Yu-suke Torisawa

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring.

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.

A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids

Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles.

Proliferation assay on a silicon chip applicable for tumors extirpated from mammalians

We describe a novel anticancer drug sensitivity assay on a silicon chip applicable for tumors extirpated from in vivo mammalians. Human promyelocytic leukemia (HL‐60) cells were subcutaneously (s.c.) inoculated in SCID mice, then removed 31 days after the inoculation. The cells were embedded in a small volume (18 nL) of a collagen‐gel matrix on a pyramid‐shaped silicon microstructure for further cultivation. The respiration activity of the cells on the chip was measured by scanning electrochemical microscopy (SECM). The proliferation behavior was continuously monitored for 6 days. It seemed that the proliferation rate of the cells removed from the mice was lower than that cultured in a flask and conformed to that in mice. The effects of cisplatin (CDDP) and etoposide (VP‐16) on the HL‐60 cultured in vivo were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the SECM‐based assay is appropriate for biopsy specimens in a relatively short‐time evaluation.