Abstract: Reservation of rare family materials is the base for us to do further research. Family of Jiang-Su Huai-Yin is one of the biggest non-syndromic deafness families in the world. In this family,deafness is maternally inherited and all the sufferers have the mitochondrial DNA 12s RNA A1555G mutation. Four methods are used in the experiments for establishing immortal lymphoblastoid cell lines of the family with non-syndromic deafness. Results were as follows: 1 cell line was from small amout of whole blood method, 1 cell line from frozen whole blood method, 14 cell lines from frozen leukocyte method, and 36 cell lines from cyclosporin A method. In this paper, we will discuss these four methods through our experiments of establishing cell lines.