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

Shoichiro Sumi, Masako Kawagoe, Rie Abe, Goichi Yanai, Kai Chiang Yang, Yasumasa Shirouzu

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)52-60
Number of pages9
JournalRegenerative Therapy
Volume7
DOIs
Publication statusPublished - Dec 1 2017

Fingerprint

Cell culture
Cell Culture Techniques
Embryoid Bodies
Suspensions
Stem cells
Nestin
Equipment and Supplies
Hydrostatic Pressure
Embryonic Stem Cells
Cell- and Tissue-Based Therapy
Tretinoin
Cell Size
Culture Media
Biomedical Research
Insulin
Hydrostatic pressure
Cell Count
Cells
Acids
Geometry

Keywords

  • Cell culture insert
  • Cell sphere
  • Embryoid body
  • Hanging drop
  • Mouse embryonic stem cell
  • Spheroid

ASJC Scopus subject areas

  • Biomedical Engineering
  • Developmental Biology
  • Biomaterials

Cite this

A multiple-funnels cell culture insert for the scale-up production of uniform cell spheroids. / Sumi, Shoichiro; Kawagoe, Masako; Abe, Rie; Yanai, Goichi; Yang, Kai Chiang; Shirouzu, Yasumasa.

In: Regenerative Therapy, Vol. 7, 01.12.2017, p. 52-60.

Research output: Contribution to journalArticle

Sumi, Shoichiro ; Kawagoe, Masako ; Abe, Rie ; Yanai, Goichi ; Yang, Kai Chiang ; Shirouzu, Yasumasa. / A multiple-funnels cell culture insert for the scale-up production of uniform cell spheroids. In: Regenerative Therapy. 2017 ; Vol. 7. pp. 52-60.
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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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