Schematic of cultured cell detachment and reculturing.

<p>In step 1, cells are pre-cultured in an alginate-coated culture dish. In steps 2–4, the alginate layer is locally isolated by applying an EDTA-containing culture medium with a micropipette, and cells cultured on the alginate are collected. In step 5, cells collected with the micropipette are recultured in another culture dish.</p

Micrographs and extracellular action potentials of cardiomyocyte clusters derived from human ES cells.

<p>Fig. A shows a micrograph of four cultured cardiomyocyte clusters derived from hES cells on the alginate thin sheet above the multielectrode array culture dish (1 day in vitro (DIV)). (A)-1 to (A)-4 are the extracellular field potentials of the cardiomyocyte clusters in (A). After the extracellular field potential measurement, the target cluster that had ventricular-type signals (cluster no. 3) was detached from the dish and recultivated on the other dish. (B) shows the remaining three clusters after the detachment of the target cluster (4 DIV). (B)-1 and (B)-2 are the extracellular field potentials. (C) shows the selected target cardiomyocyte cluster recultivated on the other multielectrode cultivation dish (4 DIV). (C)-3 is the extracellular field potential of cluster no 3. The target cluster (no. 3) was successfully picked up from the dish on which a plurality of clusters were cultivated. It retained similar characteristics of external field potential waveforms before and after the sorting procedure.</p

Micrographs of the steps from preculture, detachment, and collection to reculture of a single hippocampal neuron derived from rat E18.

<p>The time from detachment to reculture took <2 min, as with the cardiomyocytes. During and after isolation, the neuron did not shrink and the neurite retained its shape. After reculturing, the neuron immediately adhered to the culture dish and extended its neurite within a few minutes.</p

Construction of the collection dish for cardiomyocytes and neurons.

<p>First, a calcium alginate layer was formed in both culture dishes (a), (e). For the cardiomyocyte culture dish, a mixture containing a low concentration of alginate and collagen type IV extracellular matrix was layered onto the alginate sheet (b). The solution was then concentrated and dried (c), gelled with CaCl₂, and washed three times (d). For the neuron culture dish, PLL was micro-contact printed using a PDMS stamp (f). Following incubation for 5 min under high humidity conditions (similar to those in a cell incubator), the stamp was removed from the culture dish.</p

Results of quantitative gene copy number assays.

<p>(a) Results of CGH assays performed for the MAT-LyLu cell line. Liver tissue of the rat was used as a reference. Gene amplifications for <i>csrp2</i> and <i>zdhhc17</i> located on chromosome 7q13 were found. (b) Results of TaqMan copy number assays performed with clusters larger than 300 µm² collected in the collection reservoir, and cells smaller than 300 µm² collected in the discarded reservoir. Results of the assays for the MAT-LyLu cell line (positive control) and liver tissue (negative control) are also shown.</p

Summary of image processing.

<p>Firstly, photographs of both a cell and the background were taken. Next, the background image was subtracted from the cell image and holes were filled. Finally, imaging biomarkers, <i>S</i>, <i>S_n</i>, <i>N_n</i>, and <i>R</i>, were calculated. Bars, 10 µm. The hole-filling procedure is explained as indicated by an asterisk. Bar, 10 µm.</p

Typical cell images for clustered cells composed of more than three cells in cancer cell-implanted and control blood.

<p>Each data count (<i>n</i>) indicates the image number having the same cluster size and <i>N_n</i>. Bars, 20 µm.</p

Typical cell images for single and double cells in cancer cell-implanted and control blood.

<p>Each data count (<i>n</i>) indicates the image number having the same cluster size and <i>N_n</i>. Bars, 20 µm.</p

Instrumental set-up.

<p>(a) Summary of the on-chip multi-imaging flow cytometry system. The system was composed of seven major modules: (i) microchip, (ii) bright-field (BF) imaging, (iii) fluorescent (FL) detection, (iv) multi-view, (v) CCD camera, (vi) sorting, and (vii) controller, as numbered in the figure. (b) Summary of the multi-view module. (c) A photograph of the system.</p

Summary of total cell area, <i>S</i>, total nucleus area, <i>S_n</i>, number of nuclei, <i>N_n</i>, and perimeter ratio, <i>R</i>, for each cluster size.

<p>Summary of total cell area, <i>S</i>, total nucleus area, <i>S_n</i>, number of nuclei, <i>N_n</i>, and perimeter ratio, <i>R</i>, for each cluster size.</p