Characterization of the molecular mechanisms underlying increased mucus production of HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation

The multifunctional intestinal mucus layer plays a crucial role in human health. Our understanding of the human colonic mucus layer in terms of structure, function and has been largely dependent on expensive and advanced ex vivo or in vitro models, which often require high expertise. The mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used in more simple in vitro models, but produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured in Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2 and had a thicker mucus layer compared to conventional culturing methods. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased mucus production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell inserts for 15 days were subject to transcriptome analysis to investigate underlying molecular pathways at gene expression level. We also further characterized the model by measuring transepithelial resistance and pH and lactate production of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms in which mucus production is enhanced under SWMS and propose potential applications of this model in future studies. HT29-MTX-E12 and Caco-2 cells were seeded on Transwell membranes. One day after seeding, cells were further cultured for 14 days under either SWMS conditions (i.e. low apical volume and continuous shaking at 65 rpm) or static conditions (similar conditions as during seeding). After harvesting, RNA isolated from cells was subjected to microarray analysis.

多功能肠黏液层在人类健康中发挥着至关重要的作用。目前学界对人类结肠黏液层结构与功能的认知，在很大程度上依赖于成本高昂且高精尖的离体（ex vivo）或体外（in vitro）模型，这类模型往往对操作专业度要求极高。产黏液肠细胞系HT29-MTX-E12是简易体外模型中常用的细胞系，但其仅能分泌少量肠道特异性黏蛋白MUC2。此前已有研究证实，在半湿界面机械刺激（Semi-Wet interface with Mechanical Stimulation, SWMS）培养条件下培养的HT29-MTX-E12细胞，其MUC2分泌量更高，黏液层厚度也优于传统培养方法，但目前仍不清楚其所涉及的潜在分子通路。为此，本研究旨在进一步探究SWMS培养条件下HT29-MTX-E12细胞黏液分泌量提升的细胞机制。研究中将接种于Transwell插入式培养板（Transwell inserts）的细胞培养15天后，通过转录组分析从基因表达层面解析其潜在分子通路。此外，我们还通过检测条件培养液的跨上皮电阻（transepithelial resistance）、pH值及乳酸生成量，对该模型进行了更全面的表征。本研究证实了SWMS培养条件下MUC2分泌量更高，并证明该培养方法主要可促进细胞增殖。此外，我们还发现SWMS培养条件下细胞代谢更倾向于有氧模式，这与此前同类模型的研究结果一致。综上，我们提出了SWMS培养条件下黏液分泌增强的潜在作用机制，并为该模型在未来研究中的应用提供了潜在方向。本研究将HT29-MTX-E12与Caco-2细胞接种于Transwell膜上。接种1天后，将细胞分别置于SWMS培养条件（即顶膜低体积培养液并以65 rpm持续振荡）或静态培养条件（与接种时的培养条件一致）中继续培养14天。收获细胞后，提取细胞总RNA并进行微阵列（microarray）分析。