Cryopreservation of HeLa Cells at a High Hydrostatic Pressure of 1.0–1.5 kbar

Ugraitskaya, S. V.; Шишова, Н. В.; Valeeva, E. R.; Kaurova, Svetlana A.; Shvirst, N. E.; Фесенко, Е. Е.

doi:10.1134/s0006350921010140

Cited by 4 publications

(4 citation statements)

References 32 publications

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“…Indeed, the relevant studies [9-10] have reflexively resorted to media with concentrations of 30 wt % or higher, and reported ambiguous results. [11] This seems to confirm misgivings voiced in theoretical works [12] to the effect that suppression of ice-formation by CPAs might have counterproductive consequences on pressure buildup inside the capillary. The matter has however not been conclusively investigated, foremost because of lack of direct access to the pressure value as mentioned, but possibly also because the case for HPF or SPRF of live samples might seem weaker at first glance.…”

Section: Introductionsupporting

confidence: 73%

Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations

Rolle

Okotrub

Babin

et al. 2022

Preprint

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Self-pressurized rapid freezing (SPRF) has been proposed as a simple alternative to traditional high pressure freezing (HPF) protocols for vitrification of biological samples in electron microscopy and cryopreservation applications. Both methods exploit the circumstance that the melting point of ice reaches a minimum when subjected to pressure of around 210 [MPa], however, in SPRF its precise quantity depends on sample properties and hence, is generally unknown. In particular, cryoprotective agents (CPAs) are expected to be a factor; though eschewed by many SPRF experiments, vitrification of larger samples notably cannot be envisaged without them. Thus, in this study, we address the question of how CPA concentration affects pressure inside sealed capillaries, and how to design SPRF experiments accordingly. By embedding a fiber-optic probe in samples and performing Raman spectroscopy after freezing, we first present a direct assessment of pressure buildup during SPRF, enabled by the large pressure sensitivity of the Raman shift of hexagonal ice. Choosing dimethyl sulfoxide (DMSO) as a model CPA, this approach allows us to demonstrate that average pressure drops to zero when DMSO concentrations of 15 wt % are exceeded. Since a trade-off between pressure and DMSO concentration represents an impasse with regards to vitrification of larger samples, we introduce a sample architecture with two chambers, separated by a partition that allows for equilibration of pressure but not DMSO concentrations. We show that pressure and concentration in the fiber-facing chamber can be tuned independently, and present differential scanning calorimetry (DSC) data supporting the improved vitrification performance of two-chamber designs.

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Section: Introductionsupporting

confidence: 73%

Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations

Rolle

Okotrub

Babin

et al. 2022

Preprint

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“…This technique has been widely used for biospecimen fixation with applications in electron microscopy [55,56]. However, there are few studies of cryopreservation of living cells and tissues under hydrostatic pressure and ultra-rapid cooling, and further studies are required to investigate living specimens during cryopreservation [57].…”

Section: High Hydrostatic Pressurementioning

confidence: 99%

Technologies for Vitrification Based Cryopreservation

Amini

Benson

2023

Bioengineering

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Cryopreservation is a unique and practical method to facilitate extended access to biological materials. Because of this, cryopreservation of cells, tissues, and organs is essential to modern medical science, including cancer cell therapy, tissue engineering, transplantation, reproductive technologies, and bio-banking. Among diverse cryopreservation methods, significant focus has been placed on vitrification due to low cost and reduced protocol time. However, several factors, including the intracellular ice formation that is suppressed in the conventional cryopreservation method, restrict the achievement of this method. To enhance the viability and functionality of biological samples after storage, a large number of cryoprotocols and cryodevices have been developed and studied. Recently, new technologies have been investigated by considering the physical and thermodynamic aspects of cryopreservation in heat and mass transfer. In this review, we first present an overview of the physiochemical aspects of freezing in cryopreservation. Secondly, we present and catalog classical and novel approaches that seek to capitalize on these physicochemical effects. We conclude with the perspective that interdisciplinary studies provide pieces of the cryopreservation puzzle to achieve sustainability in the biospecimen supply chain.

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“…Though CPA concentrations are often low in an electron microscopy context, the matter can be quite different if we examine SPRF with a view to reversible cryopreservation of live specimens. Indeed, the relevant studies 9,10 have reflexively resorted to media with concentrations of 30 wt% or higher, and reported ambiguous 11 results. This seems to confirm misgivings voiced in theoretical works 12 to the effect that suppression of ice formation by CPAs might have counterproductive consequences on pressure build‐up inside the capillary.…”

Section: Introductionmentioning

confidence: 99%

Self‐pressurised rapid freezing at arbitrary cryoprotectant concentrations

Rolle,

Okotrub,

Zaytseva

et al. 2023

Journal of Microscopy

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Self‐pressurized rapid freezing (SPRF) has been proposed as a simple alternative to traditional high pressure freezing (HPF) protocols for vitrification of biological samples in electron microscopy and cryopreservation applications. Both methods exploit the circumstance that the melting point of ice reaches a minimum when subjected to pressure of around 210 [MPa], however, in SPRF its precise quantity depends on sample properties and hence, is generally unknown. In particular, cryoprotective agents (CPAs) are expected to be a factor; though eschewed by many SPRF experiments, vitrification of larger samples notably cannot be envisaged without them. Thus, in this study, we address the question of how CPA concentration affects pressure inside sealed capillaries, and how to design SPRF experiments accordingly. By embedding a fiber‐optic probe in samples and performing Raman spectroscopy after freezing, we first present a direct assessment of pressure buildup during SPRF, enabled by the large pressure sensitivity of the Raman shift of hexagonal ice. Choosing dimethyl sulfoxide (DMSO) as a model CPA, this approach allows us to demonstrate that average pressure drops to zero when DMSO concentrations of 15 wt % are exceeded. Since a trade‐off between pressure and DMSO concentration represents an impasse with regards to vitrification of larger samples, we introduce a sample architecture with two chambers, separated by a partition that allows for equilibration of pressure but not DMSO concentrations. We show that pressure and concentration in the fiber‐facing chamber can be tuned independently, and present differential scanning calorimetry (DSC) data supporting the improved vitrification performance of two‐chamber designs.Lay version of abstract for “Self‐pressurized rapid freezing at arbitrary cryoprotectant concentrations”Anyone is familiar with pipes bursting in winter because the volume of ice is greater than that of liquid water. Less well known is the fact that inside a thick‐walled container, sealed and devoid of air bubbles, this pressure buildup will allow a fraction of water to remain unfrozen if the sample is also cooled sufficiently rapidly far below the freezing point. This phenomenon has already been harnessed for specimen preparation in microscopy, where low temperatures are useful to immobilize the sample, but harmful if ice‐formation occurs. However, specimen preparation cannot always rely on this pressure‐based effect alone, but sometimes requires addition of chemicals to inhibit ice‐formation. Not enough is known directly about how these chemicals affect pressure buildup: Indeed, rapid cooling below the freezing point is only possible for small sample volumes, typically placed inside sealed capillaries, so that space is generally insufficient to accommodate a pressure sensor. By means of a compact sensor, based on an optical fiber, laser and spectrometer, we present the first direct assessment of pressure inside sealed capillaries. We show that addition of chemicals reduces pressure build‐up and present a two‐chambered capillary to circumvent the resulting trade‐off. Also, we present evidence showing that the two‐chambered capillary design can avoid ice‐formation more readily than a single‐chambered one.This article is protected by copyright. All rights reserved

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Cryopreservation of HeLa Cells at a High Hydrostatic Pressure of 1.0–1.5 kbar

Cited by 4 publications

References 32 publications

Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations

Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations

Technologies for Vitrification Based Cryopreservation

Self‐pressurised rapid freezing at arbitrary cryoprotectant concentrations

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