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.

An in vitro intestinal platform with a self-sustaining oxygen gradient to study the human gut/microbiome interface.

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.

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking.

BACKGROUND: The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. RESULTS: The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. CONCLUSIONS: The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function.

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer.

BACKGROUND & AIMS: The successful culture of intestinal organoids has greatly enhanced our understanding of intestinal stem cell physiology and enabled the generation of novel intestinal disease models. Although of tremendous value, intestinal organoid culture systems have not yet fully recapitulated the anatomy or physiology of the in vivo intestinal epithelium. The aim of this work was to re-create an intestinal epithelium with a high density of polarized crypts that respond in a physiologic manner to addition of growth factors, metabolites, or cytokines to the basal or luminal tissue surface as occurs in vivo. METHODS: A self-renewing monolayer of human intestinal epithelium was cultured on a collagen scaffold microfabricated with an array of crypt-like invaginations. Placement of chemical factors in either the fluid reservoir below or above the cell-covered scaffolding created a gradient of that chemical across the growing epithelial tissue possessing the in vitro crypt structures. Crypt polarization (size of the stem/proliferative and differentiated cell zones) was assessed in response to gradients of growth factors, cytokines, and bacterial metabolites. RESULTS: Chemical gradients applied to the shaped human epithelium re-created the stem/proliferative and differentiated cell zones of the in vivo intestine. Short-chain fatty acids applied as a gradient from the luminal side confirmed long-standing hypotheses that butyrate diminished stem/progenitor cell proliferation and promoted differentiation into absorptive colonocytes. A gradient of interferon-γ and tumor necrosis factor-α significantly suppressed the stem/progenitor cell proliferation, altering crypt formation. CONCLUSIONS: The in vitro human colon crypt array accurately mimicked the architecture, luminal accessibility, tissue polarity, cell migration, and cellular responses of in vivo intestinal crypts.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells.

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.

Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.

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.

Development of a protease-resistant reporter to quantify BCR-ABL activity in intact cells.

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (kcat/KM) of purified ABL kinase for the lead peptide (125 s-1 µM-1) is similar to that of the starting peptide (103 s-1 µM-1) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, the peptide is metabolized into 5 fragments, with only 2 of the fragments corresponding to those in the lysate. Thus the two environments possess differing peptidase activities, which must be accounted for when designing peptidase-resistant peptides. In both settings, the substrate is phosphorylated by BCR-ABL providing a readout of BCR-ABL activity. A small panel of tyrosine kinase inhibitors verified the substrate's specificity for BCR-ABL/ABL kinase activity in both lysates and cells in spite of the multitude of other kinases present. The designed peptide X-A acts as a long-lived BCR-ABL kinase reporter in the leukemic cells possessing the BCR-ABL mutation.

Selective single cell isolation for genomics using microraft arrays.

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.

Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α.

The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest.

Array-Based Platform To Select, Release, and Capture Epstein-Barr Virus-Infected Cells Based on Intercellular Adhesion.

Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.

Small sample sorting of primary adherent cells by automated micropallet imaging and release.

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.

Dynamics and evolution of ß-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening.

Wnt/ß-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/ß-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/ß-catenin signaling were screened over short (<6) and long (>25) generational timescales. To characterize phenotypic divergence over these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1119 clonal colonies in parallel. This screen revealed phenotypic divergence after <6 generations at a similar scale to that observed in monoclonal cell lines cultured for >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 provides insight into the pathway's mechanisms of positive feedback and self-inhibition.

Array of Biodegradable Microraftsfor Isolation and Implantation of Living, Adherent Cells.

A new strategy for efficient sorting and implantation of viable adherent cells into animals is described. An array of biodegradable micro-structures (microrafts) was fabricated using a polydimethylsiloxane substrate for micromolding poly(lactic-co-glycolic acid) (PLGA). Screening various forms of PLGA determined that the suitability of PLGA for microraft manufacture, biocompatibility and in vitro degradation was dependent on molecular weight and lactic/glycolic ratio. Cells plated on the array selectively attached to the microrafts and could be identified by their fluorescence, morphology or other criteria. The cells were efficiently dislodged and collected from the array using a microneedle device. The platform was used to isolate specific cells from a mixed population establishing the ability to sort target cells for direct implantation. As a proof of concept, fluorescently conjugated microrafts carrying tumor cells stably expressing luciferase were isolated from an array and implanted subcutaneously into mice. In vivo bio-luminescence imaging confirmed the growth of a tumor in the recipient animals. Imaging of tissue sections from the tumors demonstrated in vivo degradation of the implanted microrafts. The process is a new strategy for isolating and delivering a small number of adherent cells for animal implantation with potential applications in tissue repair, tumor induction, in vivo differentiation of stem cells and other biomedical research.

Measurement of protein tyrosine phosphatase activity in single cells by capillary electrophoresis.

A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn(2+) and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min(-1) mg(-1)) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min(-1) mg(-1)). Zn(2+), 1,2-naphthoquinone, and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 µM, and 100 nM, respectively. Dephosphorylation of the reporter, following loading into living single cells, occurred at rates of at least 2 pmol min(-1) mg(-1). When single cells were exposed to 1,2-naphthoquinone (50 µM), Zn(2+) (100 µM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.

Laser-based directed release of array elements for efficient collection into targeted microwells.

A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.

Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis.

Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH(2)PO(4) and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH(2)PO(4) concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×10(5) in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10(-18)-10(-20)mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and robust separation method for SFL and S1PFL and various metabolites of phosphoinositide-related signal transduction is expected to enable improved enzymatic assays for biological and clinical applications.

Benchtop micromolding of polystyrene by soft lithography.

Polystyrene (PS), a standard material for cell culture consumable labware, was molded into microstructures with high fidelity of replication by an elastomeric polydimethylsiloxane (PDMS) mold. The process was a simple, benchtop method based on soft lithography using readily available materials. The key to successful replica molding by this simple procedure relies on the use of a solvent, for example, gamma-butyrolactone, which dissolves PS without swelling the PDMS mold. PS solution was added to the PDMS mold, and evaporation of the solvent was accomplished by baking the mold on a hotplate. Microstructures with feature sizes as small as 3 µm and aspect ratios as large as 7 were readily molded. Prototypes of microfluidic chips made from PS were prepared by thermal bonding of a microchannel molded in PS with a flat PS substrate. The PS microfluidic chip displayed much lower adsorption and absorption of hydrophobic molecules (e.g. rhodamine B) compared to a comparable chip created from PDMS. The molded PS surface exhibited stable surface properties after plasma oxidation as assessed by contact angle measurement. The molded, oxidized PS surface remained an excellent surface for cell culture based on cell adhesion and proliferation. To demonstrate the application of this process for cell biology research, PS was micromolded into two different microarray formats, microwells and microposts, for segregation and tracking of non-adherent and adherent cells, respectively. The micromolded PS possessed properties that were ideal for biological and bioanalytical needs, thus making it an alternative material to PDMS and suitable for building lab-on-a-chip devices by soft lithography methods.

Kinetic analysis of PI3K reactions with fluorescent PIP2 derivatives.

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.

Continuous analysis of dye-loaded, single cells on a microfluidic chip.

Continuous analysis of two dyes loaded into single mammalian cells using laser-based lysis combined with electrophoretic separation was developed and characterized on microfluidic chips. The devices employed hydrodynamic flow to transport cells to a junction where they were mechanically lysed by a laser-generated cavitation bubble. An electric field then attracted the analyte into a separation channel while the membranous remnants passed through the intersection towards a waste reservoir. Phosphatidylcholine (PC)-supported bilayer membrane coatings (SBMs) provided a weakly negatively charged surface and prevented cell fouling from interfering with device performance. Cell lysis using a picosecond-pulsed laser on-chip did not interfere with concurrent electrophoretic separations. The effect of device parameters on performance was evaluated. A ratio of 2 : 1 was found to be optimal for the focusing-channel : flow-channel width and 3 : 1 for the flow-channel : separation-channel width. Migration times decreased with increased electric field strengths up to 333 V cm(-1), at which point the field strength was sufficient to move unlysed cells and cellular debris into the electrophoretic channel. The migration time and full width half-maximum (FWHM) of the peaks were independent of cell velocity for velocities between 0.03 and 0.3 mm s(-1). Separation performance was independent of the exact lysis location when lysis was performed near the outlet of the focusing channel. The migration time for cell-derived fluorescein and fluorescein carboxylate was reproducible with <10% RSD. Automated cell detection and lysis were required to reduce peak FWHM variability to 30% RSD. A maximum throughput of 30 cells min(-1) was achieved. Device stability was demonstrated by analyzing 600 single cells over a 2 h time span.