C-1009: Cryopreservation of monkey mesenchymal stem cells inside alginate 3D micro-spheres after a high voltage encapsulation

Abstract

Cryopreservation is the only one possible way to store rare cell types for the long term. Despite this, there are still challenges to preserve stem cells. In order to improve viability and proliferation of cells after cryopreservation we encapsulated them in small alginate beads. The gel-like structure and mild environment inside alginate beads may protect encapsulated cells from cryo-injury and resist the reorganization of ice crystals during thawing. The existing encapsulation methods do not provide alginate beads with narrow size distribution and are not able to generate small beads in repeatable manner. Here we applied high voltage to encapsulate cells in alginate. Such technology has been shown to be advantageous over the commonly used air flow encapsulation [Gryshkov et al., Mater Sci Eng C Mater Biol Appl 2014]. Mesenchymal stem cells (MSCs) derived from the Common marmoset Callithrix jacchus were encapsulated in 1.6% (w/v) sterile alginate at a concentration 1 ∗ 106 cells/ml using high voltage (15, 20, 25 kV). Air flow encapsulation was run as a control. MSCs in alginate beads were frozen either immediately after encapsulation or after 24 h of incubation. Cryopreservation was conducted with 1 K/min cooling rate down to −80 °C with 10% Me2SO. After storage beads were thawed at 37 °C with further removing of alginate using sodium citrate. The recovered MSCs were seeded for proliferation and metabolic activity assays either immediately after thawing or after 5 days of recovery. High voltage encapsulation method was able to generate alginate beads containing cells with narrow size distribution (3–7%) in repeatable manner. The incubation of encapsulated MSCs slightly reduced the proliferation after thawing. Immediately frozen cells recovered at the same rate as fresh control. Our results show an increased proliferation of MSCs frozen immediately after encapsulation. The viability and proliferation of encapsulated MSCs could be further improved by modifying the alginate to allow mammalian cell types to attach to alginate structure and resist ice crystal formation during freezing.