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

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.


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. Reports have, however, described zwitterionic buffers affecting biological processes; they induce morphological artefacts in fixed Drosophila tissue 4 , and alterations to autophagy and lysosomal enzyme activity in cultured cells 1 .
Lysosomes are acidic organelles, known as the recycling centre of the cell, since they breakdown cellular material. They also have important roles in cellular processes, including plasma membrane repair and cellular signalling as the second largest intracellular Ca 2+ store 5-7 . Lysosomal dysfunction is a component of multiple diseases including Alzheimer's, Parkinson's and ~70 inherited lysosomal storage diseases 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.
This study describes the effect of HEPES on lysosomal morphology and Ca 2+ levels in control cells and those null for the lysosomal protein NPC1, whose function is lost in the lysosomal storage disease Niemann-Pick Type C (NPC). The findings highlight the importance of understanding the impact of growth media components on lysosomal functions.

Buffers
All buffers (MOPS, PIPES, MES, PPB) were purchased from Sigma-Aldrich apart from HEPES (Thermofisher/Lonza) and Tris (Roche). With the exception of HEPES, which was purchased as a pre-made 1 M solution (pH 7.4), all buffers were made as 1 M stock solutions in mqH 2 O (or 1 M NaOH in mqH 2 O for PIPES), adjusted to pH 7.4 and filter sterilised through a 0.22 µm filter. PPB was adjusted to pH 7.4 by combining 1 M solutions of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate. Buffers were added to culture media to a final concentration of 10 mM unless otherwise stated.

Lysosomal measurements
Lysosomes were visualised in live cells in chamberslides (Ibidi) using 300 nM LysoTracker red or green (Life Technologies) in Dulbecco's modified phosphate buffered saline (DPBS) for 15-minutes at room temperature, washed tree times with DPBS, and imaged using a Zeiss Axio Observer inverted microscope with Colibri LED light source and Zeiss Mrm CCD camera with Axiovision 4.8 software. Lysosomal area per cell was measured from LysoTracker fluorescence images in ImageJ 1.50i and 1.52n 11 using the analyse particles function. LysoTracker fluorescence was measured in cells grown in Corning CellBIND 96-well plates (0.8x10^5 cells/well) using a SpectraMax® Gemini microplate reader (Molecular Devices).

Statistical analysis
All statistical analyses were performed in GraphPad Prism 8 software with data analysed by two-way ANOVA with Tukey's post-hoc test or unpaired t-test as appropriate and where indicated in the figure legends.

Results
In agreement with previous findings of lysosomal enzyme dysfunction 1 , we observed HEPES-mediated lysosomal dysfunction in control CHO-H1 cells that was exacerbated at high cell confluency. Namely, a concentration-dependent expansion of the lysosomal system following 3-days growth in HEPES-containing buffer observed using LysoTracker ( Figure 1A & B). Having confirmed this effect, we determined whether other zwitterionic buffers triggered similar effects. At a buffer concentration commonly found in growth media (10 mM), only PIPES, out of the six buffers tested, increased lysosomal area in control CHO-H1 cells over the 3-day treatment, that was also exacerbated by high cell confluency ( Figure 1C & D).
To determine the long-term effects of growth in HEPEScontaining media (10 mM), control CHO-H1 and the NPC1-null CHO-M12 cells were grown in this media for 12-months. When grown in HEPES-free media, there is a 4.8-fold increase in LysoTracker florescence, measured using a plate reader, in the lysosomal disease CHO-M12 cells, compared to control CHO-H1. Following 12 months of growth in media with HEPES, we observed no further increase in LysoTracker staining in NPC1-null 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). This illustrates that growth in HEPESsupplemented media impacts upon healthy lysosomal function and reduces the difference between control and lysosomal disease cells. This observation may have particular importance for cells requiring long-term growth in buffered media (e.g., iPSC-neurons).
Therefore, we tested the effect of HEPES supplementation of SynaptoJuiceB on iPSC-neurons in culture for 7 days. Again, we observed an expansion of the lysosomal system (Figure 2A). Because zwitterionic buffers may act as a "proton sponge", affecting both the volume and ion balance of lysosomes 13 , particularly lysosomal Ca 2+ content which is dependent on lysosomal acidification 14 , we measured lysosomal Ca 2+ content in these neurons. We observed significantly reduced lysosomal Ca 2+ (2.2-fold) in neurons grown in the presence of 10 mM HEPES for 7 days compared to those grown without HEPES ( Figure 2B). Raw data underlying this study are available at Figshare 15 .

Discussion and conclusions
Our findings indicate that lysosomal expansion occurs after both short-and long-term culture in HEPES-buffered media and is exacerbated at higher cellular confluency. Moreover, this expansion impacts lysosomal function, namely lysosomal ion signalling in the form of reduced lysosomal Ca 2+ content and is consistent with previous report of altered lysosomal glucosylceramidase activity in cells grown in HEPES 1 . Together, these data suggest that HEPES operates as a lysosomal proton sponge 13, 16 . These observations provide a significant note of caution for lysosomal researchers, potentially impacting on lysosomal biochemical experiments such as measurement of pH 17 or lysosomal purification methods 18 . Not all zwitterionic buffers have the same effects, only PIPES was also detrimental to lysosomal function, suggesting other zwitterionic buffers may be appropriate HEPES substitutes. Regardless, stringent consideration must be spent on buffer selection for relevant lysosomal studies.