Neonatal Cardiomyocyte isolation and culture
[Neonatal Cardiomyocyte isolation and culture
Overview
The neonatal cardiomyocytes are widely used to study and understand the biochemical pathways and properties of cardiomyocytes.
The isolation and culture of rat neonatal cardiomyocytes has some benefits over that of mouse neonatal cardiomyocytes, including higher yields of viable cells and increased transfection rates.
The following protocol describes the isolation and culture of neonatal rat, Day 1 to 3.
A) Autoclave
- Forceps
- Scissors
- Small beaker (30mL)
- Beaker (100mL)
- 0.1% gelatin (TypeA)
B) Collagenase, type 2 (Worthington)
- 1g/100mL KHBuffer, and filter Put to stock tubes, 1.5mL each, store at -20C
C) Trypsin 2.5% (Gibco, 15090-046)
Put to stock tubes, 0.6mL each, store at -20C
D) DMEM
F) Insulin-Transferrin-Selenium-Ethanolamine ITS - X (100X)
G) Fetal Bovine Serum (FBS)
Procedure
Isolation of cardiac Tissue from Neonatal Rat
1. Coat culture dish with 0.1% gelatin. After aspiration and dry, wash with sterile PBS (in hood).
2. Prepare all the buffers and autoclave
- Prepare 50ml of KH Buffer placed on ice.
- Prepare KH Buffer (35 ml per 20 neonatal rats) in 37C warm. Dissolve collagenase and Trypsin to pre-warmed KHB buffer (solution A).
3. Sterilize scissors, forceps and beakers
4. Prepare 70% ethanol in 100mL – autoclaved beaker and one ice.
5. 1-3 days old neonatal rats are rinsed quickly in 70% ethanol solution for surface sterilization. Pups are decapitated using sterile scissors, and the chest is opened along the sternum to allow access to the chest cavity and the heart. Bleeding is a sign of heart injury. Move the forceps to lower edge of chest. Catch the upper side of the chest with other forceps and push out the heart.
6. Hearts are extracted from the body and transferred immediately into the ice cold KH Buffer. All following steps are performed in the sterile cell-culture hood.
7. Throw the rat to trash bag.
8. Repeat 5)-7)
9. Wash hearts twice with ice-cold KH Buffer.
10. Use scissor to mince hearts into small pieces with scissor.
Enzymatic Tissue Digestion and culture
1. Transfer the cut heart to small baker. Remove KH Buffer and add fresh 2 mL KH Buffer.
2. Add 4mL solution A and place to 37 °C water bath for about 10 min to adjust temperature of the digestion solution with gentle agitation.
3. Take away the buffer.
4. Add 6mL solution and place to 37 °C water bath for about 15 min with gentle agitation. Caution: longer incubation periods may reduce cell viability.
5. Take the buffer (digested myocardium solution) and put into a new 50 mL conical tube. Mix with 6mL DMEM containing 10 DMEM containing 10%FBS to stop the effect of collagenase/trypsin.
6. Re-suspend the undigested tissue fragments in 6mL solution A. Gently pipetting and then incubate for additional 15 min with gentle agitation.
7. Repeat 5)-6) until heart pieces become very small and white. Usually around 4 times repeats.
8. Centrifuge at 800 rpm for 5 min.
9. Remove supernatant and resuspend the pellet with DMEM (10%FBS). Put the cell solution to culture bottle. (20 rat/10mL DMEM/1 culture bottle).
10. Culture in 5% CO2 incubator for 90min.
11. Pat the bottle and take the supernatant. Sticking cell is mainly fibroblasts. Add the bottle new DMEM with 10% FBS without BrdU. This bottle is used for cardiac fibroblasts culture.
12. The supernatant put into a new culture bottle or 50mL tube. Add DMEM containing 10% FBS with BrdU (0.1mM). Count the cell number. Usually cells from 20 rat can be diluted 50mL DMEM.
13. After cell count, dilute with DMEM/10%FBS/BrdU to appropriate concentration. Usually plating with 5X105/mL cell should be 80-90% confluence.
14. Culture in 5% CO2 incubator.
15. Culture medium can be exchanged after 24 hours if necessary.
