Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

Summary

This protocol describes the dissection of neonatal dmp1-topaz mouse calvaria and isolation of osteocytes expressing the green fluorescent protein through cell digestion and fractionation, in addition to osteocyte preparation for fluorescence activated cell sorting (FACS).

Abstract

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the spotlight and has been shown to have multiple major functions like actively modifying the extracellular matrix and forming an endocrine organ with the lacunocanalicular system that encloses it sending messages to distant sites. Owing to the methods that made it possible to test the osteocyte in vitro from isolating primary osteocytes to osteocyte-like cell lines, osteocytes are now experiencing a resounding interest and a surge of knowledge on structure and function. Many aspects of the osteocyte biology and interaction with other molecular components are yet to be discovered. In this protocol, we describe in detail the efficient isolation of primary osteocytes from dmp1-topaz neonatal mouse calvaria, which express the green fluorescent protein in osteocytes, through cell fractionation and subsequently acquiring cultures of primary osteocytes by FACS.

Introduction

Osteocytes are terminally differentiated cells from osteoblastic progenitors that became embedded in their secreted matrix¹. They are the most abundant and longest-living cells among bone cell populations. They reside within lacunae and have a characteristic stellate morphology with dendrites that extend through channels called canaliculi forming an extensive network of communication and metabolic exchange with their surrounding environment and the bone surface². Osteocytes choreograph both osteoblasts and osteoclasts roles in bone remodeling, they are the primary mechanosensory cells conferring adaptation to mechanical ....

Protocol

All animal procedures and animal care were performed in accordance with Tohoku University rules and regulations.

1. Dissection of newborn dmp1-topaz mouse calvaria

1. For this protocol, use 6‒7 day old pups of C57BL/6-Tg(Dmp1-Topaz)1Ikal/J mice. Euthanize mice with 5% isoflurane inhalation and then transfer the pups into 70% ethanol.
2. Transfer the euthanized pup into a non-treated culture dish.
3. Using scissors and tweezers, grab the skin at the base of the skull and make an incision.
4. Using the first incision as a starting point, cut on both lateral sides of the skull in front of the ears, remove th....

Results

The purpose of this protocol is to demonstrate the process of obtaining cultures of primary osteocytes from dmp1-topaz neonatal mouse calvaria through a fractionation process using collagenase to degrade the collagen matrix and EDTA for calcium chelation, after which cells are prepared for FACS to separate osteocytes from other cell populations.

Methods for obtaining primary osteocytes from neonatal mouse calvaria often describe the use of fractions (1‒8) for sorting²³........

Discussion

The first isolated osteocyte was from a chicken calvaria⁷ isolated by using (OB7.3) or the aviant variant of PHEX; however, this method is limited by the availability of workable antibodies, as osteocyte-specific antibodies that are also specie-specific have to be manufactured. Researchers used a different modification of the sequential enzymatic process to obtain osteocytes from mouse and rat long bones; the reported purity of these cultures were set at about 70%⁹. The dev.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD FACSDiva software	BD Biosciences		Data aquisition and analysis
BD Falcon Tube	BD Biosciences	352235	12 x 75 mm Tube with Cell Strainer Cap, 35 μm nylon mesh.
Bovine serum albumin (BSA)	Sigma-Aldrich, MO, USA
Collagenase	Wako, Osaka, Japan	034-22363	0.2% (w/v), crude collagenase mix sourced from C. histolyticum.
EDTA	Dojindo, Kumamoto, Japan		5mM EDTA prepared with 0.1% BSA
FACSAriaTM II	BD Biosciences
Fetal bovine serum (FBS)	Biowest, Nuaillé, France
Isolation buffer			70mM NaCl, 10mM NaHCO, 60mM sorbitol, 3mM K2HPO4, 1mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES
Millex Sterile Filter Unit	Merck Millipore, Ireland	SLGV033RS	0.22μm
Nylon cell strainer	FALCON, NY, USA		40μm
Trypsin-EDTA	Life Technologies, NY, USA		0.5% x10. diluted to x1 in PBS
α-MEM	Wako, Osaka, Japan		Containing 10% fetal bovine serum, 100 IU/ml penicillin G, and 100 μg/ml streptomycin