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Long-Term Cryopreservation Preserves Blood-Brain Barrier Phenotype of iPSC-Derived Brain Microvascular Endothelial Cells and Three-Dimensional Microvessels.
Molecular Pharmaceutics ( IF 4.9 ) Pub Date : 2020-07-28 , DOI: 10.1021/acs.molpharmaceut.0c00484
Raleigh M Linville 1, 2 , Jackson G DeStefano 1, 3 , Renée F Nerenberg 1, 2 , Gabrielle N Grifno 1, 2 , Robert Ye 1 , Erin Gallagher 1, 3 , Peter C Searson 1, 2, 3
Affiliation  

Brain microvascular endothelial cells derived from induced pluripotent stem cells (dhBMECs) are a scalable and reproducible resource for studies of the human blood–brain barrier, including mechanisms and strategies for drug delivery. Confluent monolayers of dhBMECs recapitulate key in vivo functions including tight junctions to limit paracellular permeability and efflux and nutrient transport to regulate transcellular permeability. Techniques for cryopreservation of dhBMECs have been reported; however, functional validation studies after long-term cryopreservation have not been extensively performed. Here, we characterize dhBMECs after 1 year of cryopreservation using selective purification on extracellular matrix-treated surfaces and ROCK inhibition. One-year cryopreserved dhBMECs maintain functionality of tight junctions, efflux pumps, and nutrient transporters with stable protein localization and gene expression. Cryopreservation is associated with a decrease in the yield of adherent cells and unique responses to cell stress, resulting in altered paracellular permeability of Lucifer yellow. Additionally, cryopreserved dhBMECs reliably form functional three-dimensional microvessels independent of cryopreservation length, with permeabilities lower than non-cryopreserved two-dimensional models. Long-term cryopreservation of dhBMECs offers key advantages including increased scalability, reduced batch-to-batch effects, the ability to conduct well-controlled follow up studies, and support of multisite collaboration from the same cell stock, all while maintaining phenotype for screening pharmaceutical agents.

中文翻译:

长期冷冻保存可保留 iPSC 衍生的脑微血管内皮细胞和三维微血管的血脑屏障表型。

源自诱导多能干细胞 (dhBMEC) 的脑微血管内皮细胞是研究人血脑屏障(包括药物递送机制和策略)的可扩展且可重复的资源。dhBMECs 的汇合单层在体内概括了关键功能包括紧密连接以限制细胞旁通透性和流出以及营养物质运输以调节跨细胞通透性。已经报道了 dhBMEC 的冷冻保存技术;然而,长期冷冻保存后的功能验证研究尚未广泛开展。在这里,我们使用细胞外基质处理表面的选择性纯化和 ROCK 抑制来表征冷冻保存 1 年后的 dhBMEC。一年冷冻保存的 dhBMECs 保持紧密连接、外排泵和营养转运体的功能,具有稳定的蛋白质定位和基因表达。冷冻保存与贴壁细胞产量的降低和对细胞应激的独特反应有关,导致荧光黄的细胞旁通透性改变。此外,低温保存的 dhBMEC 可靠地形成功能性三维微血管,与低温保存长度无关,渗透率低于非低温保存的二维模型。dhBMECs 的长期低温保存提供了关键优势,包括增加可扩展性、减少批次间效应、进行良好控制的后续研究的能力以及支持来自同一细胞库的多站点协作,同时保持筛选药物的表型代理商。
更新日期:2020-09-09
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