MICROFLUIDIC SPATIAL CONTROL OF STEM CELL DIFFERENTIATION

J. Kawada1,3, H. Kimura1,3, H. Akutsu2,3, Y. Sakai1,3 and T. Fujii1,3

1Institute of Industrial Science, The University of Tokyo, Tokyo, Japan 2National Research Institute for Child Health and Development,

Tokyo, Japan 3JST CREST, Tokyo, Japan

14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands

授課老師 : 劉承賢 學生 : 陳冠宇

Abstract Introduction Material and Method Experimental Results and Discussion Conclusion

Outline

Develop:A technique to control differentiation of mouse pluripotent stem cells by generating chemical distributions in the culture environment on a microfluidic device.

Chemicals injected into the lower channel are transported

through the membrane to the upper channel.

Demonstrated:The differentiation of mouse induced Pluripotent Stem Cells (iPSCs) can be spatially controlled according to the laminar flow patterns in the lower channel.

Abstract

Pluripotent stem cells (PSCs):Have the potentials to indefinitely proliferate and to differentiate into derivatives from three germ layers

For the purpose, it is required to establish the way to

intentionally induce differentiation into specific cells.

There is almost no ways to control the spatial distribution of molecules in the conventional culture vessels.

Introduction

Microfluidic method have been proposed for exposing cultured cells to chemicals in spatially resolved ways.

The present study aims at the development of a microfluidic method to generate stable chemical distribution over a long period of time.

Introduction

Material and Method

The key component:--Lower channel for controlling fluid--Upper channel for cell culture

Material and Method

Material and Method

The distribution of the chemical concentration generated in the upper channel was evaluated by measuring the fluorescence intensity of FITC and GFP.

Result of fluorescence intensity along the channel width:

Experimental

Two different patterns:

Experimental

First, the cells suspension (107 cells / mL) was injected into upper channel

Then its inlet and outlet were closed. After the seeded cells attached to the membrane coated

with fibronectin, two types of media--Leukemia inhibitory factor (LIF)--Retinoic acid (RA)were injected into the lower channel at 2 µL/min each.

The miPSCs seeded in the channel are exposed to these

factors and exhibit their response according to their positions in the channel.

Results and Discussion

On the day 4 of culture, GFP and DAPI fluorescence intensity in the upper channel were measured

--As compared with fluoresceince intensity distribution of GFP on the day 1 --miPSCs could be differentiated locally following the laminar flow patterns formed in the lower channel --while the distribution of the cells stayed spatially constant

Results and Discussion

In Figure 5(b), the fluorescence intensity is decreasing along the channel width meaning that the cells located in the left side of the channel are differentiated and the expression of Nanog is lowered.

In Figure 5(c), the fluorescence intensity in the middle of

the channel is higher than the area close to the side walls By changing the laminar flow pattern in the lower channel,

differentiation states of miPSCs could be controlled in a position-dependent manner.

Results and Discussion

Developed:A method to control the differentiation of PSCs, using a device having two compartmentalized channels (lower and upper) separated by the porous membrane.

Demonstrated by using the method:Position-dependent differentiation patterns of miPSCs were successfully controlled according to the laminar flow patterns formed in the lower channel.

Advantage of the device:To generate the static chemical distribution by using compartmental channels and the porous membrane.

Conclusion

Thank you