Osteoclast Derivation from Mouse Bone Marrow

Summary

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Abstract

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.

As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.

An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases.

Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Introduction

Bone remodeling is dynamic and involves the coupling of bone formation with bone resorption¹. This tightly regulated process is responsible for maintaining the skeleton during normal homeostasis, and in response to injury and disease.

Osteoclasts are unique, multinucleated cells that are capable of resorbing both the organic and inorganic matrices of bone. Osteoclasts are derived from the monocyte/macrophage lineage of the bone marrow^2-5. Abnormalities in the function or formation of osteoclasts can result in a variety of clinical pathologies, including common conditions like osteoporosis.

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Protocol

NOTE: Ethical statement: All research involving vertebrate animals was performed in accordance protocols approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).

1. Preparation

1. Allow 10 ml of commercially available density gradient cell separation media (which contains polysucrose and sodium diatrizoate, adjusted to a density of 1.077 g/ml) to come to RT in a 50 ml conical tube.
2. Prepare flow cytometry (FACS) buffer with 1x phosphate buffered saline (PBS) and 2% fetal calf serum (FCS). Take a 50 ml aliquot and maintain this aliquot at RT for use during the density gradient cell separation media step.
3. Have a....

Results

The aim of this method was to easily isolate large numbers of osteoclasts in vitro, typically in one week. Successful isolation of large numbers of osteoclasts was confirmed using tartrate-resistant acid phosphatase staining (Figure 1A). Large osteoclasts are visualized as large purple cells with multiple nuclei (typically ≥ 3 nuclei). Using this protocol, it is common to isolate osteoclasts with as many as 30 nuclei per osteoclast (Figure 1B).

Using .......

Discussion

An ability to easily isolate and cultivate large numbers of osteoclasts in vitro has been responsible for helping to advance understanding of bone biology and osteoclast-mediated diseases. It was the identification of RANKL that lead to this, when it was recently identified as the major regulator of osteoclast formation, differentiation and survival^16-18.

It has been our experience that the in vitro cultivation of osteoclasts from bone marrow is largely dependent o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEM, no glutamine, no phenol red	Gibco	51200-038
M-CSF, recombinant mouse	Gibco	PMC2044
Recombinant Mouse TRANCE/RANK L/TNFSF11 (E. coli expressed)	R&D Systems	462-TEC-010
Prostaglandin E2	Sigma-Aldrich
Histopaque-1077	Sigma-Aldrich	10771
Acid Phosphatase, Lekocyte (TRAP) kit	Sigma-Aldrich	387A
Osteoassay bone resorption plates, 24 well plates	Corning Life Sciences	3987