Biol. Cell (2014) 106, 1–11 DOI: 10.1111/boc.201300028 Research article Culturing muscle fibres in hanging drop: A novel approach to solve an old problem Karolina Archacka*† 1 , Michela Pozzobon* 1 , Andrea Repele*‡§, Carlo Alberto Rossi‖, Michelangelo Campanella,§#** and Paolo De Coppi*‡ 2 *Stem Cells and Regenerative Medicine Lab, Foundation Institute of Pediatic Research Citt ` a della Speranza, Padua 35127, Italy, †Department of Cytology, Faculty of Biology, University of Warsaw, Warsaw 02-096, Poland, ‡Surgery Unit, Institute of Child Health and Great Ormond Street Hospital, University College London, London WC1N 1EH, United Kingdom, §Royal Veterinary College, University of London, Royal College Street London NW1 0T, United Kingdom, ‖Department of Pediatrics, University of Padua, Padua 35100, Italy, #University College London, Consortium for Mitochondrial Research (CfMR), NW1 0TU, London, United Kingdom, and **Metabolism in Brain Diseases, EBRI-European Brain Research Institute, Rita Levi Montalcini Foundation, 64 00143, Rome, Italy Background Information. The satellite cells (SCs) associated with muscle fibres play a key role in postnatal growth and regeneration of skeletal muscle. Commonly used methods of isolation and in vitro culture of SCs lead to the mixture of their subpopulations that exist within muscle. To solve this problem, we used the well established technique, the hanging drop system, to culture SCs in a three-dimensional environment and thus, to monitor them in their original niche. Results. Using hanging drop technique, we were able to culture SCs associated with the fibre at least for 9 days with one transfer of fibres to the fresh drops. In comparison, in the classical method of myofibres culture, that is, on the dishes coated with Matrigel, SCs leave the fibres within 3 days after the isolation. Cells cultured in both systems differed in expression of Pax7 and MyoD. While almost all cells cultured in adhesion system expressed MyoD before the fifth day of the culture, the majority of SCs cultured in hanging drop still maintained expression of Pax7 and were not characterised by the presence of MyoD. Among the cells cultured with single myofibre for up to 9 days, we identified two different subclones of SCs: low proliferative clone and high proliferative clone, which differed in proliferation rate and membrane potential. Conclusions. The hanging drop enables the myofibres to be kept in suspension for at least 9 days, and thus, allows SCs and their niche to interact each other for prolonged time. In a consequence, SCs cultured in hanging drop maintain expression of Pax7 while those cultured in a traditional adhesion culture, that is, devoid of signals from the original niche, activate and preferentially undergo differentiation as manifested by expression of MyoD. Thus, the innovative method of SCs culturing in the hanging drop system may serve as a useful tool to study the fate of different subpopulations of these cells in their anatomical location and to determine reciprocal interactions between them and their niche.  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (email p.decoppi@ucl.ac.uk) Key words: Clonal analysis, Hanging drop, Myofibre, Satellite cells, Skeletal muscle. Abbreviations used: FDB, flexor digitorum brevis; HPCs, high proliferative clones; HS, horse serum; LPCs, low proliferative clones; MPCs, muscle precursor Introduction The satellite cells (SCs) are mononucleated cells that were described over 50 years ago as the cells residing cells; SCs, satellite cells; TMRM, tetramethyl rhodamine methyl-ester;  m , mitochondrial membrane potential. C  2013 Soci ´ et ´ e Franc ¸ aise des Microscopies and Soci ´ et ´ e de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd 1