Study 3D Endothelial Cell Network Formation under Various Oxygen Microenvironment and Hydrogel Composition Combinations Using Upside‐Down Microfluidic Devices

Formation of 3D networks is a crucial process for endothelial cells during development of primary blood vessels under both normal and pathological conditions. In order to investigate effects of oxygen microenvironment and matrix composition on the 3D network formation, an upside‐down microfluidic cell culture device capable of generating oxygen gradients is developed in this paper. In cell experiments, network formation of human umbilical vein endothelial cells (HUVECs) within fibrinogen‐based hydrogels with different concentrations of hyaluronic acid (HA) is systematically studied. In addition, five different oxygen microenvironments (uniform normoxia, 5%, and 1% O2; oxygen gradients under normoxia and 5% O2) are also applied for the cell culture. The generated oxygen gradients are characterized based on fluorescence lifetime measurements. The experimental results show increased 3D cell network length when the cells are cultured under the oxygen gradients within the hydrogels with the HA addition suggesting their roles in promoting network formation. Furthermore, the formed networks tend to align along the direction of the oxygen gradients indicating the presence of gradient‐driven cellular response. The results demonstrate that the developed upside‐down microfluidic device can provide an advanced platform to investigate 3D cell culture under the controlled oxygen microenvironments for various biomedical studies in vitro. An upside‐down microfluidic device capable of generating oxygen gradients for cell culture within hydrogels is developed to study 3D network formation of endothelial cells under various combinations of oxygen microenvironments and extra cellular matrix compositions. The device provides a powerful and straightforward platform to perform 3D cell culture under controlled microenvironments for various biomedical researches in vitro.