Hydrogel‐based three‐dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of tissue specific cells embedded within a support material, such as a hydrogel. As hydrogel properties directly affect cell function, hydrogel composition is often tailored to the cell type(s) of interest and the functional objective of the model. Here, we develop a parametric analysis and screening method to identify suitable encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence‐based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.

中文翻译：

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鉴定促进封装的原代成肌细胞形成肌管的水凝胶制剂的筛选方法


基于水凝胶的三维（3D）细胞模型对生物工程和药物开发很有吸引力，因为它们可以更接近地模拟体外天然组织的细胞功能。一般来说，这些模型由嵌入支持材料（例如水凝胶）内的组织特异性细胞组成。由于水凝胶特性直接影响细胞功能，因此水凝胶成分通常根据感兴趣的细胞类型和模型的功能目标进行定制。在这里，我们开发了一种参数分析和筛选方法，以确定在甲基丙烯酰明胶（GelMA）水凝胶中从原代小鼠成肌细胞形成肌管的合适封装条件。通过改变 GelMA 重量百分比（wt%，控制凝胶模量）、细胞密度和基质胶浓度来研究基质特性对肌管形成的影响。收缩肌管通过成肌细胞融合形成，其特征是肌球蛋白重链 (MyHC) 表达。为了有效筛选凝胶配方，我们开发了一种基于荧光的读板仪测定法来量化凝胶样品中的 MyHC 染色，作为肌管形成的指标。我们观察到较低的 GelMA wt% 会导致 MyHC 染色（肌管形成）增加。细胞密度并未显着影响 MyHC 染色，而基质胶的加入增加了 MyHC 染色，但未观察到浓度依赖性效应。这些发现得到了在初始筛选中选择的样本中观察到的自发收缩肌管的支持。 这项工作提供了一种快速筛选用于开发 3D 细胞模型的水凝胶配方的方法，并为 3D 中原代小鼠成肌细胞形成肌管的凝胶配方提供了具体指导。