Hypoxic cell death is reduced by pH buffering in a model of engineered heart tissue.
INTRODUCTION: In this report, we tested the ability of HEPES-buffered culture medium to reduce acidotic cell death in hypoxic monolayer cell cultures and in a diffusion-limited model of engineered heart tissue (EHT). METHODS: Neonatal rat cardiomyocytes were either plated (monolayers) or suspended in a hydrogel disc containing HEPES. RESULTS: Monolayers cultured in 0% oxygen exhibited a pH drop to 5.46 +/- 0.27 after 4 days with no HEPES, or 7.11 +/- 0.09 with 50 mM HEPES. The lowest observed pH in EHTs was estimated as 6.2 with no HEPES and 7.1 with 50 mM HEPES, which were endpoints of noticeably different pH gradients across the EHTs. Addition of HEPES to hypoxic monolayers corresponded with fewer propidium iodide-positive cells and TUNEL-positive cells; addition of HEPES to EHTs resulted in greater calcein staining and less LDH release. CONCLUSION: Effective pH buffering reduces cell death by attenuating the acidosis that accompanies anaerobic metabolism.
Brown, DA; MacLellan, WR; Dunn, JCY; Wu, BM; Beygui, RE
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