1 Review Spheroids formation on non-adhesive surfaces by Liquid Overlay Technique: considerations and practical approaches † Elisabete C. Costa 1 Duarte de Melo-Diogo 1 André F. Moreira 1 Marco P. Carvalho 1 Ilídio J. Correia 1 * 1 CICS-UBI - Health Sciences Research Centre, Universidade da Beira Interior, 6200-506 Covilhã, Portugal Correspondence: Ilídio J. Correia, CICS-UBI - Health Sciences Research Centre, Universidade da Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal E-mail: icorreia@ubi.pt Telephone: +351 275 329 055 Fax: +351 275 329 099 Keywords: 3D cell culture; High-throughput screening; Scaffold-free techniques; Liquid Overlay Technique; Tumor spheroids. Abbreviations: Embryonic stem cells: ESC, Extracellular matrix: ECM, high-throughput screening: HTS, hyaluronic acid: HA, induced pluripotent stem cells: iPSC, Liquid Overlay Technique: LOT, poly(2-hydroxyethyl methacrylate): poly-HEMA, mesenchymal stem cells: MSCs, normal human dermal fibroblasts: NHDF, polydimethylsiloxane: PDMS, reconstituted basement membrane: rBM, room temperature: RT, two-dimensional: 2D, three-dimensional: 3D. † This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/biot.201700417]. This article is protected by copyright. All rights reserved Received: June 9, 2017 / Revised: October 3, 2017 / Accepted: October 19, 2017