Three-dimensional Models of the Nasopharynx for the Study of Epstein-Barr Virus Infection

Phillip Ziegler; Alex S Reznik; Shweta P Kitchloo; Eric Wang; Stella E Lee; Anthony Green; Michael M Myerburg; Clare E Sample; Kathy Ho Yen Shair

doi:10.21769/BioProtoc.4365

Three-dimensional Models of the Nasopharynx for the Study of Epstein-Barr Virus Infection

Bio Protoc. 2022 Mar 20;12(6):e4365. doi: 10.21769/BioProtoc.4365.

Authors

Phillip Ziegler¹, Alex S Reznik¹, Shweta P Kitchloo¹, Eric Wang², Stella E Lee², Anthony Green³, Michael M Myerburg⁴, Clare E Sample⁵, Kathy Ho Yen Shair⁶

Affiliations

¹ University of Pittsburgh Medical Center (UPMC) Cancer Virology Program, Pittsburgh, PA, United States.
² UPMC Department of Otolaryngology, University of Pittsburgh, Pittsburgh, PA, United States.
³ University of Pittsburgh Research Histology Services, University of Pittsburgh, Pittsburgh, PA, United States.
⁴ Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
⁵ The Penn State Hershey Cancer Institute, Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, United States.
⁶ Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, United States.

Abstract

The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods ( Ziegler et al., 2021 ). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures. Graphic abstract: Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

Keywords: 3-D model; Air-liquid interface (ALI); Epstein-Barr virus (EBV); Nasopharyngeal epithelium; Nasopharynx; Organotypic rafts; Pseudostratified.