Formation of Capillary Bed in Micro-pore-embedded Microfluidics *Soojung Oh

1†, Hyunryul Ryu

2†, Dongha Tahk

1, Jihoon Ko

1 and Noo Li Jeon

1, 2

1 School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, KOREA

2 Institute of Advanced Machines and Design, Seoul National University, Seoul, KOREA

ABSTRACT

In this manuscript, we propose a novel microfluidic platform to culture capillary bed which can be

considered invaluable system for further experiment in the field of organ-on-a-chip. Microvessel network

is assembled within the designed micro-channel and through the micropore, cell spheroid can be accessed

having direct contact with microvessel network. Following our presented fabrication method, micro-pore-

embedded membrane covers top of microchannel structure and “Open-top” microchannel can be made.

By controlling the size of the pore and channel width, designed capillary bed can be assembled.

KEYWORDS: Capillary Bed, Microfluidics, Body-on-a-Chip

INTRODUCTION

Constant research to vascularize tissue in vitro is ongoing big research topic all over the world as

blood vessel is the vital conduit to maintain human body. Tissues and organs cultured outside our body

cannot survive for long without nutrients supplied through blood vessel. Also, it is considered impossible

to grow cell spheroid larger than few hundred microns as core cells might suffer hypoxia and lead to cell

death. For this reasons, many researchers propose novel methods to culture pre-vascularized tissues and

blood vessel itself [1, 2]. However, in vitro research have been limited due to the lack of methodology to

study the interactions between cell or tissues with blood vessel network.

Since the microfluidic system was presented as a suitable solution to narrow down the gap in

differences between in vivo and in vitro experiment, several designed organ-on-a-chip devices are

introduced [3]. Some of these platforms includes hydrogel-based three-dimensional cell culture methods

to make the in vitro environment similar to the in vivo system. Meanwhile, the microfluidic platforms to

assemble endothelial cells to blood vessel network were also established. Based on the hydrogel culture

technology, Kim et al., presented the in vitro vessel network-making protocol in the microfluidic device

[2]. Recently, these advances were about to be integrated to show the proper model for the investigation

of the trans-endothelial migration of the cancer or immune cells.

In spite of great interest in culturing

blood vessel and small tissues at the same

time, not many work have been proposed

due to high methodological barrier to

overcome. Here in this manuscript, we

proudly introduce our novel microfluidic

platform to co-culture blood vessel

network and cell spheroid enable to

observe complete contact between them.

Our device figurate capillary bed as it

provide blood vessel and enough place to

culture small tissues or cell spheroids.

Complete contact between cell spheroid

and blood vessel might give opportunity to

observe angiogenesis in nature driven

pathway. Here, we address blood vessel

formation according to channel width and

achieved long vessel up to 5 mm.

Figure 1. “Open-top” microfluidic device and experimental

scheme. (A) Real size microfluidic device compared to penny.

(B) Capillary bed is complex of dense microvessel and tissue.

(C) 6 mm diameter sink is placed on top of designed micro-

channel and micropore connects two section. HDMEC assem-

bles capillary vessel while co-cultured with LF.

603978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA

EXPERIMENTAL

The bottom line of our designed microchip is

“micropore” which can be fabricated following

our own method. This micropore connects upper

and lower section of the device allowing whole

culture platform to be synchronize in one

(Figure 1 C). Point of view from the lower

microchannel, their holed ceiling connected to

upper sink direct “Open-top” situation. Through

the “Open-top”, microchannel in the middle can

be provided with additional media perfusion and

this greatly enhance culture environment. This

enables to design wider microchannel for cells

to live.

Fabrication of the device starts with

conventional photolithography and soft

lithography method (Figure 2). Unlike usual

mold design, our mold is fabricated to produce

inverted replica PDMS pre-mold. This pre-mold

piece have micro-pillars and plasma bonded to

cover slip. Empty space is then filled with

Teflon and completely dried in dry oven. Once

more empty spaced is filled with degassed

PDMS and cured. Pre-mold is forced to detach from the cover slip. Post-mold with 6 mm diameter hole is

prepared and plasma bonded. Pre-mold can be easily removed as we have coated the mold surface with

Teflon in advance and there micro-pore-embedded membrane is derived. Size of the micropore is 200μm.

Reservoirs and holes in need is punched out and plasma bonded to glass coverslip. Completed device is

stored in dry oven for 24 hours before experiment to maintain hydrophobicity.

RESULTS AND DISCUSSION

To verify the performance of our “open-top” microfluidic device, we monitored the growth of the

blood vessel network over several days. As in previous report, human dermal microvascular endothelial

cells (HDMECs) were mixed with fibrin gel and injected within center channel. Human lung fibroblasts

(LFs) were also mixed with fibrin gel and injected in both side of channel. In the presence of LFs,

sufficient growth factors are supplied to HDMECs and they can be induced to assemble a lumen [2].

Figure 2. Fabrication procedures of micro-pore embed-

ded microfluidics.

Figure 3. Media perfusion through micropore enhance culture environment. Red dotted circle indicate the mi-

cropore. Compared to control, complete microvessel formation is observed in “open-top” device and this ena-

bles longer vessel formation up to 5mm channel width. Tubulin is stained green and nuclei is stained in blue.

604

Media is supplied to HDMECs through

media channel and also from the upper

sink. In the presence of micropore,

sufficient media supply is possible and

address cell survival through the whole

channel. This enables to design wider

channel and here we have achieved long

vessel up to 5 mm (Figure 3).

To visualize the perfusability and

chemical diffusion, rhodamin is

introduced to the inner-side of lumen

(Figure 4 A) and FITC-dextran (10kDa)

were sprayed over the sink to access the

outer-side of the lumen. Rhodamin in the

inner stream was observed to be limited to

be diffused out by the hydrostatic pressure

given from outer side. This status

sustained for more than 30 minutes which

means fluidic isolation between both sides

of the network has been performed

(Figure 4 B). This character might further

help to solve the medium supply problem

in co-culture.

CONCLUSION

Here we have proposed novel microfluidic device to address long vessel assembly. Through “Open-

top”, we were able to deliver sufficient media supply to every corner of wide channel. Different with

conventional membrane, the micropore is designed large enough for cell spheroid to settle down and

enables direct contact with cell complex lower channel. Also we have observed selected fluid delivery.

We estimate excellent potential to this platform and expect this device to be used as a new breakthrough

of science technology beyond tissue engineering.

ACKNOWLEDGEMENTS

This work was supported by the Brain Korea 21 Plus Project in 2015 (Grant No. F14SN02D1310),

the National Research Foundation funded by the Ministry of Education (Grant No. NRF-

2015R1A2A1A09005662) and the Korean Health Technology R&D Project, Ministry of Health & Wel-

fare, Republic of Korea (Grant No. HI14C14000).

REFERENCES

[1] R. K Jain, P. Au, J. Tam, D. G Duda and D. Fukumura, “Engineering vascularized tissue,” Nature

Biotechnology, 23(7) 821-823, 2005.

[2] SD Kim, HJ Lee, MH Chung and NL Jeon, “Engineering of functional, perfusable 3D microvascular

networks on a chip”, Lab Chip, 13(8), 1489-1500, 2013.

[3] D. Huh, G. A. Hamilton and D. E. Ingber, “From 3D cell culture to organs-on-chips”, Trends in Cell

Biology, 21(12) 745-754, 2011.

CONTACT

* Soojung Oh ; phone: +82-10-9420-0060; soojung@snu.ac.kr

† This authors contributed equally to this work.

Figure 4. (A) Selective fluid delivery is performed with assem-

bled microvessel. Rhodamin (Red) is introduced in the intra

side of the lumen and FITC-dextran (Green) is sprayed over

the sink to access the external side of lumen. (B) Chemicals

diffusion is partially blocked by the barrier function of mi-

crovessel and this status sustained for 30 minutes.

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