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HMSCs from UCB: isolation, characterization and determination of osmotic properties for optimal cryopreservation
Full text linkIn 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
Isolation, characterization and analysis of the osmotic behaviour of hMSCs from UCB for optimal cryopreservation
No full textDemographic 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Analysis of the osmotic behaviour for optimal cryopreservation of hMSCs from UCB
No full textIn this work, the osmotic behavior for optimal cryopreservation of the human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) of three different donors, after isolation and characterization, was investigated. The osmotic properties, i.e., namely inactive cell volume, water and cryo-protectant dimethyl sulfoxide (Me2SO) permeabilities, were evaluated by direct comparison to experimental measurements obtained at three different temperatures under hypertonic conditions (obtained adding sucrose or Me2SO to PBS solutions), and returning back to isotonic conditions. Cell size was determined using an impedance measurement device (Coulter Counter), under both equilibrium and dynamic conditions, while the classic 2-parameters bi-compartimental model was adopted for the linear and non-linear regression analyses. It was found that the inactive volume fraction of hMSCs from UCB apparently increase during swelling, thus limiting the positive excursions of cell volume. This happens either during the swelling phase of the shrink-swell dynamics during Me2SO addition, or during the return back to isotonic conditions after equilibration with a hypertonic solution of the impermeant or permeant solute. Therefore, this cell lineage does not behave as a perfect osmometer and a peculiar cell volume control system needs to be considered to model the osmotic phenomena taking place during its cryopreservation.
Source of Funding (if any)
Regione Autonoma della Sardegna (RAS) - Progetto L.7/2007 RAS, Bando 2012, CUP F71J1200090000
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
A Novel Model for the Osmotic Behaviour of Human Mesenchymal Stem Cells
No full textStem 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)
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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