Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Summary

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Abstract

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Introduction

Bone homeostasis is a complex physiological process that is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts¹. A balance between osteoblastic and osteoclastic activity mediated through osteoblasts and osteoclasts, respectively, is highly essential for maintaining bone health and homeostasis, because perturbations in bone homeostasis might lead to bone diseases, such as abnormal bone growth or loss of bone density. As unique bone-resorption cells, osteoclasts are important in diseases related to abnormal bone destruction, including osteoporosis, periodontitis, and periprosthetic osteolysis^2,....

Protocol

All the methods involving the animals described here are approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University of Chinese Medicine.

1. Setup

1. Prepare several longitudinally cut 1 mL pipette tips (1 cm) and several 1.5 mL microcentrifuge tubes. Put the pipette tips and microcentrifuge tubes at 103 kPa and 121 °C for 20 min and ensure that they are sterile.
2. Prepare a box of ice and several sterile dishes to preserve the isolated tissues during the isolation procedure.

2. Preparation of Culture Medium

1. Prepare the complete culture medium....

Results

The purpose of the protocol was to isolate and purify large numbers of osteoclast precursors conveniently and induce osteoclasts successfully. By supplementing with M-CSF and RANKL, giant osteoclasts were seen on days 5–6. The formation of osteoclasts was successfully identified by TRAP staining (Figure 1A). Large and purple cells were regarded as TRAP-positive cells with multiple nuclei (typically ≥ three nuclei). Through this method, it was typical to obtai.......

Discussion

The ability to obtain and study osteoclasts in vitro is a critical and fundamental skill for any researcher wishing to study bone metabolism, which may help understand the mechanisms of bone-absorbing diseases and develop novel therapeutic agents. The present study described a protocol with some modifications based on previous methods.

By using pipette tips and microcentrifuge tubes to obtain bone marrow, it largely reduced the operation time of obtaining bone marrow and the workload of labora.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acid Phosphatase, Lekocyte (TRAP) kit	Sigma-Aldrich	387A
Automatic Hard Tissue Slicer	Lecia	RM2265
Bovine femoral bone			Purchased by ourselves
Cell Incubator	Heraus	BB16/BB5060
Fetal Bovine Serum	Sarana	s-fbs-au-015
Goat Anti-Rabbit IgG H&L (Alexa Fluor 488)	Abcam	ab150077
Hard Tissue Grinders	Lecia	SP-2600
Histopaque Kit	TianJing Haoyang	TBD2013DR	Silicified centrifugal tube amd cell separation solution
Hochest33342	Sigma-Aldrich	B2261
Inverted Phase Contrast Microscope	Olympus	CKX31
Inverted Fluorescence Microscope	Lecia	DMI-3000
MEM, no Glutamine	Gibco	11090-081
Penicillin-Streptomycin, Liquid	Gibco	15140122
Rabbit Anti-Calcitonin receptor	Bioss	bs-0124R
Recombinant Rat M-CSF	PeproTech	400-28
Recombinant Rat sRANK Ligand	PeproTech	400-30
Red Blood Cell Lysis Buffer	Absin	abs47014932
Scanning Electron Microscopy	FEI	Quanta 200
Toluidine Blue	Sigma-Aldrich	89649