20 Growth kinetics and single-cell cloning capability of skin cell fibroblasts in dromedary camel (Camelus dromedarius)

Somatic cell nuclear transfer (SCNT) could be used for genetic modification of animals by alteration of candidate donor cell genes. Single-cell cloning is the critical step to generate, isolate and validate cells with uniform genetic backgrounds. In the present study, skin fibroblast cell lines were established from the skin biopsy samples taken from elite camels, and their growth kinetics and capability of single-cell cloning were investigated. Skin tissue biopsies were excised from the ears of four elite dromedary camels. Cultures were established using the tissue implant method with Delbucco’s Modified Eagle Medium (DMEM)/F12 supplemented with 10% fetal bovine serum, as previously published protocols from our laboratory. A total of 2 × 10⁵ cells were seeded in a 35-mm dish from each cell line with 3 replicates. The cells were passaged every 3 to 4 days and the cell population doubling time was calculated. Samples from every 5 passages were used to study their karyotype. To develop single-cell clones, several 70 cells were picked up from the sixth passage, added into 10-mL growth media, and plated individually into 96-well plates. Culture plates were observed for cell growth and the cells were counted after 2 weeks of culture. All the data is presented as mean ± standard deviation. In the present study, the derived skin fibroblast cells were cultured for over 40 passages within a duration of 5 months. No apparent differences were observed in the growth kinetics among the four cell lines up to the 20th passage. We observed an average population doubling time of 42.9 ± 3.8 and 33.4 ± 4.7 h at the 5th and 20th passages, respectively. However, in three cell lines, the doubling times increased gradually from 32.1 ± 3.1 to 428.1 ± 10.2 h from the 25th to the 40th passage, respectively. One cell line kept growing with a doubling time of 32.1 h even after 144 days of culture, which might have spontaneously transformed into an immortalized cell line. An average of 57.2 ± 8.8% (n = 94) metaphase spread showed normal karyotype with 74 chromosomes. For single-cell clones, the outgrowth rate was 47.2 ± 11.1% cells in 14 days of culture. Out of which, 21.6 ± 6.9% clones could be passaged and expanded further into several 2 × 10⁵ cells. The remaining cell clones stopped growing after their passage. In conclusion, our results demonstrate that the camel skin cells maintain growing in culture over 40 passages for ~5 months. Single-cell clones could be generated from primary culture skin cells and could be used as the candidate cells for genomic modification study.