Guzzo Sonia, De Bonis Pasquale, Pavan Barbara, Fadiga Luciano
Department of Neuroscience and Rehabilitation, Section of Physiology, University of Ferrara; Center for Translational Neurophysiology of Speech and Communication (CTNSC), Italian Institute of Technology (IIT), Ferrara, Italy.
Department of Translational Medicine and for Romagna, University of Ferrara, Ferrara, Italy.
Neural Regen Res. 2023 Feb;18(2):389-395. doi: 10.4103/1673-5374.346054.
In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier, our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants. We hypothesized that the autologous origin of human brain microvascular endothelial cells (hBMECs) is the first requirement for the suitable coating to prevent the glial inflammatory response triggered by foreign neuroprosthetics. Therefore, this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurgery patients. Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells. The addition of 10 nM β-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing, supporting the well-known protective role played by estrogens on microvessels. In particular, β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs, while it was not necessary for freshly isolated male-derived hBMECs; however, it did counteract the decay in the viability of the latter after thawing. The tumor-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed before and after two periods of cryopreservation. Despite the thermal stress, the hBMECs remained viable and suitable for re-freezing and storage for several months. This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools, offering the potential to avoid additional surgical sampling for each patient.
在当前用于构建血脑屏障体外模型的内皮细胞分离领域，我们的工作致力于通过一种用于脑慢性植入物的创新仿生涂层技术来重现神经血管单元的特征，以实现神经胶质顺应性。我们假设人脑血管内皮细胞（hBMECs）的自体来源是合适涂层的首要要求，以防止外来神经假体引发神经胶质炎症反应。因此，本研究建立了一种新方法，以保存从神经外科手术患者的灰质和白质标本中分离出的hBMECs的体外活力。由于这些细胞在体外难以存活且增殖逐渐减少，培养成人hBMECs通常被认为是一项具有挑战性的任务。研究发现，在hBMEC培养基中添加10 nM的17 - 醋酸β-雌二醇是促进分离和复苏后细胞黏附与增殖的关键且具有区分性的因素，这支持了雌激素对微血管的保护作用。具体而言，17 - 醋酸β-雌二醇对新鲜分离和复苏的雌性来源hBMECs至关重要，而对新鲜分离的雄性来源hBMECs则并非必需；然而，它确实能抵消后者复苏后活力的下降。无肿瘤的hBMECs因此培养长达2个月，并在两个冷冻保存期前后评估其生长效率。尽管存在热应激，hBMECs仍保持活力，适合再次冷冻并储存数月。这种提高hBMECs体外活力的方法为使用冷冻保存的自体hBMECs作为仿生治疗工具开辟了新的前景，有可能避免为每位患者进行额外的手术取样。
