A multiple-funnels cell culture insert for the scale-up production of uniform cell spheroids

HANDLE Open Access
  • Sumi, Shoichiro
    Laboratory of Organ and Tissue Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University
  • Kawagoe, Masako
    Laboratory of Organ and Tissue Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University･Kuraray Co., Ltd.
  • Abe, Rie
    Laboratory of Organ and Tissue Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University･Graduate School of Life and Medical Sciences, Doshisha University
  • Yanai, Goichi
    Laboratory of Organ and Tissue Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University
  • Yang, Kai-Chiang
    Laboratory of Organ and Tissue Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University･School of Dental Technology, College of Oral Medicine, Taipei Medical University
  • Shirouzu, Yasumasa
    Laboratory of Organ and Tissue Reconstruction, Institute for Frontier Life and Medical Sciences, Kyoto University･Department of Stem Cell Biology, Kansai Medical University

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Abstract

Introduction: 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.

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