背景:细胞球的形成是生物医学研究中的重要程序。基础研究和治疗应用需要大量均匀尺寸和形状的高质量细胞球。传统方法,包括悬滴法和悬浮培养,用于细胞球生产。然而,这些方法很耗时,细胞球不容易收获,并且很难控制细胞球的大小和几何形状。为了解决这些问题,一种新型的多漏斗细胞培养插入物被设计用于大小控制,容易收获,和扩大细胞球的生产。
方法:培养基质具有680个微漏斗,顶部宽度为1毫米,0.89mm深度,和0.5毫米见方的底部。小鼠胚胎干细胞用于测试新开发的设备。接种的胚胎干细胞在向下的培养基表面朝向底部开口沉降并聚集为胚状体(EB)。对于细胞球收获,将培养插入物的底部与另一个培养皿中的培养基表面接触,设备中的培养基通过静水压力随细胞球向下流动。
结果:易于收集具有均匀尺寸和形状的致密细胞球。球体的直径可以通过调节接种细胞密度来控制。与悬浮培养产生的EB相比,使用新插入物产生的EB的自发神经分化(巢蛋白和Tju1)和视黄酸诱导的内胚层分化(Pdx-1和胰岛素I)得到了改善。
结论:这种新型细胞培养插页将改善细胞球的未来研究,并有利于细胞疗法的临床应用。
BACKGROUND: Formation of cell spheres is an important procedure in biomedical research. A large number of high-quality cell spheres of uniform size and shape are required for basic studies and therapeutic applications. Conventional approaches, including the hanging drop method and suspension culture, are used for cell sphere production. However, these methods are time consuming, cell spheres cannot be harvested easily, and it is difficult to control the size and geometry of cell spheres. To resolve these problems, a novel multiple-funnel cell culture insert was designed for size controlling, easy harvesting, and scale-up production of cell spheres.
METHODS: The culture substrate has 680 micro-funnels with a 1-mm width top, 0.89 mm depth, and 0.5 mm square bottom. Mouse embryonic stem cells were used to test the newly developed device. The seeded embryonic stem cells settled at the downward medium surface toward the bottom opening and aggregated as embryoid bodies (EBs). For cell sphere harvest, the bottom of the culture insert was put in contact with the medium surface in another culture dish, and the medium in the device flowed down with cell spheres by hydrostatic pressure.
RESULTS: Compact cell spheres with uniform size and shape were collected easily. The diameter of the spheres could be controlled by adjusting the seeding cell density. Spontaneous neural differentiation (nestin and Tju1) and retinoic acid-induced endodermal differentiation (Pdx-1 and insulin I) were improved in the EBs produced using the new insert compared to those in EBs produced by suspension culture.
CONCLUSIONS: This novel cell culture insert shall improve future studies of cell spheres and benefit clinical applications of cell therapy.