Cardiomyocyte Preparation

Bettencourt-Dias & Laube F combined

1) Collect hearts in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep 2) Wash them once in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep 3) Remove the newt ventricles 4) Wash ventricles in 70% Leibovitz-15 (L15) medium (AL15) w/ pen-strep 5) Store ventricles O.N. at 25C in AL15 w/ pen-strep 6) Digest ventricles 2ml d.sol for 8h in a shaking water bath at 27C. Change the enzyme solution every 1-2h. Note: D.sol - aPBS w/ - 0.5% Bactotrypsin (Difco) - 380U/ml collagenase (Sigma) - 0.15% BSA - 0.3% glucose - gentamycin (50ug/ml)

7) Neutralize cell suspensions w/ complete 4ml AL-15 medium (AL-15 w/ 10% FBS and pen-strep) 8) Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…) 9) Centrifuge the neutralized suspensions for 10min at 500 rpm . 10) Resuspend in complete AL15 (volume???) 11) preplate in uncoated 6cm culture dishes for 3 days at 25C in a humidified CO2 incubator. Note: Blood cells, connective tissue and some pigmented cells attach to the culture dish during this step, while the cardiomyocytes remain in suspension (non-adhere) 12) Transfer the supernatant onto laminin coated dishes (15ug/ml in serum-free medium, coating for 60 min) *at a density of 4000 cells/cm2* (is it necessary at such density?) 13) Change the medium only when 3 days have passed since plating.