Nielsen Simone S E, Siupka Piotr, Georgian Ana, Preston Jane E, Tóth Andrea E, Yusof Siti R, Abbott N Joan, Nielsen Morten S
Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine, Aarhus University.
Institute of Pharmaceutical Science, King's College London.
J Vis Exp. 2017 Sep 24(127):56277. doi: 10.3791/56277.
The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 ± 80 Ω cm, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10 ± 0.13 10 cm sec (mean ± SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.
本方案的目的是提供一种优化的程序，用于纯化和培养猪脑微血管内皮细胞（pBECs），并基于pBECs建立体外血脑屏障（BBB）模型，包括单培养（MC）、添加星形胶质细胞条件培养基（ACM）的MC以及与猪或大鼠来源的星形胶质细胞进行非接触共培养（NCC）。pBECs从5 - 6月龄家猪大脑皮质的毛细血管片段中分离培养。这些片段通过仔细去除脑膜、分离和匀浆灰质、过滤、酶消化和离心进行纯化。为了进一步消除污染细胞，将毛细血管片段用含嘌呤霉素的培养基培养。当达到60 - 95%汇合度时，从毛细血管片段生长的pBECs传代至可渗透膜滤器插入物并建立模型。为了增加pBECs的屏障紧密性和BBB特征表型，用以下分化因子处理细胞：膜通透性8 - CPT - cAMP（此处简称为cAMP）、氢化可的松和磷酸二酯酶抑制剂RO - 20 - 1724（RO）。该程序持续9 - 11天，在建立NCC模型时，星形胶质细胞提前培养2 - 8周。遵循方案中所述程序能够建立细胞旁通透性高度受限的内皮细胞层，NCC模型显示平均跨内皮电阻（TEER）为1249±80Ω·cm，荧光素黄的细胞旁通透性（Papp）为0.90×10±0.13×10 cm/sec（平均值±标准误，n = 55）。对该pBEC表型的进一步评估显示紧密连接蛋白claudin 5、ZO - 1、闭合蛋白和黏附连接蛋白p120连环蛋白表达良好。所呈现的模型可用于一系列健康和疾病状态下BBB的研究，并且由于其高度受限的细胞旁通透性，该模型适用于转运和细胞内运输的研究。
