Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons.

Sophie R Cook,Nicholas D Allen,Rafael A Badell-Grau,Kimberley M Jones,Emily D Kirkham,Jincy Winston,Helen Waller-Evans,Emyr Lloyd-Evans,Brendan P Kelly

doi:10.12688/amrcopenres.12903.1

Abstract

Good's buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.

Highlights

Good’s buffers, including 4-(2-hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES), are commonly used zwitterionic buffers in cell culture[1,2,3]
This study describes the effect of HEPES on lysosomal morphology and Ca2+ levels in control cells and those null for the lysosomal protein Niemann-pick type C1 (NPC1), whose function is lost in the lysosomal storage disease Niemann-Pick Type C (NPC)
Following 12 months of growth in media with HEPES, we observed no further increase in LysoTracker staining in NPC1-null Chinese hamster ovary (CHO)-M12 cells, whereas we observed a 2.8-fold increase in LysoTracker fluorescence in control CHO-H1 compared with control cells grown in HEPES-free media (Figure 1E)

Summary

Introduction

Good’s buffers, including 4-(2-hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES), are commonly used zwitterionic buffers in cell culture[1,2,3]. These buffers were developed to be stable, membrane impermeant, and inert in biological reactions[2], leading to their widespread use. Known as the recycling centre of the cell, since they breakdown cellular material. They have important roles in cellular processes, including plasma membrane repair and cellular signalling as the second largest intracellular Ca2+ store[5,6,7]. Considering the reported impact of HEPES on lysosomal enzymes[1], it is important to understand its effects, as well as other zwitterionic buffers, on lysosomal functions

Methods

Results

Conclusion