Deterministic Single-Cell Encapsulation for in Vitro Microsystems and Therapeutic Cell Delivery

Abstract

Encapsulation of cells into microscale hydrogels (microgels) may abet a number of regenerative medicine therapies by enabling precise microscale control in assembly of complex tissue mimics and programming in vivo delivery of cells via different routes of administration. Although providing the appropriate matrix cues has been shown to be a potent method for producing desired biological phenomena of encapsulated cells, there has been little work to control local properties of hydrogels at the single cell level to influence the biological functions of encapsulated cells, either in vitro or in vivo. Here, a microfluidic system is developed and characterized that enables encapsulation of single cells in alginate hydrogel with high encapsulation efficiency and yield. The ability of this system to incorporate alginate polymers with different mechanical properties and compositional characteristics, and their baseline effect on encapsulated cells, was assessed. Manipulation of microgel physical properties through secondary crosslinking mechanisms was also explored. Finally, modification of hydrogels with binding pair molecules was tested as a method for assembling microgels into controlled structures. The utility of singly encapsulated cells was then explored in several contexts. Microwell apparatuses were used to template encapsulated mesenchymal stem cells (MSCs) into clusters of controlled size, and the relationship between MSC differentiation and cell-cell interactions was studied. These results were similar to analogous experiments in two-dimensional setup that used micropatterning techniques to control cell-cell interactions. Singly encapsulated were also used to study the effects of different stromal cell types on hematopoietic stem and progenitor cell (HSPC) maintenance and differentiation. Microfabricated surfaces were used to coculture HSPCs together with encapsulated MSCs that had undergone different differentiation regimens. The results show that, besides broadly supporting HSPCs, differentiation state of MSCs affect the fate decisions of HSPCs. Deterministic single-cell encapsulation was also assessed in the context of in vivo cell therapies. Short in vivo survival hamper many therapies that involve infusion of exogenous cells in the body. Microgel encapsulation of cells substantially extended in vivo residence time of intravenously injected cells, and was accompanied by sustained secretion of native soluble factors. Microgel material properties were shown to further influence the residence time of infused cells. To determine if encapsulation of cells would be beneficial in a therapeutic context, encapsulated MSCs were coinfused with donor cells in a hematopoietic cell transplant setting, and this strategy was found to expedite the white blood cell recovery. These studies, and the techniques developed and refined to perform them, demonstrate the utility of microencapsulation for basic science research as well as therapeutic applications, particularly in the cell therapy field.