Separation and culture of rabbit vaginal smooth muscle cells in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND:Vaginal smooth muscle cells are mainly cultured by enzyme digestion method and explant method.The former method requires a short culture time with a high output,but this method demands a large number of tissues with a high opportunity of pollution,and the optimal digestion time is difficult to control.The latter method is simple and effective,but the culture needs a long time.OBJECTIVE:To observe the culture time of rabbit vaginal smooth muscle cells using explant + enzyme digestion methods,and to compare with the explant method.DESIGN,TIME AND SETTING:The in vitro observation experiment was performed at the Burn Institute and Animal Laboratory of First Affiliated Hospital,Nanchang University from February 2005 to February 2006.MATERIALS:Female New Zealand rabbits aged 4-5 months,with the body mass of 2.0-2.5 kg were selected for this study.METHODS:Vaginal smooth muscle cells using explant method:1 mm?1 mm?1 mm explants were uniformly incubated in a culture medium,and then placed in a incubator at 37 ℃ for 3.0-4.0 hours.After tissue confluence,tissues were immersed in DMEM containing 0.1 volume fraction,stored at 37 ℃ for 3.0-4.0 days.Following cells grew from the tissue islets,the medium was changed about once every three days.Vaginal smooth muscle cells using explant + enzyme digestion methods:The tissue pieces were digested by 0.1% collagenase type Ⅳ at 37 ℃ for 0.5-1 hour,and then enzymatic digestion was interrupted when the edge of tissue pieces became coarse.These pieces were cultivated in culture dishes.The remaining steps were the same as the explant method.Serial subculture:The 1st subculture was made when 50%-60% cells were confluent.After the 2nd passage of cells was obtained,subculture was made when 80% cells were confluent.MAIN OUTCOME MEASURES:Time of primary culture;Surface structure and ultrastructure of the 3rd passage of vaginal smooth muscle cells.RESULTS:The eruption time and the confluent time of vaginal smooth muscle cells could be shortened by the explant + collagen digestion methods about 1-2 days and 3-4 days respectively compared with the explant method only.Vaginal smooth muscle cells,which were obtained by the two methods mentioned above,could propagate for 5-6 passages.The attached cells were polygonal or spindle and after confluence could be seen,the marked smooth muscle cells "Peak-Valley" structure in some domains under the inverted microscope.The third passage of smooth muscle cell nuclei were serration;the cytoplasm contained the marked smooth muscle cell structural myofilaments and dense bodies and plenty of golgi bodies;the rough endoplasmic reticulum broadened;the free ribosome were abundant under the transmission electron microscope.This indicated that vaginal smooth muscle cells with synthesis phenotype could be greatly collected.CONCLUSION:Explant + enzyme digestion methods need a short cycle of primary culture.Vaginal explants should be kept moisture during drawing materials,isolation and the whole.The time of enzymatic digestion should not be too long,to the point that palpi pilosi is seen surrounding the explants.Primary culture should remain static state and use of plastic culture dishes.