Worthington Kristan S, Wiley Luke A, Kaalberg Emily E, Collins Malia M, Mullins Robert F, Stone Edwin M, Tucker Budd A
Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, The University of Iowa, 4111 Medical Education and Research Facility, 375 Newton Road, Iowa City, IA 52242, USA.
Acta Biomater. 2017 Jun;55:385-395. doi: 10.1016/j.actbio.2017.03.039. Epub 2017 Mar 27.
Recent advances in induced pluripotent stem cell (iPSC) technology have paved the way for the production of patient-specific neurons that are ideal for autologous cell replacement for treatment of neurodegenerative diseases. In the case of retinal degeneration and associated photoreceptor cell therapy, polymer scaffolds are critical for cellular survival and integration; however, prior attempts to materialize this concept have been unsuccessful in part due to the materials' inability to guide cell alignment. In this work, we used two-photon polymerization to create 180μm wide non-degradable prototype photoreceptor scaffolds with varying pore sizes, slicing distances, hatching distances and hatching types. Hatching distance and hatching type were significant factors for the error of vertical pore diameter, while slicing distance and hatching type most affected the integrity and geometry of horizontal pores. We optimized printing parameters in terms of structural integrity and printing time in order to create 1mm wide scaffolds for cell loading studies. We fabricated these larger structures directly on a porous membrane with 3µm diameter pores and seeded them with human iPSC-derived retinal progenitor cells. After two days in culture, cells nested in and extended neuronal processes parallel to the vertical pores of the scaffolds, with maximum cell loading occurring in 25μm diameter pores. These results highlight the feasibility of using this technique as part of an autologous stem cell strategy for restoring vision to patients affected with retinal degenerative diseases.
Cell replacement therapy is an important goal for investigators aiming to restore neural function to those suffering from neurodegenerative disease. Cell delivery scaffolds are frequently necessary for the success of such treatments, but traditional biomaterials often fail to facilitate the neuronal orientation and close packing needed to recapitulate the in vivo environment. Here, we use two-photon polymerization to create prototype cell scaffolds with densely packed vertical pores for photoreceptor cell loading and small, interconnected horizontal pores for nutrient diffusion. This study offers a thorough characterization of how two-photon polymerization parameters affect final structural outcomes and printing time. Our findings demonstrate the feasibility of using two-photon polymerization to create scaffolds that can align neuronal cells in 3D and are large enough to be used for transplantation. In future work, these scaffolds could comprise biodegradable materials with tunable microstructure, elastic modulus and degradation time; a significant step towards a promising treatment option for those suffering from late-stage neurodegeneration, including retinal degenerative blindness.
诱导多能干细胞（iPSC）技术的最新进展为生成患者特异性神经元铺平了道路，这些神经元对于神经退行性疾病的自体细胞替代治疗而言是理想的。就视网膜变性及相关光感受器细胞治疗而言，聚合物支架对于细胞存活和整合至关重要；然而，此前将这一概念付诸实践的尝试部分未成功，原因在于材料无法引导细胞排列。在本研究中，我们使用双光子聚合技术创建了宽度为180μm、具有不同孔径、切片距离、填充间距和填充类型的不可降解原型光感受器支架。填充间距和填充类型是垂直孔径误差的重要因素，而切片距离和填充类型对水平孔的完整性和几何形状影响最大。我们根据结构完整性和打印时间优化了打印参数，以便创建用于细胞接种研究的1mm宽支架。我们直接在具有3μm直径孔的多孔膜上制造这些更大的结构，并接种人iPSC来源的视网膜祖细胞。培养两天后，细胞嵌套其中并沿着支架的垂直孔延伸神经突，在直径为25μm的孔中细胞接种量最大。这些结果凸显了将该技术用作自体干细胞策略一部分，为患有视网膜退行性疾病的患者恢复视力的可行性。
细胞替代疗法是旨在为神经退行性疾病患者恢复神经功能的研究人员的重要目标。细胞递送支架对于此类治疗的成功通常必不可少，但传统生物材料往往无法促进重现体内环境所需的神经元定向和紧密堆积。在此，我们使用双光子聚合技术创建原型细胞支架，其具有密集排列的垂直孔用于光感受器细胞接种，以及小的、相互连接的水平孔用于营养物质扩散。本研究全面表征了双光子聚合参数如何影响最终结构结果和打印时间。我们的研究结果证明了使用双光子聚合技术创建能够在三维空间中排列神经元细胞且足够大以用于移植的支架的可行性。在未来的工作中，这些支架可以由具有可调微结构、弹性模量和降解时间的可生物降解材料组成；这是朝着为包括视网膜退行性失明在内的晚期神经退行性疾病患者提供有前景的治疗选择迈出的重要一步。
