(765f) Deterministic Encapsulation of Single Cells in Thin Tunable Microgels for Niche Modeling and Therapeutic Delivery

Angelo S. Mao¹, Jae-Won Shin^1,2, David A. Weitz¹, David J. Mooney¹

¹School of Engineering and Applied Sciences, Harvard University, Cambridge, MA

²Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL

Introduction: High-throughput encapsulation of single cells into thin hydrogel layers has the potential to abet regenerative medicine therapies and basic science applications, for example enabling the construction of microscale assemblages in vitro and improving existing cell therapy techniques. Current methods for encapsulating cells into microscale hydrogels generally follow a Poisson distribution and possess limited control over hydrogel properties. We report a microfluidic, water-in-oil based method for deterministic encapsulation of single cells in an ~6 micron thin layer of alginate hydrogel that was used for microscale tissue construction in vitro and improved cell delivery in vivo.

Materials and Methods: A cross-junction polydimethylsiloxane (PDMS) microfluidic device was used to fabricate cell-containing alginate microgels using calcium carbonate as the polymer cross-linker. Different polymer molecular weights were tested, as were combinations of alginate with extracellular matrix proteins. Encapsulated cells were cultured in PDMS microwells with exposure to osteogenic induction media or delivered intravenously into mice. Levels of donor-derived soluble factors were measured from blood plasma of injected mice.

Results and Discussion: By adsorbing calcium carbonate nanoparticles to cells prior to encapsulation, a high percentage of alginate microgels were found to contain cells without decreasing the fraction of cells that were encapsulated. Cells exhibited viability near 90% over a three-day period. Microgels were found to be mechanically tractable and capable of forming interpenetrating hybrid microgels. Moreover, microscale cell assemblages of encapsulated mesenchymal stem cells (MSCs) were constructed with a PDMS-based microwell system. Singly-encapsulated cells were observed to differentiate more robustly than multiply-encapsulated cells in larger microgels. Finally, singly-encapsulated human MSCs were found to respond more strongly to stimulatory factors, experience more delayed clearance kinetics, and sustain greater donor-derived soluble factors in blood plasma following intravenous delivery into mice, compared to multiply-encapsulated MSCs in larger gels.

Conclusions: The improved differentiation capacity of singly-encapsulated MSCs and delayed clearance kinetics of intravenously delivered singly-encapsulated MSCs indicate the potential of this approach in a variety of regenerative medicine and tissue engineering applications.