We report a water-soluble and non-toxic method to incorporate additional extracellular matrix proteins into gelatin hydrogels, while obviating the use of chemical crosslinkers such as glutaraldehyde. Gelatin hydrogels were fabricated using a range of gelatin concentrations (4%-10%) that corresponded to elastic moduli of approximately 1 kPa-25 kPa, respectively, a substrate stiffness relevant for multiple cell types. Microbial transglutaminase was then used to enzymatically crosslink a layer of laminin on top of gelatin hydrogels, resulting in 2-component gelatin-laminin hydrogels. Human induced pluripotent stem cell derived spinal spheroids readily adhered and rapidly extended axons on GEL-LN hydrogels. Axons displayed a more mature morphology and superior electrophysiological properties on GEL-LN hydrogels compared to the controls. Schwann cells on GEL-LN hydrogels adhered and proliferated normally, displayed a healthy morphology, and maintained the expression of Schwann cell specific markers. Lastly, skeletal muscle cells on GEL-LN hydrogels achieved long-term culture for up to 28 days without delamination, while expressing higher levels of terminal genes including myosin heavy chain, MyoD, MuSK, and M-cadherin suggesting enhanced maturation potential and myotube formation compared to the controls. Future studies will employ the superior culture outcomes of this hybrid substrate for engineering functional neuromuscular junctions and related organ on a chip applications.

中文翻译：

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用于神经肌肉组织工程应用的酶交联明胶-层粘连蛋白水凝胶。

我们报告了一种水溶性且无毒的方法，将额外的细胞外基质蛋白掺入明胶水凝胶中，同时避免使用戊二醛等化学交联剂。使用一系列明胶浓度（4%-10%）制造明胶水凝胶，其弹性模量分别对应于约 1 kPa-25 kPa，这是与多种细胞类型相关的基质刚度。然后使用微生物转谷氨酰胺酶在明胶水凝胶顶部酶促交联一层层粘连蛋白，形成双组分明胶-层粘连蛋白水凝胶。人诱导多能干细胞衍生的脊髓球体很容易粘附在 GEL-LN 水凝胶上并快速延伸轴突。与对照组相比，GEL-LN 水凝胶上的轴突表现出更成熟的形态和优异的电生理特性。GEL-LN 水凝胶上的施万细胞正常粘附和增殖，表现出健康的形态，并维持施万细胞特异性标记物的表达。最后，GEL-LN 水凝胶上的骨骼肌细胞实现了长达 28 天的长期培养而没有分层，同时表达更高水平的末端基因，包括肌球蛋白重链、MyoD、MuSK 和 M-钙粘蛋白，表明成熟潜力和肌管形成增强与对照相比。未来的研究将利用这种混合基质的优异培养结果来设计功能性神经肌肉接头和相关器官芯片应用。