Biological Resource Center of Plants, Animals and Microorganisms, China National GeneBank, BGI-Shenzhen, Shenzhen, China.
Shenzhen Key Laboratory of Environmental Microbial Genomics and Application, BGI-Shenzhen, Shenzhen, China.
Cell Biol Int. 2021 Jul;45(7):1571-1578. doi: 10.1002/cbin.11596. Epub 2021 Apr 1.
The population decline of the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from an animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we established and characterized fibroblast cell cultures from the skin sample of a newborn common hippopotamus in this study. By combining the tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 34 h according to the growth curve. Karyotyping based on Giemsa staining showed that the cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. The amplified mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with that of the registered H. amphibius complete mitochondrial DNA, confirming the species of origin of the cells. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4, and vimentin. In conclusion, we generated the fibroblast cell cultures from a common hippopotamus and identified their characteristics using multiple techniques. We believe the cryopreserved cells could be useful genetic materials for future research.
由于普通河马（Hippopotamus amphibius）数量的减少，有必要保存其遗传资源，以进行物种保护和研究。在所有行动中，冷冻保存来源于动物活组织检查的成纤维细胞培养物被认为是一种简单而有效的方法。然而，据我们所知，普通河马的活细胞培养物还没有得到保存。为此，我们在这项研究中建立并鉴定了来自新生普通河马皮肤样本的成纤维细胞培养物。通过结合组织外植体直接培养和酶消化方法，我们分离出了大量具有典型成纤维细胞形态和高活力的细胞。在整个研究过程中，细胞培养物中均未检测到细菌/真菌或支原体。根据生长曲线，群体倍增时间为 34 小时。基于 Giemsa 染色的核型分析显示，培养的细胞为二倍体，共有 36 条染色体，其中一对为性染色体。培养细胞的扩增线粒体细胞色素 C 氧化酶亚基 I 基因序列与已注册的 H. amphibius 完整线粒体 DNA 的序列完全一致，证实了细胞的起源物种。流式细胞术和免疫荧光染色结果表明，检测到的细胞呈成纤维细胞标志物 S100A4 和波形蛋白阳性。总之，我们从普通河马中生成了成纤维细胞培养物，并使用多种技术鉴定了它们的特征。我们相信冷冻保存的细胞可以成为未来研究的有用遗传物质。
