Cell cultures derived from mammals are the most widely used of all the taxa for research and commercial purposes. The American Type Culture Collection (ATCC), the largest biological resource center of its kind in the world, maintains over 3,600 primary and continuous cultures representing over 150 species (www.​atcc.​org, verified January 20, 2013). In the past 50 years, researchers have established primary cultures from common and endangered breeds or strains of livestock, laboratory animal and companion animal species, including cattle, pig, sheep, rat, guinea pig, horse, dog and cat, but also from a large number of non-domestic and endangered species. San Diego Global Wildlife Conservancy’s Frozen Zoo® is one of the oldest and most diverse repositories of mammalian cell cultures from wild species (www.​sandiegozoogloba​l.​org, verified January 20, 2013). Samples representing 20 mammal orders, several hundred species and thousands of individuals have been cryo-banked since 1975 (Houck 2012). Another equally historical biobank, the National Cancer Institute’s repository of over 100,000,000 DNA, tissue and cell line samples, will be integrated into the Smithsonian Institution’s collection later this year (Table 16.1). In 2002, the Mediterranean Marine Mammal Tissue Bank (MMMTB) was established by researchers at the University of Padova, Italy, to collect tissues from stranded and captive marine mammals from the Mediterranean region (Peruffo et al. 2010). Due to the body condition of most stranded animals, the majority of the samples are stored in formalin, however, cultures have been derived from bottlenose dolphin (Tursiops truncatus), sperm whale (Physeter macrocephalus), and striped dolphin (Stenella coeruleoabla) (Peruffo et al. 2010). Other attempts to culture and cryopreserve cells from marine mammals include pygmy sperm whale (Kogia breviceps) (Mancia et al. 2012), Florida manatee (Trichechus manatus latirostris) (Sweat et al. 2001), dolphin (Lagenorhynchus obliquidens) and gray seal (Halichoerus grypus) (Cecil and Nigrelli 1970).

Fibroblast cell establishment and culture techniques in mammals are not complicated and good plating and growth can be obtained following explant or enzymatic digestion and culture at 37 °C in a 5 % CO₂ environment (Freshney 2005). Basic culture media, such as Eagle’s or Dulbecco’s Minimal Essential Media (E- or D-MEM), along with the standard supplementation of fetal bovine serum (FBS, 5–20 %) and antibiotics (penicillin/streptomycin or penicillin/streptomycin/fungizone) support cell growth in the primary and passaged cultures (Freshney 2005). Good post-thaw viability of cryopreserved cells can be obtained using a basic freezing medium containing MEM supplemented with 20 % FBS and 10 % dimethyl sulfoxide (DMSO) and a 24 h cooling period at –80 °C in an insulated container prior to immersing in liquid nitrogen. Although researchers have attempted to optimize techniques for specific species or tissue types (León-Quinto et al. 2011), a standardized protocol for mammalian fibroblast culture can be implemented in a laboratory handling a wide variety of species.

Of great importance for wildlife conservation is the ease with which samples can be collected from living mammals without greatly disrupting the animal’s function or health. Skin and ear biopsies obtained using a biopsy punch tool (1-8 mm disposable biopsy punches, Integra-Miltex Inc., Plainsboro, NJ, USA) or biopsy dart (biopsy syringe, Palmer Cap-Chur Inc., Powder Springs, GA, USA) have successfully produced fibroblast cultures from living animals in numerous species (León-Quinto et al. 2011; Torvar et al. 2008; Mastromonaco, unpublished data; Fig. 16.1). A recent study using six threatened Chilean species showed that biopsies can be stored in physiological saline solution supplemented with 1 % antibiotic-antimycotic (ABAM) at 4 °C without altering the time required for primary confluence for up to 7 days for ear punch biopsies and 3 days for skin dart biopsies (Torvar et al. 2008). In our laboratory, on-going studies in domestic and non-domestic bovine species are demonstrating that ear biopsies stored in phosphate buffered saline (PBS) supplemented with 1 % ABAM at 4 °C for 0, 3 and 7 days are capable of producing chromosomally stable primary and passaged cultures (Mastromonaco, unpublished data). Benkeddache et al. (2012) demonstrated that viable cells can be retrieved from rabbit ear biopsies stored in a physiological saline solution supplemented with 10 % DMSO at –20 °C for up to 10 days prior to freezing in liquid nitrogen at similar rates to fresh controls (86 versus 82 %). Samples stored up to 20 days prior to freezing in liquid nitrogen had significantly reduced viability (39 %), but still contained living cells (Benkeddache et al. 2012). These temporary storage conditions are ideal for field collections where access to a laboratory or liquid nitrogen can be hours or days away. If liquid nitrogen or nitrogen vapour are available, biopsies can be stored for a longer period of time prior to culture. Many repositories include pieces of tissue submersed in a freezing medium containing cryoprotectant (e.g. DMEM supplemented with 20 % FBS and 10 % DMSO) and frozen in liquid nitrogen. Work done by researchers at San Diego Zoo Global Wildlife Conservancy, USA, has shown that cultures can be obtained upon thawing of these cryopreserved tissues (M. Houck, personal communication). As with the biopsies from living animals, tissues from deceased animals can provide valuable material hours or even days after their death. Ear fibroblasts are consistently obtained from animals that have died unexpectedly and are found typically within 24-48 h (Mastromonaco, unpublished data).

Several studies have attempted to develop novel techniques for cell preservation. In an effort to reduce cellular injury caused by osmotic stress and improve membrane integrity, researchers used methods of microwave heating (dry preservation; Chakraborty et al. 2008) and spin-drying (lyopreservation; Chakraborty et al. 2011) to promote rapid evaporation of water from living cells. Loi et al. (2008) lyophilized sheep granulosa cells and stored them in the dark at room temperature for 3 years. Upon rehydration, the cells were non-viable and exhibited severe membrane damage, but were able to produce blastocysts when used for SCNT (Loi et al. 2008). Similar results were observed with mouse granulosa cells (Ono et al. 2008) and pig fetal fibroblasts (Das et al. 2010).

Commercial stakes in the aquaculture industry have been one of the driving forces behind much of the fish cell culture research. In a recent review, Lakra et al. (2011) reported that fish cells had been cultured and banked from more than 74 species with over 283 cell lines originating from various sources, including organs, embryos and fins. The majority of these cell lines were created for toxicology or disease studies. More recently, researchers interested in preserving rare and endangered fish species or strains have identified the importance of banking fish cell cultures as a source of genetic material to compensate for the difficulty with cryopreserving fish sperm, oocytes and embryos (Rawson 2012). A fish cryobank facility, established in 2008 at the Institute of Biomedical and Environmental Science and Technology, University of Bedfordshire, UK, has already banked tissues and cells from 19 IUCN-listed fish species (Rawson 2012). Cells from the Atlantic sturgeon (Acipenser oxyrhynchus), a species of conservation interest in North America and Europe, have been cultured and banked (Grunow et al. 2011). However, in these cases, the animals were sacrificed as internal organs or larvae were used for culture establishment.

Although many laboratories use mammal-based techniques for fish cell culture, including culture media (e.g. DMEM supplemented with FBS and antibiotics) and incubation in a 5 % CO₂ environment (Choresca et al. 2012; Han et al. 2011), fish cells are easy to maintain in air or tightly capped flasks at room temperature without the need for excessive feeding (Lakra et al. 2010; Lannan 1994). Studies indicate that cold-water species grow better between 15 and 21 °C, whereas warm-water species grow better between 25 and 35 °C (Lannan 1994). Bols et al. (2005) indicated that optimal growth for most fish cells lines is well above the natural temperature for the fish. The requirements for pH and ability to grow well in air have led many laboratories to use Leibovitz’s L-15 medium as the basal medium supplemented with FBS and antibiotics (Mauger et al. 2006; Lakra and Goswami 2011).

Many of the available cell lines have been established from internal organs, which can be collected aseptically and are not exposed to surface or environmental microorganisms. However, it is important to develop protocols for live animal collections, as has been done with mammals. Consequently, the use of fins, a highly regenerative tissue, for culture establishment has become very desirable. Whole fins have been used to grow cultures from glass catfish (Kryptopterus bicirrhis) (Han et al. 2011), goldfish (Carassius auratus) (Mauger et al. 2006), and pool barb (Puntius sophore) (Lakra and Goswami 2011), the latter of which grew to 100 passages. The study in goldfish tested cell growth from all fin types and found comparable results in all except the dorsal fin (Mauger et al. 2006). Caudal fin explants have been used to successfully establish cell cultures in the golden mahseer (Tor putitora), an endangered Indian fish species (Prasanna et al. 2000). However, with this tissue source comes an increased risk of microbial contamination and the need to use different or increased concentrations of antibiotics, which often result in increased cytotoxicity and slower or no growth from the tissue explants (Rathore et al. 2007). Fish cultures can be very slow growing (Lannan 1994) sometimes taking up to 3–4 weeks to reach primary confluence (Mauger et al. 2006), which makes it difficult to prevent microbial growth from overcoming cellular growth. Lakra and Bhonde (1996) tested fin culture in eight fish species with unpromising outcomes: only one species attached and grew whereas the other seven either attached but did not grow or were lost to fungal contamination. Studies in our laboratory attempting to optimize culture conditions from fin biopsies of an endangered African cichlid species, ngege (Oreochromis esculentus), have been delayed by persistent culture contamination originating from the tissue samples (Filice, unpublished data). Interestingly, fin biopsy cultures of a commercial tilapia species being used as a model are not succumbing to contamination using the same tissue disinfection, sample processing and culture techniques (Filice, unpublished data). Preliminary attempts at short-term storage of fin samples in a variety of chilled media have not yet yielded promising results (Filice, unpublished data).

Avian cell lines, primarily from domestic chicken, have been extensively used and studied by virologists and toxicologists for the benefits of both human and avian medicine (Moresco et al. 2010; Portz et al. 2008). The majority of cell culture work in bird species has been done using chicken embryos at an early stage of development (day 8–11). To date, there are very few reports in the literature regarding the preservation of avian genomes as somatic cells. Chinese researchers have been producing early embryo-derived cell cultures to preserve nationally protected domestic chicken breeds, including the Qingyuan partridge chicken (Na et al. 2010), Langshan chicken (Guan et al. 2010) and Big Bone chicken (Su et al. 2011). In all these cases, stable cell lines exhibiting good viability and chromosome normality were created. Recently, Kjelland and Kraemer (2012) used semi-mature and mature feathers containing feather pulp and post-hatch egg shells to culture fibroblasts from six domesticated bird species, including emu (Dromaius novaehollandiae) and ostrich (Struthio camelus). This study is an excellent example of the use of cast-off material and non-invasive sample collection.

The majority of researchers use mammalian-based cell culture systems for the growth of primary and passaged cells as in the studies described above. Minimal essential media supplemented with FBS and antibiotics are generally described. No evidence has been found in the published literature of attempts to optimize culture conditions or cryopreservation techniques in an effort to improve cell viability in other bird species.

Reptiles encompass the least studied taxon when it comes to in vitro techniques, such as cell culture. Lack of pressure from commercial or research interests, particularly health- and food-related industries, has resulted in a smaller number of studies investigating cultured reptile cells. Primary cell cultures have been produced from early embryos of the soft shell turtle (Pelodiscus sinensis) (Liu et al. 2012), heart, liver and muscle of the Chinese alligator (Alligator sinensis) (Zeng et al. 2011), and venom glands of the South American rattlesnake (Crotalus durissus terrificus) (Duarte et al. 1999). Embryos incubated up to 30–40 days were used to establish cultures from green sea turtles (Chelonia mydas), a species listed as endangered by the IUCN (Moore et al. 1997). In an attempt to develop non-lethal techniques for culture derivation, tail and toe clippings were used to prepare cell cultures from five species of Australian dragon lizards (Ezaz et al. 2008). The cell lines were grown for ten passages and retained their viability and normal diploidy. In our laboratory, cell cultures were produced from Komodo dragon (Varanus komodoensis), another IUCN-listed reptile species, from a thin strip of skin taken from the incision site during a surgical procedure (Mastromonaco, unpublished data).

Reptile cells can be grown in mammalian-based culture systems (e.g. DMEM supplemented with FBS and antibiotics) and a 5 % CO₂ environment (Stephenson 1966). Studies have shown that although the cells can grow at a wide range of temperatures (Stephenson 1966), most species exhibit optimal growth at 28–30 °C (Ezaz et al. 2008; Moore et al. 1997; Clark et al. 1970). Although studies have investigated the influence of temperature on in vitro cell growth rates, very little work has been done, as in birds, on investigating optimal techniques for culture establishment, culture conditions and cryopreservation methods.

Amphibians, on the other hand, have experienced a recent explosion in biobanking activity. The sudden amphibian crisis resulting from both continued habitat loss and the on-going spread of chytridiomycosis have created urgency among zoological and academic professionals to preserve genetic material from any remaining individuals and species. Fortunately, one of the most widely studied laboratory animal species is the African clawed frog (Xenopus laevis) in which numerous cell lines, primarily from embryos and tadpoles, have been developed for research purposes and thus, some basic tips for amphibian cell culture are available (Anizet et al. 1981). Tadpole hindlimbs were used to produce cell cultures for transgenic studies in a related species, Xenopus tropicalis (Sinzelle et al. 2012). The cell lines were long-lived, surviving over 60 passages, an ideal condition for transgenic studies. There are no descriptions in the scientific literature of cell cultures grown from non-lethal tissue sources or from endangered amphibian species. In November, 2011, Science News Daily reported that researchers at San Diego Zoo Global Wildlife Conservancy had managed to grow cells from frozen biopsy pieces of the endangered Mississippi gopher frog (Rana sevosa), increasing the number of banked individuals to 19 (www.​sciencenewsdaily​.​org, verified January 20, 2013).

Similar to the experience with fish tissues, amphibian samples that cannot be collected aseptically, as with internal organs, are highly susceptible to contamination from the external environment. This is especially the case with early embryos in which the jelly coats harbour microbes encountered during passage through the cloaca and from the environment (Laskey 1970). This poses a challenge when attempting to grow uncontaminated cultures and great effort is spent investigating other antibiotics (e.g. gentamycin) or combinations of antibiotics (e.g. kanamycin + polymyxin E) to increase microbicidal activity (Laskey 1970).

Spermatogonial stem cells (SSCs), the basis for continuous sperm production in males, have been investigated as a source of germline material for sperm production from genetically valuable animals. SSC techniques, originally developed in mice, have now been documented in a variety of species (reviewed by Dobrinski and Travis 2007). In the cat and dog, transplantation of SSCs, which involves characterization, isolation, and transfer of cells, has been attempted with mixed results (Travis et al. 2009). In brief, this involved isolating the SSCs followed by transfer into a germ-cell depleted (via radiation) host. On occasion, it has been possible to recover ~20 % of mature sperm cells derived from the donor (Travis et al. 2009). Others have transplanted germ cells from a wild felid (ocelot; Leopardus pardalis) into the domestic cat to produce spermatozoa successfully from the donor (Silva et al. 2012). Recent results in SSC transplantation in primates are encouraging since spermatogenesis can be resumed and lead to the production of fully functional spermatozoa (Hermann et al. 2012). In fishes, fully functional rainbow trout (Oncorhynchus mykiss) sperm were produced by masu salmon (Oncorhynchus masou) following transplantation of trout spermatogonia into the peritoneal cavity of newly hatched salmon embryos (Yoshizaki et al. 2012). These authors document similar successes in a variety of other fish species (reviewed by Yoshizaki et al. 2012). A recent study in birds showed that Japanese quail (Coturnix japonica) spermatogonia were capable of colonizing domestic chicken (Gallus gallus) testes, although the production of fully functional sperm was not detected (Pereira et al. 2012).

In a 2009 review, Tilly and Telfer discuss the exciting new findings supporting the existence of proliferative germ cells in the adult ovary, which may soon challenge the age-old belief that females are born with a non-renewable pool of oocytes at birth. These ovarian stem cells have been isolated from multiple species, including the giraffe (Giraffa camelopardalis), and successfully cultured in vitro (Telfer, personal communication).

Adult or somatic stem cells typically arise in tissues with high regenerative capacity, including blood, skin, intestine, and respiratory tract (Wagers and Weissman 2004). Although some of these tissues are difficult to obtain from living animals, especially non-invasively, the banking of a population of cells with an indefinite lifespan and the capacity to create many other cells within the organism, primarily germ cells, is highly enticing and requires further investment in wildlife species. Furthermore, studies using different cell types as nuclear donors for SCNT indicate that the less differentiated the donor nucleus, the greater the developmental potential of the SCNT embryo both pre- and post-implantation (reviewed by Hochedlinger and Jaenisch 2002), suggesting that some adult stem cell populations may be a more appropriate source of donor material for offspring production by SCNT.

The majority of studies on adult stem cells to date have been carried out in laboratory and livestock species, including mouse (brain: Gritti et al. 1996), rhesus monkey (bone marrow: Chang et al. 2006), goldfish (retina: Wu et al. 2001), zebrafish (melanocyte: Tryon and Johnson 2012), pig (skin: Dyce et al. 2004), cattle (bone marrow: Bosnakovski et al. 2005), red deer (antler: Berg et al. 2007), horse (cord blood: Koch et al. 2007), and chicken (feather follicle: Xi et al. 2003). Isolation of adult stem cells may be more difficult than differentiated somatic cells depending on their source as proportionately smaller numbers of stem cells are available in adult tissue fragments. Furthermore, in vitro culture of stem cells ranges from being as effortless as fibroblast culture (mesenchymal stem cells; Cheng et al. 2012) to requiring specialty media (neural stem cells; Hutton and Pevny 2008) and use of feeder cell layers (epithelial stem cells; Nowak and Fuchs 2009). Most importantly, proper characterization of the isolated and cultured cells is required in order to classify them as stem cells, including extended reproductive lifespan, ability to differentiate into other cell lineages, and expression of a specific array of cell surface markers (Dominici et al. 2006).

Despite the claims that biobanking facilities have started including stem cell cultures from endangered species as part of their cryopreserved inventory, very little information is available in the published literature regarding the isolation, establishment and characterization of adult stem cells from wildlife species. Stem cells derived from adipose tissue biopsies of wild Scandinavian brown bears (Ursus arctos) collected during the implantation of tracking devices were shown to differentiate into osteocytes and chondrocytes in vitro (Fink et al. 2011). As with red deer (Cervus elaphus), antler-derived stem cells were cultured from fallow deer (Dama dama) and characterized using specific mesenchymal stem cell markers, such as STRO-1 (Rolf et al. 2008).

Unlike fibroblast cell culture and banking, which can be easily undertaken by most laboratories, adult stem cell culture and banking requires a greater investment in supplies, equipment, and knowledge (e.g. molecular techniques), as well as more stringent criteria for evaluation, which in turn require greater efforts to complete (e.g. long-term culture, exposure to differentiating factors, cell surface marker identification, chromosome analysis). Although samples for adult stem cell culture can be obtained non-invasively, or opportunistically, there is a greater chance of failure to establish a stem cell line compared to fibroblast culture. Therefore, although fibroblast culture banks can become a widespread movement, stem cell banks may continue to remain in specialized centers, most of which are affiliated with academic institutions.

Mammals have been the most extensively studied taxa for SCNT with live offspring being consistently produced from rodents to primates. The application of SCNT as a method of assisted reproduction for endangered species was first done in mammals (gaur (Bos gaurus); Lanza et al. 2000) and has yet to be reported in any other taxa. Researchers faced with the task of obtaining large numbers of oocytes required for SCNT from endangered females overcame this challenge by using oocytes from related domestic species as cytoplasmic recipients; a technique called interspecies SCNT (iSCNT). This idea was supported by the study of Dominko et al. (1999), which demonstrated that the bovine cytoplasm was capable of reprogramming, at least partially, diverse donor nuclei (sheep, pig, monkey, rat) past maternal-embryonic transition and, with the exception of the rat, progress to the blastocyst stage. Other than a handful of studies on non-traditional, albeit domesticated or laboratory-based, species using intraspecies SCNT, including dromedary camel (Camelus dromedarius; Wani et al. 2010), ferret (Mustela putorius furo; Li et al. 2006b), rhesus monkey (Macaca mulatta; Zhou et al. 2006), and long-tailed macaque (Macaca fascicularis; Ng et al. 2004), all reports in wild species involve iSCNT. To date, published reports show that more than 30 wild species have been attempted with the majority of the species achieving blastocyst development, but poor post-implantation success (Table 16.2; Fig. 16.3).