My experimental approach requires the isolation of mononuclear leucocytes from the nasal polyp of CRSwNP patients, with a high yield of at least 90% viable cells.

Unfortunately, after several tests and optimization trials of my protocol, I couldn't obtain a single-cell suspension of viable lymphocytes with a good cell yield.

Here my approch in brief: 1. After mincing the nasal polyps (freshs) into very small pieces with a scalpel blade, the latter are enzymatically digested using filtred collagenase IV (from gibco) at a concentration of 1 mg/ml or 2 mg/ml (in DPBS with calcium and magnesium) for 1 hour at 37°C with 5% CO2 or in a 37°C warm bath . But at the end of every incubation, I noticed with my naked eye that the tissue pieces were still undigested. 2. After centrifugation and washing at 530g for 10 minutes, the digested material was filtered through a 50 µm cell strainer and then centrifuged. 3. Hence, I verify whether I obtained viable mononuclear leucocytes by photonic microscopy. Unfortunately, I couldn't have a monocellular suspension of mononuclear leucocytes at the end of this experiment; either I obtain a very low cell yield with a very low number of viable mononuclear leucocytes; or a suspension containing mainly dead cell clumps and cell debris (clumps of tissue stained with trypan blue) . Could anyone help me indentify the cause of the failure of this protocol ?

Thanks in advance!