Almari Bushra, Brough David, Harte Michael, Tirella Annalisa
Division of Pharmacy and Optometry, School of Health Studies, Faculty of Biology, Medicine and Health, University of Manchester.
Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester.
J Vis Exp. 2019 Jul 6(149). doi: 10.3791/59597.
According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer's disease (AD) is the accumulation of toxic amyloid-β (Aβ) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic Aβ production. Chinese hamster ovary cells previously transfected with a human APP gene, secreting Aβ (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of Aβ was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 μm for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted Aβ were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h). A concentration of 1.5 x 10 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 μm, 3) containing about 100-150 cells per microbead and 4) able to secrete Aβ. In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.
根据淀粉样蛋白级联假说，阿尔茨海默病（AD）发展过程中最早的触发因素是有毒淀粉样蛋白β（Aβ）片段的积累，最终导致该病的典型特征：淀粉样斑块、神经原纤维缠结以及突触和神经元丧失。缺乏反映疾病进展的相关非转基因临床前模型是阻碍有效药物治疗发现的主要因素之一。为此，我们开发了一种制备含有分泌淀粉样蛋白细胞的藻酸盐微珠的方案，用于研究慢性Aβ产生的影响。本研究使用了先前转染了人类APP基因并分泌Aβ的中国仓鼠卵巢细胞（即7PA2细胞）。通过将7PA2细胞封装在藻酸盐中制备了一种用于Aβ持续释放的三维（3D）体外模型。该过程经过优化，使微珠直径达到500 - 600μm，以便进一步进行体内研究。通过改变制备参数（如藻酸盐浓度、凝胶流速、静电势、头部振动频率、凝胶溶液）对7PA2细胞在藻酸盐中的封装进行了优化。随时间分析分泌的Aβ水平，并在藻酸盐微珠和标准细胞培养方法之间进行比较（长达96小时）。发现浓度为1.5×10⁷个/mL的7PA2细胞和2%（w/v）的藻酸盐浓度（用HEPES缓冲），随后在0.5M氯化钙中凝胶化5分钟，能够制备出最稳定的微珠。制备的微珠1）尺寸均匀，2）平均直径为550μm，3）每个微珠含有约100 - 150个细胞，4）能够分泌Aβ。总之，我们优化的制备含有产生淀粉样蛋白的7PA2细胞的稳定藻酸盐微珠的方法可能使AD的重要方面在体外和体内都能得以建模。
