Akiko Nakamasu

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

Engineering a three-dimensional tissue model with a perfusable vasculature in a microfluidic device

In this study, we developed a microfluidic platform for a three-dimensional tissue model with a perfusable capillary network, which will allow, for the first time, a perfusion-culture in a tissue model with a high cell density. Our group previously reported that a spheroid of lung fibroblasts induced angiogenic sprouts from microchannels [1]. In this study, we successfully connected angiogenic sprouts to the vessel-like hollow structure in a spheroid and perfused the formed vascular network through microfluidic channels to the spheroid. This model opens up new techniques for tissue-culture for long-term.

Development of three-dimensional tumor model with a perfusable vasculature using a microfluidic device

disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant embryos from 42 lethal and subviable lines, analysed at E14.5. Virtually all embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve. The most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.

Reconstitutive analyses of impacts of pericytes and blood flow on angiogenic morphogenesis using a microfluidic device

During palatogenesis, the anterior palate is covered with ortho or parakeratinized epithelium while the posterior palate is covered with non-keratinized epithelium. To elucidate the developmental mechanisms underlying these region-specific differentiation patterns of palatal epithelium along the antero-posterior axis, we employed the tissue recombination assay during in vitro organ cultivation of the developing palate at E16 for 2 days. The recombination assay results revealed that epithelial differentiation with specific localization patterns of Cytokeratin10 and Ki67 are modulated by mesenchymal tissues. Based on these results, we examined the underlying signaling regulations that modulate epithelial differentiation, using laser microdissection and genome wide screening. Our screening data suggested Meox2 (Mesenchyme homeobox 2) to be a key regulator that controls epithelial differentiation in the mesenchymal tissue. To examine the developmental function of Meox2, we employed in vitro organ cultivation along with the knockdown and overexpression of Meox2 using siRNA andMeox2 overexpression vector, respectively, at E14.5 for 2 and 4 days. After 2-day cultivation, we examined the altered expression patterns of related signaling molecules such as Shh and Bmp, using in situ hybridization and RT-qPCR. After 4-day cultivation, we examined the altered histogenesis and localization patterns of Cytokeratin10 and Ki67. Based on the restricted and specific expression of candidate mesenchymal genes and the results of the recombination assay, we conclude that posteriorly expressed Meox2 is involved in the determination of non-keratinized epithelial differentiation through complex signaling regulations in mice palatogenesis.