# Construction and Characterization of Immortalized Skin Fibroblasts from Milu Deer

## TL;DR

Scientists created an immortal cell line from Milu deer skin fibroblasts to aid conservation efforts.

## Contribution

A novel immortalized fibroblast cell line from Milu deer was successfully constructed and characterized.

## Key findings

- The ML-iSFC cell line showed strong proliferation and maintained fibroblast characteristics.
- Optimal growth was achieved with 3 ng/mL FGF2 in the culture medium.
- Karyotype analysis confirmed chromosomal stability in immortalized cells.

## Abstract

The Milu deer, also known as Père David’s deer, is an endangered species native to China. To contribute to ongoing conservation efforts, we established an immortalized cell line from the skin of a male fawn. Sustained cell division in vitro was achieved through introduction of the SV40T gene. Further, we optimized culturing conditions for growing these cells, and confirmed that cells maintain the essential characteristics of normal skin fibroblasts, with a strong capacity for cell proliferation. The availability of this immortal cell line provides a novel, valuable resource for future scientific research and conservation efforts aimed at protecting Milu deer.

Somatic cell preservation is an effective strategy for conserving the genetic potential of endangered species. To contribute to the conservation of the Milu deer (Elaphurus davidianus), this study aimed to establish and characterize an immortalized skin fibroblast cell line (ML-iSFC). The cell line is based on fibroblasts from the skin tissue of a male fawn of Milu deer. Optimal culture conditions were determined by supplementing the culture medium with different growth factors, and immortalization was achieved through simian virus 40 large T antigen (SV40T) transduction. Optimal culturing conditions for the cells were determined by adding a range of growth factors. The cellular morphology, growth characteristics, and marker expression of the cells were further evaluated. Cell cycle and proliferation were assessed by flow cytometry and CCK-8 assays, respectively. Chromosomes were determined by karyotype analysis. The highest cell growth rate was observed when the culture medium was supplemented with 3 ng/mL of FGF2. The fibroblast-specific marker vimentin (VIM) was expressed in both ML-SFC and ML-iSFC, while the epithelial marker keratin 18 (KRT18) was weakly expressed in ML-SFC cells. Cell proliferation and cell-cycle analysis revealed that ML-iSFC exhibited a higher growth rate and greater vitality compared to ML-SFC. Karyotype analysis showed that ML-iSFC maintained the same chromosome number and morphology as ML-SFC. In summary, this study reports the successful construction of an immortalized fibroblast cell line from Milu deer, which will serve as a valuable tool for Milu deer conservation.