3D Oral and Cervical Tissue Models for Studying Papillomavirus Host-Pathogen Interactions.

Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Establishing human primary cell-derived 3D organotypic raft cultures Support Protocol 1: Isolation of epithelial cells from patient-derived tissues Support Protocol 2: Growth and maintenance of primary human epithelial cells in monolayer culture Support Protocol 3: PCR-based HPV screening of primary cell cultures Basic Protocol 2: Establishing human 3D cervical tissues using the rotating wall vessel bioreactor Support Protocol 4: Growth and maintenance of human A2EN cells in monolayer culture Support Protocol 5: Preparation of the slow-turning lateral vessel bioreactor Support Protocol 6: Preparation of Cytodex-3 microcarrier beads Basic Protocol 3: Histological assessment of 3D organotypic raft tissues Basic Protocol 4: Spatial analysis of protein expression in 3D organotypic raft cultures Basic Protocol 5: Immunofluorescence imaging of RWV-derived 3D tissues Basic Protocol 6: Ultrastructural visualization and imaging of RWV-derived 3D tissues Basic Protocol 7: Characterization of gene expression by RT-qPCR.

Jackson R ，Maarsingh JD ，Herbst-Kralovetz MM ，Van Doorslaer K ... -  《-》

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

Radtke AL ，Herbst-Kralovetz MM 《Jove-Journal of Visualized Experiments》

Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

Ozbun MA ，Patterson NA 《-》

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions.

Gardner JK ，Herbst-Kralovetz MM 《Viruses-Basel》

Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle.

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.

Lee D ，Norby K ，Hayes M ，Chiu YF ，Sugden B ，Lambert PF ... -  《-》