Cryopreservation of electro-spraying alginate encapsulated mesenchymal stem cells

摘要

Perspectives of application of stem cells in regenerative medicine require improving of cryopreservation protocols for long term storage, especially for tissue-imitating 3D constructs containing cells. Encapsulation of stem cells into alginate-based 3D constructs that repeat an extracellular matrix may provide a mild environment during cryopreservation as well as the possibility for large-scale expansion. The gel-like structure and mild environment inside alginate beads may affect the metabolic activity of encapsulated cells post-cryopreservation. The effects of high voltage, alginate encapsulation and pre-incubation time on morphology, viability and proliferation of MSCs post-cryopreservation were evaluated. Mesenchymal Stem Cells (MSCs) derived from the Common marmoset monkey Callithrix jacchus (cjMSC) were encapsulated into 1.5% (w/v) sterile medium viscosity unmodified alginate (Sigma Aldrich, filter-sterilized using 0.8-0.45-0.22 μm filter set) with a concentration 1~*10~6 cells per ml of initial alginate solution using electro-spraying. Final alginate beads (300 μm in diameter) were obtained under different applied voltage (15, 20, 25 kV). As a negative control to high voltage, air flow encapsulation was run in parallel. Beads (0.5 ml) containing MSCs (0.5~*10~6 cells) were either immediately frozen after encapsulation (Oh) or cultured overnight prior to cryopreservation (24h). Cryopreservation was conducted with 1 °K/min cooling rate down to -80°C with 10% Me_2SO (v/v) as a cryoprotective agent (CPA). After 24h of storage at -80°C beads were thawed at 37°C with further removing of alginate using 55 mM sodium citrate for 2 min. Then MSCs were either seeded for MTT test at 10~4 cells/well immediately after thawing or cultured for 5 days followed by MTT. Membrane integrity of encapsulated cells before cryopreservation and after thawing was evaluated by Trypan Blue assay. Metabolic activity and proliferation activity of MSCs were measured using MTT Proliferation Assay (Promega, USA). The MTT data were normalized on respective control. MSCs can be encapsulated into alginate beads with desired concentration and without significant changes in viability and metabolic activity post-encapsulation. MSCs showed normal morphology, attached and proliferated well after thawing. The incubation of MSCs inside alginate beads prior to cryopreservation resulted in lower proliferation rate as compared to native control (18 ± 6% and 45 ± 8%) and at the same rate as frozen control. The same behavior was observed for re-passage efficiency of encapsulated and frozen MSCs. Immediately frozen cells after encapsulation recovered at the same rate as native control, indicating an effectiveness of cell cryopreservation inside alginate beads. This study shows an importance of cell cryopreservation inside unmodified alginate directly after encapsulation. Due to toxic effect of Me_2SO, cryopreservation of MSCs inside alginate beads using other less toxic CPAs will be conducted.