GSM5830301: Liver 3.B_TCR; Homo sapiens; OTHER
GSM5830301
Liver tissue and matched blood were obtained from patients (50-65 years old) undergoing liver resection for metastatic colorectal cancer (n = 1; male), haemangioma (n = 1; female), or hepatocellular carcinoma (n = 2; one male and one female). Disease-free liver tissue was collected from the resection margin. Liver tissue was cut into small pieces with a scalpel and ground through a 70 μm cell strainer. Cells were washed with R10 (RPMI-1640 [Sigma-Aldrich] + 10% FCS [Sigma-Aldrich] + 1% Penicillin-Streptomycin [Thermo Fisher Scientific]; 2000 rpm [931 g], 10 min, 4°C) and mononuclear cells isolated by density gradient centrifugation on a discontinuous 35%/70% Percoll (GE Healthcare) gradient (2000 rpm [931 g], 20 min, room temperature, no brake). Mononuclear cells were collected from the interface and washed with R10 (1600 rpm [596 g], 10 min, 4°C). Residual red blood cells were lysed with ammonium-chloride-potassium (ACK) for 3-5 min. Cells were washed twice (1600 rpm [596 g], 10 min, 4°C) and cryopreserved (90% FCS, 10% DMSO [Sigma-Aldrich]) in liquid nitrogen. PBMCs were isolated from fresh whole blood by density gradient centrifugation (Lymphoprep, Axis-Shield) at 2000 rpm (931 g) for 30 min with no brake. Cells were cryopreserved (90% FCS, 10% DMSO) in liquid nitrogen. Blood and liver cells were thawed, stained, and live MAIT cells (CD3+MR1/5-OP-RU+) and memory T cells (CD3+MR1/5-OP-RU-CCR7-; donor 2 and 3 only) sorted by fluorescence-activated cell sorting (BD FACSAria III, 70 μm nozzle). scRNA-seq and scTCR-seq libraries were generated using Chromium Single Cell V(D)J Reagent Kits (v1.0 Chemistry) following manufacturer's instructions.