Microencapsulation of Live Cells in Synthetic Polymer Capsules

Abstract

In cell therapies, it is advantageous to encapsulate live cells in protective, semipermeable microparticles for controlled release of cytokines, growth factors, monoclonal antibodies, or insulin. Here, a modified electrospraying approach with an organic solution of poly(lactide-co-glycolide) (PLGA) polymer is used to create synthetic PLGA capsules that effectively protect live cells. Using a design of experiment (DOE) methodology, the effect of governing jetting parameters on encapsulation efficiency, yield, and size is systematically evaluated. On the basis of this analysis, the interaction between bovine serum albumin concentration and core flow rate is the most dominant factor determining core encapsulation efficiency as well as the microcapsule size. However, the interaction between shell solvent ratio and shell flow rate predominantly defines the particle yield. To validate these findings, live cells have been successfully encapsulated in microcapsules using optimized parameters from the DOE analysis and have survived the electrohydrodynamic jetting process. Extending the currently available toolkit for cell microencapsulation, these biodegradable, semi-impermeable cell-laden microcapsules may find a range of applications in areas such as tissue engineering, regenerative medicine, and drug delivery.

Figure 1

Schematic illustration of coaxial electrohydrodynamic (EHD) jetting. The viscoelastic polymer shell and the cell-loaded core solutions are forced through a coaxial needle under applied electric potential resulting in cell encapsulated core–shell microparticles.

Figure 2

Determination of significant contributing factors in encapsulation efficiency. (a) Interaction plot for core encapsulation efficiency. (b) Contour plot of core encapsulation efficiency vs. core flow rate and BSA concentration, where PLGA concentration, shell solvent ratio, and shell flow rate are all held constant at 12.5% w/v, 93.5:6.5 v/v CHCl₃/DMF, and 0.3 mL h^–1, respectively. (c) Pareto chart of significant contributing factors in core encapsulation efficiency. Experimental factors are as follows: (A) BSA concentration, (B) core flow rate, (C) PLGA concentration, (D) shell solvent ratio, and (E) shell flow rate.

Figure 3

Determination of significant contributing factors in microcapsule yield. (a) Interaction plot for the yield of microparticles. (b) Contour plot of yield vs shell flow rate and shell solvent ratio, where BSA concentration, core flow rate, and PLGA concentration are all held constant at 15% w/v, 0.06 mL h^–1, and 12.5% w/v, respectively. (c) Pareto chart of significant contributing factors in determination of yield. Experimental factors are as follows: (A) BSA concentration, (B) core flow rate, (C) PLGA concentration, (D) shell solvent ratio, and (E) shell flow rate.

Figure 4

Determination of significant contributing factors in microcapsule size. (a) Interaction plot for the size of microparticles. (b) Contour plot of size vs core flow rate and BSA concentration, where PLGA concentration, shell solvent ratio, and shell flow rate are all held constant at 12.5% w/v, 93.5:6.5 CHCl₃/DMF, and 0.3 mL h^–1, respectively. (c) Pareto chart of significant contributing factors in the determination of size. Experimental factors are as follows: (A) BSA concentration, (B) core flow rate, (C) PLGA concentration, (D) shell solvent ratio, and (E) shell flow rate.

Figure 5

Fabrication of core–shell microparticles: (a) SEM image of uniform microparticles, (b-1) superimposed DAPI and Texas red channels of CLSM image of microparticles; (b-2) CLSM image of the DAPI channel, showing the PLGA shell layer containing blue dye; (b-3) CLSM image of the Texas red channel, showing the BSA core layer containing red silica microspheres; (b-4) lower-resolution superimposed CLSM image of microparticles; (b-5) zoomed-out CLSM image of the DAPI channel, showing the PLGA shell layer containing blue dye; (b-6) zoomed-out CLSM image of the Texas red channel, showing the BSA core layer containing red silica microspheres; (c-1) SEM image of cross-sectioned BSA layer containing a silica microsphere; (c-2) SEM image of cross-sectioned PLGA layer; (c-3) SEM image of cross-sectioned PLGA layer; and (c-4) SEM image of cross-sectioned BSA layer.

Figure 6

GFP-NIH3T3 cell microencapsulation. (a-1) CLSM image of GFP-NIH3T3 cell-core/PLGA-shell microparticles (superimposed green and blue channels); (a-2) CLSM image of PLGA shells (blue channel); (a-3) CLSM image of GFP-NIH3T3 cells in BSA core (green channel); (b-1) flow cytometry analysis of encapsulation efficiency of core–shell microparticles not encapsulating GFP-NIH3T3 cells with PLGA (blue dye) shell and BSA core layers (control); and (b-2) flow cytometry analysis of encapsulation efficiency of core–shell microparticles encapsulating GFP-NIH3T3 cells with PLGA (blue dye) shell and BSA core layers.

Figure 7

In vitro cell viability of NIH3T3 cells encapsulated in core–shell microparticles. (a) CLSM image of NIH3T3 cells stained with Live/Dead assay kit (green: live/red: dead) demonstrating NIH3T3 cells before encapsulation in core–shell particles. (b) CLSM image of NIH3T3 cells stained with Live/Dead assay kit (green: live/red: dead) encapsulated in core–shell microparticles.