Aortic Valve Tissue Viability In A Dynamic Pulsatile Microphysiological System - Impact Of Assay Principle And Performance
Maximilian Winkelkotte1, Stephan Behrens2, Florian Schmieder2, Anett Jannasch1, Klaus Matschke1, Frank Sonntag2, Sems-Malte Tugtekin1, Claudia Dittfeld1.
1Carl Gustav Carus Faculty od Medicine, Technische Universität Dresden, Department of Cardiac Surgery, Heart Centre Dresden, Dresden, Germany, 2Fraunhofer Institute of Materials and Beam Technology IWS, Dresden, Germany.
OBJECTIVE: Since current research models insufficiently reflect (patho)physiology of aortic valve (AV) stenosis, focus remains model adaptation and advancement. Dynamic micro-physiological systems (MPS) and tissue incubation chambers (TIC) are designed to investigate (patho)physiological processes with best possible approximation of particular in vivo conditions. Tissue viability is one basic requirement for valid results followed by the maintenance of cell differentiation status and ECM composition. Aim of the presented work is the verification of resazurin-based AV tissue viability detected in culture medium via the proof of LDH-activity in AV tissue cryocuts itself.
METHODS: Porcine AV tissue specimen of 3x5 mm2 were cultured in the TIC-MPS for 14 days within a conventional cell incubator under sterile conditions (n=6). Pulsatile flow rate of 13.4 µl/s (150 bpm) generating average shear forces of 0.017 dyn/cm2 was applied. Resazurin (300 µM; culture medium) reduction was monitored every second day. After 14 days AV tissues were snap frozen, cryo-sectioned and intracellular LDH activity was visualized using a Tetrazolium-based dye and DAPI-counterstaining of cell nuclei. Static controls were incubated in conventional cell culture plates.
RESULTS: Resazurin reduction in dynamic MPS-TIC culture was with 47.6 ± 12.8 % significantly reduced on day 12. On day 14 of incubation only 43.9 ± 10.5 % viability remained. Resazurin assay based viability of static AV tissue culture was significantly reduced to 51.9 ± 16.8 % at day 10 and lowered to 37.4 ± 10.9 % (day 14). In contrast, 14d end point analysis of equivalent setup revealed no significant LDH-viability stain reduction of viable tissue sections. Extended incubation periods are envisioned.
CONCLUSIONS: AV tissue viability testing to determine efficiency of dynamic vs. static culture depends on assay system and detected enzyme activity. LDH-viability stain offers the advantage of entire tissue sample monitoring, allowing first estimations of tissue morphology but requires experiment termination.
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