Stem cells have 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 promising candidate for many cell-based therapies. Unlike induced pluripotent stem cells and embryonic stem cells, adult hMSCs do not raise ethical and legislative issues, so their use takes advantage of an increased likelihood of authority approval and public acceptance. Even though bone marrow has been established as the primary source of adult hMSCs, peripheral sources such as Umbilical Cord Blood (UCB) are being investigated because of their non-invasive and painless collection. Storing of MSCs represents a fundamental step in tissue engineering, gene therapy and regenerative medicine applications. The most common preservation method, to make cells available on demand, consists in cooling to a cryogenic temperature. Cryopreservation allows long shelf lives and genetic stability, reduced risk of microbial contamination, and improved cost effectiveness [1]. Unfortunately, cryopreserved cells are damaged by the process itself [2]. This loss, which amounts to up to 50 %, is unacceptable for the hMSCs from UCB whose collection and isolation is known to be difficult. 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. Cryopreservation consists of different stages: cooling to sub-zero temperatures with or without permeant and non-permeant Cryo-Protectant Agents (CPAs), storage, thawing and return to physiological environment. Any of these steps can potentially lead to cell damage due to the physical and chemical phenomena involved such as intracellular ice formation, excessive solutes concentrations, cell shrinkage or swelling and CPA cytotoxicity. Due to the process complexity, a very high number of trials are required for experimental optimization, which is able to lead to suboptimal solutions only. On the contrary, mathematical modelling may provide considerable time and cost savings as well as mechanistic understanding of the underlying phenomena. In order to develop a model describing the process of cryopreservation, the osmotic behaviour needs to be investigated first to predict the osmosis-driven change in the amount of intracellular water, which can form lethal ice or glass during the process, as well as to limit excessive cell volume excursions and solute concentrations that might lead to the so-called solution injury [3, 4]. As reported in [5] and [6], we first attempted to determine the osmotic properties of hMSC from UCB making use of the typical bi-compartimental two-parameters model used in cryopreservation [7]. This attempt was not successful in simulating the osmotic response of these cells, as they do not behave as perfect osmometers, and show a peculiar osmotic response. In this work a novel approach is proposed to take this particular osmotic response into account. The model describes the volume variation of intracellular water, dimethyl sulphoxide (DMSO), and sodium chloride (NaCl). The difference of the proposed model from typical models reported in the literature consists in allowing NaCl to permeate the cell membrane. According to this assumption, incomplete volume recovery either upon restoring isotonic conditions after initial contact with hypertonic solutions of non-permeant (sucrose) CPAs or in the swelling phase of the well-known shrink-swell response in presence of permeant (DMSO) CPAs, is attributed to a net efflux of NaCl (low-permeant). The low-permeant solute efflux reduces its intracellular concentration, thus reducing the influx of water and the final equilibrium cell volume [8]. The NaCl flux can be rationalised by invoking the notion of mechanosensitive (MS) channels. There are two main theories purporting to explain mechanosensitive channel gating: (i) the bilayer model hypothesising that the tension of the lipid bilayer alone is sufficient to gate the MS channels, and (ii) the tethered model involving relative displacement of the MS channel with respect to the cytoskeleton or extracellular matrix proteins [9]. Whatever the exact microscopic mechanism involved, the overall effect is that the membrane tension is reduced in the shrinking phase leading to the closure of MS channels, while its increase during swelling results in their opening with the overall effect of changing NaCl permeability. The proposed model was found to be able to describe the osmotic behaviour of hMSC from UCB in presence of permeant and not permeant CPAs. Using this model, the permeabilities of water, NaCl and DMSO have been estimated from experimental data, while the inactive cell volume was fixed at the value previously obtained in [5]. According to the MS channels hypothesis, NaCl permeability has been found to depend on whether the cell is shrinking or swelling with higher values of permeability associated with the latter. It should be noted that different parameter values were estimated from experimental runs in presence of sucrose and DMSO. More precisely, lower values for NaCl and water permeability were found in presence of DMSO. Regarding water permeability, this behaviour is well known in literature. A decrease in the hydraulic conductivity has been found in presence of different CPAs (glycerol, ethylene glycol and propylene glycol, DMSO) and at increasing CPA concentrations [10]. It has been hypothesised that CPAs block water channels changing bilayer permeability [11]. Also molecular dynamics simulations have indicated that, depending on concentration, DMSO is able to fluidise the membrane [12] leading to the relaxation of membrane tension that could alter the permeability of MS channels. It is therefore not unlikely that NaCl permeability is affected by permeant CPAs in a similar manner, in accordance with the estimated parameter values. Further validation of the model was achieved by predicting experimental cycles of sucrose addition and restoring isotonic conditions, as well as runs with concentrations of DMSO different from those used for the determination of model parameters. 1. Karlsson, J.O.M. and M. Toner, Cryopreservation, in Principles of Tissue Engineering, 2ndEd.2000, Academic press: San Diego. p. 293-307. 2. Mazur, P., Life in Frozen State. 2004, London, UK: CRC Press. 3. Fadda, S., A. Cincotti, and G. Cao, The Effect of Cell Size Distribution During the Cooling Stage of Cryopreservation without CPA. Aiche Journal, 2010. 56(8): p. 2173-2185. 4. Fadda, S., A. Cincotti, and G. Cao, Rationalizing the Equilibration and Cooling Stages of Cryopreservation: The Effect of Cell Size Distribution. Aiche Journal, 2011. 57(4): p. 1075-1095. 5. Casula, E., et al., hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation. Chemical Engineering Transactions, 2015. 43: p. 265-270. 6. Casula, E., S. Fadda, and A. Cincotti. Isolation, Characterization and Analysis of the Osmotic Behaviour of hMSCs from UCB for Optimal Cryopreservation. in AIChE Annual Meeting. 2015. Salt Lake City, UT. 7. Kleinhans, F.W., Membrane permeability modeling: Kedem-Katchalsky vs a two-parameter formalism. Cryobiology, 1998. 37(4): p. 271-289. 8. Katkov, I.I., A two-parameter model of cell membrane permeability for multisolute systems. Cryobiology, 2000. 40(1): p. 64-83. 9. Martinac, B., Mechanosensitive ion channels: molecules of mechanotransduction. Journal of Cell Science, 2004. 117(12): p. 2449-2460. 10. Wang, J.Y., et al., Dual Dependence of Cryobiogical Properties of Sf21 Cell Membrane on the Temperature and the Concentration of the Cryoprotectant. Plos One, 2013. 8(9). 11. Gilmore, J.A., et al., Effect of Cryoprotectant Solutes on Water Permeability of Human Spermatozoa. Biology of Reproduction, 1995. 53(5): p. 985-995. 12. de Menorval, M.A., et al., Effects of Dimethyl Sulfoxide in Cholesterol-Containing Lipid Membranes: A Comparative Study of Experiments In Silico and with Cells. Plos One, 2012. 7(7).

A Novel Model for the Osmotic Behaviour of Human Mesenchymal Stem Cells

CASULA, ELISA;FADDA, SARAH;CINCOTTI, ALBERTO
2016

Abstract

Stem cells have 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 promising candidate for many cell-based therapies. Unlike induced pluripotent stem cells and embryonic stem cells, adult hMSCs do not raise ethical and legislative issues, so their use takes advantage of an increased likelihood of authority approval and public acceptance. Even though bone marrow has been established as the primary source of adult hMSCs, peripheral sources such as Umbilical Cord Blood (UCB) are being investigated because of their non-invasive and painless collection. Storing of MSCs represents a fundamental step in tissue engineering, gene therapy and regenerative medicine applications. The most common preservation method, to make cells available on demand, consists in cooling to a cryogenic temperature. Cryopreservation allows long shelf lives and genetic stability, reduced risk of microbial contamination, and improved cost effectiveness [1]. Unfortunately, cryopreserved cells are damaged by the process itself [2]. This loss, which amounts to up to 50 %, is unacceptable for the hMSCs from UCB whose collection and isolation is known to be difficult. 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. Cryopreservation consists of different stages: cooling to sub-zero temperatures with or without permeant and non-permeant Cryo-Protectant Agents (CPAs), storage, thawing and return to physiological environment. Any of these steps can potentially lead to cell damage due to the physical and chemical phenomena involved such as intracellular ice formation, excessive solutes concentrations, cell shrinkage or swelling and CPA cytotoxicity. Due to the process complexity, a very high number of trials are required for experimental optimization, which is able to lead to suboptimal solutions only. On the contrary, mathematical modelling may provide considerable time and cost savings as well as mechanistic understanding of the underlying phenomena. In order to develop a model describing the process of cryopreservation, the osmotic behaviour needs to be investigated first to predict the osmosis-driven change in the amount of intracellular water, which can form lethal ice or glass during the process, as well as to limit excessive cell volume excursions and solute concentrations that might lead to the so-called solution injury [3, 4]. As reported in [5] and [6], we first attempted to determine the osmotic properties of hMSC from UCB making use of the typical bi-compartimental two-parameters model used in cryopreservation [7]. This attempt was not successful in simulating the osmotic response of these cells, as they do not behave as perfect osmometers, and show a peculiar osmotic response. In this work a novel approach is proposed to take this particular osmotic response into account. The model describes the volume variation of intracellular water, dimethyl sulphoxide (DMSO), and sodium chloride (NaCl). The difference of the proposed model from typical models reported in the literature consists in allowing NaCl to permeate the cell membrane. According to this assumption, incomplete volume recovery either upon restoring isotonic conditions after initial contact with hypertonic solutions of non-permeant (sucrose) CPAs or in the swelling phase of the well-known shrink-swell response in presence of permeant (DMSO) CPAs, is attributed to a net efflux of NaCl (low-permeant). The low-permeant solute efflux reduces its intracellular concentration, thus reducing the influx of water and the final equilibrium cell volume [8]. The NaCl flux can be rationalised by invoking the notion of mechanosensitive (MS) channels. There are two main theories purporting to explain mechanosensitive channel gating: (i) the bilayer model hypothesising that the tension of the lipid bilayer alone is sufficient to gate the MS channels, and (ii) the tethered model involving relative displacement of the MS channel with respect to the cytoskeleton or extracellular matrix proteins [9]. Whatever the exact microscopic mechanism involved, the overall effect is that the membrane tension is reduced in the shrinking phase leading to the closure of MS channels, while its increase during swelling results in their opening with the overall effect of changing NaCl permeability. The proposed model was found to be able to describe the osmotic behaviour of hMSC from UCB in presence of permeant and not permeant CPAs. Using this model, the permeabilities of water, NaCl and DMSO have been estimated from experimental data, while the inactive cell volume was fixed at the value previously obtained in [5]. According to the MS channels hypothesis, NaCl permeability has been found to depend on whether the cell is shrinking or swelling with higher values of permeability associated with the latter. It should be noted that different parameter values were estimated from experimental runs in presence of sucrose and DMSO. More precisely, lower values for NaCl and water permeability were found in presence of DMSO. Regarding water permeability, this behaviour is well known in literature. A decrease in the hydraulic conductivity has been found in presence of different CPAs (glycerol, ethylene glycol and propylene glycol, DMSO) and at increasing CPA concentrations [10]. It has been hypothesised that CPAs block water channels changing bilayer permeability [11]. Also molecular dynamics simulations have indicated that, depending on concentration, DMSO is able to fluidise the membrane [12] leading to the relaxation of membrane tension that could alter the permeability of MS channels. It is therefore not unlikely that NaCl permeability is affected by permeant CPAs in a similar manner, in accordance with the estimated parameter values. Further validation of the model was achieved by predicting experimental cycles of sucrose addition and restoring isotonic conditions, as well as runs with concentrations of DMSO different from those used for the determination of model parameters. 1. Karlsson, J.O.M. and M. Toner, Cryopreservation, in Principles of Tissue Engineering, 2ndEd.2000, Academic press: San Diego. p. 293-307. 2. Mazur, P., Life in Frozen State. 2004, London, UK: CRC Press. 3. Fadda, S., A. Cincotti, and G. Cao, The Effect of Cell Size Distribution During the Cooling Stage of Cryopreservation without CPA. Aiche Journal, 2010. 56(8): p. 2173-2185. 4. Fadda, S., A. Cincotti, and G. Cao, Rationalizing the Equilibration and Cooling Stages of Cryopreservation: The Effect of Cell Size Distribution. Aiche Journal, 2011. 57(4): p. 1075-1095. 5. Casula, E., et al., hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation. Chemical Engineering Transactions, 2015. 43: p. 265-270. 6. Casula, E., S. Fadda, and A. Cincotti. Isolation, Characterization and Analysis of the Osmotic Behaviour of hMSCs from UCB for Optimal Cryopreservation. in AIChE Annual Meeting. 2015. Salt Lake City, UT. 7. Kleinhans, F.W., Membrane permeability modeling: Kedem-Katchalsky vs a two-parameter formalism. Cryobiology, 1998. 37(4): p. 271-289. 8. Katkov, I.I., A two-parameter model of cell membrane permeability for multisolute systems. Cryobiology, 2000. 40(1): p. 64-83. 9. Martinac, B., Mechanosensitive ion channels: molecules of mechanotransduction. Journal of Cell Science, 2004. 117(12): p. 2449-2460. 10. Wang, J.Y., et al., Dual Dependence of Cryobiogical Properties of Sf21 Cell Membrane on the Temperature and the Concentration of the Cryoprotectant. Plos One, 2013. 8(9). 11. Gilmore, J.A., et al., Effect of Cryoprotectant Solutes on Water Permeability of Human Spermatozoa. Biology of Reproduction, 1995. 53(5): p. 985-995. 12. de Menorval, M.A., et al., Effects of Dimethyl Sulfoxide in Cholesterol-Containing Lipid Membranes: A Comparative Study of Experiments In Silico and with Cells. Plos One, 2012. 7(7).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11584/195780
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