Rationalizing the equilibration and cooling stages of cryopreservation: The effect of cell size distribution

Fadda, Sarah; Cincotti, Alberto; Cao, Giacomo

doi:10.1002/aic.12320

Cited by 16 publications

(4 citation statements)

References 39 publications

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“…In silico models of ice formation (e.g. Haji-Akbari & Debenedetti, 2017) are not yet scalable to whole animals (Li & Liu, 2010), but could be used to explore cellular dehydration and IIF (see Botkin, Hoffmann & Turova, 2011;Fadda, Cincotti & Cao, 2011), and ice propagation (e.g. Lee et al, 1993;Worland et al, 2004) in real time (Table 5).…”

Section: (1) Understanding the Processes And Challenges Of Freezingmentioning

confidence: 99%

Mechanisms underlying insect freeze tolerance

2018

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Freeze tolerance - the ability to survive internal ice formation - has evolved repeatedly in insects, facilitating survival in environments with low temperatures and/or high risk of freezing. Surviving internal ice formation poses several challenges because freezing can cause cellular dehydration and mechanical damage, and restricts the opportunity to metabolise and respond to environmental challenges. While freeze-tolerant insects accumulate many potentially protective molecules, there is no apparent 'magic bullet' - a molecule or class of molecules that appears to be necessary or sufficient to support this cold-tolerance strategy. In addition, the mechanisms underlying freeze tolerance have been minimally explored. Herein, we frame freeze tolerance as the ability to survive a process: freeze-tolerant insects must withstand the challenges associated with cooling (low temperatures), freezing (internal ice formation), and thawing. To do so, we hypothesise that freeze-tolerant insects control the quality and quantity of ice, prevent or repair damage to cells and macromolecules, manage biochemical processes while frozen/thawing, and restore physiological processes post-thaw. Many of the molecules that can facilitate freeze tolerance are also accumulated by other cold- and desiccation-tolerant insects. We suggest that, when freezing offered a physiological advantage, freeze tolerance evolved in insects that were already adapted to low temperatures or desiccation, or in insects that could withstand small amounts of internal ice formation. Although freeze tolerance is a complex cold-tolerance strategy that has evolved multiple times, we suggest that a process-focused approach (in combination with appropriate techniques and model organisms) will facilitate hypothesis-driven research to understand better how insects survive internal ice formation.

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Section: (1) Understanding the Processes And Challenges Of Freezingmentioning

confidence: 99%

Mechanisms underlying insect freeze tolerance

2018

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“…While such loss is acceptable for some cell lineages for research application, it becomes unacceptable in clinical practices, especially those involving human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) whose collection and isolation is known to be difficult (Bieback et al, 2004;Casula et al, 2017;Gao et al, 1995). Thus, optimization of the operating conditions adapted to the osmotic behavior of the specific cell lineage under investigation is crucial for cryopreservation (Benson, 2021;Casula et al, 2017Casula et al, , 2019Cincotti & Fadda, 2012;Fadda et al, 2009Fadda et al, , 2010Fadda et al, , 2011aFadda et al, , 2011bHughes et al, 2012;Karlsson & Toner, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, best practices may be defined through mathematical modeling and numerical simulations (Benson, 2021;Cincotti & Fadda, 2012;Fadda et al, 2009Fadda et al, , 2010Fadda et al, , 2011aFadda et al, , 2011bKarlsson & Toner, 1996), provided that the adopted mathematical model is capable to describe properly the behavior of the system under investigation.…”

Section: Introductionmentioning

confidence: 99%

“…Unfortunately, the number of experimental variables and parameters involved in a cryopreservation protocol is prohibitively large to permit a rigorous optimization of the process (Benson, 2021; Casula et al, 2019; Karlsson & Toner, 2000): not only cooling and thawing rates need to be optimized to limit the lethal intracellular ice formation or recrystallization but also concentration, temperature, and temporal duration of CPA addition and removal usually carried out in a step‐wise fashion, demand a careful design to limit osmotic injury and cytotoxicity. On the other hand, best practices may be defined through mathematical modeling and numerical simulations (Benson, 2021; Cincotti & Fadda, 2012; Fadda et al, 2009, 2010, 2011a, 2011b; Karlsson & Toner, 1996), provided that the adopted mathematical model is capable to describe properly the behavior of the system under investigation.…”

Section: Introductionmentioning

confidence: 99%

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hMSCs in contact with DMSO for cryopreservation: Experiments and modeling of osmotic injury and cytotoxic effect

Traversari

Delogu

Aparício

et al. 2022

Biotech & Bioengineering

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In this study a combined analysis of osmotic injury and cytotoxic effect useful for the optimization of the cryopreservation process of a cell suspension is carried out. The case of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) in contact with dimethyl sulfoxide (DMSO) acting as Cryo‐Protectant Agent (CPA) is investigated from the experimental as well as the theoretical perspective. The experimental runs are conducted by suspending the cells in hypertonic solutions of DMSO at varying osmolality, system temperature, and contact times; then, at room temperature, cells are pelleted by centrifugation and suspended back to isotonic conditions. Eventually, cell count and viability are measured by means of a Coulter counter and flow‐cytometer, respectively. Overall, a decrease in cell count and viability results when DMSO concentration, temperature, and contact time increase. A novel mathematical model is developed and proposed to interpret measured data by dividing the cell population between viable and nonviable cells. The decrease of cell count is ascribed exclusively to the osmotic injury caused by expansion lysis: excessive swelling causes the burst of both viable as well as nonviable cells. On the other hand, the reduction of cell viability is ascribed only to cytotoxicity which gradually transforms viable cells into nonviable ones. A chemical reaction engineering approach is adopted to describe the dynamics of both phenomena: by following the kinetics of two chemical reactions during cell osmosis inside a closed system it is shown that the simultaneous reduction of cell count and viability may be successfully interpreted. The use of the Surface Area Regulation (SAR) model recently proposed by the authors allows one to avoid the setting in advance of fixed cell Osmotic Tolerance Limits (OTLs), as traditionally done in cryopreservation literature to circumvent the mathematical simulation of osmotic injury. Comparisons between experimental data and theoretical simulations are provided: first, a nonlinear regression analysis is performed to evaluate unknown model parameters through a best‐fitting procedure carried out in a sequential fashion; then, the proposed model is validated by full predictions of system behavior measured at operating conditions different from those used during the best‐fit procedure.

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Modelling the Cryopreservation Process of a Suspension of Cells: The Effect of a Size-Distributed Cell Population

Cincotti

Fadda

2012

Computational Modeling in Tissue Engineering

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Cryopreservation of biological material is a crucial step of tissue engineering, but biological material can be damaged by the cryopreservation process itself. Depending on some bio-physical properties that change from cell to cell lineages, an optimum cryopreservation protocol needs to be identified for any cell type to maximise post-thaw cell viability. Since a prohibitively large set of operating conditions has to be determined to avoid the principal origins of cell damage (i.e., ice formation and solution injuries), mathematical modelling represents a valuable alternative to experimental optimisation. The theoretical analysis traditionally adopted for the cryopreservation of a cell suspension addresses only a single, average cell size and ascribes the experimental evidence of different ice formation temperatures to statistical variations. In this chapter our efforts to develop a novel mathematical model based on the population balance approach that comprehensively takes into account the size distribution of a cell population are reviewed. According to this novel approach, a sound explanation for the experimental evidence of different ice formation temperatures may now be given by adopting a fully deterministic criterion based on the size distribution of the cell population. In this regard, the proposed model represents a clear novelty for the cryopreservation field and provides an original perspective to interpret system behaviour as experimentally measured so far. First our efforts to successfully validate the proposed model by comparison with suitable experimental data taken from the literature are reported. Then, in absence of suitable experimental data, the model is used to theoretically investigate system behaviour at various operating conditions. This is done both in absence or presence of a cryo-protectant agent, as well as when the extra-cellular ice is assumed to form under thermodynamic equilibrium or its dynamics is taken into account consistently by means of an additional population balance. More specifically, the effect of the cell size distribution on system behaviour when varying cooling rate and cryo-protectant content within practicable values for a standard cryopreservation protocol is investigated. It is demonstrated that, cell survival due to intra-cellular ice formation depends on the initial cell size distribution and its osmotic parameters. At practicable operating conditions in terms of cooling rate and cryo-protectant concentration, intra-cellular ice formation may be lethal for the fraction of larger size classes of the cell population whilst it may not reach a dangerous level for the intermediate size class cells and it will not even take place for the smaller ones.\u

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Rationalizing the equilibration and cooling stages of cryopreservation: The effect of cell size distribution

Cited by 16 publications

References 39 publications

Mechanisms underlying insect freeze tolerance

Mechanisms underlying insect freeze tolerance

hMSCs in contact with DMSO for cryopreservation: Experiments and modeling of osmotic injury and cytotoxic effect

Modelling the Cryopreservation Process of a Suspension of Cells: The Effect of a Size-Distributed Cell Population

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