Showing papers by "J. Christopher Love published in 2010"

Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving

Abstract: The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions of specific subsets to an immunological response. Microengraving is process that uses a dense, elastomeric array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells (∼104–105 cells/assay). In this paper, we describe an approach based on this technology to quantify the rates of secretion from single immune cells. Numerical simulations of the microengraving process indicated an operating regime between 30 min–4 h that permits quantitative analysis of the rates of secretion. Through experimental validation, we demonstrate that microengraving can provide quantitative measurements of both the frequencies and the distribution in rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells. The experimental limits of detection ranged from 0.5 to 4 molecules/s for IL-6, IL-17, IFNγ, IL-2, and TNFα. These multidimensional measures resolve the number and intensities of responses by cells exposed to stimuli with greater sensitivity than single-parameter assays for cytokine release. We show that cells from different donors exhibit distinct responses based on both the frequency and magnitude of cytokine secretion when stimulated under different activating conditions. Primary T cells with specific profiles of secretion can also be recovered after microengraving for subsequent expansion in vitro. These examples demonstrate the utility of quantitative, multidimensional profiles of single cells for analyzing the diversity and dynamics of immune responses in vitro and for identifying rare cells from clinical samples.

Massively parallel detection of gene expression in single cells using subnanolitre wells.

Abstract: The relationship between the expression of particular genes in cells and their impact on phenotypic characteristics is important for understanding how cells regulate responses to their environment. We have developed a microwell-based method to detect copies of mRNA transcripts directly from individual cells by one-step, single-cell, reverse transcription polymerase chain reaction (RT-PCR). Our approach permits the detection of mRNA transcripts of interest for more than 6000 single cells in parallel per assay with high sensitivity and specificity for constitutively active genes. This simple method was also combined with microengraving and image-based cytometry to examine the relationships between gene expression and cellular secretion of antibodies in a clonal population. We observed that most individual human B cell hybridomas transcribed a requisite gene for their antibodies, but only a subset of those cells secreted the antibody. The technique should also allow the detection of replicating intracellular pathogens such as retroviruses.

Development and optimization of a process for automated recovery of single cells identified by microengraving.

Abstract: Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost-effective manner, but connecting these systems to the existing platforms used in routine high-throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody-producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high-throughput, single-cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099-01) were sorted with 100% accuracy (50 clones of each) in ∼2 h, and the cells retained viability. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

Integrated single-cell analysis shows Pichia pastoris secretes protein stochastically.

Abstract: The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single-cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell-cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner.

On-chip activation and subsequent detection of individual antigen-specific T cells.

Abstract: The frequencies of antigen-specific CD4+ T cells in samples of human tissue have been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNγ and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical s...

Layer-by-Layer Assembly of a pH-Responsive and Electrochromic Thin Film

Abstract: This article summarizes an experiment on thin-film fabrication with layer-by-layer assembly that is appropriate for undergraduate laboratory courses. The purpose of this experiment is to teach students about self-assembly in the context of thin films and to expose students to the concepts of functional polymeric coatings. Students dip coat microscope slides and conductive glass with films containing polyaniline (PANi), a conducting polymer, and sulfonated polystyrene. Assembly of the film is characterized by visual inspection, UV−vis spectroscopy, and conductance measurements with a hand-held multimeter. Green-colored films with measurable conductance are apparent after two PANi layers. Subsequently, the students observe the pH-dependent optical properties of the films and also determine the electrochromic performance of the films using spectroelectrochemistry. The feasibility of this experiment has been tested by inclusion into an undergraduate-level polymer science laboratory with third- and fourth-year...

Method for detecting active and latent virally infected cells

Abstract: The invention provides methods for detecting virus production, determining frequency and identity of HIV reservoirs, or evaluating gene expression on a single-cell basis using microengraving and RT-PCR.

Making antibodies from scratch.

Abstract: volume 28 number 11 november 2010 nature biotechnology variability in growth and profusion of necrotic cells. CMCs appear to enhance cell culture performance in all of these areas, and notably do not require selection of specific cells and aggregates for consistent growth over repeated subcultures, thereby minimizing maintenance requirements. These improvements yield cell cultures that are substantially closer to mammalian cell cultures with regard to large-scale process considerations. CMC-based strategies should therefore facilitate the development of economically viable plant cell tissue culture processes for many natural products.

Method of performing one-step, single cell RT-PCR

Abstract: The invention provides methods for detecting virus production, determining frequency and identity of HIV reservoirs, or evaluating gene expression on a single-cell basis using microengraving and RT-PCR.