Ozbun Michelle A, Patterson Nicole A
Department of Molecular Genetics and Microbiology, The University of New Mexico School of Medicine, The UNM Cancer Center, Albuquerque, New Mexico.
Curr Protoc Microbiol. 2014 Aug 1;34:14B.3.1-18. doi: 10.1002/9780471729259.mc14b03s34.
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.
乳头瘤病毒对上皮细胞具有严格的嗜性，并且它们完全依赖细胞分化来完成其生命周期，从而产生子代病毒颗粒。因此，体外病毒完全复制的许可环境（其中病毒体形态发生在病毒和细胞的协同作用下发生）需要培养上皮组织。本单元第一部分介绍了一种培养分化上皮组织的方案，该组织模拟了正常皮肤的许多重要形态和生化特征。该技术包括在由活成纤维细胞和胶原晶格组成的真皮替代物上培养表皮细胞。当角质形成细胞 - 真皮替代物置于气液界面时，上皮分层和分化随之发生。这种方法中细胞 - 基质明显的漂浮性质导致了“筏式”培养的昵称。该一般技术可应用于正常低传代角质形成细胞、用乳头瘤病毒基因或基因组稳定转染的细胞或从肿瘤病变建立的角质形成细胞。然而，仅从用维持致癌性人乳头瘤病毒基因组处于染色体外复制形式的细胞起始的器官型上皮培养物中分离出感染性乳头瘤病毒颗粒。本单元第二部分致力于一种病毒体分离方法，该方法可最大限度地减少气溶胶和皮肤接触这些人类致癌物。尽管方案的重点是培养产生感染性乳头瘤病毒子代的组织，但这种培养系统有助于在这些挑剔病毒的复杂复制周期中对其进行研究，并且筏式组织可以在过程中的任何时间点进行操作和收获。重要的是，在此过程中实现了单步病毒生长周期，因为子代病毒颗粒不太可能释放以引发后续轮次的感染。
