Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Department of Bioengineering, University of Washington, Seattle, Washington.
Biophys J. 2021 Dec 7;120(23):5384-5394. doi: 10.1016/j.bpj.2021.10.024. Epub 2021 Oct 23.
The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.
本项目的目标是验证体外肠细胞培养模型产生的黏液的功能相关性和实用性。这是因为需要在生理上复制来自天然肠组织的健康和异常黏液条件，在这些条件下，黏液特性与肠道疾病模型有关。与从健康供体衍生的结肠细胞培养物中收集的黏液相比，比较了从手术切除的非炎症性横结肠组织中收集的黏液。使用振荡流变仪、粒子追踪微流变仪、多角度激光光散射、折射计和免疫组织化学成像比较了这些黏液样本的流变学和生物化学性质。原代人结肠上皮细胞的气-液界面培养生成了一个连续的单层，附有一层黏液层，在 1 周内（第 2 天为 1.3 ± 0.5%，第 7 天为 2.4 ± 0.3%）显示出固体重量百分比（wt%）的增加。在培养过程中观察到的全范围黏液浓度（0.9-3.3%）与离体黏液相似（1.3-1.9%）。基于重量的体外和离体黏液之间的体流变学测量显示出相似的复杂粘度，两种系统的复杂粘度都随固体重量百分比的增加而增加。粒子追踪微流变学显示，与体外黏液相比，离体黏液样品的复杂粘度更高，这可以解释为体外黏液中存在的水分数比离体黏液中多，即体外黏液比离体黏液更不均匀。折射计、多角度激光光散射和免疫染色显示，与体外黏液相比，离体黏液的黏液复杂尺寸增加，这可能是由于在离体黏液收集过程中混合了黏液和细胞碎片。气-液界面培养系统产生的肠黏液具有与天然人肠道黏液相似的组成和流变学，为分析肠道黏液的病理差异提供了一个平台。
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