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Reproducible, scalable, and cost effective fabrication and versatile characterization of tissue spheroids (TS) is highly demanded by 3D bioprinting and drug discovery. Consistent geometry, defined mechanical properties, optimal viability, appropriate extracellular matrix/cell organization are required for cell aggregates aimed for application in these fields. A straightforward procedure for fabrication and systematic multiparametric characterization of TS with defined properties and uniform predictable geometry employing non-adhesive technology is suggested. Applying immortalized and primary cells, the reproducibility of spheroid generation, the strong correlation of ultimate spheroid diameter, and growth pattern with cell type and initial seeding concentration are demonstrated. Spheroids viability and mechanical properties are governed by cell derivation. In this study, a new decision procedure to apply for any cell type one starts to work with to prepare and typify TS meeting high quality standards in biofabrication and drug discovery is suggested., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)