Malysz John, Rovner Eric S, Wake Robert, Petkov Georgi V
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center.
Department of Urology, Medical University of South Carolina.
J Vis Exp. 2020 Jan 31(155). doi: 10.3791/59884.
Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 °C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.
膀胱壁内的逼尿肌平滑肌（DSM）细胞最终促进尿液储存和排尿。制备有活力、新鲜且分离的DSM细胞是一项重要的技术挑战，成功实现这一目标可为后续的功能和分子研究提供理想的细胞。本文详细阐述并开发的方法，已被我们团队成功使用了十多年，该方法描述了从开放性膀胱手术中获取的人膀胱标本的解剖过程，随后对DSM组织块进行两步酶处理并进行机械研磨，以获得新鲜分离的DSM细胞。第一步是进行解剖，将DSM层（也称为固有肌层）与黏膜（尿路上皮、固有层和黏膜肌层）以及相邻的结缔组织、血管组织和脂肪组织分离。然后将DSM切成小块（2 - 3毫米×4 - 6毫米），置于含名义钙的解剖/消化溶液（DS）中。接下来，将DSM小块转移至含木瓜蛋白酶和胶原酶的DS中，在约37°C下分别依次处理，每步处理30 - 45分钟。用不含酶的牛血清的DS洗涤并使用火抛光移液管进行研磨后，这些组织块会释放出单个DSM细胞。新鲜分离的DSM细胞非常适合用于离子通道的膜片钳电生理和药理学特性研究。具体而言，我们发现TRPM4通道阻滞剂9 - 菲咯啉可降低用两性霉素B穿孔膜片钳方法记录的电压阶跃诱发的阳离子电流。DSM细胞也可通过其他技术进行研究，如单细胞逆转录聚合酶链反应、微阵列分析、免疫细胞化学、原位邻近连接分析和钙成像。利用单个DSM细胞的主要优点在于所做的观察直接与所揭示的单细胞特征相关。对新鲜分离的人DSM细胞的研究为表征包括膀胱中阳离子通透的各种离子通道的特性提供了重要见解，并将继续作为阐明DSM细胞特性和调节机制的金标准。
