Hirofumi Shiono

Characterization of time‐course morphological features for efficient prediction of osteogenic potential in human mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hBMSCs) represents one of the most frequently applied cell sources for clinical bone regeneration. To achieve the greatest therapeutic effect, it is crucial to evaluate the osteogenic differentiation potential of the stem cells during their culture before the implantation. However, the practical evaluation of stem cell osteogenicity has been limited to invasive biological marker analysis that only enables assaying a single end‐point. To innovate around invasive quality assessments in clinical cell therapy, we previously explored and demonstrated the positive predictive value of using time‐course images taken during differentiation culture for hBMSC bone differentiation potential. This initial method establishes proof of concept for a morphology‐based cell evaluation approach, but reveals a practical limitation when considering the need to handle large amounts of image data. In this report, we aimed to scale‐down our proposed method into a more practical, efficient modeling scheme that can be more broadly implemented by physicians on the frontiers of clinical cell therapy. We investigated which morphological features are critical during the osteogenic differentiation period to assure the performance of prediction models with reduced burden on image acquisition. To our knowledge, this is the first detailed characterization that describes both the critical observation period and the critical number of time‐points needed for morphological features to adequately model osteogenic potential. Our results revealed three important observations: (i) the morphological features from the first 3 days of differentiation are sufficiently informative to predict bone differentiation potential, both activities of alkaline phosphatase and calcium deposition, after 3 weeks of continuous culture; (ii) intervals of 48 h are sufficient for measuring critical morphological features; and (iii) morphological features are most accurately predictive when early morphological features from the first 3 days of differentiation are combined with later features (after 10 days of differentiation). Biotechnol. Bioeng. 2014;111: 1430–1439.

Flexible sheet-Type sensor for noninvasive measurement of cellular oxygen metabolism on a culture dish

A novel flexible sensor was developed for the noninvasive oxygen metabolism measurement of cultivated cells and tissues. This device is composed of a transparent double-layered polymer sheet of ethylene-vinyl alcohol (EVOH) and poly(dimethylsiloxane) (PDMS) having an array of microhole structures of 90 μm diameter and 50 μm depth on its surface. All the microhole structures were equipped with a 1-μm-thick optical chemical sensing layer of platinum porphyrin-fluoropolymer on their bottom. The three-dimensional microstructures of the sensor were fabricated by a newly developed simple and low-cost production method named self-aligned hot embossing. The device was designed to be attached slightly above the cells cultivated on a dish to form a temporarily closed microspace over the target cells during measurement. Since the change in oxygen concentration is relatively fast in the microcompartmentalized culture medium, a rapid evaluation of the oxygen consumption rate is possible by measuring the phosphorescence lifetime of the platinum porphyrin-fluoropolymer. The combined use of the device and an automated optical measurement system enabled the high-throughput sensing of cellular oxygen consumption (100 points/min). We monitored the oxygen metabolism of the human breast cancer cell line MCF7 on a Petri dish and evaluated the oxygen consumption rate to be 0.72 ± 0.12 fmol/min/cell. Furthermore, to demonstrate the utility of the developed sensing system, we demonstrated the mapping of the oxygen consumption rate of rat brain slices and succeeded in visualizing a clear difference among the layer structures of the hippocampus, i.e., the cornu ammonis (CA1 and CA3) and dentate gyrus (DG).