Isolation, characterization and analysis of the osmotic behaviour of hMSCs from UCB for optimal cryopreservation

Demographic studies have shown that population is ageing. As a consequence, degenerative events are increasing and the regenerative medicine market is growing rapidly. Within this context, stem cells possess an enormous therapeutic potential for regeneration and replacement of degenerated tissues. In particular, the ability to readily expand in culture, while maintaining a self-renewing phenotype, has made human Mesenchymal Stem Cells (hMSCs) a candidate for many cell-based therapies (Pittenger et al., 1999; Parekkadan et al., 2007). Unlike induced pluripotent stem cells and embryonic stem cells, adult hMSCs do not raise ethical and legislative issues, so that their use takes advantage of an increased likelihood for authority approval and public acceptance. Even if bone marrow has been established as the primary source of adult hMSCs, due to the invasive nature of bone marrow aspiration, the identification of other abundant and reliable sources has nowadays become a priority. Regarding this, the successful isolation based on adherence capability to tissue culture plastic of hMSCs from peripheral sources, such as Umbilical Cord Blood (UCB), has been reported, , even if, according to other contradictory studies, hMSCs in these biological samples were not found. In this framework, the ability to preserve these rare cells with high efficiency represents an even more crucial step in the regenerative medicine supply chain, since preservation now represents a core technology to bring cell-based products to market, on demand (Karlsson and Toner, 2000). The principal preservation method consists of freezing the bio-specimens to cryogenic temperature in order to take advantage of the preservative power of the cold. If compared to the other preservation methods like maintaining the bio samples in continuous culture, cryopreservation has the benefits of affording long shelf lives, genetic stability, reduced microbial contamination risks, and cost effectiveness. The other side of the coin is that cryopreserved biological material can be damaged by the cryopreservation process itself. This damage ultimately translates into a reduced number of viable or functional cells, a loss that can be as high as 50% (Wang et al., 2011). This can be tolerated for some cell lineages, but it’s unacceptable for others, as the hMSCs from UCB, whose collection and isolation is known to be difficult (Bieback et al., 2004). Even if, in principle, cell expansion/proliferation may solve the problem, an increased number of passages will inexorably lead these cells to lose their peculiar characteristics, and should be avoided (Lee et al., 2004). Cryopreservation consists of cooling to sub-zero temperatures with or without a Cryo-Protectant Agent (CPA), storage, thawing and return to physiological environment for specific usages. Moreover, the steps of addition and removal of a cytotoxic CPA as DiMethyl SulfOxide (DMSO) which permeates through cells membrane into cytoplasm, needs to be taken into account when looking for optimal cryopreservation. A part from storage, all these different stages are potentially able to damage the cells due to the physical and/or chemical phenomena involved such as intra-cellular ice formation, excessive solutes concentrations and cell shrinkage or swelling. In general, due to the high number of trials actually required for experimental optimization of the cryopreservation process, mathematical modelling is considered a practical solution (Karlsson and Toner, 2000). To this aim, the osmotic behaviour needs to be first investigated in order to be able to predict the volume of residual intra-cellular water left by osmosis to form lethal ice or glass during the cryopreservation process, as well as to limit excessive cell volume excursions and solutes concentrations that might lead to the so-called solution injury (Fadda et al., 2010; 2011). In this work, the hMSCs from UCB of three different donors were isolated by a density gradient centrifugation method, followed by plastic adherence of mononuclear cells. The isolation (20% success rate) was verified through phenotypic cytofluorimetric analysis, and adipogenesis/osteogenesis capability differentiations. The osmotic properties, namely inactive cell volume, water and CPA (DMSO) permeabilities, were determined by means of experimental runs carried out under hypertonic conditions (obtained with the addition of sucrose or DMSO to PBS, isotonic solution), at three different temperatures. To the best of authors’ knowledge, these osmotic transport parameters have never been studied before for the hMSCs from UCB. Cell size was determined using an impedance measurement device (Coulter Counter), under equilibrium and dynamic conditions. Since the impedance measuring device does not discriminate single cells by debris or cells agglomerates, the measured cell volume distributions were filtered out from the data originally provided by the Coulter Counter through a novel data treatment, proposed in this work. Linear and non-linear regression analyses were carried out to determine the adjustable parameters by means of the salt-water sack model in the 2-parameters bi-compartimental version by Kleinahns (1998), as applied to a single-sized cell population (i.e. identical cells, with size equal to the average), as classically proposed for numerous cell lineages in the technical literature addressing cryopreservation. Basically, this model addresses a suspension of cells in a liquid, ideal solution, characterized by a given osmolality; the cells are supposed to act as a perfect osmometer in response of the ruling driving force, i.e. the difference between intra- and extra-cellular solute osmolalities, where only water and DMSO are assumed to permeate through cell membrane, while neglecting ionic transfer. According to this model, in this work a rational parameter estimation was attempted by carrying out an ideal fitting procedure. It was found that the adopted model is not capable to simulate entirely the osmotic response for the cells under investigation. Specifically, only during swelling an apparent dependence of the so-called inactive cell volume from temperature and CPA concentration needs to be considered for hMSCs from UCB. Thus, these cells do not behave as a perfect osmometer, and show a peculiar osmotic response. It may be concluded that, a regulatory volume system is activated for these cells, albeit only during swelling. This control system is presumably related to the action of ion pumps and transport channels, which are well-known in the literature for conditioning cells to return to their isotonic size, thus contrasting the lethal effect produced by osmosis (Hoffmann et al., 2009). In such a case, a more complex mathematical model than the standard salt-water sack model needs to be taken into account for capturing system behaviour. Specifically, resorting to the complex Goldman-Hodgkin-Katz model of permeant ions where quantities as membrane potential and ion permeabilities are introduced (Fernandez et al., 2013) may be considered. But, this demands a complex validation through direct comparison to data measured in well-designed experiments, which may be difficult to achieve. As an alternative, or in conjunction with this ionic transport mechanism for controlling cell swelling, the extrusion of permeant osmolites/solutes (produced inside the cells to the detriment of the inactive cell volume to osmosis) may be hypothesised. This would result in a smaller deviation from the standard salt-water sack model, simpler than taking into account the electro-diffusion of ions through cell membrane. References Bieback, K., Kern, S., Kluter, H., Eichler, H., 2004. Critical Parameters for the Isolation of Mesenchymal Stem Cells from Umbilical Cord Blood. Stem Cells 22, 625–634. Fadda, S., Cincotti, A., Cao, G., 2010. The Effect of Cell Size Distribution During the Cooling Stage of Cryopreservation without CPA. AIChE Journal 56 (8), 2173-2185. Fadda, S., Cincotti, A., Cao, G., 2011. Rationalizing the Equilibration and Cooling Stages of Cryopreservation: The Effect, of Cell Size Distribution. AIChE Journal 57 (4), 1075-1095. Fernandez, J.M., Di Giusto, G., Kalstein, M., Melamud, L., Rivarola, V., Ford, P., Capurro, C., 2013. Cell Volume Regulation in Cultured Human Retinal Muller Cells Is associated with Changes in trasmembrane potential. Plos One 8 (2), e57268. Hoffmann, E.K., Lambert, I.H., Pedersen, S.F., 2009. Physiology of Cell Volume Regulation in Vertebrates. Physiological Reviews 89, 193–277. Karlsson, J.O.M., Toner, M., 2000. Cryopreservation, in: Lanza, R.P., Langer, R., Vacanti, J., (Eds.), Principles of Tissue Engineering, 2nd Ed. Academic press, San Diego, pp. 293-307. Kleinhans, F.W., 1998. Membrane Permeability Modeling: Kedem-Katchalsky vs a Two Parameter Formalism. Cryobiology 37, 271-289. Lee, M.W., Choi, J., Yang, M.S., Moon, Y.J., Park, J.S., Kim, H.C., Kim, Y.J., 2004. Mesenchymal stem cells from cryopreserved human umbilical cord blood. Biochemical and Biophysical Research Communications 320, 273–278. Parekkadan, B., Sethu, P. Van Poll, D., Yarmush, M.L., Toner, M., 2007. Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood. Tissue Engineering 13, 2465-2473. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411), 143– 147. Wang, H.Y., Lun, Z.R., Lu, S.S., 2011. Cryopreservation of Umbilical Cord Blood-Derived Mesenchymal Stem Cells Without Dimethyl Sulfoxide. CryoLetters 32 (1), 81-88.

Demographic studies have shown that population is ageing. As a consequence, degenerative events are increasing and the regenerative medicine market is growing rapidly. Within this context, stem cells possess an enormous therapeutic potential for regeneration and replacement of degenerated tissues. In particular, the ability to readily expand in culture, while maintaining a self-renewing phenotype, has made human Mesenchymal Stem Cells (hMSCs) a candidate for many cell-based therapies (Pittenger et al., 1999; Parekkadan et al., 2007). Unlike induced pluripotent stem cells and embryonic stem cells, adult hMSCs do not raise ethical and legislative issues, so that their use takes advantage of an increased likelihood for authority approval and public acceptance.