A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres

Abstract

Cell encapsulation within photopolymerized polyethylene glycol (PEG)-based hydrogel scaffolds has been demonstrated as a robust strategy for cell delivery, tissue engineering, regenerative medicine, and developing in vitro platforms to study cellular behavior and fate. Strategies to achieve spatial and temporal control over PEG hydrogel mechanical properties, chemical functionalization, and cytocompatibility have advanced considerably in recent years. Recent microfluidic technologies have enabled the miniaturization of PEG hydrogels, thus enabling the fabrication of miniaturized cell-laden vehicles. However, rapid oxygen diffusive transport times on the microscale dramatically inhibit chain growth photopolymerization of polyethylene glycol diacrylate (PEGDA), thus decreasing the viability of cells encapsulated within these microstructures. Another promising PEG-based scaffold material, PEG norbornene (PEGNB), is formed by a step-growth photopolymerization and is not inhibited by oxygen. PEGNB has also been shown to be more cytocompatible than PEGDA and allows for orthogonal addition reactions. The step-growth kinetics, however, are slow and therefore challenging to fully polymerize within droplets flowing through microfluidic devices. Here, we describe a microfluidic-based droplet fabrication platform that generates consistently monodisperse cell-laden water-in-oil emulsions. Microfluidically generated PEGNB droplets are collected and photopolymerized under UV exposure in bulk emulsions. In this work, we compare this microfluidic-based cell encapsulation platform with a vortex-based method on the basis of microgel size, uniformity, post-encapsulation cell viability and long-term cell viability. Several factors that influence post-encapsulation cell viability were identified. Finally, long-term cell viability achieved by this platform was compared to a similar cell encapsulation platform using PEGDA. We show that this PEGNB microencapsulation platform is capable of generating cell-laden hydrogel microspheres at high rates with well-controlled size distributions and high long-term cell viability.

Fig. 1

(A) Schematic of PEGNB hydrogel microsphere fabrication by emulsification in a cross-flow microfluidic channel or (B) by vortex, followed by exposure to ultraviolet radiation.

Fig. 2

(A) Size distributions and images of PEGNB hydrogel microspheres made by emulsification in a cross-flow microfluidic devices or (B) by vortex.

Fig. 3

(A) Viability of A549 cells encapsulated within PEGNB microparticles by either vortex or microfluidic emulsification. Cell residence times in the hydrogel-forming solutions prior to photopolymerization were 1 minute and 10 minutes for the vortex and microfluidic methods, respectively. (B) Images of cell-laden hydrogel microspheres made by each method (scale bar = 100 μm). Viability of cells is indicated by live/dead staining assay (live and dead correspond to green and red, respectively).

Fig. 4

Factors affecting post-encapsulation cell viability were investigated. (A) Viability of cells maintained in media within a syringe over time. (B) Viability of cells pumped through a device in culture medium with or without being formed into emulsion droplets. Flow rates for cells and oil were 2 μL/min and 10 μL/min, respectively. (C) Viability of cells maintained within a hydrogel-forming media solution incubated within a syringe over time. (D) Viability of cells encapsulated into PEGNB microspheres via microfluidic emulsification, coll ected in a vial, and exposed to UV light following specific incubation periods in culture media. Viability was measured for 30 days.

Fig. 5

Comparison between observed viability for cells encapsulated within PEGNB and PEGDA microspheres via microfluidic emulsification. (A) Images of cells encapsulated into 10 wt% PEGNB and PEGDA bulk hydrogels. Cells were mixed with PEG hydrogel-forming solution followed by exposure to UV light to polymerize the cell-laden bulk structure. (B) Cell viability was quantified for 30 days following encapsulation in bulk hydrogels. (C) Schematic illustration of the nitrogen jacketed microfluidic device used for cell encapsulation into PEGDA hydrogel microspheres. (D) Quantification of post-encapsulation viability of cells within PEGNB and RGDS-modified PEGDA hydrogel microspheres. Scale bar = 100 μm.