Development of a sensitive cell culture system to assess prion infectivity and the efficacy of prion decontamination technologies

Abstract

Creutzfeldt-Jakob disease (CJD) can be iatrogenically transmitted during transplants, grafts and transfusions from CJD infected donors and also contaminated surgical instruments. A variety of methods to amplify and detect the presence of infectious prions as disease markers are available. However, these techniques do not measure the infectivity potentially associated with these markers. Currently, animal-bioassays are used to detect infectivity; however, they have limitations in detectable prion strains, cost, ethical considerations and assay length. Novel cell-based infectivity assays offer the potential to overcome these limitations, lower the requirements for animal use and the application of different cell lines could detect a wider range of prion strains. This project utilised murine neuroblastoma, N2a #58 cells infected with 22L-murine scrapie to develop a highly sensitive assay for the in situ detection of amyloid-rich prion (PrPSc) accumulation as an indication of prion infectivity. The autofluorescence quenching properties of Sudan black (SB) were incorporated into a novel Thioflavin T (ThT) based protocol for amyloid staining with improved specificity and sensitivity. Cell passages were incorporated into the assay to increase incubation time, improve cell viability and subsequently improve assay sensitivity; thus, demonstrating the detection of infectivity from a final 10-10 dilution of 22L-infected brain homogenate. Introduction of 22L-inoculated, surgical grade stainless steel wires to the N2a #58 cells demonstrated the SB/ThT detection of prion infectivity pre and post decontamination, which was comparable to animal bioassay data. Furthermore, preliminary work on the incorporation of the SB/ThT detection of prion infectivity within neural stem cells (NSC’s), for prion propagation within a cell line that did not require genetic manipulation for increased prion susceptibility, highlighted problems with unspecific fluorescence of dead cells during NSC differentiation. Improvements in culture conditions of the NSC’s regarding atmospheric conditions and trophic support were addressed in preparation for their use in future prion infectivity assays.

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Identifiers

ORCID for Thomas Secker: orcid.org/0000-0001-7168-8592

ORCID for Charles W. Keevil: orcid.org/0000-0003-1917-7706

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