formation of capillary bed in micro-pore-embedded microfluidics · 2016. 4. 11. · formation of...
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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
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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.
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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; [email protected]
† 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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