Preferential vitrification of water in small alginate microcapsules significantly augments cell cryopreservation by vitrification.

The morphological changes of small (approximately 100 microm) alginate microcapsules and the biophysical alterations of water in the microcapsules during cryopreservation were studied using cryomicroscopy and scanning calorimetry. It was found that water in the small microcapsules can be preferentially vitrified over water in the bulk solution in the presence of 10% (v/v) or more dimethylsulfoxide (DMSO, a cryoprotectant), which resulted in an intact morphology of the microcapsules post cryopreservation with a cooling rate of 100 degrees Celsius/min. A small amount of Ca(2+) (up to 0.15 M) was also found to help maintain the microcapsule integrity during cryopreservation, which is attributed to the enhancement of the alginate matrix strength by Ca(2+) rather than promoting vitrification of water in the microcapsules. The preferential vitrification of water in small microcapsules was further found to significantly augment cell cryopreservation by vitrification at a low concentration of cryoprotectants (i.e., 10% (v/v)) using a small quartz microcapillary (400 microm in diameter). Therefore, the small alginate microcapsule could be a great system for protecting living cells that are highly sensitive to stresses due to freezing (i.e., ice formation) and high concentration of cryoprotectants from injury during cryopreservation.