Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy Leonard J Nelson, Andreas Koulovasilopoulos, Philipp Treskes, Peter C Hayes, John N Plevris, Pierre O. Bagnaninchi College of Medicine and Veterinary Medicine, The University of Edinburgh, 49 Little France Crescent, EH16 4SB ABSTRACT Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100ȝl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models. KEYWORDS: Optical coherence tomography, Phase fluctuations, cell viability, toxicity screening, tissue engineering INTRODUCTION Conventional 2D human hepatocyte cell line (C3A) cultures for drug testing rapidly lose polarity and poorly represents normal liver tissue[1, 2]. Consequently, three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. Amongst them hydrogel encapsulation is a popular method. Given established endpoint toxicity assays are often incompatible with assessing 3D tissues, we investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell viability in response to acetaminophen hepatotoxicity. Optical coherence tomography (OCT) is an optical imaging modality based on low coherence interferometry [3, 4] with clinical applications in the field of ophthalmology [5]. OCT enables in depth non-invasive, real-time, and label-free imaging of highly scattering tissues, such as tendons[6], both in vitro and in vivo. Hence OCT is very well suited to non- invasive monitoring of in vitro engineered tissues throughout tissue development. Cell distribution, growth, and migration within bioscaffolds of various architectures and chemical compositions have been investigated with optical coherence tomography ([7-14]. However a limiting factor is that no clear contrast is generally found between the bioscaffold material and biological cells. Nolte and colleagues demonstrated that optical fluctuations arise from cellular and intracellular motility ([15-17] by imaging dense multicellular tumour spheroids with digital holographic optical coherence imaging, and that these fluctuations were correlated to cellular metabolism. Similarly, we developed a common-path optical coherence phase tomography system to record interferometric phase fluctuations associated with the motility of viable cells in 2D and 3D[18-20]. Dunkers et al. [21] also showed that Please verify that (1) all pages are present, (2) all figures are correct, (3) all fonts and special characters are correct, and (4) all text and figures fit within the red margin lines shown on this review document. Complete formatting information is available at http://SPIE.org/manuscripts Return to the Manage Active Submissions page at http://spie.org/submissions/tasks.aspx and approve or disapprove this submission. Your manuscript will not be published without this approval. Please contact author_help@spie.org with any questions or concerns. 9322 - 16 V. 1 (p.1 of 6) / Color: No / Format: A4 / Date: 1/12/2015 9:49:53 PM SPIE USE: ____ DB Check, ____ Prod Check, Notes: