Efficient Embryoid Body Formation from Human iPS Cells
on Novel Microfabric Vessels

無断転載禁止Tatsuaki Miwa, Hiroki Sato, Alimjan Idiris and Hiromichi Kumagai
Kumagai Fellow Laboratory, Research Center, Asahi Glass Company, Japan

Abstract
Human iPS (induced pluripotent stem) cells have high potential
applications in regenerative medicine and drug discovery with theirability of differentiating into a wide variety of cell types. Suspension
culture of iPS cell aggregates, named as embryoid bodies (EBs), isone of effective methods for propagation and differentiation of theiPS cells. Furthermore, size and uniformity of the EBs are known to
be one of critical factors affecting the differentiation efficiency.
However, there are still technical limitations in the generation
method of large-number of EBs with uniform size by simple andeasy handling. To solve such problems, we attempted to apply novel
micro-fabricated culture wares (named EZSPHERE), on which large-

number of micro-wells are solely created by laser, followed by lowcell-adhesive coating. The diameter and depth of each micro-wellcan be altered around 200-1,000 and 100-400 micrometer,respectively. We confirmed that the EZSPHERE is very useful for
generating large-number of uniformly-sized EBs, when we inoculate
2.3 x 105 iPS cells into a standard type of the EZSPHERE (35 mm
dish with approximately 2,400 micro-wells) in differentiation medium.
After cultivation for 4 days, a typical Gaussian distribution wasobtained for diametric size (108±33 micrometer) of the generated
EBs with the total number of over 2,200 It was found that shape ofthe micro-wells is suitable for gathering inoculated cells and most ofIntroduction

the EBs were formed within 3-6 hours. In addition, it was also
confirmed that the obtained EBs could propagate at a good rate andmaintain uniformity in growth medium. Differentiation tendency of theEBs was also confirmed by induction into cardiomyocyte or nervecells. These results indicate that EZSPHERE is a useful tool for thecontrolled large-scale generation of EBs with uniform size and thedifferentiation capacity in a reproducible manner.
This study was performed as a part of the AMED (Japan Agency forMedical Research and Development) project “Research CenterNetwork for Regenerative Medicine”

Methods and Materials

Novel micro-fabricated vessels: EZSPHERE
Fabrication process

EB-formation process

CO2 gas laser

Inoculate iPSCs

Step-1:
2D culture

Step-2:EB-formation

Monolayer of iPSCs

Aggregates of iPSCs

Topography of micro-wells
Micro-wells
created on theΦ35-mm plastic
Petri dish(dissociated into singlecells before inoculation)

iPS cells & media

EB formation, proliferation & differentiation method

Inoculation

Cells drop into each micro-well
Plastic Petri dishes or plates

Differentiated cells

Differentiation(Dissociatedinto single cells)

On-feeder
orFeeder-lessiPS cells:
Human iPS cell lines 201B7 and253G1(passageuntil50),
purchased and licensed from theiPS Academia Japan , Inc.

Step-3:Differentiation

EZSPHERE

Differentiation

3~6 hours
About 100～400 μm

Abbreviations:
iPSCs: iPS cells
hiPSCs: human iPS cells
EBs: embryoid bodies

Topography of micro-wells(EZSPHERE)

Micro-wells of EZSPHERE are solely createdby CO2 gas laser beam on the plastic dishes
or plates, followed by coating with lowadhesive reagents (MPC polymer).

ResultsFigure 1. High efficient generation of EBs
with uniform size on the EZSPHERE(A) EBs formed in EZSPEHERE

Figure 2. EB size control with micro-wellsizes or inoculating cell densities

Figure 3. Improvement of EB viability by
optimizing culture condition on the EZSPHERE(A) Micro-well size and topography of two types of EZSPHERE

The following two approaches on the culture condition optimization are found to beeffective for improving formation efficiency and viability of EBs on the EZSPHERE.

#900(B) Histogram of EB size (diameter)800

155±34 (μm)(n=2411)#903
500 μm ←Well diameter→ 800 μm

100 μm ←Well depth→ 400 μm(A) EB viability

1,000After

400 cells/well

1,000 cells/well(#900)(#900)

1,000 cells/well(#903)

After
改善後

Live/Dead

Diameter (μm)(B) Diameter of EBs

Before
改善前

Scale bar: 400 μm

Two EZSPHERE models (code #900 & #903) and their micro-well sizes anddensities (A). EBs obtained by changing micro-well sizes (#900→#903) or celldensities(400→1000 cells/well) (B) . EB sizes increased by enlargement of
the micro-well size or increasing cell number inoculated into each micro-well.

Figure 5. High performance of maintaining
undifferentiation state on the EZSPHERE
Feeder-less culture

EBs on the EZSPHERE

SSEA4

SOX2

Oct 3/4

Day-2

Figure 6. Differentiation potency of EBs
evaluated by neural cell induction

Day-3

Day 0

Day-5

Inhibitors

Day-7

MediumPrimate ESCulture
condition

On-feeder

Day 1Day 8・SB-431542
・Dorsomorphin

Day 23
Dorsomorphin

Primate ES＋5% KSR

Primate ES

EZSPHERE

Chamber slide(suspension culture)(adhesion culture)(B) Confirmation of neural differentiation efficiency
EB diameter (μm)

Time courses of morphology (A) and diameter (B) of EBs created on theEZSPHERE with using the proliferation medium mTeSR1. For this analysis,
expanded iPS cells in feeder-less culture condition with mTeSR1 medium weredissociated into single cells and inoculated at density of 200 or 400 cells/microwell into EZSPHERE. Then, cultured for 7 days by half-exchange of culture
medium (mTeSR1) everyday for the analysis. The results indicated that EB size
increased effectively by time with remaining their smooth round shape and sizeuniformity.

EB formation

Differentiation

EZSPHERE
100ΦDishLow adhesion 100ΦDishImmunostaining:βIII tubulin
DAPI
Bar:400 mm

Day - 3~45 mL

10 mL
Modified
StemPro34

BMP-4
ROCKi

BMP-4, Activin AbFGF

0Bar:300 mm

Differentiation efficiency of EBs created on the EZSPHERE were determined byinduction of βIII tubulin-positive neural cells. Using the differentiation protocol (A),EBs were induced to neural cells and confirmed by Immunofluorescence staining(B). The result demonstrated differentiation ability of the EBs into βIII tubulinpositive neurites.

1IWPVEGF

3~4bFGF
VEGF

5~7Medium change every 2 or 3 days…

(B) Confirmation of cardiac differentiation efficiency
1. Contracting EBs (yellow arrow)

1000 μm(A) Schematic of the differentiation protocol

day 3(A) Schematic of the differentiation protocol

SSEA4

FACS analysis was performed for the iPS cells proliferated with mTeSR1 medium
in the conditions of feeder-less 2D culture on the dish and EB formation on theEZSPHERE for 4 days, respectively, in order to compare their maintaining levelsof undifferentiation state. In the FACS analysis, the pluripotency-associatedtranscription factors Sox2, Oct3/4 and SSEA4 were uses as undifferentiation
markers. It was found that no significant difference between two culture
conditions, indicating high capacity of maintaining undifferentiated state on theEZSPHERE.

day 2

Figure 7. Differentiation potency of EBs
evaluated by cardiac differentiation

mTeSR1
on LN521

Oct 3/4

day 1

Comparison of EB morphology & viability before and after optimizing the culture
conditions with KSR-supplementation and half-exchange of the culture medium (A).
Time course of survival cell ratio determined at day 1-3 for the EBs obtained in A (B).

2D culture

SOX250Scale bar:400 μm

400Day-1

Live/Dead0

Cell density in inoculation（cells/micro-well)
200

100

Viability (%)

200

Figure 4. Efficient proliferation of EBs in undifferentiating condition on the EZSPEHERE（Scale bar: 400 μm）(B) Ratio of survival cells

Before(B) EB sizes affected by the micro-well size or cell density

Fluorescence microscopy image of EBs obtained on the EZSPHERE (A). 2Dcultured iPS cells were inoculated into the EZSPHERE after dissociating into
single cells and stained with Calcein AM (green for living cells) next day.
Histogram of EB size (diameter) distribution (B). 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.

400

→150

(A well of 96-well plate type EZSPHERE)

200

Well number(Φ35-mm dish)4000Cell density in inoculation（cells/micro-well)←2,300(1) Supplementing the basal medium with Knockout Serum Replacement (KSR).
(2) Exchanging of a half volume of culture medium during the EB culture process.

Bright field images

Number of EBs

600(A) Morphology of EBs

Feeder-free culture medium:mTeSR1 medium (STEMCELLTechnologies), which used for
iPS cell culture on the Matrigel(CORNING)or
Laminin-521(BioLamina) .

●Step-1 : Proliferate hiPS cells with on-feeder or feeder-less condition.
●Step-2 : Dissociate hiPS cells into single cells and inoculate them intoEZSPHERE, where inoculated cells equally drop into eachmicro-wells 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.

Fluorescent images

About 200～1,000 μm

On-feeder culture medium:Primate ES medium (Reprocell).,which used for iPS cell culture on
SNL feeder cells treated withmytomycin C for inactivation.

2. α-actinin (red) DAPI(blue)

50 μm

EBs created on the EZSPHERE were differentiated to cardiomyocytes in above scheme(A). Most of EBs were contracting at day 15 (B)-1. EBs were dissociated and plated ongelatin coated slide at day14, followed by α-actinin and DAPI stains of cardiomyocytes
after 1 day culture (B)-2. Sarcomere alignment (high magnification) was observed.

Summary
The novel microfabric vessels EZSPHER, produced by laser-fabrication of usual plastic
dishes and plates, are useful for mass-production of EBs with uniform size.
Sizes of EBs created on the EZSPHERE are able to be controlled by choosing microwell size of the EZSPHERE and/or changing cell number to be inoculated.
EBs created on the EZSPHERE maintained their pluripotency state and possible to beefficiently growth with uniform size.
It was demonstrated by inductions of nerve cells and cadiomyocytes that EBs createdon the EZSPHERE kept their high differentiation potency.
Considering utilization of iPS cells for regenerative medicine, developing large-scaleand efficient iPS cell producing techniques are required. In this study, we demonstrated
that the novel micro-fabric culture vessels, EZSPHERE, enable to culture of EBs for bothcell expansion and differentiation processes as well as useful tool for the controlledlarge-scale generation of EBs with uniform size in a reproducible manner by simple andeasy handling. Furthermore, we are trying to attempt EZSPHERE techniques to morelarge-scale culture in the future.