retrograde perfusion in heart of rats with collagenase - (Apr/12/2005 )

Hi,

Does any body know how to make a retrograde perfusion in rat´s heart? i have to obtain cardiomyocytes of an adult rat for culture them in laminin-coated tissue cultureware,these cardiomyocytes will be on culture about 20 days; the only way that I´ve seen on internet is to perfuse the heart, but how to make it?

thank you
Pal

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Hi Pal:


hearts were perfused retrogradely at a pressure of 10 kPa (70 mm Hg) with Krebs-Henseleit bicarbonate-buffered saline containing (mmol/L) NaHCO3 25, NaCl 119, KCl 4.7, CaCl2 2.5, MgSO4 1.2, and KH2PO4 1.2 (pH 7.6 at 37°C) supplemented with 10 mmol/L glucose and equilibrated with 95% O2/5% CO2. The temperature of the perfusates and hearts was maintained at 37°C by the use of a water-jacketed apparatus. After equilibration (15 minutes), the perfusion was interrupted for 1 to 30 minutes by clamping the aortic perfusion line, and the hearts were thus rendered globally ischemic. Hearts ceased beating within 1 minute of ischemia. Where indicated, ischemic hearts were reperfused for 1 to 30 minutes by reopening the aortic perfusion line. Hearts resumed beating within 1 minute of reperfusion, and coronary flow returned to within 80% of control values (control, 13 mL/min per heart; reperfused, 11 mL/min per heart). Control hearts were perfused for up to 40 minutes after the preequilibration period without interruption to the perfusate flow.

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