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

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 gen...

Full description

Bibliographic Details
Main Authors: E. Casula, G. Asuni, V. Sogos, A. Cincotti
Format: Article
Language:English
Published: AIDIC Servizi S.r.l. 2015-05-01
Series:Chemical Engineering Transactions
Online Access:https://www.cetjournal.it/index.php/cet/article/view/4812
id doaj-b9fdcf877ef34a78b7233c56eaca0188
record_format Article
spelling doaj-b9fdcf877ef34a78b7233c56eaca01882021-02-20T21:16:01ZengAIDIC Servizi S.r.l.Chemical Engineering Transactions2283-92162015-05-014310.3303/CET1543045hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal CryopreservationE. CasulaG. AsuniV. SogosA. CincottiIn 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.https://www.cetjournal.it/index.php/cet/article/view/4812
collection DOAJ
language English
format Article
sources DOAJ
author E. Casula
G. Asuni
V. Sogos
A. Cincotti
spellingShingle E. Casula
G. Asuni
V. Sogos
A. Cincotti
hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation
Chemical Engineering Transactions
author_facet E. Casula
G. Asuni
V. Sogos
A. Cincotti
author_sort E. Casula
title hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation
title_short hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation
title_full hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation
title_fullStr hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation
title_full_unstemmed hMSCs from UCB: Isolation, Characterization and Determination of Osmotic Properties for Optimal Cryopreservation
title_sort hmscs from ucb: isolation, characterization and determination of osmotic properties for optimal cryopreservation
publisher AIDIC Servizi S.r.l.
series Chemical Engineering Transactions
issn 2283-9216
publishDate 2015-05-01
description 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.
url https://www.cetjournal.it/index.php/cet/article/view/4812
work_keys_str_mv AT ecasula hmscsfromucbisolationcharacterizationanddeterminationofosmoticpropertiesforoptimalcryopreservation
AT gasuni hmscsfromucbisolationcharacterizationanddeterminationofosmoticpropertiesforoptimalcryopreservation
AT vsogos hmscsfromucbisolationcharacterizationanddeterminationofosmoticpropertiesforoptimalcryopreservation
AT acincotti hmscsfromucbisolationcharacterizationanddeterminationofosmoticpropertiesforoptimalcryopreservation
_version_ 1724259347132841984