Cryopreservation and Laser Nanowarming of Zebrafish Embryos Followed by Hatching and Spawning

Abstract

protocols to bank precious germplasm from endangered species, [1,4] manage biodiversity, provide year-round access to embryos to grow and harvest important aquaculture species, [5,6] and support biomedical research [7,8] and gene banking. [9] One of the best model systems for studying teleost embryo cryopreservation is the zebrafish (Danio rerio) embryo. Since becoming an increasingly important model system for biomedical research, [10,11] researchers have enhanced this model into tens of thousands of mutant, transgenic, and wild-type zebrafish lines. [12-14] Since preserving all these valuable genotypes through live fish colonies is expensive, risky, and beyond the capacity of even the largest stock centers, cryopreservation is an important technique for insuring against the loss of genetically important lines due to catastrophic events, such as the spread of an infectious disease or reproductive failures within a breeding facility. [10,11] Further, it can reduce significant long-term cost and space needs by banking valuable zebrafish lines that are not routinely used. [10,11] In last decade many other aquatic species such as medaka, [15] Xiphophorus, [16] and Xenopus [17] have also become increasingly valuable biomedical models and require robust embryo cryopreservation protocols for long term maintenance of transgenic and mutant lines. [10] Cryopreservation relies on two key techniques. First, a certain amount of intracellular water must be replaced with viscous antifreeze molecules, or cryoprotective agents (CPA). Second, the cooling and rewarming of specimens to and from cryogenic temperatures must occur at rates faster than the rates of ice crystallization. These "critical cooling" and "critical warming" rates and the CPA type and concentration used for vitrification are inversely related, i.e., the higher the CPA concentration is, the lower the critical cooling and warming rates. In addition, critical warming rates exceed critical cooling rates by at least an order of magnitude in both cryoprotectant media and biological systems. [18,19] The main challenges with applying the these well-known principles of cryopreservation to zebrafish embryos are i) the relatively large size of the embryo (1000 times bigger in volume than the mammalian gametes that are routinely cryopreserved), resulting in a low surface-to-volume ratio that impedes water and CPA efflux/influx; ii) the presence of multiple compartments, such as the blastoderm and yolk, with differing This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a single injection of cryoprotective agents (CPAs) and gold nanorods (GNRs) into the yolk and immersion in a precooling bath to dehydrate the perivitelline space. Then embryos are encapsulated within CPA and GNR droplets, plunged into liquid nitrogen, cryogenically stabilized, and rewarmed by a laser pulse. Postlaser nanowarming, embryos (n = 282) exhibit intact structure by 1 h (40%), continued ...

Preoperative Cryopreservation Promotes Digital Survival after Digit Replantation

“…This is due to the fact that despite of the achievement of the successful replantation of cryopreserved rat ovaries and even the successful replantation of amputated and cryopreserved rat hind limb, these body parts are all small sized organs or tissues. Differently, large body parts are very tough to be revitalized after cryopreservation as a result of the constraints of permeation ability in large tissues having significantly different biological features brought by various types of cells in the tissues [30][31][32][33], while homogeneous temperature distribution cannot be achieved in more complex structures during the freezing process. In addition, myocytes tend to form smooth or striated myofilaments in tissues that are too complicated to sustain the problems triggered by the cryopreservation process.…”

Conduction Cooling and Plasmonic Heating Dramatically Increase Droplet Vitrification Volumes for Cell Cryopreservation

“…[47] To model the laser warming, a customized Matlab code was used to trace the photons interaction (i.e., scattering or absorption) with the GNRs (nanoComposix Inc.) in the droplet as reported in the authors' previous publication. [19,21] The optical properties of GNR loaded droplets were obtained from experiments stated in previous publications. [43] A distribution of specific absorption rate (SAR, W m −3 ) was generated by the Monte Carlo modeling and imported into Comsol for temperature simulation.…”

“…[14][15][16][17] For example, the common goal of reducing intracellular CPA concentration (i.e., lowering toxicity), requires high cooling and warming rates to minimize lethal ice formation to achieve a high survival rate. [17][18][19] Slow freezing is the traditional cryopreservation method after cells are equilibrated with low permeable CPA concentration (i.e., 1.4 M DMSO) in a cryovial. Slow cooling rate (i.e., 1°C min −1 ) allows the growth of extracellular ice crystals which exclude CPA molecules thereby raising the CPA concentration around cells.…”

Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation

“…A promising alternative to existing conventional cryopreservation methods is ice-free vitrification; that is, rapid cooling of a biomaterial to a glass-like state 19 , 20 . To avoid ice formation, the cooling and subsequent warming rates need to exceed the critical cooling rate (CCR) and critical warming rate (CWR), respectively.…”