EZSPHERE™ is specifically designed for creating a large number of spheroids and EBs with uniform size.

Micro-wells of EZSPHERE™ are solely created by CO2 gas laser on the plastic dishes or plates, followed by coating with low adhesive reagents (MPC polymer). Because micro-wells are closely positioned each other in the plastic culture ware, inoculated cells equally drop into each well.

Innovative culture mothod for iPSCs using the EZSPHERE™ : ZSPHERE™ can be used for dveloping large-scale and efficient iPSCs producing techniques.

Step-1 : Proliferate hiPS cells with on-feeder or feeder-less condition.

Step-2 : Dissociate hiPS cells into single cells and inoculate them into EZSPHERE™, where inoculated cells equally drop into each micro-well and form aggregate “EBs” in 3 to 6 hours.

Step-3 : Proliferate hiPS cells in the EB form with un-differentiation medium. Differentiate the EBs with using differentiation medium.

High efficient generation of EBs with uniform size on the EZSPHERE™

(A) Fluorescence microscopy image of EBs obtained ont he EZSPHERE™. 2D cultured iPScells were inoculated into the EZSPHERE™ after dissociating into single cells and stained with CalceinAM(greenforlivingcells ) next day.

(B) Histogram of EBsize (diameter) distribution. EBs created on the 35-mmΦDishtype EZSPHERE™ were imaged and analyzed with the digital image analyzing software “Image J” to determine size distribution. The Gaussian distribution of EB size indicated uniformly sized EBs in the EZSEHERE™.

EB size is controlled by changing the inoculation cell number and/or micro-well size of EZSPHERE™ 

(A) Phase contrast image of iPSC aggregate generated from different number of iPSCs as 400 or 2,000 cells per micro-well in EZSPHERE™#900 or 9,000 cells per micro-well in Larger size of microwell EZSPHERE™#905 (diameter = 1,400 μm). Scale Bar = 400 μm

(B) The size of iPSC aggregate was uniform and could be controlled by inoculation number of iPSCs and/or micro-well size.

iPSCs grown as EBs with feeder-free cell cultural medium on the EZSPHERE™ maintained their puripotency

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).

1) Dopaminergic neuron differentiation in EZSPHERE™

(A) Differentiation of hiPC aggregates into dopaminergic neuron was attempted by using the EZSPHERE™ continuously throughout a series of steps from the hiPSC aggregate-formation to induction of midbrain dopaminergic neuron.

(B) Phase-contrast images of a time course of 12 days.

(C) Immunostaining for the midbrain progenitor marker FoxA2 and neural marker βIII-tubulin at day 12.

(D) Flow cytometry analysis with Oct3/4 antibody indicated that there were almost no iPSCs remained without differentiation.

(E) Immunostaining for the TH, Tyrosine hydroxylase and relative value of dopamine secretion in Low and High-KCl measurements by ELISA.

2) Cardiomyocyte differentiation in EZSPHERE

(A) Culture scheme. Cell aggregates were cultured in EZSPHERE™#900 (100mm Dish) for 4 days, and transferred 2D low cell attach dish after medium change.

(B) The size and microscopic image of Cell aggregates harvested at day 4.

(C) Immunostaining of, dissociated and re-plated cells at day 10 after day 1 for cardiomyocyte-specific maker α-actinin and Cardiac Troponin T, cTnT. The majority of cells had cardiomyocyte-specific structures, sarcomere.

(D) Flow cytometry analysis shows over 80% cells were positive for cTnT.

  Watch the videos below to learn how to use EZSPHERE™

Referred protocol: 

Hiroki Sato, Alimjan Idiris, Tatsuaki Miwa, Hiromichi Kumagai
Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells
Scientific Reports, 2016 Aug 10:6:31063. doi:10.1038/srep31063

References: 

1) Stem Cell & Differentiation