Cardiac Myocyte Culture
Dissociate culture of rat cardiac myocytes on the MED probe.
Jong-Kook LEE, M.D., Ph,D.
Dept. Circulation, Research Institute of Environmental medicine,
Nagoya University, Nagoya, Japan
E-mail: jlee@riem.nagoya-u.ac.jp
1) Preparation of silicon ring
2) Sterilization of MED probe
3) Coating with collagen
4) Sterilization of surgical tools
5) Culturing
6) Plating of myocyte and medium exchange
7) Sample data from myocyte culture
8) References
(1) Preparation of silicon ring
1. Make a ring from silicon sheet to surround microelectrode array. The height of the ring is around 2-3 mm.
2. Glue this silicon ring on the surface of MED probe with silicon glue (Shinetsu Kagaku; KE45T). Do not touch and damage the electrodes.
3. Leave it to dry out for about 8 hours.
NOTE:
* This step may be skipped, if you can collect enough number of cells.
* Alpha MED Sciences can provide MED probe with silicon ring as your request.
(MED probe with silicon ring
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(2) Sterilization of MED probe
1. Soak MED probe with silicon ring into 70%ethanol for 15 min.
2. Dry out the probes completely under the clean bench for 30 min.
3. Irradiate UV to the probes under the clean bench for 15 min.
4. Store the sterile MED probe into the sterile 100 mm petri-dish.
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(3) Coating with collagen
1. Put the rat tail collagen solution into the MED probe to cover entire microelectrode array.
2. Incubate the MED probe into CO 2 incubator at 37C for 30 min.
3. Remove the rat tail collagen solution in a clean bench.
4. Rinse it twice with sterile PBS(-).
5. Dry out in a clean bench.
6. Put the culture media into the MED probe.
7. Remove the culture media right before use.
* Rat tail collagen solution : ~ 40 ug/ml in 0.15 M acetic acid.
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(4) Sterilization of surgical tools
Surgical scissors (2), Fine forceps (2), Beaker [ 30 ml (1), 50 ml (1) ]
1. Sterilize tools with (250C, 30 min)
2. After sterilization, pour 70% ethanol into beakers.
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(5) Culturing
* Neonatal Cardiomyocyte Isolation System (Worthington; NCIS kit)
* Following steps are modified from the manufacture's original method.
[First day]
1. Right after soaking a neonatal rat (PND 1) into 70% ethanol, take out a whole heart immediately after decapitation. And then put it into HBSS (Hank's balanced salt solution: contained in the NCIS kit). Repeat this procedure one by one (total ~10 pups).
* Put the HBSS in the 100 mm petri-dish onto the ice to keep the temperature at 4C during following procedures.
2. Separate ventricles from the heart with new scissors and put it into new HBSS.
3. Mince ventricles into small pieces.
4. Soak minced pieces into the trypsin solution (100ug/ml in HBSS).
5. Store in a refrigerator for overnight.
[Second day]
1. Make A solution, which contains one bial of collagenase in 20 ml L-15 solution.
* both collagenase and L-15 solution are included in the NCIS kit.
2. Put trypsin-treated pieces into 50 ml centrifugal tube with the trypsin solution.
3. Centrifugation (1000 rpm, 5 min, room temp).
4. Remove supernatant.
5. Add 20 ml A solution and make pipetting gently.
6. Put all solution into 75 cm 2 culture flask (Falcon: #353136, with vent cap).
7. Shake the flask into water bath at 37C for 30 - 45 min.
* The time for digestion would be modified depending on the condition of cells.
8. Make pipetting gently 10 - 20 times at room temp and leave it for 3-4 min.
9. Pour the cell suspension into cell strainer (Falcon: #352350, 70um Nylon).
10. Oxygenate the suspension (may skip this procedure).
11. leave it at room temp for 20 min (may skip this procedure).
12. Shake it gently by hand.
13. Centrifugation (1000 rpm, 5 min, room temp).
14. Remove supernatant.
15. Add 20 ml L-15 solution.
16. Make pipetting gently.
17. Centrifugation (1000 rpm, 5 min, room temp).
18. Remove supernatant.
19. Add 20 ml L-15 solution.
20. Make pipetting gently.
21. Centrifugation (1000 rpm, 5 min, room temp).
22. Remove supernatant.
23. Add culture medium (M199 with 10% FBS) (10 ml)
24. Pour the cell suspension into a 25 cm 2 culture flask (Corning: #430639, vent cap) and store it into CO 2 incubator at 37C for 30 min (Pre-plating method to remove fibroblasts).
25. Take the supernatant to cardiac myocyte culture.
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(6) Plating myocyte and medium exchange
1. Pour the cell suspension into silicon ring on the MED probe.
* Following amount of cell suspension (1.5 x 10 7 cell/ml) will be suitable for confluent monolayer culture inside the silicon ring.
- 150 um spacing probe (diameter of silicon ring is around 3 mm): 25-50 uL
- 450 um spacing probe (diameter of silicon ring is around 5 mm): 50-100 uL
2. Store it into CO 2 incubator for 30-60 min until cells adhere to the surface of the MED probe.
3. After adhesion, pour fresh culture medium (1-1.5 ml) slowly as below from outside of the silicon ring to mix the medium inside the ring.
(culture medium: M199 with 10% FBS)
M199 solution (culture medium) GIBCO 11150-059
10% fetal bovine serum (FBS) GIBCO
antibiotics ( gentamicin ) 5 0 ug/ml SIGMA
* Exchange the culture medium at least every other day ( all volume change)
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(8) Sample data from cardiac myocyte culture
1. Spontaneous activities can be observed around two days in vitro.
2. Pacing responses can be observed around 3-5 days in vitro.
(Spontaneous activities)
(Pacing responses)
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(8) References
1. Jong-Kook Lee et al, Circulation. 2002:106.II-68
(abstract: American Heart Association 75th Annual Scientific Meeting).
2. Inoue N et al, J Am Coll Cardiol 2004 Aug 18;44(4):914-22.
