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MICROFLUIDIC PRODUCTION OF YARN-BALL-SHAPE HYDROGEL BEADS AND
ITS APPLICATION TO HIGH-DENSITY CELL CULTIVATION
Ayaki Miyama, Masumi Yamada, Sari Sugaya, and Minoru SekiChiba
University, JAPAN
ABSTRACTWe present a microfluidic system to prepare
highly-unique hydrogel microbeads having yarn-ball morphology.
The
process includes the incomplete gelation of a sodium alginate
solution into a Ca-alginate hydrogel fiber, and its fragmentation
and folding in water-in-oil (W/O) droplets to form yarn-ball-shape
beads. The synthesized microbeads allow the efficient supply of
oxygen and nutrients to its center compared to homogeneous
spherical beads. We examined factors dominating the size, shape,
and uniformity of the beads, and performed high-density cell
cultivation (~1×108 cells/mL). The obtained hydrogel microbeads
would be highly useful as carriers or matrices for biological
immobilization, cultivation, and cell transplantation.
KEYWORDS: Hydrogel beads, Multiphase flow, Alginate, Cell
cultivation
INTRODUCTIONMicrofluidic technology is a powerful means to
prepare microscale objects including polymer particles and
hydrogel
substrates. Researchers have developed microfluidic systems to
prepare hydrogel particles for cell culture applications [1-3];
however, the morphologies of the obtained beads were either
spherical or planer (uniform height). Hydrogel particles having a
significantly-high surface-to-volume ratio is advantageous in terms
of the efficient supply of oxygen and nutrients to the cells
located at the center. Also, such particles would not prevent the
blood flow when they are transplanted into the blood vessels as the
cell carriers. Here we propose a unique microfluidic system to
prepare yarn-ball-shape Ca-alginate hydrogel beads by utilizing the
multiphase droplet flow and the fragmentation and the folding of
incompletely gelled hydrogel fibers. The obtained yarn-ball-shape
hydrogel beads would be more effective than spherical beads in
terms of the enhanced substrate transport because of the void
spaces. As an application, cell-incorporating beads were prepared
and used as a high-density cell cultivation matrix.
PRINCIPLEThe preparation process of the yarn-ball-shape hydrogel
beads is shown in Fig. 1. Alginate is used as the hydrogel
matrix, which forms hydrogels under the presence of multivalent
cations like Ca2+, Sr2+, and Ba2+. By continuously introducing a
sodium alginate (NaA) solution, a buffer solution, and a gelation
solution, the NaA solution is partially gelled, forming a hydrogel
fiber in the laminar flow. The introduction of the buffer solution
modulates the rapid gelation of alginate and prevents the channel
clogging. In the downstream, the incompletely-gelled fiber was then
fragmented and encapsulated into W/O droplets, by the outer
oil-flow introduced into the microchannel. The partially-gelled
fibers were folded in the droplets and completely gelled to form
yarn-ball-shape hydrogel beads.
Figure 1: Schematic diagram showing the formation of
yarn-ball-shape hydrogel beads. The incompletely-gelled hydrogel
fiber was fragmented and incorporated into the droplet, forming
yarn-ball-shape hydrogel beads.
EXPERIMENTALPDMS microfluidic devices were fabricated by using
soft lithography and replica molding techniques. The depth of
the
microchannel was uniform, 110 μm. The widths of the inlet
channels and the gelation channel were 100 μm and 400 μm,
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 825 15th
International Conference onMiniaturized Systems for Chemistry and
Life SciencesOctober 2-6, 2011, Seattle, Washington, USA
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Figure 2. The formation of hydrogel beads in PDMS micro-channels
when CaCl2 conc. was changed as indicated. At an appropriate conc.
of CaCl2 (10 mM), yarn-ball-shape beads were obtained. When lower
and higher concentra-tion of CaCl2, spherical beads and fiber
hydrogel were fab-ricated. Q1, Q2, Q3, and Q4 were 100, 10, 1, and
3 μL min-1, respectively .
Figure 3. Micrographs showing the hydrogel beads con-taining
1-μm green fluorescent particles. Microbeads were obtained (a) when
CaCl2 conc. was 5 mM, and (b, c) when it was 10 mM, (b) with and
(b) without containing 1% BSA in the gelation solution. When BSA
was added to gelation solution, the beads size became smaller. Q1,
Q2, Q3, and Q4were 100, 10, 1, and 3 μL min-1, respectively.
respectively. An aqueous solution of NaA (1.8%) was used, where
green fluorescent microbeads (Φ = 0.1 μm) were added to clearly
visualize the inner structure of the microbeads. To balance the
viscosities of the introduced solutions, a thickener (dextran, Mw
of 500,000) was added in the buffer and the gelation solutions, at
a concentration of 10%. Olive oil, the gelation solutioncontaining
Ca2+, the buffer solution, and the NaA solution were respectively
introduced from Inlets 1~4 as shown in Fig. 2 (a). The formation
process of the hydrogel beads was observed at the confluence and
the downstream areas 5 and 25 mm from the confluence. The obtained
hydrogel beads were washed with and stored in 0.1 M CaCl2 solution,
and their morphologies were observed. In the case of preparing
cell-incorporating beads, the buffer and gelation solutions were
adjusted to be isotonic by adding NaCl. NIH-3T3(mouse fibroblast)
or HeLa (human epithelial-like cell) cells were suspended in the 2%
NaA solution at a concentration of 3~5×106 cells/mL. The obtained
cell-incorporating beads were coated with poly-L-lysine membranes
to prevent the swelling and the deformation of the beads in the
medium, following the cultivation for at least 7 days.
RESULTS AND DISCUSSIONThe concentration of the gelation agent
would be a
critical factor to properly generate the yarn-ball-shape
hydrogel beads. At first, we therefore examined the effect of the
CaCl2 concentration on the formation behaviors of the beads. As
shown in Figure 2, parallelflows of the aqueous solutions were
formed at the microchannel confluence. When the CaCl2 conc. was as
high as 20 mM, the continuously formed fiber was not fragmented due
to the formation of solid hydrogel, andaqueous droplets were not
formed. On the other hand, when the CaCl2 conc. was lower than 10
mM, W/O droplets were generated at a point ~25 mm from the
confluence. The incompletely-gelled and fragmented hydrogel fibers
were observed in the droplets, when the CaCl2 conc. was 10 mM.
Figure 3 shows the hydrogel beads obtained at (a) a low (5 mM) and
(b,c) an appropriate concentration (10 mM) of CaCl2, respectively.
We confirmed that the concentration of Ca2+ was critical to produce
the yarn-ball-shape beads; when the CaCl2 concentration was too low
(5 mM),nearly-spherical beads were obtained since the droplets were
formed and the inner compositions were mixed before the formation
of the hydrogel fiber. The bead size was 300 ± 110 μm in diameter
(Fig. 3b), with the fiber width of ~30 μm. When a surfactant (1%
BSA) was added in the gelation solution (Fig. 3c), the beads size
became smaller and more uniform (180 ± 40 μm),because of the
decreased surface tension between the water and the oil phases.
Also, we could control the
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bead size and/or the fiber diameter composing the beads, by
changing the flow rates of the introduced solutions(Figure 4). The
diameter of the fiber composing the beads was changed from 10 to 30
μm. Note that the production of Ba-alginate microbeads was possible
when BaCl2 was used as the gelation agent.
As the application for the high-density cell cultivation, we
incorporated animal cells (NIH-3T3 and HeLa) into the NaA solution
and prepared the cell-containing hydrogel microbeads as shown in
Figure 5. The Initial cell densities were 4.7×106 cells/mL (HeLa)
and 3.0×106
cells/mL (NIH-3T3), respectively. The cell viabilities were kept
high (> 80%) even after the encapsulation into the beads. Cells
continuously proliferated to form spherical colonies inside the
hydrogel fiber, with the final cell densities of 2.4×107 cells/mL
(HeLa at Day 7) and 3.8×107 cells/mL (NIH-3T3 at Day 15),
respectively. This result shows that the hydrogel beads having the
void spaces would be suitable for the high-density cell
cultivation, which possibly enhance the supply of oxygen and
nutrients to the center of the beads.
CONCLUSIONA microfluidic system was presented for preparing
highly-unique hydrogel microbeads having yarn-ball morphology,
which are advantageous in terms of the effective molecular
transport compared to homogeneous spherical beads. The presented
hydrogel beads are usefulwhen used for tissue engineering, cell
transplantation, and bio-production processes.
ACKNOWLEDGEMENTSThis research was supported in part by
Grants-in-aid
for Scientific Research A (20241031) and for Young Scientists
(B) (23700554) from Japan Society for Promotion of Science (JSPS),
and for Improvement of Research Environment for Young Researchers
from Japan Science and Technology Agency (JST).
REFERENCES[1] S. Sugiura, T. Oda, Y. Izumida, Y. Aoyagi, M.
Satake, A. Ochiai, N. Ohkohchi, and M. Nakajima, “Size control of
calcium alginate beads containing living cells using micro-nozzle
array,” Biomaterials, 26, 3327-3331 (2005).[2] W. Tan and S.
Takeuchi, “ Monodisperse alginate hydrogel microbeads for cell
encapsulation,” Adv. Mater., 19, 2696-2701 (2007).[3] P. Panda, S.
Ali, E. Lo, B. G. Chung, T. A. Hatton, A. Khademhosseini and P. S.
Doyle, “Stop-flow lithography to generate cell-laden microgel
particles,” Lab Chip, 8, 1056-1061 (2008).
CONTACT*Minoru Sekitel: +81-43-290-3436; e-mail:
[email protected]
Figure 4. Fluorescence micrographs showing the obtained
yarn-ball-shape hydrogel beads, when the NaA flow rate Q4 was
changed as indicated. Q1, Q2, and Q3 were 100, 10, and 1 μL min-1,
respectively. The fiber diameters were (a) ~13, (b) ~18, and (c)
~28 μm, respectively.
Figure 5. Micrographs showing the (a) HeLa and (b) NIH-3T3 cells
encapsulated and cultivated in the yarn-ball-shape hy-drogel
microbeads. Cell viabilities were measured by Live dead assay. The
initial cell densities were 4.7×106 and 3.0×106 cells mL-1,
respectively. Q1, Q2, Q3, and Q4 were 100, 10, 0.5, and 2 μL min-1,
respectively.
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