In vitro construction of cell sheets of urine-derived cells in children with hypospadias using temperature responsive culture dishes

Abstract

Objective To investigate the feasibility of acquiring cell sheet using temperature-responsive culture dishes (Nunc Upcell) in children with hypospadias.Methods The clean urine were collected from children with hypospadias.The urine-derived cells were cultured and characterized by cell growth curve,RT-PCR and immunofluorescene staining was employed to identify the mRNA and protein expression in them.Afterwards,cells were then seeded on Nunc Upcell dishes,and acquired the cells sheet according to differential temperature.Results The phase contrast microscopic observation and cell growth curve revealed that the expansion capacity of urine-derived cells from children with hypospadias were identical to those of normal ones.Cells could actively grow to passage 10 and number is up to 109 cells,which completely satisfy the application of tissue engineering.Two groups of cells had the similar population doubling time and expressed urothelial and smooth muscle cell surface markers could be noted in RT PCR and immune-staining.Furthermore,the intact cell sheets could be obtained from the temperature-responsive culture dishes and cells were able to maintain ideal proliferation capacity after transferring.Conclusions The urine-derived cells from children with hypospadias have in vitro expand extensively and can be served as an alternative cell source for urinary tract tissue engineering.The intact cell sheets can also be obtained via temperature-responsive culture dishes,which provide a better platform for the following study of urethral reconstruction or other urological tissue repair. Key words: Regenerative medicine; Tissue engineering; Cell culture