Name: methods for analyzing the impacts of natural uranium on in vitro osteoclastogenesis
Uploaded: 2018-01-30T13:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis at JoVE.com

著者は、役に立つのテクニカル サポートにシャンタル Cro を感謝したいです。 この研究はからの補助金によって賄われていた、「兵站 à l'Energie Atomique et aux エネルギー代替"(URANOs - プログラム トランスバーサル ・ デ ・ Toxicologie ・ デュ ・ CEA と CPRR CEA アレバ)、ANR から (ウランの毒性: バイオミネラリゼーション マルチレベルのアプローチ骨、ANR-16-CE34-0003 で処理)。この作品は、CNRS ニース ・ ソフィア ・ アンティポリ大学によっても支えられました。

<ol>
	<li>Keith, S., Faroon, O., Roney, N., Scinicariello, F., Wilbur, S., Ingerman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+profile+for+uranium.">Toxicological profile for uranium.</a> Agency for Toxic Substances and Disease Registry. , (2013).</li><li>Ballou, J. E., Gies, R. A., Case, A. C., Haggard, D. L., Buschbom, R. L., Ryan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+and+early+disposition+of+inhaled+233UO2(NO3)2+and+232UO2(NO3)2+in+the+rat.">Deposition and early disposition of inhaled 233UO2(NO3)2 and 232UO2(NO3)2 in the rat.</a> Health Phys. 51 (6), 755-771 (1986).</li><li>Kathren, R. L., McInroy, J. F., Moore, R. H., Dietert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+in+the+tissues+of+an+occupationally+exposed+individual.">Uranium in the tissues of an occupationally exposed individual.</a> Health Phys. 57 (1), 17-21 (1989).</li><li>Kurttio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+effects+of+uranium+in+drinking+water.">Renal effects of uranium in drinking water.</a> Environ.Health Perspect. 110 (4), 337-342 (2002).</li><li>Leggett, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basis+for+the+ICRP's+age-specific+biokinetic+model+for+uranium.">Basis for the ICRP's age-specific biokinetic model for uranium.</a> Health Phys. 67 (6), 589-610 (1994).</li><li>Vidaud, C., Bourgeois, D., Meyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+as+target+organ+for+metals:+the+case+of+f-elements.">Bone as target organ for metals: the case of f-elements.</a> Chem. Res. Toxicol. 25 (6), 1161-1175 (2012).</li><li>Arzuaga, X., Gehlhaus, M., Strong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+action+associated+with+uranium+induced+adverse+effects+in+bone+function+and+development.">Modes of action associated with uranium induced adverse effects in bone function and development.</a> Toxicol. Lett. 236 (2), 123-130 (2015).</li><li>Ikeda, K., Takeshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+osteoclast+differentiation+and+function+in+skeletal+homeostasis.">The role of osteoclast differentiation and function in skeletal homeostasis.</a> J. Biochem. 159 (1), 1-8 (2016).</li><li>Teiltelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteoclast+and+its+unique+cytoskeleton.">The osteoclast and its unique cytoskeleton.</a> Ann N Y Acad Sci. 1240, 14-17 (2011).</li><li>Nesbitt, S. A., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+matrix+collagens+through+bone-resorbing+osteoclasts.">Trafficking of matrix collagens through bone-resorbing osteoclasts.</a> Science. 276 (5310), 266-269 (1997).</li><li>Salo, J., Lehenkari, P., Mulari, M., Metsikko, K., Vaananen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+osteoclast+bone+resorption+products+by+transcytosis.">Removal of osteoclast bone resorption products by transcytosis.</a> Science. 276 (5310), 270-273 (1997).</li><li>Pierrefite-Carle, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+natural+uranium+on+the+UMR-106+osteoblastic+cell+line:+impairment+of+the+autophagic+process+as+an+underlying+mechanism+of+uranium+toxicity.">Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.</a> Arch. Toxicol. 91 (4), 1903-1914 (2017).</li><li>Ubios, A. M., Guglielmotti, M. B., Steimetz, T., Cabrini, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+inhibits+bone+formation+in+physiologic+alveolar+bone+modeling+and+remodeling.">Uranium inhibits bone formation in physiologic alveolar bone modeling and remodeling.</a> Environ. Res. 54 (1), 17-23 (1991).</li><li>Bozal, C. B., Martinez, A. B., Cabrini, R. L., Ubios, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ethane-1-+hydroxy-1,1-bisphosphonate+(EHBP)+on+endochondral+ossification+lesions+induced+by+a+lethal+oral+dose+of+uranyl+nitrate.">Effect of ethane-1- hydroxy-1,1-bisphosphonate (EHBP) on endochondral ossification lesions induced by a lethal oral dose of uranyl nitrate.</a> Arch. Toxicol. 79 (8), 475-481 (2005).</li><li>Kurttio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+as+a+possible+target+of+chemical+toxicity+of+natural+uranium+in+drinking+water.">Bone as a possible target of chemical toxicity of natural uranium in drinking water.</a> Environ. Health Perspect. 113 (1), 68-72 (2005).</li><li>Collin-Osdoby, P., Osdoby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-mediated+osteoclast+formation+from+murine+RAW+264.7+cells.">RANKL-mediated osteoclast formation from murine RAW 264.7 cells.</a> Methods Mol. Biol. 816, 187-202 (2012).</li><li>Beranger, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+poly(ADP-Ribose)+polymerase-1+and+JunD/Fra2+accounts+for+RANKL-induced+Tcirg1+gene+expression+during+osteoclastogenesis.">Differential binding of poly(ADP-Ribose) polymerase-1 and JunD/Fra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesis.</a> J. Bone Miner. Res. 22 (7), 975-983 (2007).</li><li>Mirto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+uranium(VI)+speciation+for+the+evaluation+of+in+vitro+uranium+cytotoxicity+on+LLC-PK1+cells.">Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells.</a> Hum. Exp. Toxicol. 18 (3), 180-187 (1999).</li><li>Carri&#232;re, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+uranium+speciation+on+normal+rat+kidney+(NRK-52E)+proximal+cell+cytotoxicity.">Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.</a> Chem. Res. Toxicol. 17 (3), 446-452 (2004).</li><li>Milgram, S., Carri&#232;re, M., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+accumulation+and+distribution+of+uranium+and+lead+in+osteoblastic+cells+as+a+function+of+their+speciation.">Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.</a> Toxicology. 252 (1-3), 26-32 (2008).</li><li>Milgram, S., Carri&#232;re, M., Thiebault, C., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+and+phenotypic+effects+of+uranium+and+lead+on+osteoblastic+cells+are+highly+dependent+on+metal+speciation.">Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.</a> Toxicology. 250 (1), 62-69 (2008).</li><li>Studzinski, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Growth, Differentiation and Senescence A Practical Approach. , (1999).</li><li>Gritsaenko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+uranium+impairs+the+differentiation+and+the+resorbing+function+of+osteoclasts.">Natural uranium impairs the differentiation and the resorbing function of osteoclasts.</a> Biochim Biophys Acta. 1861 (4), 715-726 (2017).</li><li>Motiur Rahman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation-coupled+osteoclast+differentiation+by+RANKL:+Cell+density+as+a+determinant+of+osteoclast+formation.">Proliferation-coupled osteoclast differentiation by RANKL: Cell density as a determinant of osteoclast formation.</a> Bone. 81, 392-399 (2015).</li></ol>

著者がある何も開示するには

我々 の知る限り、これは天然ウランの骨細胞に及ぼす影響を研究することを目指して詳細な手順が記載されている最初の時間です。この方法は骨生理学へのウランの影響のより良い理解を達成するために役に立つでしょう、ウランのキレート剤のスクリーニングのため興味深い新しいツールを提供することがあります。さらに、ここで説明されているプロトコル可能性があります osteoclatogene...

ウランは天然に存在する放射性元素の土壌、空気、水の存在など、動物と人間は彼らの食事療法の自然なウランに公開されます。自然な源に加えてウランは環境の豊かさを高める人為的活動から起きる。ウランは化学と放射線の危険を引き起こします。ただしの場合 (これは同位体混合物含む 99.27 238U、0.72 235U、0.006 234U) 天然ウランは低特定活動 (25.103 Bq.g-1) があるのでその健康への影響に起因して、化学的毒性。
何その侵入経路 (吸入、摂取、または皮膚暴露)、ほとんど糞便と体内に侵入が排除されウランの小さい部分だけ全身循環に達する。血液中のウランの約 67% が腎臓によってフィルター処理順番と 24 h1尿中に肉体を離れます。残りは腎臓や骨、ウランの毒性2,3,4の 2 つの主なターゲット器官ほとんど堆積されています。探索するいくつかの研究が行われているスケルトンは、ウラン長期保持2,3,4,5,6のプライマリ サイトとして識別されている、ので、骨生理学7におよぼすウラン
骨は、その生涯を通じて継続的に改造は石灰化組織です。骨を改造する専門にされた細胞のタイプに依存し、ほとんどの 2 つのフェーズで構成されています複雑なプロセスである: 骨芽細胞による de novo 骨建設続いて既存の古い行列破骨細胞と骨吸収。破骨細胞は、8を骨に接続する吸収サイトに移行する原産の造血前駆細胞の融合から生じる大きな多核細胞です。愛着は、その骨格9の広汎な再編成と同時に発生します。この再編がセルとハイドロキシアパ タイトとの分解に関与するプロテアーゼの解散につながる陽子を分泌する破骨細胞を骨表面の間の分離されたコンパートメントの確立のために必要な有機マトリックス。結果として得られる劣化製品は貪食、骨表面の反対側膜領域にセルを経由、分泌、トランスサイトーシス10,11と呼ばれるプロセス。
体内と体外の調査からの結果を示すウランが骨形成を阻害して数と骨芽細胞7,12の活動を変化させます。対照的に、骨と破骨細胞に及ぼすウランは不十分な検討しています。いくつかの生体内での研究は、マウスやラット13,14硝酸ウラニル投与後骨吸収の強化を報告しています。さらに、疫学的調査は、飲料水経由でウラン摂取量の増加が男性15の骨吸収マーカーの血清レベルの増加に関連付けられている傾向があることを提案しました。一緒に取られて、これらの調査結果は、ウランは、骨に蓄積することが骨吸収を促進する結論に至った。しかし、ウランのこの潜在的な影響に関与する細胞のメカニズムは、未解決の問題を残る。このため、骨細胞を吸収に及ぼすウランの影響を調べることにしました
ここでは、設けてプロトコルについて述べるを特徴付ける、前破骨細胞の生存率と破骨細胞分化と吸収活性天然ウランの影響を定量化します。未加工 264.7 変換されたマクロファージ細胞ライン、これは容易に破 4 または 5 日間のサイトカイン RANKL の存在下で培養した場合に区別できるし、古典的な研究に用いられる実験記載が行われています。破骨細胞の分化および機能の16。開発手順は、信頼性の高い、再現性の高い結果とは完全に主要な破骨細胞を対象に与えます。これらすべての理由から、この方法論が骨におけるウランの毒性に関与する分子メカニズムのより良い理解を得ることに有用であると考えています。さらに、このアプローチは新しいウランのキレート剤を特定するためのスクリーニング ツールとして適応できると考えています。

<table><tbody><tr><td>DMEM</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Lonza</td><td>BE12-169F</td><td></td></tr><tr><td>EMEM without phenol red</td><td>Lonza</td><td>12-668E</td><td></td></tr><tr><td>Water for cell culture</td><td>Lonza</td><td>BE17-724F</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>&amp;nbsp;L-Glutamine solution</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Trypan Blue Solution 0.4%</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>HyClone fetal bovine serum</td><td>GE Life Sciences</td><td>SH30071.03</td><td></td></tr><tr><td>7.5% sodium bicarbonate aqueous solution</td><td>Sigma-Aldrich</td><td>S8761</td><td></td></tr><tr><td>Acid Phosphatase, Lekocyte (TRAP) kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT) powder</td><td>Sigma-Aldrich</td><td>M5655</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Alizarin Red S sodium salt, 1% w/v aq. sol.</td><td>Alfa Aeros</td><td>42746</td><td></td></tr><tr><td>Osteoassay bone resorption plates, 24 well plates</td><td>Corning Life Sciences</td><td>3987</td><td></td></tr><tr><td>Multiwell 24 well plates</td><td>Falcon</td><td>353504</td><td></td></tr><tr><td>Flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Falcon</td><td>353133</td><td></td></tr><tr><td>Polypropylene Conical Tubes 50 ml</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Cell scrapers 30 cm</td><td>TPP</td><td>90003</td><td></td></tr></tbody></table>

methods for analyzing the impacts of natural uranium on in vitro osteoclastogenesis

骨生理学と干渉するウランが示されているし、この金属が骨に蓄積することは周知します。しかし、少しは天然ウランの骨細胞の挙動に及ぼす影響について知られています。特に、破骨細胞、細胞の骨基質の吸収のための責任のウランの影響は記載されていません。この問題を調べるためには、破骨細胞前駆体のモデルとして天然ウランのソースとマウスの未加工 264.7 のセル行酢酸ウランを使用して新しいプロトコルを定めています。ここで、破骨細胞の前駆物質のウランの細胞毒性をテストして、破骨細胞形成と破骨細胞の吸収機能にその影響を評価するために必要なすべてのアッセイを詳しく説明します。条件を開発した、特にウラン含有培地の調製および未加工 264.7 の播種細胞を信頼性が高く、高度生殖の結果を取得する許可。さらに、破骨細胞のサイズや堤吸収症マトリックスの割合など様々 なパラメーターの解析を容易にするソフトウェア ツールの使用を最適化します。

酒石酸抵抗性酸性フォスファターゼ染色は、3 つ以上の核を持つ紫色の大型の細胞と破骨細胞を可視化する使用されました。RANKL の存在下で培養した未加工 264.7 のセルから取得した破骨細胞の代表的な画像とウラニル イオンは図 1に示します。ウランへの応答で破骨細胞の数とサイズの変更は、全体の井戸の複合画像と拡大写真で簡単に表示さ?...

Watch this Scientific Journal Video about Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis at JoVE.com

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

ウランは、骨代謝に影響を与える知られています。ここでは、担当骨細胞破骨細胞の機能や分化、生存率に及ぼす天然ウランの影響の調査を目的としたプロトコルを提案する.

培養破骨細胞形成に天然ウランの影響を分析するための方法

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

methods-for-analyzing-impacts-natural-uranium-on-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

8.1K ビュー.  Université Nice Sophia-Antipolis. ウランは、骨代謝に影響を与える知られています。ここでは、担当骨細胞破骨細胞の機能や分化、生存率に及ぼす天然ウランの影響の調査を目的としたプロトコルを提案する.

ビデオ: Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

ウランの酸化銅ナノ粒子による微量元素の除去<em&gt;その場での</em&gt;回復ブリード水と細胞生存率への影響

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

環境学

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

破骨細胞形成乳房癌細胞と共培養末梢血単球からの人間の前臨床モデルの開発

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

がん研究

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

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL による破骨細胞文化アッセイのマウス骨髄破骨細胞形成における mTORC1 の役割を調査するには

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

発生生物学

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

小説<em&gt; in vivoで</em&gt;遺伝子導入技術と<em&gt; in vitroで</em筋骨格障害における骨損失の検討のための&gt;細胞ベースのアッセイ

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

医学

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

破骨細胞形成と培養破骨細胞前駆体の増殖に及ぼす抗 c-fms 抗体

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

免疫・感染学

Osteoclast Derivation from Mouse Bone Marrow

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.

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.

マウス骨髄からの破骨細胞の導出

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

生物学

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

インビトロで破骨細胞再吸収を可視化および定量するためのシンプルなピットアッセイプロトコル

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

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

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.

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.

ラット骨髄からの破骨細胞前駆体の分離・精製・分化

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Source: Cen, W., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.</a> J. Vis. Exp. (2022)
In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.

Resorption Pit Assay:リン酸カルシウムコーティング培養プレートを使用して成熟破骨細胞のカルシウム吸収活性を定量化するIn Vitro技術

Source: Cen, W., et al. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (2022)
In this video, we ...

JoVE Encyclopedia of Experiments

生物学的手法

アッセイ技術

細胞ベースのアッセイ

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

Source: Marahleh, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59089">Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the effect of different anti-c-fms antibody concentrations on osteoclastogenesis, which is the process of osteoclast formation from osteoclast progenitors.

In vitroでのマウス骨髄細胞の破骨細胞形成に対する抗C-fms抗体の効果

Source: Marahleh, A., et al. Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro. J. Vis. Exp. 
...