Do physical forces contribute to cryodamage?

Abstract

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component--mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 microm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37 degrees C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing.

Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

“…Anywhere else is potentially damaging. The major damaging factors, which occur during cryopreservation, are associated with chilling injury, osmotic stress, CP toxicity, and ice crystallization , Quinn 1985, Saragusty et al 2009). In general, we are trying to reduce these damages by increasing cooling and warming rates using vitrification.…”

Cryopreservation of Porcine Gametes, Embryos and Genital Tissues: State of the Art

“…Freezing injury can then be related to high electrolyte concentration effects (solute effects), presence of intracellularly ice (formed direct or eutectic) and also the pressure of large extracellular crystals on the veins of concentrated (i.e. vitrified) extender and cells (Saragusty et al 2009). See Figures 1and 2 for an illustration of these events.…”

“…The FlatPack™ was considered as cryobiologically convenient (very thin and with a large surface) to dissipate heat during cooling and warm rapidly, as those small containers tested. It is important to remember that the freezing in these containers, with high heat dissipation, inflicts less damage to the cells by intra-container mechanical pressure (Saragusty et al 2009). Figure 4 shows cryological differences in shape and size of frozen water lakes between mini-straws and FlatPack™.…”

Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds ofAcer saccharinum

“…The large size, irregular geometry and high water content of recalcitrant-seeded explants promote intracellular ice formation at subfreezing temperatures, an event regarded as invariably lethal (Wesley-Smith et al, 2014). Why ice formation is lethal remains an unanswered question in cryobiological research, with two major schools of thought relating to membrane damage (Steponkus et al, 1993) or enigmatic damage associated with compression of cellular constituents reminiscent of desiccation stress Rubinsky, 1994, 1998;Hubel et al, 2007;Saragusty et al, 2009). This paper represents the second in a two-part series that investigates the damaging nature of intracellular ice.…”

“…In addition, mechanical compression within cells, which is an hypothesized effect of freezing stress, can trigger PCD in animal tissues (Loening et al, 2000;Hunter et al, 2002) and affects morphology of plants (Robinson et al, 2013). Consolidation of cytoplasm by water removal or an advancing ice front may result in compressive stresses Rubinsky, 1994, 1998;Hubel et al, 2007;Saragusty et al, 2009), which induce subtle disruption of the spatial organization within the cytoplasm to elicit signals for rapid metabolism in response to environmental cues (Hyman and Simons, 2012). Permealization of the outer mitochondrial membrane (MOMP) appears to be an initial checkpoint that irreversibly induces PCD in animal cells (Danial and Korsmeyer, 2004).…”