Establishment of immortalized lymphoblastoid cell lines of the Goldenhar syndrome family and sporadic cases

BACKGROUND: At present, the first choice method of eternally preserving a whole genome is to establish immortalized cell lines of various diseases, especially rare diseases, using Epstein-Barr virus for transforming B lymphocytes,. OBJECTIVE: To establish the immortalized lymphoblastoid cell lines from a family and sporadic patients with Goldenhar syndrome. DESIGN, TIME AND SETTING: A single sample observation was performed in the laboratory of the Department of Histology and Embryology, Central South University (Changsha, China) from March to October in 2008. PARTICIPANTS: The peripheral blood samples were collected from 20 surviving members in three generations of a Goldenhar syndrome family in Huaihua of Hunan province, 4 sporadic cases and their parents. METHODS: The peripheral blood samples were collected to isolate B lymphocytes, followed by transformation with Epstein-Barr virus to establish immortalized cell lines. The cyclosporine A was also added to inhibit T lymphocytes growth. Each blood sample was divided into two groups to be cultured simultaneously. Then, the transformed cells were cryopreserved and then resuscitated. Their genetic stability was detected by Karyotype analysis before and after the establishment of the cell lines. MAIN OUTCOME MEASURES: The transformation of B lymphocytes was observed under a light microscope. The recovery rate of the immortalized cell lines after cryopreservation was calculated and the chromosomes were analyzed through G-banding staining. RESULTS: After about 1-week culture, it was seen under the inverted microscope that B lymphocyte blastogenesis appeared and lymphocytes were enlarged. There were visible cytoplasmic prominences, with various shapes, gathering around the cells. About 15 days later, a few white cell colonies were visible with the naked eyes. And about 1 month later, the cells proliferated and formed dense colonies. Finally, a total of 8 immortalized cell lines from the patients with Goldenhar syndrome were established successfully, with the success rate of 100%. Among all the subjects, the success rate of establishing immortalized cell lines was 95.8 % (23/24). The recovery rates of all the established immortalized cell lines were 100% after 1, 3 or 6 months of cryopreservation. Moreover, the genetic integrity of the cell lines was stable according to the Karyotype analysis through G-banding staining. All of these findings show that this is a reliable method to establish a cell line. CONCLUSION: The immortalized cell lines from Goldenhar syndrome patients were established successfully in this study.