Biofabrication of injectable fibrin microtissues for minimally-invasive therapies: application of surfactants

Microtissues created from the protein fibrin and containing embedded cells can be used in modular tissue engineering approaches to create larger, hierarchical and complex tissue structures. In this paper we demonstrate an emulsification-based method for the production of such fibrin microtissues containing fibroblasts (FB) and endothelial cells (EC) and designed to promote tissue vascularization. Surfactants can be beneficial in the microtissue fabrication process to reduce aggregation and to facilitate recovery of microtissues from the emulsion, thereby increasing yield. The nonionic surfactants Pluronic L101 (R) and Tween 20 (R) both increased microtissue yield in a dose-dependent fashion. Cell viability of both human FB and human EC remained high after exposure to low surfactant concentrations but decreased with increasing surfactant concentration. L101 was markedly less cytotoxic than Tween, and therefore was the surfactant of choice in this application. The yield of cell-laden microtissues increased with increasing L101 concentration, though microtissues were slightly larger at low concentrations. The total metabolic activity of cells in retrieved microtissues was bimodal and was highest at an L101 concentration of 0.10% wt/vol. Network formation by EC in microtissues embedded in surrounding 3D fibrin hydrogels was also most extensive in microtissues made using an L101 concentration of 0.10% wt/vol. Minimally-invasive delivery of microtissue populations was demonstrated by injection through a standard 18 G needle, and the ability to form robust endothelial networks was maintained in injected microtissue populations. Taken together, these data demonstrate a facile emulsification-based method to create modular, cell-laden hydrogel microtissues that can be delivered by injection to promote tissue regeneration. Appropriate selection of the type and concentration of surfactant used in the process can be used to maximize viability and specialized function of the embedded cells. Such biomaterial-based microtissues may have broad applicability in cell-based therapies and tissue engineering.