Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps)

https://doi.org/10.1016/j.cbpc.2011.04.002Get rights and content

Abstract

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a “living” tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.

Introduction

Biorepositories continue to increase in number and size throughout the world, and their contents are essential for pathologic, toxicologic and molecular analyses (Ayers, 2011, Cortes et al., 2010, Moritz and Labbe, 2008, Troyer, 2008). Techniques such as laser capture microdissection and gene expression analysis on formalin fixed paraffin-imbedded tissues have also considerably improved the value of available biospecimens. However, among the vertebrate biological materials available, the banking of viable primary cells and tissues is very limited compared to the much greater number of immortalized cell lines. For example, the American Type Culture Collection (ATCC), established in 1914, has available only a dozen primary cell types, most of which are human, and no model tissues (www.atcc.org). Here, we describe a straightforward approach to the cryopreservation of viable lung tissue from both rare and common organisms. Such an approach will be highly valuable for the establishment of primary cells from unique model strains (e.g., transgenic mouse lines) and unusual or poorly studied organisms. Among the numerous advantages of viable tissue specimens is that multiple tissue-specific cell types could be retrieved and reconstituted for ex vivo organ studies, which is especially important in regard to rare species where tissue acquisition is limited.

The pygmy sperm whale (PSW), Kogia breviceps, inhabits all temperate and tropical waters and is the second most commonly stranded cetacean in the southeastern United States (Odell et al., 2004, Scott et al., 2001). Due to its deep water pelagic lifestyle (including limited periods at sea level), most information about the PSW has been obtained from stranded animals and very little is known about these cetaceans in the wild, including behavior and accurate population estimates (Odell et al., 2004, Santos et al., 2006). Based on stomach contents of stranded animals, we know that this species feeds in deep water, primarily on cephalopods and, less often, on deep-sea fishes and shrimp (McAlpine et al., 1997). Toothed whales (order Cetacea, suborder Odontoceti) are the deepest diving marine mammals known and include beaked whales and sperm whales, which are believed to reach depths of up to 2000 m (Watwood et al., 2006). These deep diving marine mammals are inspired divers, inhaling air then collapsing their lungs during dives to reduce nitrogen intake and decrease oxygen consumption (Kooyman and Ponganis, 1998, Tyack et al., 2006). The process of resurfacing, lung expansion, exhalation and inhalation is highly efficient, with approximately 90% of lung volume gas exchanged (compared to 15–20% exchange volume for humans) (Tyack et al., 2006).

The isolation and analysis of different cell types will provide a wealth of information about the health status of stranded animals. Additionally, the data obtained may improve the understanding of the adaptations of marine mammals to extreme conditions as well as environmental involvement in stranding events. Here, we present a method to cryopreserve viable lung tissue from a stranded pygmy sperm whale, we show that this method can be applied to other model organisms, and that multiple viable cell types can be retrieved from long-term frozen samples. This method has direct applications in studies on normal lung function and responses to environmental stressors in rare and/or endangered species and may have direct relevance to human lung health.

Section snippets

Tissue origin

Lung tissue was isolated from a female pygmy sperm whale (Kogia breviceps; ID no. SC0934) stranded on Myrtle Beach, SC, USA, on July 24, 2009 as part of the Southeastern US Marine Mammal Stranding Network. After examination and poor prognosis at the stranding location, the whale was subjected to humane sacrifice according to the American Veterinary Medical Association (AVMA) standards and the general guidelines from the National Marine Fisheries Service (NMFS). The whale was then transported by

Selective growth of lung cells derived from cryopreserved lung tissues

Cryopreserved PSW and mouse lung tissues were thawed and dissociated as described in Materials and methods. After initial expansion in DMEM-F medium, aliquots of cells were plated in either alveolar type II cell (SAEC) or vascular endothelial cell (EGM-2) specialized media and monitored for selective cell growth and morphology. Qualitative PSW lung cell growth curves (Fig. 1) demonstrated a stable growth trend for cells cultured in DMEM-F medium and overall diminished growth rates for cells

Discussion

Since strandings of PSWs that permit tissue isolation are rare, we performed complimentary experiments (preservation and cell isolations) on both PSW and mouse lung tissue. This parallel approach permitted us to demonstrate the broad capabilities and efficaciousness of our cryopreservation/rederivation method. Thus, the well-characterized mouse model system was used as a control to validate the procedures employed and to demonstrate “proof of principle” for potential utility of this

NOAA disclaimer

This publication does not constitute an endorsement of any commercial product or intend to be an opinion beyond scientific or other results obtained by the National Oceanic and Atmospheric Administration (NOAA). No reference shall be made to NOAA, or this publication furnished by NOAA, to any advertising or sales promotion which would indicate or imply that NOAA recommends or endorses any proprietary product mentioned herein, or which has as its purpose an interest to cause the advertised

Acknowledgments

The authors would like to thank Deirdre Myroslawa Soloshchenko for technical assistance in genotypic analyses and maintenance of the mouse colony, and D. Walker for technical assistance with the counting of cells and maintenance of records. This work was conducted with support from the National Institutes of Health, Grant Number HL085738.

References (20)

There are more references available in the full text version of this article.

Cited by (15)

  • Immunotoxic Effects of Environmental Pollutants in Marine Mammals

    2018, Marine Mammal Ecotoxicology: Impacts of Multiple Stressors on Population Health
  • Marine mammal cell cultures: To obtain, to apply, and to preserve

    2017, Marine Environmental Research
    Citation Excerpt :

    The blood should be injected into tubes containing anticoagulants (ethylenediaminetetraacetic acid or sodium heparine) and kept cool during shipment to the laboratory within 24–30 h for further processing. A deceased animal can also be a source of tissues for deriving viable cell cultures from any organ depending on the time passed since death: from several minutes to 6 h for blood, bone marrow, and lungs (Frouin et al., 2010; Hymery et al., 2013; Larsen et al., 2013); up to 12 h for kidney, bronchial epithelium, and testes (Carvan et al., 1994; Sweat et al., 2001, 2003; Wise et al., 2008); and up to 24 h or more for skin and blubber (Mancia et al., 2012; our unpublished data). The isolation of cells from solid tissue samples is usually performed as for any other mammalian species.

  • Induced pluripotent stem cell technology and aquatic animal species

    2014, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
    Citation Excerpt :

    Current methods for banking of tissues from aquatic organisms, including stranded marine mammals are for the most part inadequate, failing to maintain viable tissues and/or cells necessary for stem cell isolation and/or induction. Established cryopreservation techniques (Annalaura et al., 2012) should be considered as part of standard procedures involving strandings to preserve cells of rare species to allow for future primary and stem cell studies. Contrasting with these banking inadequacies, sophisticated toxicological testing of samples and tracking the health status and exposure history of individuals are very well developed (Fair et al., 2013).

View all citing articles on Scopus

This paper is based on a presentation given at the 5th Aquatic Annual Models of Human Disease conference: hosted by Oregon State University and Texas State University-San Marcos, and convened at Corvallis, OR, USA September 20–22, 2010.

1

These authors contributed equally to this work.

View full text