An insect antifreeze protein from Anatolica polita enhances the cryoprotection of Xenopus laevis eggs and embryos

Cryoprotection is of interest in many fields of research, necessitating a greater understanding of different cryoprotective agents. Antifreeze proteins have been identified that have the ability to confer cryoprotection in certain organisms. Antifreeze proteins are an evolutionary adaptation that contributes to the freeze resistance of certain fish, insects, bacteria, and plants. These proteins adsorb to an ice crystal's surface and restrict its growth within a certain temperature range. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in the frog Xenopus laevis. X. laevis eggs and embryos microinjected with ApAFP752 exhibited reduced damage and increased survival after a freeze/thaw cycle in a concentration-dependent manner. We also demonstrate that ApAFP752 localizes to the plasma membrane in eggs and embryonic blastomeres and is not toxic for early development. These studies show the potential of an insect antifreeze protein to confer cryoprotection in amphibian eggs and embryos.

Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

The development in cryobiology in animal breeding had revolutionized the field of reproductive medicine. The main objective to preserve animal germplasm stems from variety of reasons such as conservation of endangered animal species, animal diversity, and an increased demand of animal models and/or genetically modified animals for research involving animal and human diseases. Cryopreservation has emerged as promising technique for fertility preservation and assisted reproduction techniques (ART) for production of animal breeds and genetically engineered animal species for research. Slow rate freezing and rapid freezing/vitrification are the two main methods of cryopreservation. Slow freezing is characterized by the phase transition (liquid turning into solid) when reducing the temperature below freezing point. Vitrification, on the other hand, is a phenomenon in which liquid solidifies without the formation of ice crystals, thus the process is referred to as a glass transition or ice-free cryopreservation. The vitrification protocol applies high concentrations of cryoprotective agents (CPA) used to avoid cryoinjury. This chapter provides a brief overview of fundamentals of cryopreservation and established methods adopted in cryopreservation. Strategies involved in cryopreserving germ cells (sperm and egg freezing) are included in this chapter. Last section describes the frontiers and advancement of cryopreservation in some of the important animal models like rodents (mouse and rats) and in few large animals (sheep, cow etc).

Thermal Analyses of Nanowarming-Assisted Recovery of the Heart From Cryopreservation by Vitrification

This study explores thermal design aspects of nanowarming-assisted recovery of the heart from indefinite cryogenic storage, where nanowarming is the volumetric heating effect of ferromagnetic nanoparticles excited by a radio-frequency electromagnet field. This study uses computation means, while focusing on the human heart and the rat heart models. The underlying nanoparticle loading characteristics are adopted from a recent, proof-of-concept experimental study. While uniformly distributed nanoparticles can lead to uniform rewarming, and thereby minimize adverse effects associated with ice crystallization and thermomechanical stress, the combined effects of heart anatomy and nanoparticle loading limitations present practical challenges which this study comes to address. Results of this study demonstrate that under less-than-ideal conditions, nonuniform nanoparticles warming may lead to a subcritical rewarming rate in some parts of the domain, excessive heating in others, and increased exposure potential to cryoprotective agents (CPAs) toxicity. Nonetheless, results of this study also demonstrate that computerized planning of the cryopreservation protocol and container design can help mitigate the associated adverse effects, with examples relating to adjusting the CPA and/or nanoparticle concentration, and selecting heart container geometry and size. In conclusion, nanowarming provides superior conditions for organ recovery from cryogenic storage, which comes with an elevated complexity of protocol planning and optimization.

Vitrification Ability of Combined and Single Cryoprotective Agents

Cryoprotective agents (CPA) are an important part of many current vitrification methods. The vitrification ability of CPAs influences the probability of the glass transition and water crystallization occurrence. Thermal characteristics and the vitrification ability of two combined CPAs (PVS2 and PVS3), common plant vitrification solutions, and four single CPAs (ethylene glycol, DMSO, glycerol, and sucrose), the components of the mentioned PVSs, were evaluated utilizing a differential scanning calorimetry (DSC) during standard cooling/warming rates of 10 °C min−1. The effect of solute concentration on their vitrification ability was shown in the CPAs tested. Four typical concentration regions at which the glassy state and/or crystallization occurred were defined. We suggest the solute concentration of 0.7 g g−1 as the universal vitrification concentration, characterized by an actual Tg of CPA solution and limited water crystallization. Knowledge of the thermal properties of CPAs allows the design of new combined CPAs with the required vitrification ability respecting the cryopreservation method used and the characteristics of the cryopreserved sample.

Cryopreservation of Gametes and Embryos and Their Molecular Changes

The process of freezing cells or tissues and depositing them in liquid nitrogen at –196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.

Loading equine oocytes with cryoprotective agents captured with a finite element method model

Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane was used to simulate oocyte volume responses and concomitant intracellular CPA concentrations during the exposure of oocytes to CPA solutions. The results of the analytical simulations were subsequently used to develop a phenomenological finite element method (FEM) continuum model to capture the response of oocytes exposed to CPA solutions with spatial information. FEM simulations were used to depict spatial differences in CPA concentration during CPA permeation, namely at locations near the membrane surface and towards the middle of the cell, and to capture corresponding changes in deformation and hydrostatic pressure. FEM simulations of the multiple processes occurring during CPA loading of oocytes are a valuable tool to increase our understanding of the mechanisms underlying cryopreservation outcome.

Ultra-Rapid Laser Calorimetry for the Assessment of Crystallization in Low-Concentration Cryoprotectants

Cryoprotective agents (CPAs) are routinely used to vitrify, attain an amorphous glass state void of crystallization, and thereby cryopreserve biomaterials. Two vital characteristics of a CPA loaded system are the critical cooling and warming rates (CCR and CWR), the temperature rates needed to achieve and return from a vitrified state respectively. Due to the toxicity associated with CPAs, it is often desirable to use the lowest concentrations possible, driving up CWR and making it increasingly difficult to measure. This paper describes a novel method for assessing CWR between the 0.4×105-107 °C/min in microliter CPA loaded droplet systems with a new ultra-rapid laser calorimetric approach. Cooling was achieved by direct quenching in liquid nitrogen, while warming was achieved by the irradiation of plasmonic gold nanoparticle-loaded vitrified droplets by a high-power 1064 nm millisecond pulsed laser. We assume "apparent" vitrification is achieved provided ice is not visually apparent (i.e. opacity) upon imaging with a camera during cooling or highspeed camera during warming. Using this approach, we were able to investigate CWR in single CPA systems such as glycerol, PG, and Trehalose in water, and mixtures of glycerol-trehalose-water and propylene glycol-trehalose-water CPA at low concentrations (20-40 wt%). Further, an phenomenological model for determining the CCRs and CWRs of CPA was developed which allowed for predictions of CCR or CWR of single component CPA and mixtures, providing an avenue for optimizing CCR and CWR and perhaps future CPA cocktail discovery.