A Study On The Culture Of Myocardial Cells In Newly-born SD Rats

Objective: To explore the appropriate cultural condition and subculture's condition of myocardial cells of SD rats and to provide the necessary conditions for the establishment of conditional culture medium of embryonic stem cell by collecting metabolic liquid of myocardial cells. Methods: Two methods were taken. (I) Digestive method: The single myocardial cell was gained by dissociating cardiac muscle tissue with enzyme and was put into the 6-well cultural plate by using different removal time of fibroblast of cardiac muscle. Added Ara-C (Inhibiting the growth of fibroblast) , the plate was put into the circumstance of CO2 at 5%, temperature at 37℃ and saturated humidity for culture and subculture. The impact of different enzyme, different digestive time, different removal time, and different dosage of Ara-C on the myocardial cells' culture was compared. (II) Tissue method: Cardiac muscle tissue was cut into debris in 1mm3 size and the debris was put into 6-well cultural plate. Added Ara-C (Inhibiting the growth of fibroblast) , the plate was put into the circumstance of CO2 at 5%, temperature at 37℃ and saturated humidity for culture and subculture. The efficacy of different dosage of Ara-C on the myocardial cells of rats was compared. During the experiment, the cells' metabolic liquid was collected. Result: (I) Digestive method: A significant difference was seen in the number of the survival myocardial cells between cell group digested with collagenase (type II) and cell group digested with trypsin, which means that collagenase has less damage to the cells; When the impacts of digestion of collagenase at 5min, 15min, 30min on the existing of myocardial cells were compared, it was found it's better to digest with collagenase for 5min; When different dosages of Ara-C (OuL, 20μL, 50μL, l00μL respectively)were added, it was found that the efficacy of culture of myocardial cells was much better when the added Ara-C was at the dosage of 20μL; The efficacy of using removal time for 30min group and for 60min group was better than that for the 15min group, but there was no significant difference of efficacy between 30min group and 60min group. (II) Tissue method: All of the different dosage of Ara-C (OuL, 20μL, l00μL respectively)had effect on the growth of cardiac cells andthe group without the addition of Ara-C .grew faster than the other groups. A total of 200ml metabolic liquid of myocardial cells was collected when the experiment was finished. Conclusion: With the digestive method, the culture efficacy of myocardial cells of rats is better when digesting for 5min with collagenase (type II), removal time being 30-60min and 20L of Ara-C added. With the tissue method, it's suitable to add 20L of Afa-C to inhibit the growth of fibroblast.