Abstract

In vitro gut models, such as the mucosal artificial colon (M-ARCOL), provide timely and cost-efficient alternatives to in vivo assays allowing mechanistic studies to better understand the role of human microbiome in health and disease. Using such models inoculated with human fecal samples may require a critical step of stool storage. The effects of preservation methods on microbial structure and function in in vitro gut models have been poorly investigated. This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at − 80 °C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors (e.g., fresh stool, raw stool stored at − 80 °C, stool stored at − 80 °C with glycerol and lyophilized stool). Microbiota composition and diversity (qPCR and 16S metabarcoding) as well as metabolic activity (gases and short chain fatty acids) were monitored throughout the fermentation process (9 days). All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at − 80 °C without cryoprotectant as the most efficient method. These results will help scientists to determine the most accurate method for fecal storage prior to inoculation of in vitro gut microbiome models.

Key points

• In vitro ARCOL model reproduces luminal and mucosal human microbiome.

• Short-term storage of fecal sample influences microbial stabilization and activity.

• 48 h freezing at − 80°C: most efficient method to preserve microbial ecosystem.

• Scientific and technical requirements: influencers of preservation method.

中文翻译：

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在人类结肠体外模型中粪便样品保存以保留肠道微生物结构和功能的比较方法

摘要

体外肠道模型，例如粘膜人工结肠（M-ARCOL），为体内测定提供了及时且经济高效的替代方法，从而使机理研究可以更好地了解人类微生物组在健康和疾病中的作用。使用接种了人类粪便样本的模型可能需要粪便储存的关键步骤。保存方法对体外肠道模型中微生物结构和功能的影响研究很少。这项研究旨在评估三种常用的保存方法（与用作对照的新鲜粪便样品相比）对M-ARCOL模型中管腔和与黏液相关的微生物群落定殖的动力学的影响。来自两个健康供体的粪便在有或没有冷冻保护剂（10％甘油）的条件下在− 80°C下冷冻48 h，或者在接种四个平行的生物反应器（例如新鲜粪便，未加工粪便在− 80°C下储存）之前用麦芽糖糊精和海藻糖冻干。 ，粪便在-80°C下用甘油和冻干粪便保存）。在整个发酵过程（9天）中监测微生物群组成和多样性（qPCR和16S元条形码）以及代谢活性（气体和短链脂肪酸）。所有防腐处理均允许在M-ARCOL内部维持复杂且功能齐全的微生物群，但要考虑到微生物特征和活性的稳定时间（而不是与冷冻材料供应相关的技术限制），我们的结果表明，在不使用防冻剂的情况下，在− 80°C下冷冻48小时是最有效的方法。这些结果将有助于科学家在接种体外肠道微生物组模型之前确定最准确的粪便储存方法。

关键点

•体外ARCOL模型可复制腔和粘膜人类微生物组。

•粪便样品的短期保存会影响微生物的稳定性和活性。

•在-80°C下冷冻48小时：最有效的微生物生态系统保护方法。

•科学技术要求：影响保存方法的因素。