Biotransport Phenomena in Freezing Mammalian Oocytes

Abstract

Water transport across the cell plasma membrane and intracellular ice formation (IIF)-the two biophysical events that may cause cell injury during cryopreservation-were studied by cryomicroscopy and modeling using mammalian (Peromyscus) oocytes. Unusually high activation energy for water transport across the cell plasma membrane was identified indicating that the water transport process is unusually sensitive to temperature (and cooling rate). Although literally all studies on IIF were conducted using protocols with ice-seeding (seeding extracellular ice usually at ≥-7 °C), it is not used for cell cryopreservation by vitrification that is becoming increasingly popular today. In this article, we show that ice-seeding has a significant impact on IIF. With ice-seeding and cooling at 60 °C/min, IIF was observed to occur over a wide range from approximately -8 to -48 °C with a clear change of the ice nucleation mechanism (from surface- to volume-catalyzed nucleation) at approximately -43 °C. On the contrary, without ice-seeding, IIF occurred over a much narrower range from approximately -19 to -27 °C without a noticeable change of the nucleation mechanism. Moreover, the kinetics of IIF without ice-seeding was found to be strongly temperature (and cooling rate) dependent. These findings indicate the importance of quantifying the IIF kinetics in the absence of ice-seeding during cooling for development of optimal vitrification protocols of cell cryopreservation.

Three-dimensional numerical simulation of the effects of fractal vascular trees on tissue temperature and intracelluar ice formation during combined cancer therapy of cryosurgery and hyperthermia

“…IIF could be due to both surface-catalyzed nucleation (SCN) and volume-catalyzed nucleation (VCN) [31,43,44]. Therefore, the tumor damage could be determined by the probability of ice formation (PIF) as a result of both SCN and VCN as follows [44,45]:…”

“…where B is cooling rate in C min À1 ,t is time, T 0 is the equilibrium freezing temperature under isotonic solutions (À0.5 C), A and V are cell surface area and volume, respectively, I SCN and I VCN are nucleation rates which are calculated as follows [44,45]:…”

Preservation of Embryonic Stem Cells

“…The two model parameters (constants) under isotonic solution state (Ω 0 and κ 0 ) need to be determined a priori by experimental studies and have been reported in the literature for a number of cell types as summarized elsewhere (He and Bischof 2003;Toner 1993;Yang et al 2010b). The cumulative probability of intracellular ice formation (P IIF ) can then be calculated as follows (Toner 1993;Toner et al 1990):…”

“…Significant IIF is usually manifested as darkening of the cell cytoplasm when observed under a bright field of light microscopy, which has been used widely to quantify the kinetics of IIF (Diller 2005;Toner et al 1991;Yang et al 2010b). …”

“…Cell dehydration during freezing can be measured using either a specialized cryostage mounted on a light microscope (Diller 2005;Toner et al 1991) or differential scanning calorimetry (Bischof 2000;Devireddy et al 2001;Devireddy et al 1998;Luo et al 2002). Many studies have been performed to determine the two model parameters for various cells, which have been reviewed and tabulated elsewhere (Bischof 2000;Han and Bischof 2004;He and Bischof 2003;Yang et al 2010b). Although cell dehydration and intracellular ice formation (IIF) can be predicted using the above models, a quantitative understanding of the mechanistic link between the two biophysical events and cell injury has not been well established despite some early efforts in this respect as reviewed in (He and Bischof 2003).…”