Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system

Issue title: 40th Conference of the German Society for Clinical Microcirculation and Hemorheology, 5-6 November 2021, Senftenberg, Germany

Guest editors: J.-H. Küpper, A. Krüger-Genge and F. Jung

Article type: Research Article

Authors: Dittfeld, C.^{a; *} | Winkelkotte, M.^a | Behrens, S.^b | Schmieder, F.^b | Jannasch, A.^a | Matschke, K.^a | Sonntag, F.^b | Tugtekin, SM.^a

Affiliations: [a] Department of Cardiac Surgery, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Heart Centre Dresden, Dresden,Germany | [b] Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany

Correspondence: [*] Corresponding author: Dr. Claudia Dittfeld, Department of Cardiac Surgery, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Heart Centre Dresden, Dresden, Germany. E-mail: [email protected].

Abstract: BACKGROUND/AIM:Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS:A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50–300μM), incubation times and tissue masses and was consequently adapted. RESULTS:Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9–13.4μl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300μM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS:Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Keywords: Aortic valve stenosis, dynamic tissue culture, tissue viability, resazurin reduction, microphysiological system, human aortic valve

DOI: 10.3233/CH-219112

Journal: Clinical Hemorheology and Microcirculation, vol. 79, no. 1, pp. 167-178, 2021