Isolation of Biopsy-Derived, Human Cervical Keratinocytes Propagated as Monolayer and Organoid Cultures

Abstract

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.

Combined Transcriptome and Proteome Analysis of Immortalized Human Keratinocytes Expressing Human Papillomavirus 16 (HPV16) Oncogenes Reveals Novel Key Factors and Networks in HPV-Induced Carcinogenesis

“…We intentionally chose immortalized cells over primary cells, allowing a certain number of cell doublings to guarantee sufficient amino acid incorporation for stable isotope labeling by amino acids in cell culture (SILAC). Furthermore, the use of immortalized cells also permits an unlimited number of follow-up experiments in the HPV-negative parental cell line without bias being created by differentiation of primary cells or by variations among different donors (30, 31). Using this in vitro model, transcriptome and quantitative proteome data were combined for the first time to identify novel regulators that putatively contribute to host cell transformation.…”

From Donor to the Lab: A Fascinating Journey of Primary Cell Lines

“…In that case, cell survival and tissue dissociations were the highest and the most satisfying (Volovitz et al, 2016). Isolation of primary cancer cells with dispase is also an effective procedure in prostate, ovarian and cervical cancer (Pribyl et al, 2014;Villa et al, 2018;Wang et al, 2019). The use of dispase enables the effective recovery of viable, fibroblast-free epithelial ovarian cancer cells (EOC).…”

An epithelial organoid model with Langerhans cells for assessing virus-host interactions

“…We have tried near-diploid immortalized keratinocytes (NIKS, [55]), primary human foreskin/epidermal keratinocytes (PHFKs or HEKs) and primary human oral/gingival keratinocytes (HGKs). Also, we have generated epithelial organoids from patient-derived cervical biopsies (via suspected lesions at colposcopy, [34]). Primary cervical cells (both keratinocytes and fibroblasts from the uterine cervix) are now commercially available.…”

“…We will employ healthy mucosal or skin tissue including epidermis and dermis either from the uterine cervix (non-keratinizing, cervical keratinocytes), the oropharyngeal area (non-keratinizing, gingival keratinocytes) or the skin (keratinizing epithelial keratinocytes) with matching fibroblasts. Fortunately, these cells can now be bought, avoiding the barrier of lengthy procurement processes [34]. Epidermis on its own or combined with dermis models are also commercially available.…”

“…So long as the research question is focused on the difference between the viral variants, a simple yet effective design would be to replicate the experiment with independent transfections using the same donor pool of cells (controlling this background if possible, to some extent, given inherent variability in passaging cells over time). If the research question was broader, by including donor/host variability, then the level of interrogation would correspondingly be at the host-level, requiring unique donors such as via patient-derived samples [34]. Once the experimental design has been established, the next stage of the approach is to perform material calculations and establish the organoid cultivation time-course.…”