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DOI: 10.1002/adma.200700433

Monodisperse Alginate Hydrogel Microbeads for CellEncapsulation**

ByWei-Heong TanandShoji Takeuchi*

This Communication describes the production of monodis-

perse alginate hydrogel microbeads (94150 lm) using a

method that combines internal gelation method with T-junc-

tion droplet formation in microfluidic devices. The use of cal-

cium carbonate (CaCO3) nano-particles allows internal gela-

tion method to be applied to micro-scale production for the

first time, and microfluidic devices allow us to produce

microbeads with narrow size distributions. Our approach not

only allows easy control over bead size by varying flow

parameters, but also allows better monodispersity and controlover the shape of the hydrogel beads compared to conven-

tional external gelation methods performed in microfluidic

devices. Both blank and cell encapsulating alginate hydrogel

beads of various shapes were successfully produced using this

approach in non-silanized/silanized poly(dimethylsiloxane)

(PDMS) devices. Also, we demonstrated that the gelation

conditions in our approach were mild enough to encapsulate

mammalian cells (Jurkat) without loss of their viability, and

studied the effect on cell viability with varying concentrations

of CaCO3.

Alginates are anionic polysaccharides extracted from sea-

weeds composing of b-D-mannuronic acid and a-L-guluronic

acid, and they form hydrogels with multivalent cations such asCa2+, Ba2+, or Fe3+. Due to their resemblance to the natural

extracellular matrix, alginate hydrogels have been employed

successfully in three-dimensional cell-hydrogel scaffolds for

tissue engineering,[1,2] and in the encapsulation of trans-

planted (allogenic or xenogeneic) cells in alginate hydrogel

beads. The alginate hydrogel provides an immunoisolation

barrier for the cells,[36] potentially allowing transplantation

without the need for immunosuppression. However, there

remain several issues that limit the use of cell encapsulation

technology for transplantation, namely: (i) Large bead size.

Size of these beads should approach that of the cells/tissues to

be encapsulated to allow a high degree of permeability for

rapid exchange of nutrients into and waste out of the beads by

diffusion, thereby improving viability of cells/tissues.[4] How-

ever, conventional techniques[7] can only produce beads in the

order of several hundred micrometers; (ii) Wide size distribu-

tion. Monodisperse beads are important to accurately esti-

mate the amount of transplanted cells introduced into a pa-

tient; (iii)Deformed beads. Morphology of the beads is also

an important parameter as deformed beads might reduce the

biocompatibility or make implantation more difficult sincebeads carrying a tail are reported to cause fibrotic overgrowth

on surround tissue;[4,5] (iv)Process repeatability and reproduc-

ibility[6] in the production of uniform capsules have to be im-

proved for actual clinical applications.

Highly reproducible monodisperse droplets can be obtained

using T-junction[8] or flow-focusing methods[9] in micro-de-

vices, and several groups[10,11] have attempted to utilize such

techniques to produce alginate hydrogel beads; their strate-

gies typically involved using micro-devices to form emulsions

of Na-alginate droplets in an organic phase, followed by the

external addition of Ca2+ ions in the form of calcium chloride

(CaCl2) solution. Rapid gelling behavior of alginate in this

external gelation approach made it difficult to produce well-defined and homogenous alginate hydrogel beads, as evident

from their images of the final alginate beads obtained. [10,11]

Moreover, the absence of surfactants in such systems often re-

sulted in undesired fusion of Na-alginate droplets before gela-

tion, resulting in high polydispersity of the beads. [10]

Here, we employ the internal gelation method[1214] to pro-

duce alginate hydrogel beads. This method involves dispersing

an insoluble (or slowly soluble) calcium complex in the Na-algi-

nate solution. Upon pH reduction, Ca2+ ions are released from

the calcium complex, crosslinking the alginate to form a homo-

geneous hydrogel. Various groups[12,13] had tried but were un-

able to produce monodispersed alginate hydrogel beads with in-

ternal gelation using conventional emulsification methods. To

date, none had attempted to apply internal gelation in micro-

devices for the following reasons: (i) CaCO3 was often chosen

as the calcium complex for internal gelation. Commercial

CaCO3 powder (q= 2830 kg m3), consisting of grains with

> 5 lm, will sediment 100lm (typical microchannel dimen-

sions) in water in a few seconds, rendering it an unsuitable tech-

nique for microfluidic devices; (ii) Large CaCO3 particle sizes

make it difficult to ensure a homogeneous dispersion within the

alginate, which is important for the production of small mi-

crobeads (

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(Fig. 2a, b, and d), where AR is defined as the ratio of the

length (l) to the width (w) of the bead. These discoid beads

exhibit both narrow polydispersity in size (C.V. < 3.6 %,

Fig. 2a) and great uniformity in shape (C.V.AR = 2.65.1 %,

Fig. 2b), which is not possible with conventional methods.

With increasingQc, size of the beads gradually decreased and

the beads adopted a more spherical shape (AR approaches

unity); increase inQcshortenedsand offset the effect of high-

erv, as a result spherical beads were produced at high Qc. By

tuning flow parameters and concentration of the reactants, we

can control both size and morphology of the alginate hydrogel

beads. These shape-controllable monodisperse hydrogel beads

can be used as carriers in drug delivery systems [16,17] when

enzymes and proteins are immobilized in the hydrogel.

Production of blank alginate microbeads was performed in

PDMS devices without surface modifications to the channel

walls (Fig. 3a). However, when we at-

tempted to encapsulate mammalian

cells (Jurkat) in alginate hydrogel beads

with the same system, we observed wet-

ting of the PDMS channel (Fig. 3b) by

the Na-alginate solution. Initially, drop-

lets were sheared off at the T-junction,

but as the front of the dispersed phase

gradually advanced downstream due to

wetting, droplets were sheared off

farther and farther downstream. Even-

tually, the stream of acidic oil caused

the front of the Na-alginate stream to

gelate and droplets were randomly

pinched off, producing polydisperse

droplets. This phenomenon was ob-

served at all the flow rates tested. As

the main difference between producing

blank and cell encapsulating hydrogelbeads was the presence of cells, we pos-

tulate that this wetting of the channel

walls is due to the deposition of pro-

teins, carbohydrates and cell debris,

which changes the surface energy of the

channel walls gradually from hydropho-

bic to hydrophilic. Silanizing the walls

of the microchannel solved this problem

(Fig. 3c). Figure 4a shows how the size

of the cell encapsulating hydrogel beads

varies withQc. Beads with length/diam-

eter ranging from 100150 lm were ob-

tained for the range of flow rates tested.All the beads were spherical

(AR< 1.03, C.V.AR < 3.0%) with a nar-

row size distribution (C.V. < 3.2 %)

apart for those produced at

Qc =0.2 mLh1, which were discoidal

(AR= 1.2, C.V.AR = 5.9%) with a wider

size distribution (C.V.= 5.7 %). Al-

though the dispersed phase did not wet

the channel walls at Qc = 0.2 mLh1, we observed from high

speed camera images that the droplet shear-off point fluctu-

ated between the corner of the T-junction, and at a point

slightly downstream of the T-junction. The increase in polydis-

persity of the beads at Qc =0.2 mLh1 is probably due to the

fluctuation of the droplet shear-off point, although the reason

behind this fluctuation is not clear and the phenomenon is not

observed at higher Qc. The procedure for extracting cell en-

capsulating beads from corn oil was the same as that for blank

beads except hexadecane was used instead of hexane. Hexane

is an excellent solvent for corn oil and has a low boiling point

(69 C). Any residual hexane that had not been aspirated off

would evaporate away, and thus less rinsing was required

when hexane was used. Nevertheless, when it was used to ex-

tract cell encapsulating hydrogel beads, all the cells died. To

maintain the viability of the cells, we had to replace hexane

698 www.advmat.de 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater.2007,19, 26962701

Figure 2. a) Plot of the size of alginate hydrogel microbeads versus flow rate of the continuous

phase (corn oil with lecithin (2 % w/w)) for different concentrations of reactants. The rate of flowof the disperse phase, Qd, was 20 lL h1. b) Plot of the AR of alginate hydrogel microbeads versus

flow rate of the continuous phase for different concentrations of reactants. AR is defined as the ra-tio of the length (l) to the width (w) of the bead. (see image (d)) Images (c)-(f) show the beadsformed at various flow rates indicated in (a). These beads were extracted from corn oil and resus-pended in cell culture medium. Inset in (d) shows 3 beads being stacked together, and the thick-ness of each bead is 50 lm, which is close to the height of the channel. Scale bar applies to all im-ages. g) Size distribution of the beads shown in (d). h) Size distribution of the beads shown in (f).Both histograms were fitted with a Gaussian distribution.

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with hexadecane. Hexadecane (solubility = 9.0 108 g/100 g

water at 25 C[18]) is almost completely immiscible with water

when compared to hexane (solubility = 9.5 104 g/100 g at

25C[19]), which may explain why cells remained viable when

hexadecane was used. We also studied how the concentration

of CaCO3affected the viability of the cells (Fig. 4b). Percent-age of cells that remained alive after the encapsulation pro-

cess increased from 19.3% to 74.3 % when the CaCO3 con-

centration was increased from 1.14 to 9.10 mg mL1 solution.

Here, trypan blue was used to differentiate live from dead

cells. Figure 4c shows the cell encapsulating alginate hydrogel

beads, and Figure 4d shows the close-up of alginate beads

containing live/dead Jurkat cells after trypan blue was added.

Cells are selective in the compounds that pass through the

membrane; trypan blue is not absorbed in a live cell, but it tra-

verses the membrane in a dead cell. Hence, only dead cells

will exhibit a distinctive blue color. Higher loading of CaCO3leads to higher crosslink densities, resulting in stiffer alginate

hydrogels[20] that may help protect encapsulated cells from

mechanical stresses during preparation. Increase in viability

of the cells with the increase in CaCO3 concentration is also

attributed to the dual role played by CaCO3. Besides releas-

ing Ca2+ when the pH lowers, CO32 is also released from

CaCO3. CO32 acts as a base, regulating the pH inside the

beads. Beads prepared with a higher concentration of CaCO3essentially have a larger reserve of CO3

2 to prevent it from

becoming overly acidic. Thus, we believe that both the in-

crease in mechanical strength of the hydrogel and the milder

internal environment subsequently translate to higher viabili-

ty of the encapsulated cells.

The use of CaCO3nano-particles enabled us to combine in-

ternal gelation method with T-junction droplet formation to

produce alginate hydrogel microbeads, and modifications in

channel geometry should allow beads with a wider range of

sizes to be produced. The strategy described in the present

work has four significant advantages: 1) it offers easy control

over morphology and size by tuning flow parameters or con-

centration of reactants, 2) it allows beads with a narrow size

distribution and high uniformity in morphology to be pro-

Adv. Mater.2007,19, 26962701 2007 WILEY-VC H Verlag GmbH & Co . KGaA, Weinh eim www.advmat.de 269

Figure 3. Optical microscopy images of droplet formation in the micro-fluidic device taken with high speed camera. Droplet formation of (a)Na-alginate solution containing only CaCO3 for the production of blankalginate hydrogel microbeads in a non-silanized PDMS device.Qc =20 mLh

1 and Qd = 20 lL h1. b) Na-alginate solution containing

both CaCO3and Jurkat cells for the production of cell encapsulating algi-nate hydrogel microbeads in a non-silanized PDMS device. Wetting ofthe PDMS channel caused the front of the Na-alginate stream to gradual-ly advance downstream. Eventually, the front of the stream gelated anddroplets were randomly sheared off. Qc =20 mLh

1 and Qd = 10 lL h1.

c) Na-alginate solution containing both CaCO3 and Jurkat cells for theproduction of cell encapsulating alginate hydrogel microbeads in a sila-nized PDMS device.Qc =60 mLh

1 andQd = 20 lL h1.

Figure 4.a) Plot of the size and AR of cell encapsulating alginate hydro-gel microbeads versus flow rate of the continuous phase (corn oil with le-cithin (2 % w/w)). The rate of flow of the disperse phase, Qd, was20 lL h1. The acid concentration was 1 lL acetic acid/mL oil. b) Graphshowing how the concentration of CaCO3 affects the viability of the en-capsulated cells. Beads were produced at Qc =30 mLh

1 andQd = 20 lL h

1 for all the cases. In all cell cultures, a certain proportion ofthe cells will be dead. Here, the percentage of live cells after process isdefined as (Nalive,after/Nalive,before) 100% whereNalive,beforeandNalive,afterrefers to the percentage of cells alive before the experiment and percent-age of cells alive after the experiment, respectively. Optical microscopyimage of (c) cell encapsulating beads produced at Qc =30 mLh

1 andQd = 20 lL h

1, and d) cell encapsulating beads when trypan blue wasadded to test for cell viability. Dead cells exhibit a distinctive blue color.

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[8] S. Okushima, T. Nisisako, T. Torii, T. Higuchi,Langmuir 2004, 20,

9905.

[9] S. Takeuchi, P. Garstecki, D. B. Weibel, G. M. Whitesides,Adv. Ma-

ter.2005,17, 1067.

[10] S. Sugiura, T. Oda, Y. Izumida, Y. Aoyagi, M. Satake, A. Ochiai,

N. Ohkohchi, M. Nakajima,Biomaterials2005,26, 3327.

[11] K. Liu, H.-J. Ding, J. Liu, Y. Chen, X.-Z. Zhao, Langmuir2006, 22,

9453.[12] D. Poncelet,Ann. N. Y. Acad. Sci.2001,944, 74.

[13] L. W. Chan, H. Y. Lee, P. W. S. Heng,Int. J. Pharm. 2002,242, 259.

[14] X. D. Liu, W. Y. Yu, Y. Zhang, W. M. Xue, W. T. Yu, Y. Xiong, X. J.

Ma, Y. Chen, Q. Yuan,J. Microencapsulation2002,19, 775.

[15] S. Xu, Z. Nie, M. Seo, P. Lewis, E. Kumacheva, H. A. Stone, P. Gar-

stecki, D. B. Weibel, I. Gitlin, G. M. Whitesides, Angew. Chem. Int.

Ed.2005,44, 724.

[16] W. R. Gombotz, S. F. Wee,Adv. Drug Delivery Rev.1998,31, 267.

[17] K. Y. Lee, M. C. Peters, D. J. Mooney,Adv. Mater.2001,13, 837.

[18] C. Sutton, J. A. Calder,Environ. Sci. Technol.1974,8, 654.

[19] C. McAuliffe,J. Phys. Chem.1966,70, 1267.

[20] N. G. Genes, J. A. Rowley, D. J. Mooney, L. J. Bonassar,Arch. Bio-chem. Biophys.2004,442, 161.

[21] J. A. Rowley, Z. Sun, D. Goldman, D. J. Mooney, Adv. Mater. 2002,

14, 886.

[22] W.-H. Tan, S. Takeuchi,Proc. Natl. Acad. Sci. USA 2007,104, 1146.

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