37. Cell Encapsulation into Alginate Micro-Capsules Provides Living Cells with a Mild Environment During Cryopreservation

Abstract

Cell encapsulation technology has been proposed to treat a variety of chronic disorders. However, the availability of some cell types such as human stem cells, in turn, promotes the need for such cells to be preserved for longer periods of time. For this, cryopreservation procedures are commonly used, which can cause injuries to the cells. A semi-permeable membrane might protect the cells during cryopreservation, serving as reservoir for cryoprotective agents (CPAs) and further additives [1] . Furthermore, smaller alginate-based micro-capsules (diameter 250μm) offer additional advantages over larger ones for transplantation and cryopreservation owing to higher specific surface area, less water content as well as improved heat and mass transfer [2] . In this work we indicate on the possibility to encapsulate living cells into semi-permeable alginate micro-capsules via electrospraying in order to improve viability of living cells post-cryopreservation as well as to support their normal proliferation rate after thawing. NIH 3T3 fibroblasts (5∗106 cells/ml) were encapsulated into 1,5% or 2,0% (w/v) alginate micro-capsules (diameter 250μm) under sterile conditions using high-voltage processes based on previously optimized parameters. Cryopreservation was conducted under 2 K/min to − 30°C and 5 K/min from − 30°C to − 80°C freezing protocol using DMEM, 20% FCS, 10% DMSO as freezing medium. Thawing was performed at 20°C using standardized equipment. Proliferation and viability of encapsulated cells before cryopreservation and after thawing was measured using MTT and Calcein AM/Ethd assays respectively. The change in morphology of alginate micro-capsules was observed under Carl Zeiss Axiovert 200 M microscope using 5x or 10x magnifications and AxioVision V 4.8.2.0 built-in software. Alginate micro-capsules have been entrapped with NIH 3T3 fibroblasts cells at a density of 130 ± 24 cells per capsule. Preliminary results on cell survival after encapsulation showed that electrospraying is a suitable technique for living cells entrapment. Microscopic observations revealed that low temperature treatment and further thawing did not significantly affect the morphology of alginate capsules as they appeared to be stable and round in shape. The results of cell viability assays indicated an increase in viability of encapsulated cells post-cryopreservation as compared to non-encapsulated by 10%. The MTT proliferation assay showed that cells proliferate well after thawing. In order to further improve the viability of cells, the most suitable freezing and thawing protocol as well as the finding of alternative and less-harmful for the cells CPAs are of necessary and will supplement this study.