Artefacts and lies: understanding the limitations of your biochemical assays when studying biomaterials

Abstract

Event Abstract Back to Event Artefacts and lies: understanding the limitations of your biochemical assays when studying biomaterials Marianne Ariganello1 1 Independent Consultant, Canada Introduction: The use multiplexing (collecting multiple endpoints from a single sample) has increased in a number of fields (such as pharmaceuticals and toxicology), however many biomaterial “cytotoxicity” assays are performed in the more traditional singleplex manner (one sample = one data point). In order to economically and effectively screen novel biomaterials for incompatibility, a more universal adoption of multiplex assays is required. This poster will highlight assays that can be multiplexed for the purposes of initial screening of biocompatibility and also highlight some of their pitfalls (or shortfalls). Using personal data and data available from published literature, this poster will additionally highlight some of the false positives that can occur with biomaterials and nanoparticles when appropriate controls are not included. The purpose of this poster is twofold: an opportunity for discussion of lessons learned (aka "negative results"), and a resource of contemporary cytotoxicity assays used for high-throughput biomaterial analysis. Materials and Methods: Multiple cell lines (Saos-2, MC-3T3, J774 and A549) were assessed with a variety of biochemical and functional assays. Human epithelial cells (A549) were exposed to a surrogate biomaterial (a commercially available carbon nanotube, CNT) and analyzed using multiple markers of viability: lactate dehydrogenase (LDH), AlamarBlueTM and CellToxTM Green. MC-3T3 and Saos-2 cells were evaluated for mineralization following exposure to various phosphate sources (including polyphosphate (PolyP17) using energy-dispersive spectroscopy (EDS), Alizarin Red and von Kossa staining[1]. Results and Discussion: More accurate interpretations of the biological effect of CNTs on cells were demonstrated only when multiple assays were done in parallel (Figure 1); observations based on only one assay (e.g. AlamarBlue alone), would have been misleading. Optimization of these assays was required because the presence of particulate matter (like CNTs or microparticles) was found to interfere with multiple spectrophotometric methods[2],[3]. In studies looking at mineralization involving a common biomaterial: polyphosphate, a false-positive Alizarin Red staining was identified only after both von Kossa and Alizarin Red staining were performed on parallel samples. Conclusions: Strategic multiplexing of assays would improve the analysis of biomaterial cytotoxicity screening. An added advantage is that this multiple methodology minimizes the use of potentially expensive or difficult to generate biomaterials while optimizing cell culture resources and time. However, when choosing a particular biochemical assay, care must be taken to precisely understand what the assay measures as well as its limitations. In this way, conclusions regarding the cytotoxicity of a biomaterial can be made efficiently and accurately, minimizing biomaterial development costs and health risks for humans.