Application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

Deborah Mattinzoli, Piergiorgio Messa, Alessandro Corbelli, Masami Ikehata, Anna Mondini, Cristina Zennaro, Silvia Armelloni, Min Li, Laura Giardino, Maria P ia Rastaldi

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6 Citations (Scopus)

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.

Original languageEnglish
JournalJournal of visualized experiments : JoVE
Issue number87
DOIs
Publication statusPublished - 2014

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Osteocytes
Osteoblasts
Tretinoin
Cell culture
Cells
Acids
Monitoring
Bone
Microscopes
Molecules
Extracellular Matrix
Dendrites
Skull
Population
Up-Regulation
Down-Regulation
Cell Count
Research Personnel
Bone and Bones
Cell Line

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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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.",
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AU - Mattinzoli, Deborah

AU - Messa, Piergiorgio

AU - Corbelli, Alessandro

AU - Ikehata, Masami

AU - Mondini, Anna

AU - Zennaro, Cristina

AU - Armelloni, Silvia

AU - Li, Min

AU - Giardino, Laura

AU - Rastaldi, Maria P ia

PY - 2014

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