Development and Optimization of a Lactate Dehydrogenase Assay Adapted to 3D Cell Cultures

Organoids Pub Date : 2024-06-05 DOI:10.3390/organoids3020008
Héloïse Castiglione, Lucie Madrange, Thomas Lemonnier, Jean-Philippe Deslys, Frank Yates, Pierre-Antoine Vigneron
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Abstract

In recent years, 3D cell culture systems have emerged as sophisticated in vitro models, providing valuable insights into human physiology and diseases. The transition from traditional 2D to advanced 3D cultures has introduced novel obstacles, complicating the characterization and analysis of these models. While the lactate dehydrogenase (LDH) activity assay has long been a standard readout for viability and cytotoxicity assessments in 2D cultures, its applicability in long-term 3D cultures is hindered by inappropriate normalization and low LDH stability over time. In response to these challenges, we propose an optimization of LDH assays, including a crucial normalization step based on total protein quantification and a storage method using an LDH preservation buffer. We applied it to compare unexposed cerebral organoids with organoids exposed to a toxic dose of valproic acid, and showed efficient normalization of cellular viability as well as enhanced LDH stability within the buffer. Importantly, normalized LDH activity results obtained were independent of organoid dimension and cell density. This refined LDH assay, tailored to address 3D culture constraints, allows for the transposition of this routine test from 2D to 3D cultures.
开发并优化适用于三维细胞培养的乳酸脱氢酶检测方法
近年来,三维细胞培养系统已成为复杂的体外模型,为人类生理学和疾病提供了宝贵的见解。从传统的二维培养过渡到先进的三维培养,引入了新的障碍,使这些模型的表征和分析变得更加复杂。虽然乳酸脱氢酶(LDH)活性测定长期以来一直是二维培养物存活率和细胞毒性评估的标准读数,但其在长期三维培养物中的适用性却因归一化不当和 LDH 随时间变化稳定性低而受到阻碍。为了应对这些挑战,我们提出了一种优化 LDH 检测的方法,包括基于总蛋白定量的关键归一化步骤和使用 LDH 保存缓冲液的储存方法。我们将其应用于比较未暴露的脑器官组织和暴露于毒性剂量丙戊酸的器官组织,结果表明细胞活力的有效归一化以及缓冲液中 LDH 稳定性的增强。重要的是,归一化的 LDH 活性结果与类器官尺寸和细胞密度无关。这种经过改进的 LDH 检测方法专门针对三维培养的限制因素而定制,可将这一常规检测从二维培养转移到三维培养。
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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