method for the determination of SK activity is by radiometric assay.26–28 There are two widely used protocols. The first protocol involves incubation of the purified enzyme or cell lysate with sphingosine and [γ−32P]ATP. Organic solvents are used to extract the sphingoid base 1-phosphates under acidic conditions with recoveries of at best 50%.25,28 The products are separated by thin-layer chromatography (TLC), and [32P]S1P is identified by autoradiography or phosphorimaging.28 An alternative, albeit even less sensitive, assay uses 3H-labeled sphingosine followed by extraction in CHCl3 under basic conditions.26,27 The reaction product [3H]S1P is recovered in an aqueous phase and separated by TLC or directly quantified using liquid-scintillation counting. Major drawbacks of radiometric assays for SK are their very limited sensitivity, semiquantitative results, use of organic solvent extraction, poor resolution of structurally similar radiolabeled species, and intense investment of time and labor.25 Other techniques developed for determination of SK activity include HPLC and mass spectrometry (MS).29–32 To detect endogenous SK activity in cell and tissue extracts, these techniques still require nonquantitative extraction techniques to remove interference from cellular proteins, and their sensitivity remains limited.29 In addition, MS is an expensive technique which may not be readily available to many biology laboratories. Newly developed fluorescence-based assays for the determination of SK activity appear promising.33–35 These nonradioactive assays are simpler, faster, and more amenable to high-throughput analyses. For biological samples, the assays still require organic solvent extractions and are more suited for quantifying the activity of enzymes in purified state or in bulk cell lysates. All of the current platforms lack the sensitivity for analysis of cellular samples on the order of tens to hundreds of cells. A direct approach for measurement of the interconversion of sphingosine and S1P in cells would be of benefit, particularly if a highly sensitive means with the potential for single-cell analysis could be achieved.36 Chemical cytometry, the application of high-sensitivity chemical separations to characterize single or small numbers of cells, is emerging as an important approach for biochemical studies.37 A number of techniques including MS, electrochemistry, and capillary electrophoresis (CE) are the fundamental analytical tools for such studies. CE using laser-induced fluorescence detection (LIF) is the most sensitive of these techniques, and numerous groups have used this separation technique for a variety of biochemical studies in single cells, including assays of enzymatic activity.37 The objective of the current work was to develop a sensitive, fluorescent CE-based assay for SK and sphingoid phosphatase activity in small numbers of cells. To achieve this goal, a CE-based separation of fluorescein-labeled sphingosine (SphFl) and the phosphorylated product fluorescein-labeled sphingosine-1-phosphate (SphFl-1-P) was used to measure the activity of purified enzyme. These results demonstrated the utility of this technique for in vitro biochemical determination of kinase and phosphatase activity. Furthermore, it was demonstrated that SphFl was readily taken up by mammalian cells without the need for rigorous loading protocols. The technique was then applied to study endogenous SK activity in erythrocytes using both bulk cell lysates and very small numbers of whole cells. The highly sensitive and quantitative method was fast, simple, and robust for both in vitro and cell-based assays. The methodology described should prove to be a valuable analytical tool for quantitative cellular studies of the sphingosine kinases and phosphatases, enzymes of intense interest in both cell biology and clinical medicine.

Background: A host of technologies exists for the separation of living, nonadherent cells, with separation decisions typically based on fluorescence or immunolabeling of cells. Methods to separate adherent cells as well as to broaden the range of possible sorting criteria would be of high value and complementary to existing strategies. Methods: Cells were cultured on arrays of releasable pallets. The arrays were screened and individual cell(s)/pallets were released and collected. Results: Conventional fluorescence and immunolabeling of cells were compatible with the pallet arrays, as were separations based on gene expression. By varying the size of the pallet and the number of cells cultured on the array, single cells or clonal colonies of cells were isolated from a heterogeneous population. Since cells remained adherent throughout the isolation process, separations based on morphologic characteristics, for example cell shape, were feasible. Repeated measurements of each cell in an array were performed permitting the selection of cells based on their temporal behavior, e.g. growth rate. Conclusions: The pallet array system provides the flexibility to select and collect adherent cells based on phenotypic and temporal criteria and other characteristics not accessible by alternative methods. © 2007 International Society for Analytical Cytology

The fertilization Ca 2+ wave in Xenopus laevis is a single, large wave of elevated free Ca 2+ that is initiated at the point of sperm–egg fusion and traverses the entire width of the egg. This Ca 2+ wave involves an increase in inositol-1,4,5-trisphosphate (IP 3 ) resulting from the interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca 2+ release machinery. The extraordinarily large size of this cell (1.2 mm diameter) together with the small surface region of sperm-receptor activation makes special demands on the IP 3 -dependent Ca 2+ mobilizing machinery. We propose a detailed model of the fertilization Ca 2+ wave in Xenopus eggs that requires an accompanying wave of IP 3 production. While the Ca 2+ wave is initiated by a localized increase of IP 3 near the site of sperm–egg fusion, the Ca 2+ wave propagates via IP 3 production correlated with the Ca 2+ wave—possibly via Ca 2+ -mediated PLC activation. Such a Ca 2+ -mediated IP 3 production wave has not been required previously to explain the fertilization Ca 2+ wave in eggs; we argue this is necessary to explain the observed IP 3 dynamics in Xenopus eggs. To test our hypothesis, we have measured the IP 3 levels from 20 nl “sips” of the egg cortex during wave propagation. We were unable to detect the low IP 3 levels in unfertilized eggs, but after fertilization, [IP 3 ] ranged from 175 to 430 nM at the sperm entry point and from 120 to 700 nM 90° away once the Ca 2+ wave passed that region about 2 min after fertilization. Prior to the Ca 2+ wave reaching that region the IP 3 levels were undetectable. Since significant IP 3 could not diffuse to this region from the sperm entry point within 2 min, this observation is consistent with a regenerative wave of IP 3 production.

Background & Aims Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. Methods The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. Results The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. Conclusions This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies., Graphical abstract

The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm, depth = 150 μm) followed by addition of Matrigel that formed micropockets locking the crypts within the microwells. Embedded crypts first underwent 3D expansion inside the wells. After the cells filled the microwells, they migrated onto the surrounding surface forming a 2D monolayer in the array regions without Matrigel. This unique 2D/3D hybrid culture generated a continuous, millimeter-scale colonic epithelial tissue in vitro, which resembled the polarized architecture (i.e. distinct proliferative and non-proliferative zones) and geometry of the colonic epithelium in vivo. This work initiates the construction of a “colon-on-a-chip” using primary cells/tissues with the ultimate goal of producing the physiologic structure and organ-level function of the colon.

We demonstrate a new method for the simultaneous measurement of the activation of key regulatory enzymes within single cells. To illustrate the capabilities of the technique, the activation of protein kinase C (PKC), protein kinase A (PKA), calcium-calmodulin activated kinase II (CamKII), and cdc2 protein kinase (cdc2K) was measured in response to both pharmacological or physiological stimuli. This assay strategy should be applicable to a broad range of intracellular enzymes, including phosphatases, proteases, nucleases, and other kinases.

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. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.

There is a need in biological research for tools designed to manipulate the environment surrounding microscopic regions of tissue. In the current work, a device for the oriented capture of an important and under-studied tissue, the colon crypt, has been designed and tested. The objective of this work is to create a BioMEMs device for biological assays of living colonic crypts. The end goal will be to subject the polarized tissue to user-controlled fluidic microenvironments in a manner that recapitulates the in vivo state. Crypt surrogates, polymeric structures of similar dimensions and shape to isolated colon crypts, were used in the initial design and testing of the device. Successful capture of crypt surrogates was accomplished on a simple device composed of an array of micron-scale capture sites that enabled individual structures to be captured with high efficiency (92 ± 3%) in an ordered and properly oriented fashion. The device was then evaluated using colon crypts isolated from a murine animal model. The capture efficiency attained using the fixed biologic sample was 37 ± 5% due to the increased variability of the colon crypts compared with the surrogate structures, yet 94% ± 3% of the captured crypts were properly oriented. A simple approach to plug the remaining capture sites in the array was performed using inert glass beads. Blockage of unfilled capture sites is an important feature to establish a chemical gradient across the arrayed crypts. A chemical concentration gradient (Cluminal/Cbasal > 10) was demonstrated across the arrayed crypts for over 8 h. Finally unfixed colon crypts were demonstrated to be effectively captured by the micromesh array and to remain viable on the capture sites at 5 h after mouse sacrifice. The present study demonstrates the feasibility and potential for rationally microengineered technologies to address the specific needs of the biologic researcher.

Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface-roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Qualitative and quantitative analyses of sorting, collection efficiency, and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications.

Single-cell analyses have found increasing importance in biological investigation. Recent technical advances have made it possible to perform chemical separations of cellular constituents at the level of the individual cell. In this chapter, a laser-based method for the rapid sampling of living cells for chemical analysis using capillary electrophoresis is described. The platform technology consists of ultrasensitive laser-induced fluorescence detection in a capillary mated with a microscope integrated with a pulsed Nd:YAG laser. This platform provides a flexible system for the development of new single-cell biochemical assays of a variety of intracellular enzymes.

Translocation of membrane impermeant molecules to the interior of living cells is a necessity for many biochemical investigations. Myristoylation was studied as a means to introduce peptides into living cells. Uptake of a myristoylated, fluorescent peptide was efficient in the B lymphocyte cell line BA/F3. In contrast, this cell line was resistant to peptide uptake using a cell penetrating peptide derived from the TAT protein. In BA/F3 cells, membrane association was shown to be rapid reaching a maximum within 30 minutes. Cellular uptake of the peptide lagged the membrane association, but occurred within a similar time frame. Experiments performed at 37°C vs. 4°C demonstrated profound temperature dependence in the cellular uptake of myristoylated cargo. Myristoylated peptides with either positive or negative charge were shown to load efficiently. In contrast to TAT-conjugated cargo, pyrenebutyrate did not enhance cellular uptake of the myristoylated peptide. The myristoylated peptide did not adversely affect cell viability at concentrations up to 100 μM. This assessment of myristoyl-based transport provides fundamental data needed in understanding the intracellular delivery of myristoylated peptide cargoes for cell-based biochemical studies.

The Xenopus oocyte is a widely used model cell for studies of signal transduction mechanisms. Advances in microanalytical methods have made it feasible to perform rapid, localized collection of cytoplasm from individual Xenopus oocytes. Analytes contained in the cytoplasmic sample are separated by electrophoresis in a capillary and simultaneously transferred to a detection region. The development of bioengineered cells as sensitive detectors of intracellular components made quantitative measurements of native signaling molecules within the electrophoresed sample possible. Local determination of the second messenger inositol 1,4,5-trisphosphate is described to illustrate the methods for the sampling, electrophoresis, detection, and quantification of signaling molecules in single oocytes.

We report on a novel system for the analysis of adherent cells. Using standard lithography techniques, cell pallets are fabricated upon which cells are cultured and subsequently transported through a microfluidic system. This technology eliminates the need for the release of a cell from its growth surface, which can lead to membrance damage, and/or disruption of normal cell functions. We have demonstrated RBL (rat basophil leukemia) cells will grow on SU8 pallets and remain viable after the pallet is released. Cells on pallets can then be sorted based on pre-selected criteria and sent to a docking station for cell assays. Once completed, the cells may be interrogated and retained for further processing or disposal.

The introduction of peptides into living cells for the purpose of manipulating cellular biochemistry has become widespread throughout biology. However, little is known about the behavior of these short sequences of amino acids within cells, particularly those used as substrates or inhibitors for kinases and other enzymes. We utilized a quantitative, single-cell assay to demonstrate that an 11-amino acid peptide was efficiently phosphorylated by intracellular protein kinase B (PKB) in fibrosarcoma cell line HT1080 and in NIH-3T3 cells. The phosphorylated peptide was also readily dephosphorylated by intracellular phosphatases. Assays of the peptide's phosphorylation in single, living cells measured the balance of the activities of PKB and phosphatases in that cell. At a peptide concentration below the K M of PKB and the phosphatases, the ratio of phosphorylated to nonphosphorylated peptide at the steady state was independent of the peptide concentration. A single-cell assay utilizing this peptide revealed the existence of two subpopulations of cells whose unique activities had hitherto been obscured by population averaging. Additional studies of cells stimulated by PDGF demonstrated that a quantitative analysis of PKB activation in response to a physiological stimulus was possible. These studies demonstrated that short peptides can remain specific within the complex intracellular milieu and function as sensitive reporters of the activation state of native kinases within live cells.

Recent advances in analytical techniques have made the performance of biochemical assays on individual mammalian cells possible. Of particular interest is the ability to measure the activation of kinases, enzymes with critical roles in virtually every aspect of cell physiology. Single-cell kinase assays promise to deliver a newfound understanding of the molecular mechanisms responsible for cellular control and behavior by revealing the dynamic nature of signal transduction networks in living cells. A recent exciting development is the potential to perform assays of multiple kinases simultaneously in a single cell.

Molecularly targeted therapies are at the forefront of clinical science, and are expected to lead to personalization of medical treatments for each patient. Most such therapies are directed at inhibiting specific signal transduction enzymes or pathways, thus creating a critical need for assays capable of measuring the activities of these proteins in disease models and in patient samples. The ability to measure relevant enzyme activity in primary cell samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. Here we report our most recent efforts to create the analytical and chemical tools needed to directly measure the enzymatic activities of therapeutic targets including protein kinases, lipid-modifying enzymes and the proteasome. Fluorescent reagents are under development that report the activity of these various enzymes with the goal of performing biochemical assays in primary cells. The basic design incorporates enzyme substrates that are modified to create compounds which can be loaded into cells where they are modified by the enzyme of interest. Work has included modification of peptides to confer membrane permeability and to achieve long intracellular lifetimes. Microelectrophoretic separations combined with low-level fluorescence detection enable the quantitative analysis of these compounds from single mammalian cells. This capability addresses three major issues currently faced in the biochemical analysis of clinical samples: the need for direct measurement of the enzymatic activity of target proteins; sample size requirements that are feasible for clinical implementation; and sample heterogeneity that can mask pertinent aspects related to therapeutic response.

To measure the concentration of inositol 1,4,5-trisphosphate ([IP3]) in small regions of single Xenopus oocytes, a biological detector cell was combined with capillary electrophoresis. This method is 10,000 times more sensitive than all existing assays enabling subcellular measurement of [IP3] in Xenopus oocytes. Upon addition of lysophosphatidic acid to an oocyte, [IP3] increased from 40 to 650 nm within 2 min. IP3concentrations as high as 1.8 μm were measured after activation with lysophosphatidic acid, suggesting that the physiologic concentration of IP3 ranges from the tens of nanomolar to a few micromolar in Xenopus oocytes. Since the IP3 receptor in Xenopus oocytes is nearly identical to the type I receptor of mammalian cells, the range of [IP3] in most mammalian cells is likely to be similar to that in the oocyte. By selecting or engineering the appropriate detector cell, this strategy should be applicable to cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate, and to the discovery of new Ca2+-releasing second messengers.

The pathway and kinetics of inositol 1,4,5-trisphosphate (IP3) metabolism were measured in Xenopus laevis oocytes and cytoplasmic extracts of oocytes. Degradation of microinjected IP3 in intact oocytes was similar to that in the extracts containing comparable concentrations of IP3 ([IP3]). The rate and route of metabolism of IP3 depended on the [IP3] and the intracellular free Ca2+ concentration ([Ca2+]). At low [IP3] (100 nM) and high [Ca2+] (>/=1 microM), IP3 was metabolized predominantly by inositol 1,4, 5-trisphosphate 3-kinase (3-kinase) with a half-life of 60 s. As the [IP3] was increased, inositol polyphosphate 5-phosphatase (5-phosphatase) degraded progressively more IP3. At a [IP3] of 8 microM or greater, the dephosphorylation of IP3 was the dominant mode of IP3 removal irrespective of the [Ca2+]. At low [IP3] and low [Ca2+] (both

Receptor tyrosine kinases (RTKs) are key components of physiological and pathological cell signaling, and are normally subject to tight regulation by protein tyrosine phosphatases (PTPs). Irregularities on either side of this equilibrium can lead to inflammation, metabolic disease, and cancer. While many established and emerging techniques have been used to characterize these signaling pathways, few approaches are well adapted to the analysis of single primary cells, and fewer still measure enzyme activity directly. We report a new method for measuring the balance between RTK and PTP activity using fluorescent peptide substrates in conjunction with capillary electrophoresis. Briefly, live cells are visualized on an inverted microscope, microinjected with a fluorescent peptide substrate which is acted upon by RTKs and/or PTPs. After the desired time interval, a single cell is lysed using a microsecond laser pulse and cell contents are aspirated into a fused-silica capillary where electrophoresis is performed and reporter species are separated and quantified based on phosphorylation status. We have applied this technique to the characterization of PTP inhibition in single A431 cells by several components of diesel exhaust particles, an important component of air pollution linked to cancer. We have also adapted this system to measure the effects of small-molecule epidermal growth factor receptor (EGFR) inhibitors on RTK activity in tumor cell lines as well as tumor xenograft models, also at the single cell level. This approach provides a unique set of advantages over alternative techniques and is a valuable tool for the study of cell signaling in cancer. The integrated microscopy permits selection of cells for analysis based on morphology, viability, or vital staining, an important consideration for analysis of heterogeneous samples such as tumor specimens, especially when total sample size is small. High-sensitivity detection allows chemical cytometry data from individual cells to be gathered and the presence of phenotypic subpopulations is not lost by observing only the average behavior of a large population. Additionally, peptide substrate reporters can be tuned for enzyme specificity, and the analytical separation allows multiple reporter pairs to be separated and detected in a single experiment, permitting simultaneous analysis of multiple enzymes. The introduction of reporter peptides by microinjection preserves cellular architecture, while being equally applicable to immortalized and primary cells since no genetic manipulation is necessary. This array of advantages makes our approach uniquely well suited to the study of RTK and PTP signaling in both cultured tumor cell lines and patient specimens and a valuable new tool for cancer research. Citation Format: Ryan M. Phillips, David S. Lawrence, Christopher E. Sims, Nancy L. Allbritton. A novel approach to the measurement of tyrosine phosphorylation dynamics in intact single cells using capillary electrophoresis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2013-4128

Crypts are the basic structural and functional units of colonic epithelium and can be isolated from the colon and cultured in vitro into multi-cell spheroids termed “colonoids”. Both crypts and colonoids are ideal building blocks for construction of an in vitro tissue model of the colon. Here we proposed and tested a microengineered platform for capture and in vitro 3D culture of colonic crypts and colonoids. An integrated platform was fabricated from polydimethylsiloxane which contained two fluidic layers separated by an array of cylindrical microwells (150 μm diameter, 150 μm depth) with perforated bottoms (30 μm opening, 10 μm depth) termed “microstrainers”. As fluid moved through the array, crypts or colonoids were retained in the microstrainers with a >90% array-filling efficiency. Matrigel as an extracellular matrix was then applied to the microstrainers to generate isolated Matrigel pockets encapsulating the crypts or colonoids. After supplying the essential growth factors, epidermal growth factor, Wnt-3A, R-spondin 2 and noggin, 63 ± 13% of the crypts and 77 ± 8% of the colonoids cultured in the microstrainers over a 48–72 h period formed viable 3D colonoids. Thus colonoid growth on the array was similar to that under standard culture conditions (78 ± 5%). Additionally the colonoids displayed the same morphology and similar numbers of stem and progenitor cells as those under standard culture conditions. Immunofluorescence staining confirmed that the differentiated cell-types of the colon, goblet cells, enteroendocrine cells and absorptive enterocytes, formed on the array. To demonstrating the utility of the array in tracking the colonoid fate, quantitative fluorescence analysis was performed on the arrayed colonoids exposed to reagents such as Wnt-3A and the γ-secretase inhibitor LY-411575. The successful formation of viable, multi-cell type colonic tissue on the microengineered platform represents a first step in the building of a “colon-on-a-chip” with the goal of producing the physiologic structure and organ-level function of the colon for controlled experiments.

Molecularly targeted therapies are at the forefront of clinical science, and are expected to lead to personalization of medical treatments for each patient. Most such therapies are directed at inhibiting specific signal transduction enzymes or pathways, thus creating a critical need for assays capable of measuring the activities of these proteins in disease models and in patient samples. The ability to measure relevant enzyme activity in primary cell samples at baseline and/or after treatment would provide the ability to tailor patient therapy based on aberrant signal transduction, validate mechanisms of resistance in patients, and would offer an invaluable pharmacodynamic tool to assess whether resistance is associated with inadequate target inhibition. Here we report our current efforts to create the analytical and chemical tools needed to directly measure the enzymatic activities of therapeutic targets including protein kinases, lipid modifying enzymes and the proteasome. Fluorescent reagents are under development that report the activity of these various enzymes in model cells lines and primary cells. The basic design incorporates enzyme substrates that are modified to create compounds which can be loaded into cells where they are modified by the enzyme of interest. Work has included modification of peptides to confer membrane permeability and to achieve long intracellular lifetimes. Microelectrophoretic separations combined with low-level fluorescence detection enable the quantitative analysis of these compounds from single mammalian cells. This capability addresses three major issues currently faced in the biochemical analysis of clinical samples: the need for direct measurement of the enzymatic activity of target proteins; sample size requirements that are feasible for clinical implementation; and sample heterogeneity that can mask pertinent aspects related to therapeutic response.