Hi everyone, I am doing 3D cell culture. I have seen random holes in the tumour spheroids. Instead of forming a big necrotic core in the centre, they had holes distributed throughout the spheroids.

We did live/dead staining and found the random holes were dead cells (shown in the picture attached, which is the composition of brightfield and DRAQ7 stain, and the blue dots were dead cells). This has happened to us consistently since the last august.

Medium is Tu4%; the cell line in this picture is a human melanoma cell line.

Does anyone know what caused this and how we can stop it?