X-VIVOTM 10 Serum-free Hematopoietic Cell Medium

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	Seed 2 × 105 thymic epithelial cells in collagen type I scaffolds

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Seed 2 × 105 thymic epithelial cells in collagen type I scaffolds

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TEC Isolation, Enrichment, and Culture: Thymus was isolated from 4‐ to 6‐week‐old WT mice. Thymus was cleaned up from fat and stromal tissues under the microscope and then digested at 37°C (three steps of 5 minutes) with a solution containing Liberase (Roche, Germany) and DNAse I (Roche). Digested thymus was recovered in Iscove medium (Sigma–Aldrich, Germany) supplemented with 10% fetal bovine serum (FBS; Euroclone, Italy), 1% penicillin/streptomycin (Lonza, Switzerland), and 2% glutamine (Lonza). TECs were further enriched by depleting CD45+ cells with the AutoMACS Separator (Miltenyi Biotec, Germany) after incubating thymic cells with anti‐CD45 microbeads (Miltenyi Biotec). The purity of enriched TECs (CD45‐EpCam+) was then checked by multicolor flow cytometric analyses. Enriched TECs were cultured in CnT‐57.S (CELLnTEC Advanced Cell Systems, Switzerland) supplemented with Rho‐associated, coiled‐coil containing protein kinase (ROCK) inhibitor 1 μg/ml (Y‐27632, Merck Millipore) for 7–9 days after isolation; afterward, culture medium was switched to X‐VIVO10 (Lonza) supplemented with the ROCK inhibitor. Scaffolds Seeding and In vivo Engraftment: In the experiments showed in the present article, 2 × 105 TECs were cultured in scaffolds, unless otherwise stated. For in vivo experiments, mice were anesthetized by injecting intraperitoneally 0.5 ml of avertin 240 mg/kg (Sigma–Aldrich) and scaffolds were transplanted subcutaneously (s.c.) in the inguinal area or under the kidney capsule of CD1 athymic nude mice. Wounds were sealed with silk suture (Ethicon) and reinforced by metal clips (CellPoint Scientific, Gaithersburg, MD).

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