Tumor dissociation kit

CDX tumors were grown to approximately 800 mm³ and the mice were killed by schedule 1 method. The tumors were removed and dissociated into single cells using the Miltenyi Biotec tumor dissociation kit (#130-095-929 [Miltenyi Biotec, Germany]) following the manufacturer’s instructions on a gentleMACS octo dissociator (#130-096-427 [Miltenyi Biotec]), as previously described.⁸ Single cells were incubated with anti-mouse anti-MHC1 antibody (eBioscience clone, 34-1-2s [ThermoFisher Scientific, Waltham, MA), anti-mouse anti–immunoglobulin G (IgG) 2a+b microbeads and dead cell removal microbead set (Miltenyi Biotec #130-090-101) and applied to an LS column in a MidiMACS Separator (Miltenyi Biotec) for immunomagnetic depletion of mouse cells and dead cells. CDX ex vivo cultures were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with the following components: 10 nM hydrocortisone, 0.005 mg/mL Insulin, 0.01 mg/mL transferrin, 10 nM β-estradiol, and 30 nM sodium selenite; 5 μM Rho kinase inhibitor added fresh (Selleckchem, Y27632 [Houston, TX]), and 2.5% fetal bovine serum added after 1 week at 37°C and 5% carbon dioxide.

The cryopreserved tumors were mechanically and enzymatically dissociated using the Miltenyi Biotec tumor dissociation kit (#130-095-929) and the GentleMACS ™ Dissociator (#130-093-235) based on the manufacturer’s protocol. To generate the multicellular microcancer (immunotumoroid), 1.5 x 10 ⁴ cells/well were loaded on a 96-well GravityPLUS Hanging Drop plate (InSphero, Schlieren, Switzerland) and incubated at 37°C. Cells were seeded at 1.5 x 10 ⁴ cells/well to increase the inclusion of non-tumor cells, such as stromal cells and tumor-infiltrating lymphocytes. After 4 days of incubation (day -4), the aggregated microcancers were dropped into a 96-Well clear round bottom ultra-low attachment (ULA) plate (#7007 Corning) and cultured in DMEM/F12 media supplemented with 10% heat-inactivated human AB sera, primocin, glutamax, hepes, N-Acetyl-L-cysteine, Nicotinamide, B-27 Supplement, A83-01, insulin, EGF, and the therapeutic agents. The total volume of media per well was 240 μL. Cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C.

The tumors were cut into small pieces and digested with Tumor Dissociation Kit (#130-096-730; Miltenyi) at 37°C for 40 min with periodic agitation. An equal volume of RPMI-1640 medium containing 10% FBS was added to stop the enzymatic reaction, and the tumor samples were filtered through the 70-μM cell strainer (#352350; Falcon). The tumor-infiltrating leukocytes were isolated with the Percoll density gradient (#17-0819-01; GE) and then analyzed by flow cytometry.

Human primary CAFs were obtained as previously described (Leca et al., 2016). Briefly, the tumors were cut into small pieces of 1 mm3 using a razor blade. The tissue pieces were dissociated using the Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s recommendations. Cells were then resuspended, passed through a cell strainer (100 μM), and finally plated. Primary CAF features were verified by flow cytometry with a positive α-SMA and FAP staining. Immortalized CAFs were generated from primary CAFs of limited passage via retrovirus-mediated expression of human telomerase reverse transcriptase (hTERT). All CAFs were cultured in DMEM F-12 medium (Gibco) supplemented with 10% fetal bovine serum, 1% L-glutamine (Gibco), 1% antibiotic-antimycotic, and 0.5% sodium pyruvate (Gibco). Cells were detached and passaged using StemPro accutase cell dissociation reagent (Gibco).
All cultured cells were tested for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).

Fresh tumors from PDAC patients (Additional file 1: Table S1) were collected at HonorHealth Research Institute (Scottsdale, AZ, USA) and Samsung Medical Center (Seoul, South Korea) under Institutional Review Board-approved protocols. Signed informed consent was obtained from each patient. Primary tissues from ten different patients with localized PDAC were obtained during surgical resections, and the biopsies of six patients with metastatic PDAC (5 liver metastases and 1 omentum metastasis) were obtained by 19 gauge needles. Freshly harvested tissues were mechanically and enzymatically dissociated using a tumor dissociation kit (Cat #130-095-929, Miltenyi Biotec, Bergisch Gladbach, Germany) on a gentleMACS™ Dissociator (Miltenyi Biotec). After dissociation, single-cell suspensions were filtered using a 40-μm cell strainer to remove large pieces of debris. Red blood cells (RBC) were removed by incubating the cells with RBC lysis buffer (ThermoFisher Scientific, Carlsbad, CA). Cells were then counted and evaluated for viability using the trypan blue (0.4%) staining assay.