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Scaffold-free in vitro organogenesis exploits the innate ability of cells to synthesise and deposit their own extracellular matrix to fabricate tissue-like assemblies. Unfortunately, cell-assembled tissue engineered concepts require prolonged ex vivo culture periods of very high cell numbers for the development of a borderline three-dimensional implantable device, which are associated with phenotypic drift and high manufacturing costs, thus, hindering their clinical translation and commercialisation. Herein, we report the accelerated (10 days) development of a truly three-dimensional (338.1 ± 42.9 μm) scaffold-free tissue equivalent that promotes fast wound healing and induces formation of neotissue composed of mature collagen fibres, using human adipose derived stem cells seeded at only 50,000 cells/cm 2 on an poly (N-isopropylacrylamide-co-N-tert-butylacrylamide (PNIPAM86-NTBA14) temperature-responsive electrospun scaffold and grown under macromolecular crowding conditions (50 μg/ml carrageenan). Our data pave the path for a new era in scaffold-free regenerative medicine., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In vivo, the interior and exterior of cells is populated by various macromolecules that create an extremely crowded milieu. Yet again, in vitro eukaryotic cell culture is conducted in dilute culture media that hardly imitate the native tissue density. Herein, the concept of macromolecular crowding is discussed in both intracellular and extracellular context. Particular emphasis is given on how the physicochemical properties of the crowding molecules govern and determine kinetics, equilibria and mechanism of action of biochemical and biological reactions, processes and functions. It is evidenced that we are still at the beginning of appreciating, let alone effectively implementing, the potential of macromolecular crowding in permanently differentiated and stem cell culture systems., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)