A07 Session 2: Biopreservation of biologics: Translation from stem cells to cancer : Cryopreservation of alginate encapsulated monkey stem cells under controlled induced nucleation

Abstract

On-shelf availability of rare cell types requires their long-term preservation. The cryopreservation procedures which are commonly used for long-term preservation of living cells involve high concentrations of cryoprotective agents (CPAs), such as Me2SO, which protect cells at low temperatures from cryoinjury. Despite of this, the introduction of such CPA for cryopreservation does have pronounced toxic effects to the cells. To solve such a problem, a decrease in concentration of CPA is required. This may be achieved by encapsulation of living cells into gel-like alginate structures which mimic an extra-cellular matrix also providing 3D arrangement for cells. However, the efforts being made so far were mainly applied to control the cooling and warming temperatures during freezing but only a few publications are known being tried to control the nucleation during cryopreservation. Furthermore, there is no information about controlled nucleation during cryopreservation of encapsulated cells [1]. In the case of cryopreservation of encapsulated cells, the size of such constructs is the main property which restricts the diffusion of CPA as well as heat and mass transport to encapsulated cells [2]. In this work we introduce the electro-spraying encapsulation technology to produce small-sized (less then 200 μm) alginate 3D constructs with entrapped living cells under the high voltages. Alginate solution (1.6% (w/v))) is mixed with mesenchymal stem cells derived from a Common marmoset (cjMSCs) and pumped at 10 ml/h flow rate through the nozzle where the high voltage is applied (>15 kV). When the strength of electric field exceeds the surface tension, the spraying into cross-linking bath (100 mM CaCl2) is observed. After being gelled for 10 min, beads were washed with PBS and immediately cryopreserved using electro-freezing and seeding under controlled nucleation using optimal protocol (5% DMSO (v/v) with cooling rate 7.5 K/min down to −30 °C and 3 K/min to −80 °C). The commonly used freezing protocol (1 K/min, CM 2000, Carburos Metallicos) served as a control. Samples were kept at −80°C for a week followed by rapid warming at 37°C. Membrane integrity of cells was assessed using Trypan Blue assay. Proliferation activity of cells which undergone encapsulation and cryopreservation procedures was measured using MTT proliferation assay (ProNova, USA). High-voltage electro-spraying allows encapsulation of living cells into small alginate 3D constructs without significant influence on viability and proliferation of cells post-encapsulation. The MSCs encapsulated and frozen under controlled nucleation showed normal morphology, attached and proliferated the same as compared to control. The viability and proliferation activity of cells post-cryopreservation may be improved by controlling the nucleation temperature. These results may serve as a background for further development of cryopreservation of encapsulated cells with controlled induced nucleation for long-term storage. We will also describe different methods and devices for induction of ice nucleation. Acknowledgements: This work is supported by funding from the Cluster of Excellence REBIRTH (DFG EXC 62/1).