In this research, we successfully cultured and cryopreserved the muscle cells of post-mortem critically endangered Yangtze Sturgeon.
Eight kinds of tissues from wild Yangtze Sturgeon after death have been attempted for primary culture. The results revealed that the rate of apoptosis varies among different tissues after death for the Yangtze sturgeon. Compared with other tissues, only muscle tissue shows better viability and viable cells can migrated from wild Yangtze Sturgeon after death at room temperature. Moreover, muscle cells could be subcultured and cryopreserved. On the contrary, tissues such as the brain, heart, and kidneys seem to be deactivated soon after death and unable to migrate out of cells, or even if cells could be observed migrating, as in spleen tissue, they could not been subcultured and cryopreserved. These results suggested that muscle, located between skin and visceral tissue, more likely to avoid suffering the effect from environmental oxidation and autolysis of internal organs, thus have the greater potential to maintain viability.
WYSM cells showed a stable growth and have not shown significantly morphological changes up to passage 25. The passage number was restricted to 25th since biological characteristics of cells preserved as germplasm resources may be adversely affected by more passages and repeated digestion of trypsin (Yang et al. 2018; Kumar et al. 2019).
Unlike previously reported cell lines such as fins, kidneys and gonads (Pao et al. 2019; Suryakodi et al. 2021; Xu et al. 2022), the cultured cells were not heterogeneous consisted of a mixture of polymorphic cell, but instead exhibited mostly of fibroblast morphology during the initial passages with only a few epithelial cells. Similar phenomenon was described in the muscle cells derived from turbot and humpback grouper (Gao et al. 2019; Wang et al. 2020). A homogeneous population of fibroblasts cells was observed after the 8th generation. Furthermore, the immunofluorescence staining results supported the opinion that the WYSM cells were fibroblastic in nature due to its strong immunoreaction to fibroblastic marker (fibronectin) and a negative reaction to epithelial marker (pancytokeratin), which was different from the positive expression of mesenchymally-derived cell marker (vimentin) in some other fibroblasts reported (Vo et al. 2019). Myosatellite cell is a kind of muscle derived stem cells that is able to self-renew and have the potential to differentiate, however, it makes up less and less of a fish's body weight as it ages (Koumans et al. 1991; Wang et al. 2012). What is noteworthy, although WYSM cells derived from the aged wild Yangtze sturgeon (over 35 years old), we found that desmin as a myogenic cell protein marker was expressed in WYSM cells at a highly level in the cytoplasmic compartment.
Cryopreservation of cell lines plays an important role in preserving the genetic resources of threatened species (Cetinkaya et al. 2011). In our study, the obtained muscle cells showed more than 90% viability after post-thaw and demonstrated a relatively moderate population doubling times (52.98h) compared to other muscle cell lines, derived from Giant Panda (33.8h) (Yu et al. 2015) and triploid olive flounder (69.88h) (Peng et al. 2016), respectively. These results indicated that the WYSM cells have been successfully cryopreserved and revived. In addition, to ensure the quality of cell cryopreservation, microbial contamination of muscle cells was detected using standard test methods. Expectedly, the contamination testing results suggested that the WYSM cell lines were free of bacteria, fungi and mycoplasma contamination. Therefore, the cells were suitable for conservation as germplasm resources. This is a prerequisite for the preservation of cell line resources.
Furthermore, karyotype analysis revealed that the number of chromosomes increased and decreased in some WYSM cells. Although the chromosome mode number of muscle cells was 264, and 39% metaphase cells exhibited normal chromosomes, most of the WYSM cells obtained from the of 12 ~ 14 hour postmortem wild Yangtze sturgeon were aneuploidy. The loss of chromosomes in sturgeon cell lines may be an adaptation to the environment in vitro culture, and cells preserved as germplasm resources should be avoid excessive subculture as much as possible.
Mitochondrial gene such as 12S rRNA, 18S rRNA and COI genes (Wei et al. 2018; Ahmed et al. 2009) have been commonly used to identify various established cells origin, in addition to chromosome analysis. Here, mitochondrial gene 12S rRNA and COI gene were amplified and sequenced. Not surprisingly, the data obtained from sequencing and BLAST showed that the WYSM cell line was completely originated from Yangtze sturgeon and not cross-contaminated by other cell lines.
It has always been difficult to transfect foreign plasmids with fish cell lines (Schiøtz et al. 2011). In this study, exogenous pEGFP-N3 plasmid was transfected into WYSM cells by lipofectamine 2000, and the foreign GFP gene could be expressed in WYSM cells with 15% transfection efficiency, suggesting that WYSM cell lines could be used as an in vitro study model for functional gene analysis and gene targeting.