PDF(Pubmed)
Abstract：
UNASSIGNED: Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. To offer greater throughput for functional validation studies, we miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of in vivo muscle endogenous repair.
UNASSIGNED: The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7+ cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair \"in a dish\". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines.
UNASSIGNED: The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7+ cell expansion) to a known modulator of MuSC mediated repair (p38/β MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu.
UNASSIGNED: The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.
UNASSIGNED: The online version contains supplementary material available at 10.1186/s44330-024-00005-4.
UNASSIGNED: The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7+ cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair \"in a dish\". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines.
UNASSIGNED: The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7+ cell expansion) to a known modulator of MuSC mediated repair (p38/β MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu.
UNASSIGNED: The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.
UNASSIGNED: The online version contains supplementary material available at 10.1186/s44330-024-00005-4.
摘要：
■预测促进干细胞介导的内源性修复的分子的功能评估通常需要低通量并阻碍发现率的体内移植研究。为功能验证研究提供更大的吞吐量，我们小型化了，简化并扩展了先前开发的肌肉内源性修复（MEndR）体外试验的功能，该试验显示可捕获体内肌肉内源性修复的重要事件。
■mini-MEndR测定由设计用于96孔板的小型化纤维素支架组成，其孔隙被包封在基于纤维蛋白的水凝胶中的人成肌细胞浸润以形成工程化的骨骼肌组织。将凝血酶预吸附到纤维素支架上有助于原位组织聚合，一个关键的修改，使新用户能够迅速获得检测专业知识。生成3D肌管模板后，肌肉干细胞（MuSCs），使用改进的磁激活细胞分选方案从消化的小鼠骨骼肌组织中富集，被雕刻在工程模板内。鼠MuSCs被荧光标记，能够共同评估人和小鼠Pax7+细胞对药物治疗的反应。通过用肌毒素损伤肌肉组织以启动“在培养皿中”的内源性修复来引入再生环境。使用高含量成像系统在终点收集表型数据，并使用基于ImageJ的图像分析管道进行分析。
■小型化的格式和改进的制造方案将试剂成本降低了一半，动手播种时间减少了三倍，而图像分析管道节省了40小时的劳动力。通过评估2D和3D培养中的多个市售人类原代成肌细胞系，我们建立了细胞系选择的质量保证指标，使Myotube模板质量标准化。在微型化培养测定中概述了已知的MuSC介导的修复调节剂（p38/βMAPK抑制）的体内结果（增强的肌肉产生和Pax7细胞扩增）。但只有在干细胞和再生环境的存在下。
■微型化预测分析提供了一种简单的，缩放平台共同研究人和小鼠骨骼肌内源性修复分子调节剂，因此是加速肌肉内源性修复发现管道的有前途的策略。
■在线版本包含10.1186/s44330-024-00005-4提供的补充材料。
■mini-MEndR测定由设计用于96孔板的小型化纤维素支架组成，其孔隙被包封在基于纤维蛋白的水凝胶中的人成肌细胞浸润以形成工程化的骨骼肌组织。将凝血酶预吸附到纤维素支架上有助于原位组织聚合，一个关键的修改，使新用户能够迅速获得检测专业知识。生成3D肌管模板后，肌肉干细胞（MuSCs），使用改进的磁激活细胞分选方案从消化的小鼠骨骼肌组织中富集，被雕刻在工程模板内。鼠MuSCs被荧光标记，能够共同评估人和小鼠Pax7+细胞对药物治疗的反应。通过用肌毒素损伤肌肉组织以启动“在培养皿中”的内源性修复来引入再生环境。使用高含量成像系统在终点收集表型数据，并使用基于ImageJ的图像分析管道进行分析。
■小型化的格式和改进的制造方案将试剂成本降低了一半，动手播种时间减少了三倍，而图像分析管道节省了40小时的劳动力。通过评估2D和3D培养中的多个市售人类原代成肌细胞系，我们建立了细胞系选择的质量保证指标，使Myotube模板质量标准化。在微型化培养测定中概述了已知的MuSC介导的修复调节剂（p38/βMAPK抑制）的体内结果（增强的肌肉产生和Pax7细胞扩增）。但只有在干细胞和再生环境的存在下。
■微型化预测分析提供了一种简单的，缩放平台共同研究人和小鼠骨骼肌内源性修复分子调节剂，因此是加速肌肉内源性修复发现管道的有前途的策略。
■在线版本包含10.1186/s44330-024-00005-4提供的补充材料。
