Dear Cristina,

One possibility could be to use DNaseI with TrypLE to avoid the clumping if you consider the DNA from dead cells to be the major reason behind it. I have not tried this myself for hiPSC CM so cannot gurantee if it will work here. However we do use it during neonatal rat CM isolation and gives us decent results.

best

Shambhabi

Shambhabi Chatterjee

I have been told by verbal communication from colleague this is one option but instead of adding it to TrypLE Express to add it to plating medium.

I have read that Liberase TH plus DNase I being added together to TrypLE Express (Burridge et al 2015) but have not tried it.

Do you know at what concentration the DNase I is added to TrypLE?

Reagrding the liberase TH plus DNaseI

Stock is DNase I, 277 U/μL (Life Technologies, cat. no 18047-019)

And as mentioned in the paper (Material section, Page6) -

Working solution is:

" For cells later than day 15, add 0.5 U/mL Liberase TH and 50 U/mL DNase I to the TrypLE to break down deposited

collagen. "

I have never tried it myself so cannot tell you how much it improves the attachment of cells as single monolayer.

We prepare 2mg/ml stock solutions of DNAseI in 0.15M NaCl and store it at -20oC in 2ml aliquots. We use 1.28 mg DNAse I / 100 ml of the digestion medium for our NRCM preparations.

For hiPSC CM I generally use 0.5X Trypsin when I passage iPSC CMs. [0.5% Trypsin-EDTA (10x), no phenol red, Invitrogen #15400-054 diluted 1:1 in 1X PBS]

However, regarding iPSC CM I also have noticed similar clumpings ONLY IF the differentiation efficiency is low- I have tried passing the cells through a sterile mesh [ also mentioned in the Burridge et al, 2015 paper- 100 μm cell strainer (Corning Falcon, cat. no. 352360) ] and also performed metabolic selection prior to digetsing/passaging the CMs. They seem to give better results but I definitely lose some cells and they take more time to recover from the stress. So you could try this out depending on how many cells you can afford to lose.

Good luck !

Hi Cristina, this happens at times when you are having enough cardiomyocytes CMs (like 2-3 million) in a single falcon tube as the pellet obtained from CMs is hard to resuspend.

Here are few tips which might be helpful:

• Prepare a mixture of 5% Chicken Serum(CS) and 0.05% Tris -EDTA(TE) that is 0.5ml CS and 10 ml Tris-EDTA. Tris -EDTA(TE) (Life Technologies) supplemented with 5% Chicken Serum(CS) (Sigma-Aldrich) for 10–15 min at 37°C. The trypsin can be inactivated with DMEM containing 10% fetal bovine serum. Add 1 ml mixture of (Chicken Serum and Tris -EDTA 0.5ml CS and 10 ml Tris-EDTA) to each well of a (6 well plate) and keep it in an incubator at 37 for 10 mins.
• Flush the cells gently with P-1000 so that all the cells are detached so at this point try resuspending the cells like five to six times in each well in the six-well plate and once the cells from each well are in the neutralizing solution (DMEM+FBS). Using the 5/10 ml pipette resuspend them twice my observation is the better you resuspend them here lesser are the chances that they will be clumped post centrifugation while you are reseeding them). Moreover, do not pool many cells in one falcon tube split them into two tubes. So, for each well resuspend them into 6 ml of neutralizing solution overall for a six-well plate, you would need 30 ml of neutralizing solution.
• Post centrifugation once you aspirate the medium first add 2-3 ml of your maintenance media and mix using P1000 then finally dilute them adding more maintenance medium and further resuspend them with 5-10 ml transfer pipette. Additionally, in my experience, those clumps will detach off during the purification process, which I am not aware of what you are using?
• Additionally, passing them through the 40µm-70µm filter will get rid of clumps, but eventually, you will end up losing CMs. I would recommend following the above points (as it works in my case) and please optimize a seeding number as the key is that should form a monolayer, or they will detach off my suggestion is that from one completely confluent six-well plate you can reseed into two six well plates.

Hope it will help

Good luck