Elzinga Janneke, van der Lugt Benthe, Belzer Clara, Steegenga Wilma T
Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands.
Division of Human Nutrition and Health, Wageningen University and Research, Wageningen, The Netherlands.
PLoS One. 2021 Dec 20;16(12):e0261191. doi: 10.1371/journal.pone.0261191. eCollection 2021.
The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level 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 by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.
肠道黏液层在人类健康中起着至关重要的作用。为了在体外研究肠道黏液的功能和结构，通常使用产生黏液的肠道细胞系HT29-MTX-E12。然而，该细胞系仅产生少量的肠道特异性MUC2。先前的研究表明，与传统培养的细胞相比，在半湿界面机械刺激（SWMS）条件下培养的HT29-MTX-E12细胞产生的MUC2量更高，同时黏液层更厚。然而，尚不清楚涉及哪些潜在途径。因此，我们旨在进一步探索在SWMS条件下生长的HT29-MTX-E12细胞中MUC2产量增加的细胞过程。在静态和SWMS条件下（细胞接种和附着后）在Transwell膜上生长14天的细胞进行转录组分析，以研究基因表达水平上的潜在分子途径。将Caco-2和LS174T细胞系作为对照。我们通过测量跨上皮电阻来表征SWMS条件如何影响HT29-MTX-E12细胞的上皮屏障完整性，并通过监测条件培养基中每分子葡萄糖的pH值和乳酸产量来表征细胞代谢。我们在基因和蛋白质水平上证实了SWMS条件下MUC2产量更高，并证明这种培养方法主要刺激细胞生长。此外，我们还发现了SWMS条件下细胞代谢更偏向有氧的证据，正如之前在类似模型中所显示的那样。总之，我们提出了在SWMS条件下MUC2产量增加的不同机制，并提出了该模型在未来研究中的潜在应用。
