Masete Kopano Valerie, Günzel Dorothee, Schulzke Jörg-Dieter, Epple Hans-Jörg, Hering Nina A
Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology and Rheumatology, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
Department of Gastroenterology, Rheumatology and Infectious Diseases, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
BMC Biol. 2025 Jan 5;23(1):2. doi: 10.1186/s12915-024-02105-7.
Traditionally, transformed cell line monolayers have been the standard model for studying epithelial barrier and transport function. Recently, intestinal organoids were proposed as superior in recapitulating the intestine. Typically, 3D organoids are digested and seeded as monolayers on gelatinous matrix pre-coated surfaces for anchorage. As this coat could potentially act as a diffusion barrier, we aimed to generate robust human duodenum-derived organoid monolayers that do not need a gelatinous matrix for anchorage to improve upon existing models to study epithelial transport and barrier function.
We characterized these monolayers phenotypically regarding polarization, tight junction formation and cellular composition, and functionally regarding uptake of nutrients, ion transport and cytokine-induced barrier dysfunction. The organoid monolayers recapitulated the duodenum phenotypically as well as functionally regarding glucose and short-chain fatty acid uptake. Tumour necrosis factor-alpha induced paracellular transport of 4-kDa Dextran and transcytosis of 44-kDa horseradish peroxidase. Notably, forskolin-stimulated chloride secretion was consistently lower when organoid monolayers were seeded on a layer of basement membrane extract (BME).
BME-free organoid monolayers represent an improved model for studying transcytotic, paracellular but especially transcellular transport. As BME is extracted from mice, our model furthers efforts to make organoid culture more animal-free.
传统上，转化细胞系单层一直是研究上皮屏障和转运功能的标准模型。最近，肠道类器官被认为在模拟肠道方面更具优势。通常，三维类器官被消化并作为单层接种在预涂有凝胶状基质的表面上以实现锚定。由于这种涂层可能会起到扩散屏障的作用，我们旨在生成强大的源自人十二指肠的类器官单层，其无需凝胶状基质即可实现锚定，以改进现有模型来研究上皮转运和屏障功能。
我们从表型上对这些单层的极化、紧密连接形成和细胞组成进行了表征，并从功能上对营养物质摄取、离子转运和细胞因子诱导的屏障功能障碍进行了研究。类器官单层在表型上以及在葡萄糖和短链脂肪酸摄取功能方面模拟了十二指肠。肿瘤坏死因子-α诱导了4 kDa葡聚糖的细胞旁转运和44 kDa辣根过氧化物酶的转胞吞作用。值得注意的是，当类器官单层接种在一层基底膜提取物（BME）上时，福斯可林刺激的氯离子分泌始终较低。
无BME的类器官单层代表了一种改进的模型，用于研究转胞吞、细胞旁尤其是跨细胞转运。由于BME是从小鼠中提取的，我们的模型进一步推动了使类器官培养更加无动物化的努力。
