Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system

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

Fabrication of 3D Microstructures from Interactions of Immiscible Liquids with a Structured Surface

A new lithography technique is presented that exploits the interactions of immiscible liquids with a structured surface. This highly parallel, "low-tech" method requires no dedicated equipment and easily produces curved and/or multi-level structures out of a variety of photoactive and non-photoactive materials

Coaxial Flow System for Chemical Cytometry

Over the past decade, chemical cytometry performed by capillary electrophoresis (CE) has become increasingly valuable as a bio-analytical tool to quantify analytes from single cells. However, extensive use of CE-based chemical cytometry has been hindered by the relatively low throughput for the analysis of single adherent cells. In order to overcome the low throughput of CE-based analysis of adherent cells and increase its utility in evaluating cellular attributes, new higher throughput methods are needed. Integration of a coaxial buffer exchange system with CE-based chemical cytometry increased the rate of serial analyses of cells. In the designed system, fluid flow through a tube coaxial to the separation capillary was used to supply electrophoretic buffer to the capillary. This sheath or coaxial fluid was turned off between analysis of cells and on during cell sampling and electrophoresis. Thus, living cells were not exposed to the nonphysiologic electrophoretic buffer prior to lysis. Key parameters of the system such as the relative capillary-sheath positions, buffer flow velocities, and the cell chamber design were optimized. To demonstrate the utility of the system, rat basophilic leukemic cells loaded with Oregon Green and fluorescein were serially lysed and loaded into a capillary. Separation of the contents of 20 cells at a rate of 0.5 cells/min was demonstrated

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

Automated Capillary Electrophoresis System for Fast Single-Cell Analysis

Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. 88% of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena

Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays

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

Single-cell sphingosine kinase activity measurements in primary leukemia

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39 ± 26% per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16 ± 25%, 11 ± 26%, and 13 ± 23% in a chronic myelogenous leukemia (CML) patient, an acute myeloid leukemia (AML) patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87% of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99% of the cell population, 19 ± 36% of the loaded sphingosine was phosphorylated. Thus the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12 ± 0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer

A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies

Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as to achieve tighter control of experimental variables than can be achieved with animals. Thus, development of a primary in vitro colonic epithelium cultured as high resistance with transport protein expression and functional behavior similar to that of a native colonic would be of enormous value for pharmaceutical research. A collagen scaffold, in which the degree of collagen cross-linking was present as a gradient, was developed to support the proliferation of primary colonic cells. The gradient of cross-linking created a gradient in stiffness across the scaffold, enabling the scaffold to resist deformation by cells. mRNA expression and quantitative proteomic mass spectrometry of cells growing on these surfaces as a monolayer suggested that the transporters present were similar to those in vivo. Confluent monolayers acted as a barrier to small molecules so that drug transport studies were readily performed. Transport function was evaluated using atenolol (a substrate for passive paracellular transport), propranolol (a substrate for passive transcellular transport), rhodamine 123 (Rh123, a substrate for P-glycoprotein), and riboflavin (a substrate for solute carrier transporters). Atenolol was poorly transported with an apparent permeability (Papp) of < 5 × 10-7 cm s-1, while propranolol demonstrated a Papp of 9.69 × 10-6 cm s-1. Rh123 was transported in a luminal direction (Papp,efflux/Papp,influx = 7) and was blocked by verapamil, a known inhibitor of P-glycoprotein. Riboflavin was transported in a basal direction, and saturation of the transporter was observed at high riboflavin concentrations as occurs in vivo. It is anticipated that this platform of primary colonic epithelium will find utility in drug development and physiological studies, since the tissue possesses high integrity and active transporters and metabolism similar to that in vivo

An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State

Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles