McKinnon Daniel D, Kloxin April M, Anseth Kristi S
Department of Chemical and Biological Engineering, the BioFrontiers Institute, and the Howard Hughes Medical Institute, University of Colorado at Boulder, Jennie Smoly Caruthers Biotechnology Building, 3415 Colorado Ave, 596 UCB, Boulder, CO 80303, USA.
Biomater Sci. 2013 May 2;1(5):460-469. doi: 10.1039/c3bm00166k. Epub 2013 Jan 24.
Culturing mammalian neurons in three-dimensional (3D) microenvironments that more closely recapitulate critical biochemical and biophysical aspects of the developing or adult central nervous system (CNS) milieu remains a significant challenge in neurobiological studies and in regenerative medicine. Here, we aimed to exploit recent advances in poly(ethylene glycol) (PEG) hydrogel chemistries to define a synthetic niche capable of supporting the culture and axonal outgrowth of both aggregated and dissociated mouse embryonic stem cell-derived motor neurons (ESMNs). Using thiol-ene click chemistry to create peptide crosslinked PEG hydrogels, we identified a hydrogel formulation that promotes neuronal survival and axon outgrowth through cell-extracellular matrix interactions, such as those between the laminin-derived peptide YIGSR and its integrin, and that allows neurons to remodel their extracellular environment through matrix metalloproteinase (MMP)-mediated polymer network degradation. Our results demonstrate a 3D platform for culture of both aggregated and single mammalian motor nerve cells that not only permits cell survival over more than a week of culture, but also allows for the robust extension of motor axons. In addition, the optical transparency of the hydrogel allows simultaneous imaging of live cell functions, and as such, this material system should prove useful for studying fundamental aspects of neuronal development.
在三维（3D）微环境中培养哺乳动物神经元，使其更紧密地模拟发育中的或成体中枢神经系统（CNS）环境的关键生化和生物物理方面，仍然是神经生物学研究和再生医学中的一项重大挑战。在此，我们旨在利用聚乙二醇（PEG）水凝胶化学领域的最新进展，来定义一种能够支持聚集型和解离型小鼠胚胎干细胞衍生运动神经元（ESMN）的培养及轴突生长的合成微环境。我们利用硫醇-烯点击化学来制备肽交联PEG水凝胶，确定了一种水凝胶配方，该配方可通过细胞-细胞外基质相互作用（如层粘连蛋白衍生肽YIGSR与其整合素之间的相互作用）促进神经元存活和轴突生长，并允许神经元通过基质金属蛋白酶（MMP）介导的聚合物网络降解来重塑其细胞外环境。我们的结果展示了一个用于培养聚集型和单个哺乳动物运动神经细胞的3D平台，该平台不仅能使细胞在超过一周的培养期内存活，还能促进运动轴突的强劲延伸。此外，水凝胶的光学透明性允许对活细胞功能进行同步成像，因此，这种材料系统应被证明对研究神经元发育的基本方面有用。
