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™

 EZSPHERE™ Variety Pack infomation

Protocol 

1. Protocol and case studies (iPS spheroid formation in EZSPHERE devices)
2. Protocol for generating and inducing a large number of uniformly sized hiPSC spheroids them for cardiac differentiation

Technical Information

1. Test of anticancer drug sensitivity 
2. Growth and ATP activity of cancer cell spheroid
3. Examples of Culturing 10 cancer cell lines

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