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

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 un-derlying mechanism was unclear. Here we report that riboflavin (RF, component of cell culture medium) gen-erates 1O2, center dot OH, and O2 center dot-under visible light exposure during regular cell manipulation. These short half-life reactive oxygen species (ROS) react with tertiary amine groups of HEPES, producing 106.6 mu M of H2O2. Or-ders 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.

Automated summary

Good's buffers have been widely used in cell/organ culture as biocompatible pH stabilizers. However, the emergence of adverse effects such as cellular uptake, autophagic activation, and cytotoxicity under light exposure raises concerns over its biocompatibility. Riboflavin in the cell culture medium produces reactive oxygen species (ROS) under light exposure, causing severe toxicity and disruption of normal cell functions. Zwitterionic betaines have been identified as a new generation of biocompatible pH buffers that do not exhibit these adverse effects.