Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD, USA.
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA.
Fluids Barriers CNS. 2018 Feb 20;15(1):7. doi: 10.1186/s12987-018-0092-7.
BACKGROUND: Transwell-based models of the blood-brain barrier (BBB) incorporating monolayers of human brain microvascular endothelial cells (dhBMECs) derived from induced pluripotent stem cells show many of the key features of the BBB, including expression of transporters and efflux pumps, expression of tight junction proteins, and physiological values of transendothelial electrical resistance. The fabrication of 3D BBB models using dhBMECs has so far been unsuccessful due to the poor adhesion and survival of these cells on matrix materials commonly used in tissue engineering. METHODS: To address this issue, we systematically screened a wide range of matrix materials (collagen I, hyaluronic acid, and fibrin), compositions (laminin/entactin), protein coatings (fibronectin, laminin, collagen IV, perlecan, and agrin), and soluble factors (ROCK inhibitor and cyclic adenosine monophosphate) in 2D culture to assess cell adhesion, spreading, and barrier function. RESULTS: Cell coverage increased with stiffness of collagen I gels coated with collagen IV and fibronectin. On 7 mg mL collagen I gels coated with basement membrane proteins (fibronectin, collagen IV, and laminin), cell coverage was high but did not reliably reach confluence. The transendothelial electrical resistance (TEER) on collagen I gels coated with basement membrane proteins was lower than on coated transwell membranes. Agrin, a heparin sulfate proteoglycan found in basement membranes of the brain, promoted monolayer formation but resulted in a significant decrease in transendothelial electrical resistance (TEER). However, the addition of ROCK inhibitor, cAMP, or cross-linking the gels to increase stiffness, resulted in a significant improvement of TEER values and enabled the formation of confluent monolayers. CONCLUSIONS: Having identified matrix compositions that promote monolayer formation and barrier function, we successfully fabricated dhBMEC microvessels in cross-linked collagen I gels coated with fibronectin and collagen IV, and treated with ROCK inhibitor and cAMP. We measured apparent permeability values for Lucifer yellow, comparable to values obtained in the transwell assay. During these experiments we observed no focal leaks, suggesting the formation of tight junctions that effectively block paracellular transport.
背景：基于 Transwell 的血脑屏障（BBB）模型，其中包含源自诱导多能干细胞的人脑微血管内皮细胞（dhBMEC）的单层，显示出 BBB 的许多关键特征，包括转运体和外排泵的表达、紧密连接蛋白的表达以及跨内皮电阻的生理值。迄今为止，使用 dhBMEC 制造 3D BBB 模型尚未成功，因为这些细胞在组织工程中常用的基质材料上的粘附和存活能力较差。
方法：为了解决这个问题，我们系统地筛选了广泛的基质材料（I 型胶原、透明质酸和纤维蛋白）、成分（层粘连蛋白/entactin）、蛋白质涂层（纤连蛋白、层粘连蛋白、IV 型胶原、perlecan 和 agrin）和可溶性因子（ROCK 抑制剂和环磷酸腺苷）在 2D 培养中评估细胞粘附、扩散和屏障功能。
结果：细胞覆盖率随涂有 IV 型胶原和纤连蛋白的 I 型胶原凝胶的刚度增加而增加。在涂有基底膜蛋白（纤连蛋白、IV 型胶原和层粘连蛋白）的 7mg/mL I 型胶原凝胶上，细胞覆盖率很高，但不能可靠地达到汇合。涂有基底膜蛋白的 I 型胶原凝胶的跨内皮电阻（TEER）低于涂有 Transwell 膜的 TEER。脑基底膜中的硫酸乙酰肝素蛋白聚糖 agrin 促进了单层的形成，但导致跨内皮电阻（TEER）显著降低。然而，添加 ROCK 抑制剂、cAMP 或交联凝胶以增加刚度可显著提高 TEER 值并形成致密的单层。
结论：我们已经确定了促进单层形成和屏障功能的基质成分，成功地在涂有纤连蛋白和 IV 型胶原的交联 I 型胶原凝胶中制造了 dhBMEC 微血管，并使用 ROCK 抑制剂和 cAMP 进行了处理。我们测量了 Lucifer yellow 的表观渗透率值，与 Transwell 测定中获得的值相当。在这些实验中，我们没有观察到焦点泄漏，这表明形成了有效的阻止细胞旁转运的紧密连接。
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