Comparison of Four Different Preparation Methods for Making Injectable Microgels for Tissue Engineering and Cell Therapy

Purpose One of the major challenges in cell-laden microgel bioprocessing is to design an effective method of cell encapsulation in the biomaterial carrier while retaining high cell viability and ensuring small enough particles for injectability. In this study we aim to compare four bioprocessing techniques for making hydrogel microcarriers, including by emulsification gelation and dropwise gelation approaches. Methods A Pluronic-Fibrinogen (FF-127) hydrogel biomaterial was used to make the microgels based on a lower critical solubility temperature (LSCT) phase transition. Additional cross-linking of the hydrogels was achieved using light-activated photochemistry (i.e., photopolymerization). The four bioprocessing methodologies include emulsification gelation in oil (with and without dual photo-initiator free-radical polymerization), reverse thermal gelation (in warm cell culture media), dropwise gelation through a vibrating needle device, and dropwise gelation through an atomization device (in warm cell culture media gelation baths). The microgels made with each method were characterized with and without cells; comparisons of microgel size and cell growth were reported. Results The dual photo-initiator emulsification technique produced FF-127 spherical microgels with an average diameter of 222 and 256 mu m, with and without cells, respectively. The reverse thermal encapsulation produced irregularly shaped microgels with an average diameter of 241 and 702 mu m, with and without cells, respectively. The vibrating needle and atomization techniques produced irregularly shaped microgels with an average diameter of 195 and 151 mu m without cells, respectively, and 464 and 332 mu m with cells, respectively. The viability of fibroblasts in the microgels was high after 24 h, except for those treatments that underwent photo-polymerization (i.e., emulsification photo-polymerization and vibrating needle with photo-polymerization). The cells remained viable for up to 3 weeks in culture and spread three-dimensionally in the microgels over this time course. Conclusions The rapid temperature-induced phase transition of the FF-127 enables the formation of microgels either through dropwise gelation or by emulsification, both through physical cross-linking. The use of a free-radical polymerization cross-linking reaction was more cyto-toxic to the cells as compared to the physical cross-linking by reverse thermal gelation alone. The average microgel size in all the techniques was significantly smaller and more uniform when producing the microgels without cells as compared to with cells. The reverse thermal gelation technique produced cell-laden microgels with the least amount of specialized equipment and bioprocessing steps of all the methods reported. Lay Summary This study provides the framework for producing cell-laden microgels that are of a sufficiently small diameter to be used for injectable cell therapy. The challenge in this regard is to design a simple, scalable, and efficient methods of cell encapsulation in the biomaterials, retaining high cell viability and ensuring small enough particles for injectability. For this purpose, we evaluated four methods that are commonly applied in microgel bioprocessing, and tested these with two types of cells using hydrogels that exhibit lower critical solubility temperature (LCST) properties. This investigation has enabled us to identify advantages and disadvantages for each system of bioprocessing of cell-laden microgels.