Mesenchymal stromal cell encapsulation in uniform chitosan beads using microchannel emulsification

Mesenchymal stromal/stem cells hold potential in repairing damaged tissue through paracrine effects. Their delivery though injectable biodegradable microbeads can improve cell retention and survival at the infusion site. A stirred emulsion process was previously implemented to immobilize these cells in injectable chitosan microbeads for cell therapy applications, but this process leads to broad bead size distribution (coefficient of variation > 40 %). Polydisperse beads may negatively affect the viability of the entrapped cells through oxygen limitations, damage to larger beads during injection, and reduced control over the cell payload and treatment reproducibility. The objective of this work was to modify a microchannel emulsification system initially designed for alginate-based encapsulation to immobilize mesenchymal stromal/stem cells in monodisperse chitosan microbeads. The main factors (e.g., microchannel geometry, chitosan solution viscosity, interfacial tension and flow rate) affecting droplet generation and diameter were investigated. The adapted process enabled the production of monodisperse chitosan microbeads with controlled sizes ranging from 600 {micro}m - 1500 {micro}m in diameter at a coefficient of variation less than 10 %. In a single pass through a 21 G syringe needle (ID: 513 {micro}m), the fraction of ruptured beads was significantly reduced for microchannel-generated vs stirred emulsion-generated beads with matching volume-weighed bead diameter (D[4,3]). The viability of the immobilized cells immediately after the process was 95 % {+/-} 2 % and no significant difference in cell survival and growth factor secretion was observed between microchannel and stirred emulsion-generated beads over 3 days of culture. Future directions include channel multiplexing to increase throughput for clinical applications. Although the device was developed for cell encapsulation, this process could be implemented for encapsulation of other biomolecules, bioactive or living cell agents for applications in the food and drug industry.