A Microfluidic Approach to Encapsulate Living Cells in Uniform Alginate Hydrogel Microparticles

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Original Entry by Ryan Truby

AP 225 - Introduction to Soft Matter

October 15, 2012

Reference Information

Fig. 1, reproduced from [1]

Authors: C. J. Martinez, J. W. Kim, C. Ye, I. Ortiz, A. C. Rowat, M. Marquez, D. Weitz

Citation: C. J. Martinez, et al. A Microfluidic Approach to Encapsulate Living Cells in Uniform Alginate Hydrogel Microparticles. Macromol. Biosci. 2012, 12, 946-951.

Related Course Keywords: surface tension, wetting

Background and Introduction

Fig. 2, reproduced from [1]

Cell encapsulation is a challenge that sits at the intersection of biology, biomedical engineering, and soft matter physics. Engineers and scientists alike have strived to develop methods of encapsulating individual or groups of cells within microscale biocompatible matrices that can serve as controlled, custom environments. Encapsulated cells could offer a means for precisely studying the metabolism of cells, prompt novel tissue engineering applications, and function as microscale bioreactors for biomolecular engineering applications.

Producing monodisperse samples of encapsulated cells, post-encapsulation processing, and maintaining the viability of the encapsulated cells remain pertinent challenges in devising cell encapsulation strategies. Encapsulating cells via emulsions created with microfluidic devices has become a popular approach to cell encapsulation, as microfluidic-based strategies typically yield highly monodisperse populations of encapsulated cells. Martinez et al use microfluidics to create tear-drop shaped alginate matrices from water-oil-water double emulsions that encapsulate several yeast cells. Alginate is a biopolymer that forms hydrogels upon exposure to divalent ions (such as Ca2+). Through soft matter physics, the production of these alginate matrices can be thoroughly understood, as the presented cell encapsulation scheme is mediated by liquid surface tensions and wetting phenomena.

Summary

The authors constructed a microfluidic device from various glass capillary tubes to create water-oil-water double emulsions (see Fig. 1). The double emulsions are created in a single step: an “inner fluid” is flowed into a cylindrically shaped input capillary before it flows into a square capillary into which a “middle fluid” and “outer fluid” are flowed in opposing directions; surface tensions and wetting phenomena cause double emulsion droplets contained within the “outer fluid” to pass into an outlet capillary that leads to a 0.1 M solution of CaCl2. The “inner” and “outer fluids” were aqueous suspension of yeast cells (up to ~1x107) of sodium alginate (2 wt%, the volume was not specified in the paper) and a mixture of Tween 20 (0.5 wt%) and glycerol (40 wt%), respectively. The “middle fluid” constituted the oil phase necessary for creating the double emulsions and was comprised of 0.4 wt% Span 80 in mineral oil. Successful formation of the double emulsion droplets was the result of precise control of the individual flows for the “inner,” “middle,” and “outer fluids” flowed into the microfluidic device. (See Fig. 2.)

Fig. 3, reproduced from [1]

As the double emulsion droplets encapsulating the yeast cells were flowed into the CaCl2 solution, gravity caused the denser inner drop containing sodium alginate and yeast cells to fall towards the exterior interface of the surrounding Span 20 in mineral oil shell (see Fig. 3). When the exterior interface of the inner drop meets the shell’s exterior interface, the inner drop comes in contact with the aqueous CaCl2 solvent. The dissociated Ca2+ ions immediately begin binding with the alginate polymer chains, forming an interfacial alginate hydrogel layer between the non-polymerized sodium alginate in the inner drop and the CaCl2 solvent. Gravity continues to exert a downward force on the inner drop, evermore separating the inner drop from the mineral oil shell, while newly exposed sodium alginate instantly binds with Ca2+ ions from the solvent. These interactions continue until the sodium alginate core completely leaves the mineral oil phase and becomes a tear-drop-shaped alginate hydrogel matrix, as shown in Fig. 4. The mean volume of the alginate hydrogel drops was ~186 pL, and with a volume distribution associated with CV = 6% (where CV is the coefficient of variation), the alginate drops were relatively monodisperse.

Fig. 4, reproduced from [1]
Fig. 5, reproduced from [1]

The authors found that yeast cell viabilities following encapsulation as well as after one week of encapsulation in the alginate hydrogels were 98% and 65%, respectively. The 65% viability of the encapsulated yeast cells after one week is substantial, but one might have hoped that this percentage would have been higher. As shown in Fig. 5, the authors also observed a yeast cell count of up to 6 cells per alginate drop across various cell concentrations, with a significant number of drops not possessing any cells at all when the cell concentration of the “inner fluid” is too low. All in all, the authors were successful in their attempt to encapsulate yeast cells in microscale alginate hydrogel matrices.

Discussion and Relevance to Soft Matter

The double emulsion droplets formed in the microfludic device remain in tact as they pass through the outlet capillary and on into the calcium chloride solution due to dewetting by the "outer fluid". The "spreading coefficient," <math>S</math>, reveals whether or not the double emulsion drops will be ruptured by the "outer fluid:" if <math>S > 0</math>, the surface tension at the "middle-outer fluid" interface (<math>\sigma_{m, o}</math>) is lower than the difference between the surface tensions at the "inner-outer fluid" (<math>\sigma_{i, o}</math>) and "inner-middle fluid" (<math>\sigma_{i, m}</math>) interfaces,

<math> S = \sigma_{i,o} - \sigma_{i,m} - \sigma_{m, o} </math>

If this were so, <math>\sigma_{m, o}</math> would not be high enough to maintain the spherical shape of the double emulsion droplets, and the authors would have seen their droplets rupture. However, by tuning the compositions of their "inner," "middle," and "outer fluids," the authors were able to have <math>\sigma_{m, o} > \sigma_{i,o} - \sigma_{i,m}</math>. In turn, <math>S < 0</math>, and "dewetting" by the "outer fluid" allowed the authors to produce double emulsion droplets that maintained a spherical morphology.

Interfacial surface tensions - or in this case, a near lack thereof - between the sodium alginate inner droplet and the surrounding CaCl2 solution are responsible for the alginate hydrogels droplets' tear-drop shape. As the sodium alginate center of a double emulsion drop come in contact with Ca++ ions, there is no substantial surface tension between the sodium alginate solution and the CaCl2 solution. Therefore, as the sodium alginate phase is exposed to the CaCl2 solution, it is not deformed in any way, and the alginate gels immediately in the shape in which it leaves the "middle fluid" phase. However, there is between the sodium alginate center and the "middle fluid" shell. As the alginate center is pulled from the "middle fluid" phase, the "middle fluid" layer continues to try and enclose the un-gelled sodium alginate. This gives the final alginate hydrogel drops their tear-drop-like shape (see Fig. 3).

The microfluidic approach to encapsulating cells presented here is also beneficial in that the alginate hydrogel droplets produced will sink to the bottom of the CaCl2 solution, allowing for their facile collection and complete separation from the "middle fluid" phase. Given that i) a full understanding of the role of interfacial tensions involved in this system was required of the authors and ii) cell-encapsulation is an interesting engineering challenge that could lend itself to many potential biomedical/biological applications, one sees that the problem of microfluidics-based cell encapsulation schemes are important to the field of soft matter.

References

[1] C. J. Martinez, et al. A Microfluidic Approach to Encapsulate Living Cells in Uniform Alginate Hydrogel Microparticles. Macromol. Biosci. 2012, 12, 946-951.