BACKGROUND Animal cell-based systems have been critical tools in understanding tissue development and physiology, but they are less successful in more practical tasks, such as predicting human toxicity to pharmacological or environmental factors, in which the congruence between in vitro and clinical outcomes lies on average between 50 and 60%. Emblematic of this problem is the high-density micromass culture of embryonic limb bud mesenchymal cells, derived from chick, mouse, or rat. While estimated predictive value of this model system in toxicological studies is relatively high, important failures prevent its use by international regulatory agencies for toxicity testing and policy development. A likely underlying reason for the poor predictive capacity of animal-based culture models is the small but significant physiological differences between species. This deficiency has inspired investigators to develop more organotypic, 3-dimensional culture system using human cells to model normal tissue development and physiology and assess pharmacological and environmental toxicity. METHODS We have developed a modified, miniaturized micromass culture model using adult human bone marrow-derived mesenchymal progenitor cells (hBM-MPCs) that is amenable to moderate throughput and high content analysis to study chondrogenesis. The number of cells per culture was reduced, and a methacrylated gelatin (gelMA) overlay was incorporated to normalize the morphology of the cultures. RESULTS These modified human cell-based micromass cultures demonstrated robust chondrogenesis, indicated by increased Alcian blue staining and immunodetectable production of collagen type II and aggrecan, and stage-specific chondrogenic gene expression. In addition, in cultures of hBM-MPCs transduced with a lentiviral collagen type II promoter-driven GFP reporter construct, levels of GFP reporter activity correlated well with changes in endogenous collagen type II transcript levels, indicating the feasibility of non-invasive monitoring of chondrogenesis. CONCLUSIONS The modified hBM-MPC micromass culture system described here represents a reproducible and controlled model for analyzing mechanisms of human skeletal development that may later be applied to pharmacological and environmental toxicity studies.

中文翻译：

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用成人骨髓源间充质祖细胞修饰的高密度微团培养物对阑尾骨骼软骨发育进行建模。

背景基于动物细胞的系统已经成为了解组织发育和生理学的关键工具，但是它们在更实际的任务中不太成功，例如预测人类对药理或环境因素的毒性，其中体外和临床结果之间的一致性在于平均介于50％和60％之间。这个问题的体现是源自鸡，小鼠或大鼠的胚胎肢芽间充质细胞的高密度微团培养。尽管此模型系统在毒理学研究中的估计预测价值相对较高，但重大故障阻止国际监管机构将其用于毒性测试和政策制定。基于动物的培养模型的预测能力差的潜在根本原因是物种之间的微小但显着的生理差异。这种缺陷激发了研究人员开发更多的器官型3维培养系统，该系统使用人类细胞来模拟正常组织的发育和生理以及评估药理和环境毒性。方法我们使用成人骨髓源间充质祖细胞（hBM-MPCs）开发了一种改良的微型微团培养模型，该模型适于中等通量和高含量分析，以研究软骨形成。减少每种培养的细胞数，并加入甲基丙烯酸明胶（gelMA）覆盖物以标准化培养的形态。结果这些经过修饰的基于人类细胞的微团培养物表现出强大的软骨生成能力，阿尔辛蓝染色的增加和II型胶原蛋白和聚集蛋白聚糖的免疫检测产生以及阶段特异性软骨生成基因的表达表明了这一点。此外，在用慢病毒II型胶原启动子驱动的GFP报告基因构建体转导的hBM-MPC的培养物中，GFP报告基因活性的水平与内源II型胶原转录物水平的变化密切相关，这表明无创性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。通过增加的Alcian蓝染色和II型胶原蛋白和聚集蛋白聚糖的免疫检测产生以及阶段特异性软骨生成基因表达来表明。此外，在用慢病毒II型胶原启动子驱动的GFP报告基因构建体转导的hBM-MPC的培养物中，GFP报告基因活性的水平与内源II型胶原转录物水平的变化密切相关，这表明无创性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。通过增加的Alcian蓝染色和II型胶原蛋白和聚集蛋白聚糖的免疫检测产生以及阶段特异性软骨生成基因表达来表明。此外，在用慢病毒II型胶原启动子驱动的GFP报告基因构建体转导的hBM-MPC的培养物中，GFP报告基因活性的水平与内源II型胶原转录物水平的变化密切相关，这表明无创性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。此外，在用慢病毒II型胶原启动子驱动的GFP报告基因构建体转导的hBM-MPC的培养物中，GFP报告基因活性的水平与内源II型胶原转录物水平的变化密切相关，这表明无创性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。此外，在用慢病毒II型胶原启动子驱动的GFP报告基因构建体转导的hBM-MPC的培养物中，GFP报告基因活性的水平与内源II型胶原转录物水平的变化密切相关，这表明无创性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。说明了非侵入性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。说明了非侵入性监测软骨形成的可行性。结论本文所述的改良的hBM-MPC微质量培养系统代表了可重现和受控的模型，可用于分析人体骨骼发育的机制，以后可用于药理和环境毒性研究。