Hi all,

In my lab we are currently differentiating neurons from hiPSCs according to the protocol used in Satir, T et al, 2020 (doi: 10.1038/s41598-020-57516-7). We have been successful in the past getting them to d70 without much cell death and/or detaching but lately, our cultures are detaching from the middle of the well, not on the edges.

It has happend when cells were forming rosettes (so they are clumped-ish) and also after the accutase passage which results in 'single cell' neuroprogenitor culture. We coat Corning primaria P6 plates with L2020 from sigma, at a [L2020]=10 ug/mL overnight at 37ºC. Cells after passage look fine but on the 3rd day they detach from the center.

We were thinking about convection flows that may occur in the well, making cells in the center more vulnerable to detach or the laminin batch being defective but we tried two different batches of L2020 and the outcome stayed the same. We also plate in 2 mL of medium per well and we think less than that may leave the center of the well with a very thin layer of medium that would stress the culture more than the convection currents.

Has this ever happened to you? Any advice on how to address this issue?

Thanks in advance for your expertise! :)