**6. Concluding remarks**

278 Current Frontiers in Cryobiology

Another alternative diploid material often sought to be cryopreserved is the primordial germ cells. Compared with the blastomere, primordial germ cells are developmentally closer to the cell type to be differentiated *in vivo*, i.e. the germ cells. The first success in transplantation of primordial germ cells was demonstrated on rainbow trout (*Oncorhynchus mykiss*), a model with a relatively larger body size. Green fluorescent protein expressing primordial germ cells isolated from the genital ridge of hatchlings were injected into the peritoneal cavities of a wild type hatchling. The maker-labeled primordial germ cells were able to colonize the genital ridge of the recipient animal and transmit the donor characteristic to the next generation through sperm and eggs at a rate of up to about 4%

A similar operation in the smaller teleost species such as zebrafish is more challenging. A single primordial germ cell isolated from the pearl *Danio* (*Danio albolineatus*) at ten- to fifteen-somite stage was transplanted into the marginal region of each zebrafish (*Danio rerio*) embryo at the blastula stage and *vice versa*. The development of host germ cells was prevented in advance by injection of an antisense *dead end* morpholino oligonucleotide at an earlier embryonic stage (Slanchev et al., 2005). In the host, the transplanted primordial germ cell developed into a single gonad, making the animal regain fertility and transmit the donor genotype to the progenies. This complete germline replacement procedure can be applied to both goldfish (*Carassius auratus*) and loach (*Misgurnus anguillicaudatus*) (Saito et al., 2008). The success of these cases suggests that cryopreservation of primordial germ cells is a feasible approach to preserve the diploid germplasm. As the reservoirs of primordial germ cells, genital ridges from Rainbow trout (*Oncorhynchus mykiss*) embryos were cryopreserved by cooling in dry ice and then liquid nitrogen after treating with 1.8M ethylene glycol. About 51% of primordial germ cells survived. Fifteen to twenty surviving primordial germ cells were transplanted to the peritoneal cavity of each newly hatched animal. Germline transmission of the donor genotype could be found in 7.8% of the hosts and the germline transmission frequency was from 0.1 to 13.5%. (Kobayashi et al., 2007).

Later, Higaki et al. (2010b) vitrified whole zebrafish (*Danio rerio*) embryos at 14- to 20-somite stage with an optimized vitrification solution to cryopreserve primordial germ cells. With the use of 3M ethylene glycol and 0.5M sucrose, about 4 primordial germ cells, about 40% of all, survived in each embryo, after thawing. To increase cryo-survival, yolk-removed zebrafish (*Danio rerio*) embryos were vitrified in 20% ethylene glycol, 20% DMSO and 0.5M sucrose. Up to 90% live primordial germ cells were obtained. Half of the primordial germ cells retained pseudopodial movement. After transplanting the motile primordial germ cells into sterilized golden-type zebrafish blastulae, about 2.8% of the recipients developed

Unless there is a breakthrough in cryopreserving and recovering whole fish embryos, cryopreservation of blastomeres or primordial germ cells seem to be the only methods for cryopreserving the fish diploid germplasm. Blastomeres may have advantages over primordial germ cells in generating germline transmitting chimera. Firstly, identification and isolation of primordial germ cells relies on a readily observable transgenic marker (Higaki et al., 2010b; Kobayashi et al., 2007). Breeding of a strain to a marker transgenic strain or freshly injecting DNA constructs is required before cryopreservation procedures, making the procedures more complicated. Removal of the marker from the recovered animals may also be required in some applications. Secondly, for germ-line replacement, the

normally and produced progenies with the donor's genotype (Higaki et al., 2010a).

(Takeuchi et al., 2003).

Successful cryopreservation relies on a number of conditions and properties of the embryos or larvae to be fulfilled. The conditions, which may be interdependent on each other, are (1) the chilling sensitivity of the embryos/larvae; (2) the permeability of the embryos/larvae to cryoprotectant and water; and (3) the sensitivity of the embryos/larvae to the cryoprotectant toxicity. The permeability of the embryos/larvae can be a function of size and structural heterogeneity. The toxicity of the cryoprotectant to the embryos/larvae can be a function of permeability at a particular developmental stage. Although a cryopreservation protocol can be as simple as slow freezing *Caenorhabditis elegans* in 15% glycerol, most of the other organisms require extensive optimization before being cryopreserved efficiently. Understanding the behavior of the interacting conditions can help initiate the development of cryopreservation of other model animals.

*Chilling injury* We learned from classical model organisms that chilling sensitivity coupled with a slow cooling procedure could be detrimental and vitrification can be a shortcut or even a better starting point to achieve the same goal. Vitrification of highly chilling-sensitive insect embryos is an excellent example. On the other hand, we have to keep in mind that vitrification requires a relatively high concentration of permeating cryoprotectant(s). If a new model organism to be cryopreserved is highly sensitive to the cryoprotectant(s) and has relatively low permeability, vitrification may not be feasible. Slow cooling, which requires a lower concentration of cryoprotectant, thus also allowing longer time for permeation, may be considered.

*Permeability* The permeability of a sample towards cryoprotectants is the major barrier to cryopreservation of *Drosophila melanogaster*, *Danio rerio* and probably some other model organisms. Understanding the complexity and structural properties of the embryo/larvae can make cryopreservation possible by developing a corresponding strategy to manage the flow of cryoprotecting agents and water at will. Although the studies on *Danio rerio* embryo complexity and development did not bring about successful cryopreservation of the whole embryo, they helped development of alternatives for cryopreserving diploidic germplasms. Cryopreservation of blastomeres and primordial germ cells using the optimized conditions leads to generation of germline-transmitting chimera after transplantation of cells.

*Toxicity of cryoprotectants* Knowing the toxicity of cryoprotectants at different developmental stages of an organism is critical in determining the combination of cryoprotectants with embryonic/larval stages to be chosen for effective cryopreservation during protocol development. For example, glycerol and dimethylformamide are very toxic to fish embryos and mammalian morulae, respectively (Higaki et al., 2010b; Kasai et al., 1981).

Cryopreservation of Embryos from Model Animals and Human 281

value for short term research purposes and daily operations. It is also beneficial for a longer term establishment of these models as alternative platforms for biomedical investigations.

We would like to thank Miss Mandy Chan and Prof. Andrew L. Miller (HKUST) for contribution to the photograph of a zebrafish embryo in Figure 3C. FlyMove, an internet educational resource (Weigmann 2003), is acknowledged for the permission in reproducing an image of a fly embryo in Figure 3B. WHT is a postdoctoral fellow supported by HKUST postdoctoral fellowship. This work was conducted and supported by Research Grants

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**8. References** 

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Model organisms offer a platform to address biological issues of a broad range of interests with ease. An ideal platform must have specific traits allowing convenient manipulations in a manner beneficial to specific fields of study. Use of classical model organisms such as the house mouse *Mus musculus*, zebrafish *Danio rerio*, fruit fly *Drosophila melanogaster* and nematode *Caenorhabditis elegans* for studying physiology, genetics, genomics, behavior, human diseases and their treatments, etc is well established. These are attractive model organisms from their representative evolutionary position. They are relatively more readily available, tractable, small in body size, rapid in development and have short reproductive cycles. They are still popular models because of the establishment of transgenic technologies related to these animals (Fire, 1986; Gordon et al., 1980; Rubin & Spradling, 1982; Zelenin et al., 1991). Such genome manipulation technologies make reverse genetics possible, allowing studies to be amendable for these model organisms.

Knowledge gained on the above-mentioned classical models and other less popular model organisms has been expanding for the past few decades. Thorough comparative studies in various fields of research will benefit translational research and our understanding of the evolutionary tree of life. More interdisciplinary studies on model organisms representing animals in various branches of the phylogenetic tree will enhance our comparative study. Promising transgenic techniques have been recently established in respect of some of these animals, e.g., planaria *Girardia tigrina*, brine shrimp *Artemia sinica*, amphipod crustacean *Parhyale hawaiensis*, red flour beetle *Tribolium castaneum*, sea anemone *Nematostella vectensis*, Mollusk dwarf surfclam *Mulinia lateralis*, sea squirt *Ciona intestinalis*, channel catfish *Ictalurus punctatus*, frog *Xenopus laevis* and *Xenopus tropicalis*, chicken *Gallus gallus*, Japanese quail *Coturnix japonica*, goat *Capra hircus*, dog *Canis familiaris* and marmoset *Callithrix jacchus*, (Berghammer et al., 1999; Chang et al., 2011; Dunham et al., 2002; Gonzalez-Estevez et al., 2003; Hong et al., 2009; Houdebine, 2009; Huss et al., 2008; Lu et al. 1996; Macha et al., 1997; Mozdziak & Petitte 2004 ; Pavlopoulos and Averof, 2005; Renfer et al., 2010; Sasaki et al., 2009; Sasakura et al., 2007; Wheeler, 2003). The development of genome manipulation techniques for these emerging animal models will open the door to unlimited possibilities of *in vivo* investigations.

The cost of the knowledge explosion and scientific advancement will be the handling of an enormous number of transgenic strains generated. The cost of managing these invaluable resources can be a substantial burden on research laboratories or institutions world wide, which may impede further development. So far cryopreservation has been developed for embryos/larvae from non-classical model animals, including oyster *Crassostrea gigas*, hard clam *Meretrix lusoria*, sea urchin *Loxechinus albus*, amphioxus *Branchiotoma belcheri*, brine shrimp *Artimia franciscana*, euryhaline rotifer *Brachionus plicatilis* and marmoset *Callithrix jacchus*, etc. (Barros et al., 1997; Chao et al., 1997; Summers et al., 1987; Sun et al., 2007; Toleda & Kurokura, 1990; Yoshida et al., 2011).

The experience in cryopreserving embryos from such a broad evolutionary range will benefit the development of cryopreservation techniques in other emerging model organisms. The parameters highlighted in this review represent some keys for developing an effective cryopreservation protocol for any organisms for experimental use. The thorough understanding of these parameters in different model systems, the optimization therein, and improved procedures to store transgenic strains will not only release the management stress caused by the need for keeping the live animals but also eliminate the risk of their being affected by disease outbreaks and genetic drifts. It has great practical value for short term research purposes and daily operations. It is also beneficial for a longer term establishment of these models as alternative platforms for biomedical investigations.
