A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Qinggang Dai; Yujiao Han; Furong Xie; Xuhui Ma; Zhan Xu; Xiao Liu; Weiguo Zou; Jun Wang

doi:10.3791/56468

Un base di RANKL Osteoclast cultura Assay di midollo osseo del topo per studiare il ruolo di mTOR...

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Developmental Biology

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JoVE Journal Developmental Biology

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Un base di RANKL Osteoclast cultura Assay di midollo osseo del topo per studiare il ruolo di mTORC1 nella formazione degli osteoclasti

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09:37 min

March 15, 2018

DOI: 10.3791/56468-v

Qinggang Dai*¹, Yujiao Han*², Furong Xie*¹, Xuhui Ma³, Zhan Xu², Xiao Liu¹, Weiguo Zou², Jun Wang¹

¹Department of Pediatric Dentistry, Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology,National Clinical Research Center of Stomatology, ²State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology,University of Chinese Academy of Sciences, ³Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital,Shanghai Jiaotong University School of Medicine

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Overview

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, focusing on the role of mTORC1 in osteoclast formation. This method allows for the generation of a large number of giant osteoclasts within one week, aiding in the understanding of osteoclast differentiation.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Bone Biology

Background

Osteoclasts are essential for bone resorption.
The mammalian target of rapamycin complex 1 (mTORC1) plays a significant role in cell differentiation.
Understanding osteoclast formation is crucial for addressing osteoporosis.
This protocol provides insights into osteoclast biology.

Purpose of Study

To isolate and culture osteoclasts from mouse bone marrow.
To investigate the role of mTORC1 in osteoclast differentiation.
To develop a method that yields a high number of osteoclasts efficiently.

Methods Used

Isolation of bone marrow from mouse tibia.
Culturing of osteoclast precursors in vitro.
Assessment of mTORC1's role in differentiation.
Observation of osteoclast morphology and function.

Main Results

Successful isolation of osteoclasts from mouse bone marrow.
Demonstration of mTORC1's involvement in osteoclast formation.
Generation of a large number of giant osteoclasts within one week.
Insights into the mechanisms of osteoclast differentiation.

Conclusions

This protocol is effective for studying osteoclast biology.
Understanding mTORC1's role can inform osteoporosis therapies.
The method can be applied to further research in bone diseases.

Frequently Asked Questions

What are osteoclasts?

Osteoclasts are specialized cells responsible for bone resorption, playing a key role in bone remodeling.

Why is mTORC1 important in osteoclast formation?

mTORC1 regulates various cellular processes, including differentiation and growth, which are crucial for osteoclast development.

How long does it take to culture osteoclasts using this protocol?

The protocol allows for the generation of osteoclasts within one week.

What implications does this research have for osteoporosis?

Understanding osteoclast differentiation can lead to better therapeutic strategies for osteoporosis treatment.

Can this method be applied to other types of cells?

While this protocol is specific to osteoclasts, similar techniques may be adapted for other cell types.

Is the isolation of osteoclasts from mouse bone marrow ethical?

Yes, as long as it follows ethical guidelines for the use of animals in research.

Questo manoscritto descrive un protocollo a isolare e cultura gli osteoclasti in vitro da midollo osseo di topo e di studiare il ruolo della destinazione dei mammiferi/meccanicistica della rapamicina complesso 1 nella formazione degli osteoclasti.

L'obiettivo generale di questo protocollo è quello di isolare e coltivare osteoclasti in vitro da midollo osseo di topo e di studiare il ruolo del bersaglio mammifero del complesso rapamicina uno nella formazione degli osteoclasti. Questo metodo può aiutare a rispondere a domande chiave nella formazione degli osteoclasti, come il ruolo di mTORC1 nella differenziazione degli osteoclasti. Il vantaggio principale di questa tecnica è quello di produrre un gran numero di osteoclasti giganti entro una settimana.

Le implicazioni di questa tecnica si estendono alla terapia dell'osteoporosi perché è utile studiare il meccanismo con cui gli osteoclasti si differenziano dai loro precursori. Per iniziare questa procedura, praticare un'incisione verticale all'estremità distale della tibia di un topo soppresso e sezionare la pelle lungo gli arti posteriori. Quindi, taglia il legamento articolare delle articolazioni dell'anca con le forbici e disloca gli arti posteriori dal tronco.

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