LBPO.TB03 · 肿瘤生物学 · Late-Breaking
用于胶质母细胞瘤治疗发现的血脑屏障球体模型的建立与优化
Origination and refinement of blood brain barrier spheroid model for glioblastoma therapy discovery
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
胶质母细胞瘤（GBM）具有普遍致命性，5年生存率约为5%。治疗选择仍然陈旧且无效。大多数药物无法穿透血脑屏障（BBB），从而无法到达肿瘤。在为GBM寻找新型治疗药物所面临的诸多挑战中，尤为突出的是开发一种高通量、体外的完整BBB模型。BBB具有选择性，阻止大多数物质通过，既提供了对毒素的保护，同时也造成潜在毒素（治疗药物）无法穿越并进入脑肿瘤。我们开发了一种可重复、稳健且有效的原代人脑细胞模型，该模型在正确培养时能形成紧密连接和不可渗透的屏障。最终，该模型将用于评估细胞外囊泡（EV）、小分子和其他治疗机会，从而能够设计和测试新型疗法。简言之，将人原代星形胶质细胞（HA）、血管周细胞（vP）和脑微血管内皮细胞（EC）培养形成球体。球体核心由星形胶质细胞组成，EC和vP分层结合于外部，形成不可渗透膜的紧密连接。对细胞数量、细胞分层及各细胞加入球体的时机进行了多种迭代尝试和优化。在功能上，使用不同大小（50 nm至200 nm）荧光标记的纳米塑料和FITC-葡聚糖进行的渗透性检测显示其无法穿越BBB层。在定性方面，使用紧密连接标志物ZO-1、S100-A10、Claudin-5和细胞特异性标志物（GFAP、CD31、PDGFR-beta）的广泛免疫细胞化学染色表明，这些细胞的关系和排列产生并维持了紧密连接。球体在10天内保持健康、不可渗透和功能正常，为药物渗透性检测提供了有效的时间进程。未来的实验将评估装载新型治疗化合物的独特纳米颗粒和细胞外囊泡的局限性和尺寸选择性。
查看英文原文 English abstract
Glioblastoma (GBM) is uniformly fatal with a 5-year survival rate of approximately 5%. Treatment options remain antiquated and ineffective. Most drugs fail to penetrate the blood brain barrier (BBB) and never reach the tumor. Among myriad challenges present with identifying novel therapeutics for GBM, not least is development of a high-throughput, in vitro model of an intact BBB. The BBB is selective and prevents most substances from crossing, providing both protection from toxins and simultaneously, the inability to get potential toxins (therapies) across and into a brain tumor. We have developed a reproducible, robust, and working model of primary human brain cells that, when correctly cultured, form tight junctions and an impermeant barrier. Ultimately, the model will be utilized to assess extracellular vesicle (EV), small molecule, and other therapeutic opportunities, providing the ability to devise and test novel therapies. Briefly, human primary astrocytes (HA), vascular pericytes (vP), and brain microvascular endothelial cells (EC) are cultured to form a spheroid. The spheroid core is composed of astrocytes and the ECs and vPs are layered to combine on the exterior to form tight junctions of an impermeant membrane. Various iterations of cell number, cell layering and the timing of each cells addition to the spheroid have been tried and optimized. Functionally, permeability assays using various sized (50 nm to 200 nm) fluorescently labeled nano plastics and FITC-dextran show an inability to cross the BBB-layer. Qualitatively, extensive immunocytochemical staining using tight junction markers ZO-1, S100-A10, Cloudin-5 and cell-specific markers (GFAP, CD31, PDGFR-beta) demonstrate that the relationship and arrangement of these cells produce and maintain tight junctions. Spheroids remain healthy, impermeant and functional for 10 days, providing a valid, time course for drug permeability assays. Future experiments will assess the limitations and size-selectivity of unique nanoparticles and extracellular vesicles loaded with novel therapeutic compounds.
利益披露 Disclosure
R. Toedebusch, None..
C. Toedebusch, None..
R. Carney, None.