Linnea Gustafsson, Savvini Gkouma, Alexander Jönsson, Martin Dufva, My Hedhammar
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引用次数: 0
摘要
复制组织屏障对于生成评估新型疗法的相关体外模型至关重要。目前,通常使用带有塑料膜的组织培养插片来实现这一目的,塑料膜可产生顶端和基底面。除了为细胞提供支持外,这些膜远不能模拟其原生对应物--基底膜,后者是一种基于蛋白质的纳米纤维基质。在这项工作中,我们展示了一种简单的方法,即用纯重组功能化蜘蛛丝蛋白制成的膜取代塑料膜,从而大大提高组织培养插片的生物学相关性。蛛丝膜通过自组装形成,会自发附着在无膜组织培养插片上,为细胞提供支持。定制设计的组织培养插片可以使用标准 3D 打印机,按照协议中提供的说明打印出来,也可以购买商业化的组织培养插片来代替。本方案展示了如何在插入物中设置带丝膜的培养系统,以及随后如何实施与传统商用插入物相同的细胞培养技术。
Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts.
Replicating tissue barriers is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.
期刊介绍:
JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.