Non-physiological potassium concentrations in commercial culture media trigger acute seizure-like activity in human iPSC-derived neurons

TL;DR

Common cell culture media cause abnormal seizure-like activity in human neurons due to incorrect potassium levels.

Contribution

The study reveals that standard culture media have non-physiological potassium levels, leading to abnormal neuronal activity.

Findings

Commercial culture media have higher potassium than human CSF, causing seizure-like activity in neurons.

Neurons in human CSF show different activity than those in standard culture media.

This suggests in vitro experiments may not accurately reflect physiological brain activity.

Abstract

Neuronal in vitro cultures are pivotal for studying brain electrophysiological function and dysfunction. Neuronal activity and communication are regulated by extracellular ion concentrations. Therefore, cell culture medium ion concentrations should ideally mimic those of cerebrospinal fluid (CSF)—considered as the milieu for brain cells in vivo. In this study, we demonstrate that commonly used cell culture media, including NeurobasalTM (+ /− A), Neurobasal PlusTM, and BrainPhysTM media, do not accurately replicate human CSF ion concentrations. Using human iPSC-derived neuronal networks on microelectrode arrays, we compare neuronal activity in standard culture media with that in physiological artificial cerebrospinal fluid (aCSF) and in human cerebrospinal fluid. We show that supraphysiological potassium levels in culture media acutely induce seizure-like neuronal network activity compared with both physiological aCSF and human CSF. Importantly, human neurons exposed to human CSF obtained from healthy individuals show fundamentally different neuronal network activity than neurons exposed to currently used cell culture media. These findings raise a critical question: How can human in vitro neuronal activity be defined as physiological and reliably distinguished from pathophysiological activity, if the routinely used ion concentrations in in vitro experiments are causing aberrant neuronal activity that is fundamentally different from that of human neurons exposed to human CSF?

The online version contains supplementary material available at 10.1038/s41598-026-43094-7.

Figures