Cardiomyocyte Preparation

(Bettencourt-Dias & Laube F combined)

==Procedure:

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)

1. Neutralize cell suspensions w/ complete 4ml AL-15 medium (AL-15 w/ 10% FBS and pen-strep)
2. Pass through a 100um microsieve (reason for this???? And what to collect – flowthrough or the retenate… cardiomyocytes are min 150um in length…)
3. Centrifuge the neutralized suspensions for 10min at 500 rpm .
4. Resuspend in complete AL15 (volume???)
5. 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)
6. 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?)
7. Change the medium only when 3 days have passed since plating.