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Abstract It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principal component of the blood‐brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human‐induced pluripotent stem cell (iPSC)‐derived BMEC‐like cells cultured under serum‐free conditions in DMEM/F12 and Neurobasal media, which have fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays. image
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Low‐Glucose Culture Conditions Bias Neuronal Energetics Towards Oxidative Phosphorylation
ABSTRACT Neurons are almost exclusively cultured in media containing glucose at much higher concentrations than found in the brain. To test whether these “standard” hyperglycemic culture conditions affect neuronal respiration relative to near‐euglycemic conditions, we compared neuronal cultures grown with minimal glial contamination from the hippocampus and cortex of neonatal C57BL/6NCrl mice in standard commercially available media (25 mM Glucose) and in identical media with 5 mM glucose. Neuronal growth in both glucose concentrations proceeded until at least 14 days in vitro, with similar morphology and synaptogenesis. Neurons grown in high glucose were highly dependent on glycolysis as their primary source of ATP, measured using ATP luminescence and cellular respirometry assays. In contrast, neurons grown in 5 mM glucose showed a more balanced dependence on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), greater reserve mitochondrial respiration capacity, and increased mitochondrial population relative to standard media. Our results show that neurons cultured in artificially high glucose‐containing media preferentially use glycolysis, opposite to what is known for neurons in vivo as the primary pathway for ATP maintenance. Changes in gene and protein expression levels corroborate these changes in function and additionally suggest that high glucose culture media increases neuronal inflammation. We suggest using neuronal culture systems in 5 mM glucose to better represent physiologically relevant neuronal respiration.image
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- Award ID(s):
- 1943514
- PAR ID:
- 10607985
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Journal of Neurochemistry
- Volume:
- 169
- Issue:
- 6
- ISSN:
- 0022-3042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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