| The long-term failure rate of cancer chemotherapy is still very high (90% or higher). The main reason is multidrug resistance (MDR) of cancer cells against drugs such as doxorubicin (DOX). Although the detailed mechanisms of MDR are still unclear, extensive studies have led to the conclusion that the observed decrease in intracellular drug concentration in MDR cells was related to the overexpression of transport membrane proteins and/or exocytosis pathways. Experiments done to further elucidate the MDR mechanisms, however, have lacked the following aspects: (1) While drug sequestration was frequently studied by using fluorescence microscopy, taking advantage of the fluorescence nature of most anticancer drugs, quantitative evaluation of drug concentrations has been difficult due to fluorescence changes within the environment where the drug resides. (2) Although constructing well-defined spatial distribution of cells is crucial to ensure reproducible experimental conditions, the majority of MDR studies have been done on randomly distributed cell preparations such as suspended cells or non-confluent monolayers.; To address first issue, we first developed image processing techniques to assess fluorescence distributions in micro-scale droplets. We established accurate, precise, and efficient algorithms to calculate droplet size and fluorescence intensities in the bulk and at the interface, from confocal fluorescence images taken at the equatorial plane of the droplet. Fluorescence intensities of DOX measured in the bulk and at the interface were then calibrated with respect to concentrations in each region, aiming to use them as calibrations in the cytoplasm and at the membrane, respectively, in actual cells. The developed techniques were also applied to study physicochemical behavior of DOX at the immiscible liquid-liquid interface.; After noticing that hydrophilic sites ensure stronger attachment than hydrophobic sites even in the five minute time range of cell-substrate contact, we addressed the second issue by fabricating BioMEMS chips which accommodate hydrophilic/hydrophobic micron-scale patterns. We were able to realize selective and minutes-scale cell attachment onto predetermined hydrophilic sites. To navigate single cells onto the attachment sites and control such selective attachments, a hydrodynamic micromanipulation technique was invented and applied. This technique allowed construction of a well-defined spatial distribution of single cells in several minutes. |
