Hello everyone,
I am trying to isolate cardiomyocytes from a day old rat pups for patch clamping. I use trypsin 0.1% and 50microgram/ml DNAse 1 in HBSS to digest the ventricle pieces. Cells are isolated by 5-minute rounds of digestion for 10 times in 37degree c.
Unfortunately after 2-3 rounds of digestions the tissues pieces start to clamp together and become a blob of transparent jelly. It's very hard to break it up by triturating with a pipette. Also the cell yield is very low .
Will it help if I add EDTA to the trypsin solution? Also is coating the culture surface necessary for cardiomyocytes? I use Corning T-75 culture flasks.
Thanks in advance for your kind response.
Hello Sayeman,
I did isolate cardiomyocytes from e12.5 embryonic mouse hearts last year for my RNA-seq experiment. Here is the procedure I used. It should work for neonatal heart.
*Prepare 20 ml solution containing 9mg collagenase (Sigma), 20mg Pancreatin (Sigma) in HBSS containing penicillin/streptomycin
*Filter the enzymatic solution through 20um filter
*Mince 5-10 embryonic hearts and place in enzymatic solution
*Incubate at 37C incubator
*Every 10 minutes take the dish/tube out and pass the mixture through pasteur pipette up and down to make the pieces thinner
*Incubation upto 90 minutes in total
*Wash cells in you media 3 times to get rid of enzymes by centrifugations
*Finally filter the cell suspension through 70Um filter to get single cell suspension
Hope that helps
Hi,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541493/
Chapter Neonatal Rat Cardiomyocytes Isolation, Culture, and Determin...
These might help !!
All the best
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