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<p><?tk 1?>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 Neurobasal<sup>TM</sup> (+ /− A), Neurobasal Plus<sup>TM</sup>, and BrainPhys<sup>TM</sup> 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?</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年3月18日

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  • Tim Lyckenvik,
  • Julia Izsak,
  • Erik Arthursson,
  • My Forsberg,
  • Kalle Johansson,
  • Henrik Zetterberg,
  • Markus Axelsson,
  • Pontus Wasling,
  • Eric Hanse,
  • Stephan Theiss,
  • Sebastian Illes

摘要

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?