Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model
Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model
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DOI：
10.1021/acsbiomaterials.0c01378
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发表时间：
2021-06-29
影响因子：
5.8
通讯作者：
Hagendoorn, Jeroen
中科院分区：
文献类型：
--
作者：
Frenkel, Nicola;Poghosyan, Susanna;Hagendoorn, Jeroen
淋巴系统对于维持组织液稳态以及抗原和免疫细胞运输至淋巴结至关重要。此外，淋巴管系统在各种病理过程中发挥着重要作用，例如癌症。该研究领域的基础是具有代表性的体外模型。在这里，我们提出了一种微流体淋巴管模型，使用新开发的淋巴内皮细胞（LEC）系来研究淋巴管生成及其与结肠癌类器官的相互作用。我们通过将人端粒酶 (hTERT) 和 BMI-1 表达盒慢病毒转导至原代 LEC 中，产生永生化人 LEC。永生化 LEC 显示出增加的生长潜力、减少的衰老和延长的寿命，并且在保持不转化的同时保持典型的 LEC 形态和标记表达超过 12 个月。永生化 LEC 被引入微流控芯片中，该芯片包含独立的细胞外基质，它们在细胞外基质上形成可灌注的血管样结构。细胞外基质凝胶上淋巴管生成因子的梯度诱导发光芽的形成。在淋巴管附近添加小鼠结肠癌类器官，形成稳定的长寿命共培养模型，可以研究癌细胞诱导的淋巴管生成和癌细胞运动。因此，在无膜灌注微流控芯片中开发稳定的永生化淋巴管内皮细胞系可产生高度标准化的淋巴管生成和淋巴管-肿瘤细胞共培养测定。
The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel-tumor cell coculture assay.