Cryostor cs10 medium

Prior to mononuclear cell (MNC) enrichment, the bone marrow aspirate was filtered and washed with Phosphate Buffered Saline (PBS, Braun Melsungen, Germany). Thereafter, the same procedures were followed for peripheral blood MNCs (PBMCs) and bone marrow mononuclear cells (BM-MNCs). After removal of platelets by low-speed centrifugation (200 × g, 20 min) PBMCs/BM-MNCs were isolated by density gradient centrifugation using Ficoll-plaque (GE Healthcare, Diegem Belgium) and SepMate™ tubes (Stemcell Technologies, Vancouver, Canada). PBMCs and BM-MNCs were washed twice in cold PBS (Braun Melsungen) and resuspended in low glucose culture medium: RPMI 1640, no glucose culture medium (Life Technologies, Carlsbad, CA, USA) supplemented with gentamycin 50 μg/ml, pyruvate 1 mM, glutamine 2 mM, glucose 5 mM and HEPES 10 mM (Life Technologies). Before and after each isolation step, cells were counted using the Sysmex-XN 450 hematology analyzer (Sysmex, Norderstedt, Germany). 20–40 × 10⁶ cells were cryopreserved in CryoStor CS10 medium (Stemcell Technologies) until single-cell RNAseq analysis.
Classical monocytes (CD16⁻ CD14⁺) were isolated within the PBMC fraction by depletion of CD16⁺ cells using CD16 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by positive selection of CD14⁺ cells with CD14 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions.

On day 15 of differentiation, all cells in suspension were collected and washed once with FACS buffer (PBS, 0.5% BSA, 2 mM EDTA). Then, CD14+ cells were isolated using CD14 MicroBeads (Miltenyi Biotec) following the manufacturer's instructions; 60 μL of MicroBeads were used for 1 × 10⁷ total cells. Isolated CD14+ cells were cryopreserved in CryoStor CS10 medium (STEMCELL Technologies) or further differentiated into macrophages.
To isolate human Blood-mono, PBMCs were first isolated using Ficoll-Paque PLUS (GE Healthcare) from healthy donor blood. PBMCs were cryopreserved in CryoStor CS10 medium at a density of 20 million/mL. CD14+ monocytes were isolated from cryopreserved PBMCs using CD14 MicroBeads following the manufacturer's instructions.

DNA from fresh frozen biopsy samples, cryopreserved crypts and organoid specimens was extracted using AllPrep DNA/RNA Mini Kit (80204, Qiagen). Briefly, frozen biopsy samples were lysed on MagNA Lyser (Roche Diagnostics) (6000 rpm, twice for 15 s with a 15 s break) using Lysing Matrix D tubes (116913050-CF, MP Biomedicals) and 350 µl buffer RLT Plus. Cryopreserved pellets of crypts and organoids in CryoStor® CS10 medium (07930, StemCell Technologies) were gently thawed at + 4 °C and centrifuged for 5 min at 400 × g at + 4 °C. Supernatant was removed and pellets was lysed in 350 µl buffer RLT Plus. The following steps of DNA extraction from the lysates of biopsies, crypts and organoids were performed in line with the manufacturer’s instructions.