Isolation of Ca^(2+) Tolerant Cardiomyocytes from Aadult Rats for Patch Clamp Studies

膜片钳研究中的钙耐受大鼠心肌细胞分离方法(英文)

Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.

目的 :探讨膜片钳实验中大鼠心肌细胞分离过程中影响细胞活性和钙耐受性的各种影响因素。方法 :采用Langendorff灌流 ,先用台氏液灌流 30s ,再用无钙台氏液灌流 5～ 7min ,其后换含胶原酶Ⅰ 0 .1～ 0 .2g/L的低钙酶液消化约 8～ 30min ,最后用无钙台氏液冲洗心脏 2min ,剪下心肌组织 ,KB液中 37℃剪碎吹打温育 5min后 ,用 2 0 0μm筛网过滤 ,室温静置换液后进行膜片钳实验。 结果 :注意控制水 ,酶 ,温度等各种因素后 ,可得到杆状、横纹清晰、膜良好的耐钙心肌细胞。结论 :大鼠心肌细胞分离过程中各种因素控制好后可以保证一定的细胞成活率及其质量 。