Hypoxic cell death is reduced by pH buffering in a model of engineered heart tissue.
BACKGROUND
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.


CONCLUSIONS
Effective pH buffering reduces cell death by attenuating the acidosis that accompanies anaerobic metabolism.
Brown
David A
DA
Department of Bioengineering, University of California, Los Angeles, CA 90095, USA. dabrown@uci.edu
MacLellan
William R
WR
Dunn
James C Y
JC
Wu
Benjamin M
BM
Beygui
Ramin E
RE
D000138:Acidosis / Q000378:metabolism / Q000517:prevention & control; D000818:Animals; D002021:Buffers; D016923:Cell Death / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D003470:Culture Media; D006531:HEPES / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D020287:In Situ Nick-End Labeling; D007526:Isoelectric Point; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D054328:Proton Pump Inhibitors; D017494:Proton Pumps / Q000187:drug effects; D051381:Rats; D046509:Tissue Culture Techniques; D023822:Tissue Engineering
D002021:Buffers; D003470:Culture Media; D054328:Proton Pump Inhibitors; D017494:Proton Pumps; D007770:L-Lactate Dehydrogenase; D006531:HEPES