(H-293) Portable and Remotely Monitored Smart Hypoxia Chamber for invitro Studies of Peripheral Artery Disease and Therapeutic Testing

Introduction::
Peripheral artery disease (PAD) is a progressive, low blood perfusion condition caused by atherosclerosis in the peripheral arteries, especially in lower limbs affecting approximating 8.5 million Americans, yet undiagnosed until critical stages. The disease can progress to critical limb ischemia, which leads to high morbidity and mortality rates. Currently researchers in PAD use the range 1-5% O₂ (hypoxia) for in vitro models. There arises a need for extensive research to establish reliable in vitro hypoxic models, to accurately mimic the hypoxic environment in ischemic conditions due to narrowed or blocked arteries.The endothelial progenitor cells (EPCs) used in this study are a bone marrow–derived progenitor cell subtype and are involved in neovasculogenesis and maintain vascular integrity. Additionally, we studied the effects of Erythropoietin (Epo), which is significantly known to stimulate proliferation, migration, and protection of endothelial cells from hypoxic environments and aid to promote vascularization. Herein, we propose our smart, cost-effective hypoxia chamber as a platform to precisely induce a wide range of experimental O₂ conditions for ischemic hypoxia studies in vitro in endothelial progenitor cells (EPCs) and further extend its application for in vitro therapeutic study of Epo loaded nanoparticle system (ENPs) in PAD.




Materials and Methods::
The precision and robustness of our novel hypoxia chamber to maintain the set O₂ levels was established and validated by the stability studies performed at 1%, 2%, 3%, 4%, and 5 % O₂, over a 48-hour time duration. The system was controlled by real-time O₂ sensing within the chamber using an O₂ sensor (SST Sensing Ltd.) backed by a feedback loop system (Arduino and Raspberry-pi). Remote online monitoring (web server) was established using Dash Framework based on Python to stream the real-time sensing data over the Internet so that multiple computing devices can access the data visually. As proof of concept, the system was first applied to investigate the HIF1- α expression on A549 cells. We then used our chamber to create the in vitro hypoxia microenvironment to study the therapeutic effects of ENPs on EPCsusing our novel hypoxia chamber under 1% and 5% O₂ at 4 hours (acute hypoxia), 24 hours (chronic hypoxia), and extended hypoxia ( >24 hours) for in vitro studies. HIF1- α were quantified using qRT-PCR to confirm the successful hypoxic environment induction. Cell protection, cell proliferation, cell migration and tube formation studies were performed to study the therapeutic outcomes of ENPs.




Results, Conclusions, and Discussions::
The 48-hour stability studies for our smart hypoxia chamber showed a significantly low error rate of 0.2% and real-time O₂ percent in the chamber was visualized remotely (Figure 1). Our chamber in a preliminary study successfully induced a hypoxia condition in vitro study with a high fold change of a 1.9-fold increase in HIF-1α expression compared to the uncontrolled conventional system (0.6-fold change). ENPs formulated were characterized and found to be 164±69 nm with a Polydispersity Index of 0.170. Further studies will be performed under varying, 5% and 1% O₂ conditions with time dependent studies on EPCs to investigate the in vitro therapeutic effects of ENPs (Figure 1).
Our novel hypoxia chamber is designed to control a wide range of O₂ concentrations accurately and precisely in real-time to aid in an extensive study of in vitro hypoxic models. It provides a controlled environment to closely mimic in vivo conditions in ischemic tissues. This study with our novel chamber will therefore provide a multifaceted and robust platform for extensive study of PAD in vitro models and a potential for therapeutic screening in treating various ischemic diseases under precise microenvironment conditions with continuous and remote monitoring systems.




Acknowledgements (Optional): :

References (Optional): :