title: Application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts.
abstract: The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Deborah
authors.initials: D
authors.lastname: Mattinzoli
authors.affiliation: Department of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Piergiorgio
authors.initials: P
authors.lastname: Messa
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Corbelli
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Masami
authors.initials: M
authors.lastname: Ikehata
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Anna
authors.initials: A
authors.lastname: Mondini
authors.affiliation: Renal Physiopathology Laboratory, Department of Medical, Surgical and Health Sciences, University of Trieste.
authors.forename: Cristina
authors.initials: C
authors.lastname: Zennaro
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Silvia
authors.initials: S
authors.lastname: Armelloni
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.
authors.forename: Laura
authors.initials: L
authors.lastname: Giardino
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico; mariapia.rastaldi@policlinico.mi.it.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Rastaldi
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects*; D010011:Osteocytes / Q000166:cytology* / Q000187:drug effects*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D014212:Tretinoin