111 Cryopreservation of stem cells inside alginate 3D constructs after high-voltage encapsulation

Abstract

Application of stem cells in regenerative medicine require improving 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. Furthermore, smaller alginate micro-beads ⩽3 μm offer additional advantages over larger ones due to higher specific surface area, less water content and improved heat and mass transfer. The gel-like structure and mild environment inside alginate micro-beads may affect the metabolic activity of encapsulated cells post-cryopreservation. The effects of high voltages, alginate encapsulation and pre-incubation time on morphology, viability and proliferation of MSCs post-cryopreservation were evaluated. Mesenchymal Stem Cells derived from the Common marmoset monkey Callithrix jacchus (MSC) were encapsulated into 1.5% (w/v) sterile medium viscosity unmodified alginate (Sigma Aldrich, filter-sterilized using 0.8–0.45–0.22 mm filter set) with a concentration 1*106 cells per ml of alginate using electro-spraying method. Final alginate micro-beads were obtained with less then 300 μm in diameter under the next process parameters: applied voltages 15, 20 and 25 kV, spraying distance 10 cm, and alginate flow rate 10 ml/h. As a negative control to high voltages, the air flow encapsulation was run in parallel. Micro-beads (0.5 ml) containing MSCs (0.5*106 cells) were either immediately frozen after encapsulation (0 h) or cultured overnight prior to cryopreservation (24 h). Cryopreservation was conducted with 1 °C/min cooling rate down to −80 °C with 10% Me2SO (v/v) as a cryoprotective agent. After 24 h of storage at low temperatures micro-beads were thawed at 37 °C with further removing of alginate using 55 mM sodium citrate for 2 min with gentle shaking. Then MSCs were either seeded for MTT test at 104 cells/well immediately after thawing (protocol from manufacturer) or cultured for 5 days followed by MTT assay. Membrane integrity of encapsulated cells before cryopreservation and after thawing was evaluated by Trypan Blue exclusion method. Metabolic activity and proliferation efficiency of MSCs were evaluated using MTT Nonradioactive Proliferation Assay (CellTiter 96, Promega, USA). The MTT data were normalized on respective control groups. The one-way ANOVA was run for statistical analysis of obtained results. MSCs can be encapsulated into alginate micro-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 micro-beads prior to cryopreservation resulted in lower proliferation rate as compared to immediately frozen (18 ± 6% and 45 ± 8% in respect to native control) and at the same rate as frozen control. The same behavior was observed for re-passage efficiency of encapsulated and frozen MSCs. However, immediately frozen cells after encapsulation recovered at the same rate as native control, indicating an effectiveness of cell cryopreservation inside alginate micro-beads. This study shows an importance of cell cryopreservation inside unmodified alginate directly after encapsulation. Due to toxic effect of Me2SO, cryopreservation of MSCs inside alginate 3D structures using other less toxic CPAs will be conducted. Source of funding: This work is supported by funding from the Cluster of Excellence REBIRTH (DFG EXC 62/1). Conflict of interest: None declared. gryshkov@imp.uni-hannover.de