HEPES in Cell Culture Alters the Multi-Omics Profile Exhibited by Gaucher Disease Fibroblasts

Abstract

Lysosomal function can be affected by components in cell culture. This in turn may influence cellular metabolism and, consequently, research and diagnostics outcomes. One such component is the commonly used pH buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). HEPES specifically impacts the trafficking of the lysosomal enzyme glucocerebrosidase, which is deficient in Gaucher disease (GD). Understanding how HEPES affects cellular models of GD is essential, since glucocerebrosidase is central to diagnostic testing and the investigation of GD pathophysiology. Therefore, we examined the broader effects of HEPES on cultured fibroblasts from individuals with GD and healthy controls. We cultured dermal fibroblasts of eight adults with GD and seven healthy age- and sex-matched controls. The cells were cultured in two culture media, Ham's F10 and DMEM, both with and without HEPES. We assessed glucocerebrosidase enzyme activity and sphingolipid concentrations using a quantitative UPLC-MS/MS method. Additionally, we conducted multi-omics analyses, consisting of lipidomics, metabolomics and proteomics, to explore the broader impact of HEPES in cell culture on fibroblasts. Glucocerebrosidase activity in cell lysates increased after HEPES exposure in both GD and control fibroblasts, to an extent that may influence diagnostic outcomes. In GD fibroblasts, substrate accumulation was absent and not altered by HEPES exposure. GD fibroblasts exhibited a multi-omics profile largely overlapping with healthy controls and lacking the typical pathological features associated with GD in other cell types, such as mitochondrial dysfunction, dysregulated autophagy, disruption of intracellular calcium homeostasis, ER stress and chronic oxidative stress. In addition, the multi-omics profile was altered by HEPES, however in a non-specific manner. In conclusion, HEPES influences fibroblasts in culture, both from healthy controls and from patients with GD. Furthermore, GD fibroblasts lack a specific disease-related profile. This renders cultured fibroblasts unsuitable for studying pathophysiological processes in GD. Culturing GD fibroblasts with HEPES may compromise the reliability of diagnostics.