Human tumor dissociation buffer

The mesothelioma tumor biopsies and blood were collected from two patients who underwent surgical removal of the tumors. Tumor samples were collected and delivered directly from HUS Hospital and patients gave their written consent. Sex or gender were not considered relevant in the current study.
Cells were isolated from the original tissue after surgery by physically mincing the resected tumor into small pieces using a scalpel; subsequently, the tissue was treated using human tumor dissociation buffer (Miltenyi Biotech) following manufacturer instructions and the Gentle MACS dissociator (using the default human tumor program).

A total of 40 immunodeficient NSG mice (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ, the Jackson Laboratory), were received and acclimatized for a week. NSG mice were then injected with 0.5 × 10⁶ CD22+ RAJI and CD22− RAJI tumor cells on left and right flank, respectively. The tumor cells were allowed to expand until mouse randomization, which was performed at day 4 based on the degree of tumor cell outgrowth, measured using bioluminescence imaging (BLI) (XenoLight D-luciferin (PerkinElmer). The next day, mice were adoptively transferred (i.v.) with either 8 × 10⁶ viable mock-transduced T cell or 8 × 10⁶ viable TRAC_CAR positive T cells (7 mice per group, total 5 groups). The mice were then re-imaged at day 6 of TRAC_CAR T cell injection and sacrificed to harvest serum, spleen, and right and left tumors. The splenocytes were prepared by mechanical disruption of the organ followed by 70 µm-filtering and a Ficoll density gradient purification. The cell suspensions were analyzed by flow cytometry. The tumors were dissociated using Miltenyi’s human tumor dissociation buffer according to the manufacturer’s protocol. The cells were then filtered using a 40 µm filter and analyzed by flow cytometry.