title: Culturing muscle fibres in hanging drop: a novel approach to solve an old problem.
abstract: BACKGROUND
  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.
authors.affiliation: Stem Cells and Regenerative Medicine Lab, Foundation Institute of Pediatic Research Città della Speranza, Padua, 35127, Italy; Department of Cytology, Faculty of Biology, University of Warsaw, Warsaw, 02-096, Poland.
authors.forename: Karolina
authors.initials: K
authors.lastname: Archacka
authors.forename: Michela
authors.initials: M
authors.lastname: Pozzobon
authors.forename: Andrea
authors.initials: A
authors.lastname: Repele
authors.forename: Carlo Alberto
authors.initials: CA
authors.lastname: Rossi
authors.forename: Michelangelo
authors.initials: M
authors.lastname: Campanella
authors.forename: Paolo
authors.initials: P
authors.lastname: De Coppi
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000378:metabolism; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology* / Q000378:metabolism
chemical_list: D017570:MyoD Protein; C530821:PAX7 protein, rat; D051761:Paired Box Transcription Factors
keywords: Clonal analysis; Hanging drop; Myofibre; Satellite cells; Skeletal muscle