10X ADS                                                                   Digestion Media

116mM NaCl                                                            80 U/ml Collagenase Type II

20mM HEPES                                                         0.6 mg/ml Pancreatin (S#P7545)

1mM Na2PO4                                                           Dissolve separately in 25ml 1X ADS

5.5mM Glucose                                                        Combine (50ml) and filter

5.4mM KCl

0.8mM MgSO4

pH to 7.2

Plating Media                                                                                   Culture Media

67% DMEM                                                                                      73% DMEM

17% M199                                                                                        20% M199

5% FBS (not heat inactivated for cardioids)                               7% FBS

10% Horse Serum (not heat inactivated for cardioids)                        1% ABAM

1% ABAM                                                                             

Before Starting

1.  Thaw 1 x 35ml aliquot of horse serum

2.  Set shaker to 37C

3.  Put 2 x 100mm dishes on ice with 10ml of 1X ADS

Heart Isolation

1.    Grab neonate behind shoulders and dip in 70% ETOH

2.    Decapitate with large scissors

3.    Insert small scissors into chest and cut along sternum (Heart should be extruded)

4.    Remove the heart and place in the 100mm dish containing ice cold 1X ADS

5.    After collecting all of the hearts, transfer them to the fresh plate of ice cold 1X ADS

Heart Cell Dissociation

1.    In the hood, aspirate the ADS and cut the hearts into fine pieces

2.    Transfer the hearts to a 50ml conical containing 10ml of digestion media (DM)

3.    Incubate 5 minutes at 37C and ~80rpm

4.    Pour off the cell-free media and add 10ml fresh DM

5.    Incubate 20 minutes at 37C and ~80rpm

6.    Pipette the pieces up and down 5-10 times and then allow the tissue to settle

7.    Pipette off ~10ml of the cells and add to a 50ml conical containing 2ml horse serum

8.    Pellet the cells for 5 minutes at 1000rpm

9.    Add 10ml fresh DM and incubate another 20 minutes at 37C and ~80rpm

10. Aspirate the DM off of the pelleted cells

11. Resuspend the cells in 5ml of horse serum

12. Repeat 3 times

13. Upon completion of digestion, combine all of the cells and pellet for 5 minutes at 1000rpm

14. Aspirate the horse serum and add ~1ml plating media per heart

15. Count an aliquot of the cells

16. Dilute the cells to 2 million cells per ml

17. Plate 2ml per 35mm plate

Cardioid Culture

1.    The first media change should occur at ~48 hours

2.    Aspirate the plating media and add 2ml of culture media

3.    Change the media every 24 hours after that point until the constructs have rolled up

4.    Delamination should start on day 7-8

5.    Cardioids should have completely formed by day 10

6.    Testing should begin on day 12