One contribution of 16 to a theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.Persistent infection with oncogenic human papillomavirus (HPV) may lead to cancer in mucosal and skin tissue. Consequently, HPV must have developed strategies to escape host immune surveillance. Nevertheless, most HPV infections are cleared by the infected host. Our laboratory investigates Langerhans cells (LCs), acting at the interface between innate and adaptive immunity. We hypothesize that this first line of defence is vital for potential HPV elimination. As an alternative to animal models, we use smaller-scale epithelial organoids grown from human primary keratinocytes derived from various anatomical sites. This approach is amenable to large sample sizes-an essential aspect for scientific rigour and statistical power. To evaluate LCs phenotypically and molecularly during the viral life cycle and onset of carcinogenesis, we have included an engineered myeloid cell line with the ability to acquire an LC phenotype. This model is accurately tailored for the crucial time-window of early virus elimination in a complex organism and will shed more light on our long-standing research question of how naturally occurring HPV variants influence disease development. It may also be applied to other microorganism -host interaction research or enquiries of epithelium immunobiology. Finally, our continuously updated pathogen -host analysis tool enables state-of-the-art bioinformatics analyses of next-generation sequencing data.This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20180288
The successful isolation and propagation of patient-derived keratinocytes from cervical lesions constitute a more appropriate model of cervical disease than traditional cervical cancer-derived cell lines such as SiHa and CaSki. Our aim was to streamline the growth of patient-obtained, cervical keratinocytes into a reproducible process. We performed an observational case series study with 60 women referred to colposcopy for a diagnostic biopsy. Main outcome measures were how many samples could be passaged at least once (n = 11), and where enough cells could be established, to precisely define their proliferation profile over time (n = 3). Altering cell culture conditions over those reported by other groups markedly improved outcomes. We were also successful in making freeze backs which could be resuscitated to successfully propagate multi-layered, organoids from cervical keratinocytes (n = 3). For best results, biopsy-intrinsic factors such as size and tissue digestion appear to be major variables. This seems to be the first systematic report with a well characterized and defined sample size, detailed protocol, and carefully assessed cell yield and performance. This research is particularly impactful for constituting a sample repository-on-demand for appropriate disease modelling and drug screening under the umbrella of personalized health.
The successful isolation and propagation of patient-derived keratinocytes from cervical lesions constitute a more appropriate model of cervical disease than traditional cervical cancer-derived cell lines such as SiHa and CaSki. Our aim was to streamline the growth of patient-obtained, cervical keratinocytes into a reproducible process. We performed an observational case series study with 60 women referred to colposcopy for a diagnostic biopsy. Main outcome measures were how many samples could be passaged at least once, and where enough cells could be established, to precisely define their proliferation profile over time. Altering cell culture conditions over those reported by other groups markedly improved outcomes. We were also successful in making freeze backs which could be resuscitated for additional experiments. For best results, biopsy-intrinsic factors such as size and tissue digestion appear to be major variables. This seems to be the first systematic report with a well characterized and defined sample size, detailed protocol, carefully assessed cell yield and performance, and to successfully grow multi-layered, organoid cultures from cervical keratinocytes. This research is particularly impactful for constituting a sample repository-on-demand for appropriate disease modelling and drug screening under the umbrella of personalized health.
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