Pandemic-ready organ-chips: Engineering vessel-chips for service in biosafety level 3 facilities

Introduction:: Over the last decade, bioengineered experimental platforms, such as, microphysiological systems (MPS), have shown a remarkable potential as disease models and drug discovery tools, reducing our requirement of using animals, that can also be poor in physiological-relevance. But when COVID-19 struck and there was an urgent need to understand the mechanisms of the disease and discover therapeutic options, the full potential of these systems could not be immediately come to much use widely. This was so because SARS-Cov-2 virus requires a biosafety level 3 (BSL3) facility that has significant containment and operational requirements, and while MPS are routinely used in BSL2 labs, these devices cannot be translated to BSL3 as is. In fact, there is a significant need for new technologies to translate MPS and similar technologies for applications in studying human pathogens, hazardous materials, and others requiring special research environments. For example, the need to integrate MPS with pumps that can provide continuous perfusion and mimic hemodynamics presents a considerable set of challenges in BSL-3. Further, users must be able to perfuse fluids and subsequently introduce pathogen-containing solutions without requiring any substantial dexterity or exposure of fluid to atmosphere (creating aerosol risk). While COVID-19 often results in vasculopathy and thrombosis, and there are no animal models to reproduce COVID-19 induced vascular disease, we have created a BSL-3 ready vessel-chip platform that enables us to introduce the living SARS-Cov-2 virus in these systems, which then can potentially infect the vascular cells, replicate and recreate the clinically observed endotheliopathy and thrombosis.


Materials and Methods:: To address the challenges associated with studying human physiology and testing drugs in hazardous containment environments, we have developed a platform integrating four key components. First, a pumping system has been constructed using extrusion 3D-printing and basic machining consisting of plastic (polylactic acid, PLA), high-strength steel, and inexpensive polyethylene parts. This pump is controlled by an Arduino microcontroller with a PID control function fed by a flow sensor on the MPS outlet. Second, dual automated three-way valves were created using commercially available plastic valves and a 3D-printed housing and control unit connected to and controlled by a microcontroller programmed by a basic button-press function. Third, a MPS vascular tissue model (which can easily be exchanged for other models), fabricated using methods described previously, is interfaced directly with the fluid handling elements for reliable control of fluid perfusion. This system can include human endothelial cells from a range of sources, including primary cells and stem-cell derived cell lines, to model various sections of the human cardiovascular system for research at various scales ranging from pooled experiments for high-level uniformity to single patient resolution for population representation or treatment pre-screening. Finally, a commercially available collection reservoir sealed with an air-permeable membrane is included on the immediate outlet of the MPS device to allow for cell or lysate extraction with sufficient sample concentration for downstream genomics, transcriptomics, and proteomics assays. All components are linked by biocompatible Tygon tubing and tight-fit luer connectors to maintain a completely sealed system.


Results, Conclusions, and Discussions:: The pumping apparatus is designed with a footprint compatible with space requirements of standard incubators. The three-way valves and tubing connectors are contained in the 3D-printed housing unit controlled using a microcontroller, which can be maintained outside of the incubator. Multiple automated valve units can be arranged in parallel and synchronized on the microcontroller. A sealed injection port for pathogen sample introduction and a waste stream connected to a sealed reservoir are connected to the three-way valves. The compiled BSL-3 MPS deployment system operates using three sequential steps based on valve position and pump operation (Fig. 1). First, the dual three-way valves are positioned such that fluid (media or biological fluid) perfusion can progress from the pump directly to the MPS while the sample injection line and waste line are closed. Second, by a button-press, the user can switch the positions of both valves to close the perfusion line and MPS access to prevent any disturbances of cells on the platform by inadvertent pressure waves. With the valves in this position, the user can introduce the viral sample by connecting a standard syringe to the injection port and pushing the plunger. The fluid containing the test sample will displace the fluid between the three-way valves pushing it into the waste container (note that tubing lengths are dictated by the volume of the test sample allowing for precise loading of the system). Finally, the valves are switched back to their original positions, the pump is activated again, and the test sample is pumped to the MPS system (with a small, easily calculable, delay). The pumping instrumentation first offers a unique ability to match the physiological flow patterns known to be vital in modeling vascular diseases. The injection port remains sealed until the moment of sample introduction while the waste container is sealed by an air-permeable membrane thereby preventing any chance of fluid or aerosol exposure. Ultimately, this system represents a valuable tool for the future implementation of MPSs in controlled environments for standard research activities, rapid deployment needs in response to crises, and large-scale drug and therapeutics testing.

Acknowledgements (Optional): : This material is based upon work partially supported by the NASA, BARDA, NIH, and USFDA, under Contract No. 80ARC023CA002; NHLBI of NIH under Award Number R01HL157790, NSF CAREER Award number 1944322, and Texas A&M University President’s Excellence in Research Award (X-Grant) to A.J.

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