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Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering.
Biological Procedures Online ( IF 6.4 ) Pub Date : 2016-08-16 , DOI: 10.1186/s12575-016-0047-9
Marta Pokrywczynska 1 , Daria Balcerczyk 1 , Arkadiusz Jundzill 1 , Maciej Gagat 2 , Monika Czapiewska 1 , Tomasz Kloskowski 1 , Maciej Nowacki 3 , Agata M Gastecka 1 , Magdalena Bodnar 4 , Alina Grzanka 2 , Andrzej Marszalek 5 , Tomasz Drewa 6
Affiliation  

BACKGROUND A key requirements for therapy utilizing the tissue engineering methodologies is use of techniques which have the capability to yield a high number of cells, from small tissue biopsy in a relatively short time. Up to date there was no optimal methods of isolation and expansion of urinary bladder smooth muscle cells (UB-SMCs). The aim of this study was to compare isolation and expansion techniques of UB-SMCs to select the most repeatable and efficient one. METHOD Five protocols of porcine UB- SMCs isolation including enzymatic and explant techniques and three expansion techniques were compared. Isolation effectiveness was evaluated using trypan blue assay. Cell phenotype was confirmed by immunofluorescence staining. Proliferation rate was analyzed using MTT and X- Celligence system. Cellular senescence was assessed measuring β-galactosidase activity. RESULTS Enzymatic methods using collagenase with dispase (method I) or collagenase only (method III) allowed to isolate much larger number of cells than the methods using trypsin with collagenase (method II) and collagenase after digestion with trypsin (method IV). The success rate of establishment of primary culture was the highest when the isolated cells were cultured in the Smooth muscle Growth Medium-2 (SmGM-2). Expression of the smooth muscle markers- alpha smooth muscle actin and smoothelin was the highest for cells isolated by enzymatic method I and cultured in SmGM-2. There was no significant signs of cell senescence until the 8th passage. CONCLUSION The most efficient method of establishment of porcine UB-SMCs culture is enzymatic digestion of urinary bladder tissue with collagenase and dispase and culture of isolated cells in SmGM-2. This method was up to 10 times more efficient than other methods used for isolation and culture of UB-SMCs. This is an easy and consistent method for obtaining high numbers of urinary bladder smooth muscle cells.

中文翻译:

猪膀胱平滑肌细胞的分离,扩增和鉴定,用于组织工程。

背景技术利用组织工程学方法进行治疗的关键要求是使用能够从相对短的组织活检中产生大量细胞的技术。迄今为止,还没有最佳的分离和扩增膀胱平滑肌细胞(UB-SMCs)的方法。这项研究的目的是比较UB-SMC的隔离和扩展技术,以选择最可重复和最有效的技术。方法比较了猪UB-SMCs分离的五种方法,包括酶法和外植体技术,以及三种扩增技术。使用锥虫蓝测定法评估分离效果。通过免疫荧光染色确认细胞表型。使用MTT和X-Celligence系统分析增殖速率。通过测量β-半乳糖苷酶活性来评估细胞衰老。结果使用胶原酶和分散酶(方法I)或仅使用胶原酶(方法III)的酶法比用胰蛋白酶和胶原酶(方法II)和胶原酶消化(方法IV)的胶原酶分离的细胞数量大得多。当在平滑肌生长培养基2(SmGM-2)中培养分离的细胞时,建立原代培养的成功率最高。平滑肌标志物的表达-α平滑肌肌动蛋白和平滑肌蛋白对于通过酶法I分离并在SmGM-2中培养的细胞最高。直到第8代才有明显的细胞衰老迹象。结论建立猪UB-SMCs培养最有效的方法是用胶原酶和分散酶酶消化膀胱组织,并在SmGM-2中培养分离的细胞。该方法的效率比用于UB-SMC分离和培养的其他方法高10倍。这是获得大量膀胱平滑肌细胞的简便且一致的方法。
更新日期:2019-11-01
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