A Study Of Culture Of Rabbit Bladder Smooth Muscle In Vitro And On Acellular Matrix Graft

Objective: To study the technique of culture of bladder smooth muscle cells in vitro;and to explore the methods and feasibility of culture of smooth muscle cells on bladder acellular matrix(BAMG) in vitro as an experimental basis for bladder regeneration in vivo by tissue engineering.Methods: (1)Each bladder of 14 rabbits was bisected into two halves which were cultured by enzymatic digestion and explant technique respectively.We observed primary culture time of these two methods. To identify of smooth muscle cells in culture , HE staining was used and immunohistochemical analysis was performed with antibody to α-smooth muscle actin.；(2)The cultured cells between passages 3 and 4 were frozen by liquid nitrogen cryopreservation .After three months ,morphological and proliferative changes of cells recovered from freezing were observed under invert microscope and the cells were identified by immunohistochemical analysis；(3)The BAMG was prepared by biochemical technique and identified by HE staining and scanning electron microscopy；(4)The cultured cells between passages 3 and 5 were seeded on BAMG and their growth characteristics were observed by light and scanning electron microscopy.Results: (1)Of primary cultures,fourteen were successes and one was a failure by explant technique while seven successes and seven failures by enzymatic digestion technique. There were significant difference of success rates between the two methods(P<0.05). The primary culture time were 10.08±0.72 days by explant technique and 4.27±0.38 days by enzymatic digestion technique.Therewere significant difference of primary culture time between these two methods. Immunohistochemical staining with an α-actin antibody was positive for all passages of cells；(2)Cells of recovery from freezing could still be alive and grow rapidly with normal morphology, the results of immunohistochemical analysis were almost as same as the cells before freezing；(3)BAMG was made up of numerous fiber meshes with no evidence of cells remained through light and scanning electron microscopic examination；(4)The HE staining results of BAMG-cell constructs showed that smooth muscle cells grew in several layers with a small number of cells penetrance into matrix and the scanning electron microscope results of BAMG-cell constructs showed that there were many cells growing on the BAMG with limited areas of matrix penetrance .Conlusions: (1)Enzymatic digestion and explant techniques have individual characteristics, the primary culture time of explant technique was longer than enzymatic digestion, but the explant technique had higher success rate;(2)The biological characteristics of cells of recovery from freezing were almost as same as the cells before freezing ,which demonstrated that freezing cells may provide a good method to solve the problem about the storage of seeded cells using in tissue engineering；(3)BAMG supports the growth of the cultured bladder smooth muscle in vitro,which provide a good basis for coculture of bladder urothelial and smooth muscle cells on BAMG and transplanting them into body in future.