hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation

Abstract

In tissue engineering, storing of biological material represents a fundamental step to bring cell-based medical devices to market on demand - Karlsson and Toner (2000) and more recently Fadda et al. (2009). Compared to other methods, freezing to cryogenic temperatures allows long shelf lives and genetic stability (Karlsson and Toner, 2000). Unfortunately, cryopreserved cells are damaged by the cryopreservation process itself (Mazur, 2004). This loss (up to 50 %) can be tolerated for some cell lineages, but it’s unacceptable for others, as the human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB), whose collection and isolation is known to be difficult (Bieback et al., 2004). In this case, an optimal cryopreservation protocol is mandatory. Due to the high number of trials actually required for experimental optimization, mathematical modelling is considered a practical solution. To this aim, the osmotic properties need to be first estimated in order to determine the volume of residual intra-cellular water left by osmosis to form lethal ice or glass. In this work, the hMSCs from UCB of three different donors, after informed consent, have been isolated by a density gradient centrifugation method. The successful isolation has been verified through phenotypic cytofluorimetric analysis, and adipogenesis/osteogenesis capability differentiations. Osmotic properties, namely inactive cell volume, water and CPA (DMSO) permeabilities, have been determined by means of experimental runs carried out under hypertonic conditions (obtained with the addition of sucrose or DMSO), at three different temperatures. Cells volumes excursions have been measured by a potenziometric device (Coulter Counter) under equilibrium and dynamic conditions. Linear and non-linear regression analyses have been carried out to determine the adjustable parameters by means of the two parameters bi-compartimental model by Kleinahns (1998), as applied to a single-sized cell population (i.e. identical cells with size equal to the average). It is found that, the inactive volume fraction of hMSC from UCB apparently changes (increase) when DMSO is used instead of sucrose, thus limiting cell volume excursion during swelling. It is hypothesized that, a cell volume control system is activated during swelling, probably due to the action of ion pumps.