用于定向骨骼肌肌管长期培养的微模塑明胶水凝胶的制备

Fabrication of Micromolded Gelatin Hydrogels for Long-Term Culture of Aligned Skeletal Myotubes.

作者信息

Suh Gio C, Bettadapur Archana, Santoso Jeffrey W, McCain Megan L

机构信息

Laboratory for Living Systems Engineering, Department of Biomedical Engineering, USC Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA.

Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA.

出版信息

Methods Mol Biol. 2017;1668:147-163. doi: 10.1007/978-1-4939-7283-8_11.

DOI:10.1007/978-1-4939-7283-8_11

PMID:28842908

Abstract

Cultured skeletal myotubes are a powerful in vitro system for identifying mechanisms of skeletal muscle development and disease. However, skeletal myotubes routinely delaminate from conventional culture substrates after approximately 1 week, which significantly hampers their utility for in vitro disease modeling and drug screening. To address this problem, we fabricated micromolded gelatin hydrogels as culture substrates that are more biomimetic than conventional substrates. On micromolded gelatin hydrogels, C2C12 skeletal myoblasts align and differentiate into skeletal myotubes that are stable in culture for multiple weeks. With this protocol, we detail three key steps: (1) Fabrication of micromolded gelatin hydrogels; (2) Culture of mouse C2C12 myoblasts and differentiation into myotubes; and (3) Quantification of myotube morphology. These substrates have many applications for skeletal muscle disease modeling and drug screening over longer time scales.

摘要

培养的骨骼肌管是用于识别骨骼肌发育和疾病机制的强大体外系统。然而，骨骼肌管通常在大约1周后会从传统培养底物上脱层，这显著阻碍了它们在体外疾病建模和药物筛选中的应用。为了解决这个问题，我们制备了微模塑明胶水凝胶作为比传统底物更具仿生学特性的培养底物。在微模塑明胶水凝胶上，C2C12骨骼肌成肌细胞排列并分化为骨骼肌管，这些骨骼肌管在培养中可稳定存在数周。通过本方案，我们详细介绍三个关键步骤：(1) 微模塑明胶水凝胶的制备；(2) 小鼠C2C12成肌细胞的培养及分化为肌管；(3) 肌管形态的量化。这些底物在更长时间尺度的骨骼肌疾病建模和药物筛选中有许多应用。