Screening Technologies for Emerging Pathogens
Above: Lung microtissue stained for live cells (green) and dead cell nuclei (red).
Creation of a Lung Microtissue Bank
The novel SARS-CoV-2 zoonotic pathogen and its rapid spread exposed the need for accurate human tissue models capable of quantifying host-pathogen interactions, pathogenesis, disease progression, immune response, and screening of potential therapeutics. In March 2020 we began addressing this unmet need by developing microtissue lung models collected from properly consented donors and performed SARS-CoV-2 infection and drug studies in a collaborating Biosafety Level 3 (BSL-3) facility using Darcy perfusion culture devices.
Left: Lung microtissue stained for live cells (green) and dead cell nuclei (red).
Above: Infected human lung microtissue stained for SARS-CoV-2 (green), actin (red), and nuclei (blue).
In Vitro SARS-CoV-2 Infection and Pathogenesis
Microtissues were mechanically disaggregated to a characteristic size approximating 500 micrometers. Liquid-Like Solid (LLS™) 3D culture medium was used to support the delicate alveolar structure, cellular heterogeneity (including resident immune cells), and enable perfusion through its permeable network of culture media supplemented with SARS-CoV-2 at stock concentrations of 10²-10⁴ Plaque Forming Units (PFU) per mL in Pneumacult media. Cultures were performed in hyperoxic conditions (>90% O₂ partial pressure). Microtissues were readily infected over 48 hours and demonstrated donor-dependent host response.
Right: Infected human lung microtissue stained for SARS-CoV-2 (green), actin (red), and nuclei (blue).
Above: Infection and host response of microtissues (Top), Chloroquine and Metformin inhibition of viral RNA was anti-correlated between microtissue and monolayer culture using VERO-E6 cells (bottom).
Evaluating Drug Response In Situ
Infected samples were treated in situ via perfusion using chloroquine, β-d-N4-hydroxycytidine, water-soluble dexamethasone, metformin, remdesivir, and sirolimus to investigate efficacy and reductions in viral replication. Flow cytometry characterization of the uninfected microtissues was performed using a FACSAria™ II and analyzed using FlowJo™ X. In contrast to VERO-E6 cells cultured in monolayer the lung microtissue models showed no response to chloroquine and remdesivir but did show responses to metformin and sirolimus in some samples suggesting suppression of viral growth. SARS-CoV-2infection led to apoptosis, actin depolymerization, and the formation of SARS-CoV-2-rich blebbing captured within the pleural effusion fluid implicating a transmission mode associated with the epithelial mucinated network. 10-100X increases in pro-inflammatory IL6 cytokine secretion illustrate the inflammatory hallmark of COVID-19.
Left: Infection and host response of microtissues (Top), Chloroquine and Metformin inhibition of viral RNA was anti-correlated between microtissue and monolayer culture using VERO-E6 cells (bottom).
Impact
The 3D human lung microtissue models provided timely data during the COVID-19 pandemic, including: infection via the novel pathogen SARS-CoV-2, inflammatory host responses, lead candidates for therapeutics, and importantly demonstrated an anti-correlation with 2D cell culture studies.
