Under-oil neuronal cell culture: Enhanced system stability, yield, and modulated oxygen environment

IF 2.7 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-06-02 DOI:10.1016/j.jneumeth.2025.110511

Nai-Wen Liang , Ligi Milesh , Karina N. Buttram , Jessica L. Park , Jing Zhang , Luke A. Summey , Christian Franck , David J. Beebe , Hau D. Le , Chao Li

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引用次数: 0

Abstract

Background

Culturing neuronal cells in vitro, especially at smaller scales with reduced media volumes, has been challenging due to the limited proliferation of mature neurons and the inherent high sensitivity of neuronal cells to environmental fluctuations.

New method

In this study, we report a neuronal cell culture method that leverages oil overlay and an autonomously regulated oxygen microenvironment (AROM), in which primary rat cortical cells and human neural progenitor cells (NPCs) were cultured in standard well plates with an oil overlay on top of the media layer. The oil overlay prevents evaporation and achieves in vivo-like oxygen concentrations without the use of glove boxes or hypoxic chambers.

Results

This oil overlay method achieved > 95 % yield of viable replicates after up to 30 days. Human NPCs cultured under the oil overlay for 15 days exhibited sustained viability without requiring media change. Additionally, oil overlays create a modulated oxygen microenvironment (i.e., AROM) that mimics in vivo conditions, capable of maintaining and restoring optimal oxygen concentrations after disturbances.

Comparison with existing method

In contrast, existing method (no-oil controls) resulted in < 20 % yield, low viability for human NPCs (11 % versus 89 % with oil overlay), and oxygen concentrations that returns to ambient levels (21 % oxygen).

Conclusion

Overall, these results support the oil overlay method as a robust small-scale neuronal cell culture system, offering improved stability and higher yield. The results also underscore the critical role of the oxygen microenvironment in supporting neuronal cell viability, maintenance, and growth.

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油下神经细胞培养：增强系统稳定性、产量和调节氧环境。

背景：由于成熟神经元的增殖有限以及神经元细胞固有的对环境波动的高度敏感性，在体外培养神经元细胞，特别是在更小的培养皿中培养神经元细胞一直具有挑战性。新方法：在这项研究中，我们报告了一种利用油覆盖层和自主调节氧微环境（AROM）培养神经细胞的方法，在标准孔板中培养大鼠皮层细胞和人类神经祖细胞（npc），培养基层顶部有油覆盖层。油覆盖层防止蒸发，达到体内类似的氧气浓度，而无需使用手套箱或缺氧室。结果：采用油覆盖法培养30 d后，活产率达到95%。在油覆盖层下培养15天的人类npc在不需要更换培养基的情况下表现出持续的活力。此外，油覆盖层创造了一个模拟体内条件的调节氧微环境（即AROM），能够在干扰后维持和恢复最佳氧浓度。结论：总体而言，这些结果支持油覆盖法作为一种强大的小规模神经细胞培养系统，具有更好的稳定性和更高的产量。结果还强调了氧微环境在支持神经元细胞活力、维持和生长中的关键作用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.