Knodle S, Anderson S, Papaioannou S
In Vitro Cell Dev Biol. 1986 Jan;22(1):23-7. doi: 10.1007/BF02623437.
Rabbit aortic smooth muscle cells were prepared by enzymatic digestion of the aortic smooth muscle layer. The cells were subcultured up to Passage 22 starting from a cryogenically preserved stock (approximately 10(10) cells, Passage 8) and characterized morphologically and for 45Ca++ uptake. Microscopically the cells demonstrated the characteristics of vascular smooth muscle cells. 45Ca++ uptake by the cells plated on tissue culture flasks (25 cm2) was determined at 25 degrees C in physiological salt solution (PSS) containing 45Ca++ in low (5 mM) or high (50 mM) KCl concentrations. At the end of the incubation period (0 to 30 min), PSS was aspirated and the cells quickly washed, digested with 0.5 N NaOH, and counted for 45Ca++. High K+ increased the 45Ca++ uptake by 100% or more compared to the low K+ uptake of 45Ca++. This K+-induced 45Ca++ uptake was eliminated in osmotically shocked cells, and inhibited by nifedipine, verapamil, and diltiazem, in a dose-dependent manner. The extent of 45Ca++ uptake and the inhibitory activity of nifedipine were retained up to Passage 22. It is concluded that the developed methodology for scaled-up cultures of rabbit aortic smooth muscle cells provides morphologically intact and biochemically functioning cells suitable for calcium channel studies.
兔主动脉平滑肌细胞通过酶消化主动脉平滑肌层制备。细胞从低温保存的原代培养物（约10¹⁰个细胞，第8代）传代培养至第22代，并进行形态学和⁴⁵Ca²⁺摄取特性分析。显微镜下，细胞表现出血管平滑肌细胞的特征。在含有低（5 mM）或高（50 mM）KCl浓度的⁴⁵Ca²⁺的生理盐溶液（PSS）中，于25℃测定接种在组织培养瓶（25 cm²）上的细胞的⁴⁵Ca²⁺摄取。在孵育期结束时（0至30分钟），吸出PSS，快速洗涤细胞，用0.5 N NaOH消化，然后计数⁴⁵Ca²⁺。与低K⁺摄取的⁴⁵Ca²⁺相比，高K⁺使⁴⁵Ca²⁺摄取增加100%或更多。这种K⁺诱导的⁴⁵Ca²⁺摄取在渗透性休克细胞中消失，并被硝苯地平、维拉帕米和地尔硫䓬以剂量依赖的方式抑制。直至第22代，⁴⁵Ca²⁺摄取程度和硝苯地平的抑制活性均得以保留。结论是，所建立的兔主动脉平滑肌细胞大规模培养方法提供了形态完整且具有生化功能的细胞，适用于钙通道研究。
