Co‐Culture Systems to Study Epithelial‐Immune Interactions During SARS‐CoV‐2 Infection

Robust inflammatory responses to viral infection are mediated by immune cell populations, including monocytes and dendritic cells. However, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) does not replicate efficiently in these cell types and instead preferentially infects epithelial cell subsets in the airway. Because of this, stimulation of inflammatory cytokine responses from immune cell populations during SARS-CoV-2 infection depends not only on interactions with viral particles but also interactions with infected epithelial cells. In this article, we describe two co-culture systems to study inflammatory cytokine responses generated by epithelial-immune interaction during SARS-CoV-2 infection in vitro. Basic Protocol 1 describes how to set up a partially primary co-culture system consisting of SARS-CoV-2-infected Vero-E6 cells and primary human peripheral blood mononuclear cells (PBMCs) to observe release of the inflammasome-regulated cytokine interleukin-1β (IL-1β). Basic Protocol 2 details a primary, human co-culture system that consists of SARS-CoV-2-infected primary human airway epithelia (HAE) grown at an air-liquid interface (ALI) and primary human PBMCs, and how to observe IL-1β and IL-6 crosstalk between these cell populations during infection. In these Basic Protocols, we include a description of the use of inhibitors in these systems to perturb cytokine responses. We also provide Support Protocols for the culture of HAE and Vero-E6 and for the isolation, storage, and preparation of PBMCs prior to use in these systems. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: SARS-CoV-2 infection in a Vero-E6+PBMC co-culture system Support Protocol 1: Vero-E6 culture and passaging Support Protocol 2: Isolation and cryopreservation of PBMCs for use in co-culture Basic Protocol 2: SARS-CoV-2 infection in a primary HAE+PBMC co-culture system Support Protocol 3: Establishment and maturation of HAE grown at an ALI.