Single lymphocyte analysis with a microwell array chip

Abstract

Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single-cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live-cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope-based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen-specific B-cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus-neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody-based therapeutics and diagnosis for infectious diseases such as hepatitis viruses. '

Identification of antigen‐specific B cells by concurrent monitoring of intracellular Ca2+ mobilization and antigen binding with microwell array chip system equipped with a CCD imager

“…Cytomics is becoming increasingly important for analyzing cell conditions and cellular responses to stimuli from extracellular milieu, which will contribute to drug discovery as well as predictive medicine. Cell microarray-based analysis offers one way for analyzing cytomics; it has been conventionally performed with a single parameter (11)(12)(13)(14)(15)(16)(17)(18). Here, for the first time, we present a method of two-parameter cell microarray-based time-lapse analysis.…”

“…After that, the MAC-CCD system detected the position of each microwell on the chip again. Fifteen seconds after the stimulation, the fluorescence images of the fluo-4 signals were acquired on the whole microwell area every 3 s for an additional 45 s (scanning points [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The cells inside the wells did not move out of the wells after four gentle washes.…”

“…To date, some cell microarray technologies using microwells have been reported for analyzing a large number of nonadherent cells (11)(12)(13)(14)(15)(16). These microwells are designed to capture single cells, enabling the tracking and analyzing of cellular functions at the single-cell level.…”

High-throughput Screening of Erratic Cell Volume Regulation Using a Hydrogel-based Single-cell Microwell Array

“…24 Traditional microfabrication technology that combines photolithography with chemical etching or deep ion reactive etching was used to create microwells on a glass 25 or Si substrate. 26 Alternatively, a microwell array based on photoresist 27 or photocrosslinkable hydrogel 28 could be directly constructed on a glass substrate without the use of a harsh chemical etchant. Polydimethyl siloxane (PDMS) microwell arrays were also prepared by casting PDMS prepolymer against a master that was made of a photoresist polymer 29 or selfassembled 2D polystyrene microbeads.…”

An electroactive microwell array for trapping and lysing single-bacterial cells

“…[2][3][4][5] Especially, microwell arrays enable large scale arraying and high-throughput single-cell measurements of cellular responses. 6 Previous microwell array methods used gravity as a simple mechanism to trap single cells; [6][7][8][9] however, this method remains in the applications to mammalian cells for which typical volume is in the range of picoliters. To the best of our knowledge, no single-cell arraying method for bacteria such as Escherichia coli ͑E.…”