Establishment of perfusable Blood-Brain Barrier (BBB) models for tackling Glioblastoma (GBM)

血脑屏障（BBB）的生理保护功能阻碍药物递送到脑中，这代表了有效治疗脑肿瘤如胶质母细胞瘤（GBM）和几种肿瘤实体的脑转移的治疗剂的主要挑战。克服高选择性和受限的BBB渗透性的靶向策略是目前治疗脑癌的研究的主要焦点。目前，测试新疗法和BBB渗透性的金标准仍然是使用动物模型，伴随着高成本和对人类的翻译挑战。目前的体外替代方案是基于半透膜的无灌注简化2D测试模型或部分模拟组织功能的可灌注器官芯片模型。然而，它们是基于塑料的，缺乏对体内情况的准确模拟。需要更精确的3D体外模型来准确预测生理BBB渗透性并提高临床前成功率的转化，因此，该提议提供了在先进GBM模型的首次应用中开发结合通道灌注和合适的3D组织尺寸的创新的3D体外人BBB模型。在本文中，将应用并进一步开发两种用于在3D组织模型中生成可灌注通道的新型内部开发的基于生物制造的方法，以匹配BBB建模的特征，如组织层次结构、灌注和适应更复杂的三细胞培养。详细地说，这两种方法都利用熔融电写入（MEW）通过牺牲模板来制造双层血管移植物或微通道网络。将这些BBB结构整合在水凝胶基质内将允许与肿瘤细胞组合，并因此在高级GBM模型内进行首次应用。总的来说，该提议为创新的3D体外BBB平台铺平了道路，其具有几种可能的应用，不仅与GBM有关，而且还与其他疾病如多发性硬化症、阿尔茨海默病和脑转移有关，例如，乳腺癌细胞。为了简单起见，这个第一个项目将首先用细胞系如人微血管脑内皮细胞、脑星形胶质细胞和周细胞进行。然而，在该提议中获得的结果可以作为后续研究的基础，以使模型适应临床上更相关的细胞来源（例如，原代GBM细胞和诱导的多能干细胞（iPSC）以建立BBB）和整合，例如，免疫细胞将增加模型的复杂性。

The physiological protective function of the blood-brain barrier (BBB) hinders drug delivery into the brain, which represents a major challenge for therapeutics to effectively treat brain tumors such as glioblastoma (GBM) and brain metastases of several tumor entities. Targeting strategies to overcome the highly selective and restricted BBB permeability are a major focus of current research to cure brain cancer. Currently, the gold standard for testing novel therapeutics and BBB permeability is still using animal models accompanied by high costs and translation challenges to humans. The current in vitro alternatives are either perfusion-free simplified 2D test models based on semi-permeable membranes or perfusable Organ-on-a-Chip models, which partially emulate tissue function. However, they are plastic-based and lack accurate mimicry of the in vivo situation. More precise 3D in vitro models are demanded to accurately predict physiological BBB permeability and to improve the translation of pre-clinical success rates.Thus, this proposal offers to develop innovative 3D in vitro human BBB models combining channel perfusion and suitable 3D tissue size in a first application of an advanced GBM model. Herein, two novel in-house developed biofabrication-based methods for the generation of perfusable channels in 3D tissue models will be applied and further developed to match the characteristics for BBB modeling, like tissue hierarchy, perfusion, and the adaption to a more complex triple cell culture. In detail, both methods utilize melt electrowriting (MEW) to either fabricate a bilayered vascular graft or microchannel networks via sacrificial templating. Integration of these BBB structures within a hydrogel matrix will allow for the combination with tumor cells and, thus, to make a first application within an advanced GBM model. In general, this proposal paves the way for an innovative 3D in vitro BBB platform, with several possible applications not only related to GBM, but also other diseases like Multiple Sclerosis, Alzheimer's disease, and brain metastasis, e.g., of breast cancer cells. For the sake of simplicity, this first project will be initially performed with cell lines such as human microvascular brain endothelial cells, brain astrocytes, and pericytes. However, the results obtained in this proposal could be the basis for in follow-up studies to adapt the model to clinically more relevant cell sources (e.g., primary GBM cells and induced pluripotent stem cells (iPSCs) to establish the BBB) and integration of, e.g., immune cells will increase the model´s complexity.