Please, take a look at this ref: J Vis Exp. 2014 Dec 6;(94). doi: 10.3791/52392. Garaud S, et al, A simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes.

Tanos et al. 2013. Science Translational Medicine

Tissue was minced manually to pieces of about 5-mm diameter and digested overnight at 37°C with 2% collagenase A (10 ml/g) (Roche) in Dulbecco’s modified Eagle’s medium (DMEM)/F12. After enzymatic digestion, the tissue was centrifuged at 250g for 4 min at 4°C. Fat was removed, and the pellet containing tissue microstructures was washed with phosphatebuffered saline.

Sflomos et al. 2016. Cancer Cell

Normal breast tissue was obtained from women undergoing reduction mammoplasties with no previous history of breast cancer, as described by Tanos et al. (2013), and freshly resected tumor material of pinhead size was obtained from the pathologist. Human tissue was mechanically dissociated, digested overnight at 37 C with 10 mg/ml collagenase A (11088793001; Roche) in DMEM/F-12 (11039-021; Gibco) supplemented with 1% penicillin-streptomycin (15070-063; Thermo Fisher Scientific) and

1% fungizone (cat. #15290-018; Thermo Fisher) in continuous agitation (40 rpm) as described by Sflomos et al. (2015). Samples were rinsed and erythrocytes lysed with Red Blood Cell Lysis Buffer (R7757; Sigma) and dissociated to single cells with 0.25% Gibco Trypsin-EDTA (15400-054; Thermo Fisher) for 2 min. Trypsin was inactivated with PBS/2% calf serum (CS) followed by incu- bation with 5 mg/ml DNase (1284932; Roche) in L-15 medium (11415; Gibco) at 37 C for 2 min. 2% CS in PBS was added, and the cells were filtered through a 70-mm pore size filter (cat. #352350; BD Falcon) and counted.

These are two published protocols from my lab. Hope it helps.

Patrick