The need for novel cryoprotectants and cryopreservation protocols: Insights into the importance of biophysical investigation and cell permeability

Raju, Rekha; Bryant, Saffron J.; Wilkinson, Brendan L.; Bryant, Gary

doi:10.1016/j.bbagen.2020.129749

Cited by 67 publications

(61 citation statements)

References 153 publications

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“…82,83 On a similar basis, macromolecular solutes that are excluded from the hydration shell of proteins have increased protein stability during the freezing process. 17 Protein denaturation can result from both heat and cold. Heat denaturation of proteins occurs above 50 1C, whereas cold denaturation occurs in the range of 0-20 1C.…”

Section: Mechanisms Of Cryoprotection Using Macromolecular Cryoprotectantsmentioning

confidence: 99%

“…Osmotically inactive volume specifies the 'safe' volume changes a cell may undergo upon the addition of osmotically active polymeric solutes, without compromising cell viability. 17 Long-term cryoprotection is usually achieved by employing strategies that avoid intracellular ice formation. During the freezing process, an increase in the osmotic pressure due to the concentrated and partially frozen extracellular CPA solution dehydrates the cell.…”

Section: Perspective On Factors Influencing Macromoleculemediated Cryoprotectionmentioning

confidence: 99%

“…However, the use of serum in cell culture introduces batch-to-batch variability as well as raises ethical and immunological constraints, especially for cell culture products intended for clinical applications. 17 Efforts have been made to use human serum albumin (HSA) as an alternative. However, HSA poses a risk of infection, has limited supply, and is also extremely expensive.…”

Section: Introductionmentioning

confidence: 99%

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Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

et al. 2022

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Section: Mechanisms Of Cryoprotection Using Macromolecular Cryoprotectantsmentioning

confidence: 99%

Section: Perspective On Factors Influencing Macromoleculemediated Cryoprotectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

et al. 2022

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“…The study of cryopreservation is a rich and active field of research that has extensively explored many different cryopreserving reagents and formulations since the birth of cryobiology in the 1950s. Cooling methods have also been explored to improve the storage of cells and tissue for research and therapeutic purposes ( Raju et al, 2021 ) ( Hornberger et al, 2019 ). Much of the reported work is centered around reducing intracellular ice crystal growth, considered to be the most damaging effect of cryogenic preservation ( Mazur 2004 ).…”

Section: Introductionmentioning

confidence: 99%

Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

Overall

Barnes

2021

Front. Mol. Biosci.

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In-cell DNP is a growing application of NMR to the study of biomolecular structure and function within intact cells. An important unresolved question for in-cell DNP spectroscopy is the integrity of cellular samples under the cryogenic conditions of DNP. Despite the rich literature around cryopreservation of cells in the fields of stem cell/embryonic cell therapeutics, cell line preservation and in cryo-EM applications, the effect of cryopreservation procedures on DNP parameters is unclear. In this report we investigate cell survival and apoptosis in the presence of cryopreserving agents and DNP radicals. We also assess the effects of these reagents on cellular enhancements. We show that the DNP radical AMUPol has no effect on membrane permeability and does not induce apoptosis. Furthermore, the standard aqueous glass forming reagent, comprised of 60/30/10 d8-glycerol/D2O/H2O (DNP juice), rapidly dehydrates cells and induces apoptosis prior to freezing, reducing structural integrity of the sample prior to DNP analysis. Preservation with d6-DMSO at 10% v/v provided similar DNP enhancements per √unit time compared to glycerol preservation with superior maintenance of cell size and membrane integrity prior to freezing. DMSO preservation also greatly enhanced post-thaw survival of cells slow-frozen at 1°C/min. We therefore demonstrate that in-cell DNP-NMR studies should be done with d6-DMSO as cryoprotectant and raise important considerations for the progression of in-cell DNP-NMR towards the goal of high quality structural studies.

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“…For this reason, cryoprotectant-free vitrification methods have been attempted in human spermatozoa [ 3 ] and equine oocytes [ 4 ] without success, and so far, it has never been performed in porcine. Moreover, results from previous studies demonstrated the need for CPAs [ 3 – 6 ], and recently new nontoxic CPAs has been proposed [ 7 ]. For oocyte cryopreservation, ethylene glycol (EG) and dimethylsulfoxide (DMSO) have been the most widely used permeable CPAs.…”

Section: Introductionmentioning

confidence: 99%

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

et al. 2021

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Background The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. Results The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. Conclusion This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

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The need for novel cryoprotectants and cryopreservation protocols: Insights into the importance of biophysical investigation and cell permeability

Cited by 67 publications

References 153 publications

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

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