Features

EZSPHERE is a unique micro-fabricated plastic vessel and very useful for mass-production of EBs with uniform size.

EZSPHERE Variety Pack (35 mm Dish)

Product No.	4000-900SP	4000-901SP	4000-902SP
Diameter (µm)	approx. 500	approx. 200	approx. 500
Depth (µm)	approx. 100	approx. 100	approx. 200
No. of wells	approx. 2,700	approx. 9,200	approx. 2,700

Product No.	4000-903SP	4000-904SP	4000-905SP
Diameter (µm)	approx. 800	approx. 800	approx. 1,400
Depth (µm)	approx. 400	approx. 300	approx. 600
No. of wells	approx. 1,300	approx. 700	approx. 260

EB formation, proliferation and differentiation protocols

EB formation, proliferation and differentiation protocols

Fluorescence microscopy image of EBs obtained on the EZSPHERE (A). EBs created on the 35 mm dish type EZSPHERE were imaged and analyzed with the digital image analyzing software “Image J” to determine size distribution. Histogram of EB size (diameter) distribution. Fluorescence microscopy revealed that the almost EBs were alive with uniform size (B).

EB-size control with micro-well sizes or inoculating cell demsities

Two types of EZSPHERE #900 and #905 with micro-wells about 500 µm and 1,400 µm in diameter, respectively, were used to analysis the effect of the micro-well size or inoculated cell density on the EB sizes (A). Inoculation of iPSCs as 400 or 2,000 cells per micro-well on the same type of EZSPHERE #900 resulted in the formation of EBs with different sizes, while inoculating iPSCs as 9,000 cells per micro-well on the another EZSPHERE #905 resulted larger size of EBs (B). (Scale bars: 400 µm)

Proliferation of iPSCs with maintaining pluripotency

When iPSCs were cultured on EZSPHERE with feeder-free cell culture medium (mTeSR1), the formed EBs could proliferate at a good rate (A) with high viability (B). In the flow cytometry, these cells maintained high capacity of undifferentiated state (C).

Induction of dopaminergic neurons from the EBs

Differentiation of EBs into dopaminergic neuron was attempted by using the EZSPHERE continuously throughout a series of steps from the EB-formation to induction of midbrain dopaminergic neuron (A). Immunofluorescence staining revealed differentiation of the EBs into midbrain neuron, which tyrosin hydroxylase (TH: white arrows) and FOXA2 positive (B). Flow cytometry analysis (FACS) with Oct3/4 antibody indicated that there was almost no iPSCs remained without differentiation (C).

Induction of cardiomyocyte from the EBs

Differentiation of EBs, which prepared on the EZSPHERE, into cardiomyocytes was attempted (A). It was observed that most of EBs showed contracting (indicated with yellow arrow) at day 15 (B)-(1). EBs were dissociated and plated on gelatin coated slide, followed by α-actinin staining and sarcomere alignment (high magnification) was observed (B-2). Flow cytometry for cardiac troponin T (cTnT) positive cells revealed high differentiation efficiency (C).

1. Hiroyuki Koike, Ran-Ran Zhang, Yasuharu Ueno, Keisuke Sekine, Yun-Wen Zheng, Takanori Takebe, Hideki Taniguchi Nutritional modulation of mouse and human liver bud growth through a branched-chain amino acid metabolism Development 2017 144: 1018-1024; doi: 10.1242/dev.143032
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4. Taku Nakayama, Shimpei Otsuka, Tatsuya Kobayashi, Hodaka Okajima, Kentaro Matsumoto, Yuichiro Hagiya, Keiji Inoue Taro Shuin, Motowo Nakajima, Tohru Tanaka, Shun-ichiro Ogura Dormant cancer cells accumulate high protoporphyrin IX levels and are sensitive to 5-aminolevulinic acid-based photodynamic therapy Scientific Reports 6, Article number: 36478 (2016) doi:10.1038/srep36478
5. Ryohichi Sugimura, Deepak Kumar Jha, Areum Han, Clara Soria-Valles, Edroaldo Lummertz da Rocha, Yi-Fen Lu, Jeremy A. Goettel, Erik Serrao, R. Grant Rowe, Mohan Malleshaiah, Irene Wong, Patricia Sousa, Ted N. Zhu, Andrea Ditadi, Gordon Keller, Alan N. Engelman, Scott B. Snapper, Sergei Doulatov, George Q. Daley Haematopoietic stem and progenitor cells from human pluripotent stem cells Nature volume 545, pages 432–438 (25 May 2017) doi:10.1038/nature22370
   Application references of AGC tissue culture products Sep. 2015
6. Hiroki Sato, Alimjan Idiris, Tatsuaki Miwa, Hiromichi Kumagai
   Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells
   Scientific Reports 6, Article number: 31063 (2016) doi:10.1038/srep31063
7. Matsuura K, Seta H, Haraguchi Y, Alsayegh K, Sekine H, Shimizu T, Hagiwara N, Yamazaki K, Okano T
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   Scientific Reports 6, Article number: 21747 (2016) doi:10.1038/srep21747
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   A Simple Protocol for the Myocardial Differentiation of Human iPS Cells
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Brochures

EZSPHERE^® for Spheroid Formation (PDF 1.2MB)

Posters

Efficient Embryoid Body Formation from Human iPS Cells using Novel Microfabric Vessels (Poster from the 18th Takeda Science Foundation Symposium on Bioscience)
(PDF 5.2MB)

An Innovative Method of Embryoid Body Formation Using Novel Microfabric Vessels
(Poster from World Conference on Regenerative Medicine, Leipzig 2015)
(PDF 4MB)

Protocol

iPS spheroid formation in EZSPHERE devices

iPS spheroid formation in EZSPHERE (pdf)