A novel microfluidic platform for high-resolution imaging of a three-dimensional cell culture under a controlled hypoxic environment

Low oxygen tensions experienced in various pathological and physiological conditions are a major stimulus for angiogenesis. Hypoxic conditions play a critical role in regulating cellular behaviour including migration, proliferation and differentiation. This study introduces the use of a microfluidic device that allows for the control of oxygen tension for the study of different three-dimensional (3D) cell cultures for various applications. The device has a central 3D gel region acting as an external cellular matrix, flanked by media channels. On each side, there is a peripheral gas channel through which suitable gas mixtures are supplied to establish a uniform oxygen tension or gradient within the device. The effects of various parameters, such as gas and media flow rates, device thickness, and diffusion coefficients of oxygen were examined using numerical simulations to determine the characteristics of the microfluidic device. A polycarbonate (PC) film with a low oxygen diffusion coefficient was embedded in the device in proximity above the channels to prevent oxygen diffusion from the incubator environment into the polydimethylsiloxane (PDMS) device. The oxygen tension in the device was then validated experimentally using a ruthenium-coated (Ru-coated) oxygen-sensing glass cover slip which confirmed the establishment of low uniform oxygen tensions (<3%) or an oxygen gradient across the gel region. To demonstrate the utility of the microfluidic device for cellular experiments under hypoxic conditions, migratory studies of MDA-MB-231 human breast cancer cells were performed. The microfluidic device allowed for imaging cellular migration with high-resolution, exhibiting an enhanced migration in hypoxia in comparison to normoxia. This microfluidic device presents itself as a promising platform for the investigation of cellular behaviour in a 3D gel scaffold under varying hypoxic conditions

Stiffness of primordial germ cells is required for their extravasation in avian embryos

細胞の血行性転移の新たな仕組みを発見 --世界初、新たながん転移抑止戦略の開発にも期待--. 京都大学プレスリリース. 2022-12-13.Unlike mammals, primordial germ cells (PGCs) in avian early embryos exploit blood circulation to translocate to the somatic gonadal primordium, but how circulating PGCs undergo extravasation remains elusive. We demonstrate with single-cell level live-imaging analyses that the PGCs are arrested at a specific site in the capillary plexus, which is predominantly governed by occlusion at a narrow path in the vasculature. The occlusion is enabled by a heightened stiffness of the PGCs mediated by actin polymerization. Following the occlusion, PGCs reset their stiffness to soften in order to squeeze through the endothelial lining as they transmigrate. Our discovery also provides a model for the understanding of metastasizing cancer extravasation occurring mainly by occlusion

Oxygen-dependent contraction and degradation of the extracellular matrix mediated by interaction between tumor and endothelial cells

Understanding the mechanisms of cancer development and metastasis requires comprehensive analyses of interactions between normal and tumor cells and the extracellular matrix (ECM) in hypoxic tumor microenvironments. However, the scope of many tumor microenvironment studies is limited to verifying the development and performance of microenvironment-producing cell culture platforms. This study describes the effects of co-culture and hypoxia on contraction and degradation of the ECM. Collagen type I gel was placed in the gel channel of an oxygen tension–controllable microfluidic device as a tumor tissue substrate. MDA-MB-231 human breast cancer cells and/or human umbilical vein endothelial cells (HUVECs) were cultured inside the gel or on the adjacent media channels, respectively. Changes in the collagen gel were evaluated by generating normoxic (21% O2) or hypoxic (1% O2) conditions via variation of the supply of gas mixture. HUVECs induced collagen gel contraction and degradation more strongly than MDA-MB-231 cells. Although interaction between co-cultured MDA-MB-231 cells and HUVECs promoted gel contraction and degradation, hypoxia attenuated the effect. Immunofluorescence staining indicated decreased expression of secretory matrix metalloproteinase-7 (MMP-7) inside the collagen gel under hypoxic conditions, but no morphologic changes in cells were observed. Separate Western blot analyses using cells cultured on cell culture dishes confirmed reduced endogenous MMP expression in hypoxia-exposed HUVECs. These results demonstrate that hypoxic conditions affect collagen gel contraction and degradation by altering the expression of MMP-7 in co-cultured MDA-MB-231 cells and HUVECs

Ultrasound Imaging of Mouse Fetal Intracranial Hemorrhage Due to Ischemia/Reperfusion

Despite vast improvement in perinatal care during the 30 years, the incidence rate of neonatal encephalopathy remains unchanged without any further Progress towards preventive strategies for the clinical impasse. Antenatal brain injury including fetal intracranial hemorrhage caused by ischemia/reperfusion is known as one of the primary triggers of neonatal injury. However, the mechanisms of antenatal brain injury are poorly understood unless better predictive models of the disease are developed. Here we show a mouse model for fetal intracranial hemorrhage in vivo developed to investigate the actual timing of hypoxia-ischemic events and their related mechanisms of injury. Intrauterine growth restriction mouse fetuses were exposed to ischemia/reperfusion cycles by occluding and opening the uterine and ovarian arteries in the mother. The presence and timing of fetal intracranial hemorrhage caused by the ischemia/reperfusion were measured with histological observation and ultrasound imaging. Protein-restricted diet increased the risk of fetal intracranial hemorrhage. The monitoring of fetal brains by ultrasound B-mode imaging clarified that cerebral hemorrhage in the fetal brain occurred after the second ischemic period. Three-dimensional ultrasound power Doppler imaging visualized the disappearance of main blood flows in the fetal brain. These indicate a breakdown of cerebrovascular autoregulation which causes the fetal intracranial hemorrhage. This study supports the fact that the ischemia/reperfusion triggers cerebral hemorrhage in the fetal brain. The present method enables us to noninvasively create the cerebral hemorrhage in a fetus without directly touching the body but with repeated occlusion and opening of the uterine and ovarian arteries in the mother