Zwitterionic betaines over HEPES as the new generation biocompatible pH buffers for cell culture.

Abstract

Good's buffers have been widely applied in cell/organ culture over the past half a century as biocompatible pH stabilizers. However, the emergence of severe adverse effects, such as cellular uptake, lysosomal autophagic activation, and visible light-induced cytotoxicity, raises serious questions over its biocompatibility while underlying mechanism was unclear. Here we report that riboflavin (RF, component of cell culture medium) generates 1O2, ·OH, and O2 •- under visible light exposure during regular cell manipulation. These short half-life reactive oxygen species (ROS) react with tertiary amine groups of HEPES, producing 106.6μM of H2O2. Orders of magnitude elevated half-life of ROS in the medium caused severe cytotoxicity and systematic disorder of normal cell functions. We have further designed and validated zwitterionic betaines as the new generation biocompatible organic pH buffers, which is able to completely avoid the adverse effects that found on HEPES and derivate Good's buffers. These findings may also open a new avenue for zwitterionic betaine based materials for biomedical applications.