本研究の一部は、日本学術振興会のJSPS科研費(日本学術振興会)の助成を受けて行われました(第19K10397号、第18K09862号)。

すべての動物の手順と動物の世話は、東北大学の規則と規則に従って行われました。
1. 新生児dmp1-トパーズマウス頭蓋冠の解剖
<ol><li>このプロトコルでは、C57BL/6-Tg(Dmp1-トパーズ)1Ikal/Jマウスの6〜6日齢の子犬を使用します。5%イソフルラン吸入でマウスを安楽死させ、次に子犬を70%エタノールに移します。</li>
	<li>安楽死させた子犬を未処理の培養皿に移します。</li>
	<li>はさみとピンセットを使用して、頭蓋骨の付け根の皮膚をつかみ、切開します。</li>
	<li>最初の切開を出発点として、耳の前の頭蓋骨の両側を切り取り、皮膚を取り除き、頭蓋冠を露出させます。</li>
	<li>鼻梁から頭を持ち、ラムドイド縫合糸に沿って頭蓋冠を切ります。頭頂骨の外側の縁に沿って切り、前部を含むように伸ばします。</li>
	<li>下にある脳組織から頭蓋冠を分離します。 注意: 軟部組織の付着を最小限に抑えたきれいな骨の頭蓋冠を確保するために、骨の奥深くまで切り込まないでください。参考までに、骨の下のハサミの影が見えるはずです。</li>
	<li>頭蓋冠をリン酸緩衝生理食塩水(PBS、プロトコル全体でpH 7.4)に移します。凹面部分が上を向いていることを確認してください。</li>
</ol>2. 新生マウス頭蓋冠の分画
<ol><li>5 mLの2 mg/mLコラゲナーゼ溶液を含む50 mLのコニカルチューブに最大5つの頭蓋骨を移します。使用直前に調製した新鮮なコラゲナーゼ溶液を使用してください。<ol><li>新鮮なコラゲナーゼ溶液を調製するには、コラゲナーゼ粉末を単離バッファー(70 mM NaCl、10 mM NaHCO 3、60 mM ソルビトール、3 mM K 2 HPO4、1 mM CaCl 2、1 mg/mLウシ血清アルブミン(BSA)、5 mg/mLグルコース、25 mM HEPES)に2mg/mLで添加します。コラゲナーゼ粉末をマグネチックスターラーで溶解する。</li>
		<li>コラゲナーゼ溶液を0.22 μmの滅菌フィルターユニットに通し、50 mLチューブに入れ、プロトコル全体を通して氷上に保ちます。</li>
	</ol></li>
	<li>カルバリアを37°Cで300rpmの振とう機上で20分間インキュベートし、画分1を得た。</li>
	<li>画分1の消化物を廃棄し、5 mLのリン酸緩衝生理食塩水(PBS)で頭蓋冠を洗浄し、洗浄液を廃棄します。.</li>
	<li>頭蓋冠を5 mLの5 mLの5 mMエチレンジアミン四酢酸(EDTA)溶液に含み、PBS、pH 7.4のPBS溶液、pH 7.4、37°Cで300 rpmのシェーカーで15分間インキュベートします。</li>
	<li>ダイジェストを50 mLのコニカルチューブに集めます。頭蓋冠を5 mLのPBSで洗浄し、洗浄液をダイジェストに加えます。 注:消化物を収集して洗浄する各ポイントで、溶液中の骨をピペットで移動して、細胞を骨から切り離し、ダブレットと細胞凝集を防ぎます。</li>
	<li>画分2を得るために、消化物を300 x g で4°Cで5分間遠心分離します。上清を廃棄し、10%ウシ胎児血清(FBS)、100 IU/mLペニシリンG、および100 μg/mLストレプトマイシンを含む8 mL αの最小必須培地(MEM)にペレットを再懸濁します。10 cmの培養皿に種をまきます。画分2を5%CO2下で37°Cでインキュベートします。</li>
	<li>頭蓋骨を5 mLのコラゲナーゼ溶液中で37°C、300 rpmのシェーカーで20分間インキュベートします。</li>
	<li>分数3を収集するには、手順2.5〜6を繰り返します。画分3を5%CO2下で37°Cでインキュベートします。 注:この時点で、骨は小さくなり、断片に縮小されます。骨が誤って消化物に吸引されてプロセス中の細胞損失につながることがないようにするには、消化物を収集するための小さなチップピペット(1000〜200μLのピペットチップ)を使用します。</li>
	<li>頭蓋骨を5 mLのコラゲナーゼ溶液中で37°C、300 rpmのシェーカーで20分間インキュベートします。</li>
	<li>分数4を収集するには、手順2.5〜6を繰り返します。画分4を5%CO2下で37°Cでインキュベートする。</li>
	<li>頭蓋冠を5 mLの5 mL BSAを含む5 mL の PBS (pH 7.4) 溶液 PBS (pH 7.4) 中、37 °C でシェーカー上で 300 rpm のシェーカーで 15 分間インキュベートします。</li>
	<li>分数5を収集するには、手順2.5〜6を繰り返します。画分5を5%CO2下で37°Cでインキュベートする。骨細胞は、同じ日に選別できるように準備することも、選別前に24時間まで培養することもできます。</li>
</ol>3. 蛍光活性化細胞ソーティングのための骨細胞の調製
<ol><li>各細胞画分の培地を吸引し、10 mLのPBSで2回穏やかに洗浄します。</li>
	<li>PBSに5 mLの0.5%トリプシン-EDTAを加え、細胞を37°Cで5分間インキュベートします。 5 mLの10%FBS α-MEMとピペットを加えて、細胞を穏やかに剥離します。40 μmのセルストレーナーを通して50 mLの円錐形遠心管にふるいにかけることにより、各画分の細胞を結合します。</li>
	<li>細胞を10 mLのPBSで洗浄し、40 μmのセルストレーナーを通して50 mLのコニカル遠心管にふるいにかけて回収します。細胞を300 x g で4°Cで5分間遠心分離します。</li>
	<li>上清を吸引し、ペレットに10%FBS α-MEMを加えます。細胞濃度を約1 x 107 細胞/mLに調整します。</li>
	<li>35 μmナイロンメッシュキャップ付きチューブで細胞をろ過します。これで、セルはFACSの準備が整いました。</li>
	<li>このプロトコルでは、100 μmサイズのノズルを使用します。収集液は10%FBS α-MEMで構成されている必要があります。可能であれば、攪拌中および仕分け全体を通して4°Cでサンプルを選別してください。</li>
	<li>ソートの前に、側方散乱(SSC)領域と前方散乱(FSC)領域を調整して、アーティファクトと細胞を除去するようにゲーティングを最適化します。SSC幅とSSC高さ、およびFSC面積とFSC幅を調整してダブレットを排除します。GFP陰性骨細胞は、ソート機器のパラメータを調整するためのコントロールとして使用する必要があります。</li>
	<li>細胞回収が完了したら、細胞を300 x g 、4°Cで10分間遠心分離します。PBSで細胞を洗浄します。細胞を300 x g で4°Cで5分間遠心分離します。</li>
	<li>細胞を再懸濁し、10%FBS α-MEMを用いて所望の数を調整する。</li>
</ol>

<ol>
	<li>Manolagas, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+and+death+of+bone+cells:+basic+regulatory+mechanisms+and+implications+for+the+pathogenesis+and+treatment+of+osteoporosis.">Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.</a> Endocrine Reviews. 21 (2), 115-137 (2000).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocytes+as+Dynamic+Multifunctional+Cells.">Osteocytes as Dynamic Multifunctional Cells.</a> Annals of the New York Academy of Sciences. 1116 (1), 281-290 (2007).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensation+and+Transduction+in+Osteocytes.">Mechanosensation and Transduction in Osteocytes.</a> BoneKEy osteovision. 3 (10), 7-15 (2006).</li><li>Feng, J. Q., 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=Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism.">Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.</a> Nature Genetics. 38 (11), 1310-1315 (2006).</li><li>Lane, N. 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=Glucocorticoid-Treated+Mice+Have+Localized+Changes+in+Trabecular+Bone+Material+Properties+and+Osteocyte+Lacunar+Size+That+Are+Not+Observed+in+Placebo-Treated+or+Estrogen-Deficient+Mice.">Glucocorticoid-Treated Mice Have Localized Changes in Trabecular Bone Material Properties and Osteocyte Lacunar Size That Are Not Observed in Placebo-Treated or Estrogen-Deficient Mice.</a> Journal of Bone and Mineral Research. 21 (3), 466-476 (2005).</li><li>Dallas, S. L., Prideaux, M., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Osteocyte:+An+Endocrine+Cell+...+and+More.">The Osteocyte: An Endocrine Cell ... and More.</a> Endocrine Reviews. 34 (5), 658-690 (2013).</li><li>van der Plas, A., Nijweide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+osteocytes.">Isolation and purification of osteocytes.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 7 (4), 389-396 (1992).</li><li>Westbroek, I., De Rooij, K. E., Nijweide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte-specific+monoclonal+antibody+MAb+OB7.3+is+directed+against+Phex+protein.">Osteocyte-specific monoclonal antibody MAb OB7.3 is directed against Phex protein.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 17 (5), 845-853 (2002).</li><li>Gu, G., Nars, M., Hentunen, T. A., Metsikk&#246;, K., V&#228;&#228;n&#228;nen, 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=Isolated+primary+osteocytes+express+functional+gap+junctions+in+vitro.">Isolated primary osteocytes express functional gap junctions in vitro.</a> Cell and Tissue Research. 323 (2), 263-271 (2006).</li><li>Kramer, I., 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=Osteocyte+Wnt/&#946;-Catenin+Signaling+Is+Required+for+Normal+Bone+Homeostasis.">Osteocyte Wnt/&#946;-Catenin Signaling Is Required for Normal Bone Homeostasis.</a> Molecular and Cellular Biology. 30 (12), 3071-3085 (2010).</li><li>Kato, Y., Windle, J. J., Koop, B. A., Mundy, G. R., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+Osteocyte-like+Cell+Line,+MLO-Y4.">Establishment of an Osteocyte-like Cell Line, MLO-Y4.</a> Journal of Bone and Mineral Research. 12 (12), 2014-2023 (1997).</li><li>Kato, Y., 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=Establishment+of+an+osteoid+preosteocyte-like+cell+MLO-A5+that+spontaneously+mineralizes+in+culture.">Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 16 (9), 1622-1633 (2001).</li><li>Zhang, C., Bakker, A. D., Klein-Nulend, J., Bravenboer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Osteocytes+in+Their+3D+Native+Matrix+Versus+2D+In+Vitro+Models.">Studies on Osteocytes in Their 3D Native Matrix Versus 2D In Vitro Models.</a> Current Osteoporosis Reports. 17 (4), 207-216 (2019).</li><li>Woo, S. M., Rosser, J., Dusevich, V., Kalajzic, I., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+IDG-SW3+replicates+osteoblast-to-late-osteocyte+differentiation+in+vitro+and+accelerates+bone+formation+in+vivo.">Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 26 (11), 2634-2646 (2011).</li><li>Spatz, J. 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=The+Wnt+Inhibitor+Sclerostin+Is+Up-regulated+by+Mechanical+Unloading+in+Osteocytes+in+Vitro.">The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.</a> The Journal of biological chemistry. 290 (27), 16744-16758 (2015).</li><li>Gillet, J. P., Varma, S., Gottesman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+relevance+of+cancer+cell+lines.">The clinical relevance of cancer cell lines.</a> Journal of the National Cancer Institute. 105 (7), 452-458 (2013).</li><li>Sandberg, R., Ernberg, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+tumor+characteristic+gene+expression+in+cell+lines+using+a+tissue+similarity+index+(TSI).">Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (6), 2052-2057 (2005).</li><li>Kalajzic, I., 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=Dentin+matrix+protein+1+expression+during+osteoblastic+differentiation,+generation+of+an+osteocyte+GFP-transgene.">Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene.</a> Bone. 35 (1), 74-82 (2004).</li><li>Paic, F., 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=Identification+of+differentially+expressed+genes+between+osteoblasts+and+osteocytes.">Identification of differentially expressed genes between osteoblasts and osteocytes.</a> Bone. 45 (4), 682-692 (2009).</li><li>Nakashima, 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=Evidence+for+osteocyte+regulation+of+bone+homeostasis+through+RANKL+expression.">Evidence for osteocyte regulation of bone homeostasis through RANKL expression.</a> Nature Medicine. 17 (10), 1231-1234 (2011).</li><li>Kalajzic, I., 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=In+vitro+and+in+vivo+approaches+to+study+osteocyte+biology.">In vitro and in vivo approaches to study osteocyte biology.</a> Bone. 54 (2), 296-306 (2013).</li><li>Chia, L. Y., Walsh, N. C., Martin, T. J., Sims, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+gene+expression+of+haematopoietic-cell-free+preparations+of+highly+purified+murine+osteocytes.">Isolation and gene expression of haematopoietic-cell-free preparations of highly purified murine osteocytes.</a> Bone. 72, 34-42 (2015).</li><li>Halleux, C., Kramer, I., Allard, C., Kneissel, M., Helfrich, M. H., Ralston, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bone Research Protocols. , 55-66 (2012).</li><li>Bernhardt, A., Wolf, S., Weiser, E., Vater, C., Gelinsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+isolate+primary+human+osteocytes+from+bone.">An improved method to isolate primary human osteocytes from bone.</a> Biomedizinische Technik (Berlin). 65 (1), 107-111 (2020).</li></ol>

著者は開示するものは何もありません。

最初に単離された骨細胞は、(OB7.3)またはPHEXの鳥類変異体を使用して単離されたニワトリ頭蓋冠7 からのものでした。しかしながら、この方法は、種特異的でもある骨細胞特異的抗体を製造しなければならないので、実行可能な抗体の利用可能性によって制限される。研究者らは、マウスおよびラットの長骨から骨細胞を得るために、連続酵素プロセスの異なる改変を使用し...

骨細胞は、それらの分泌マトリックス1に埋め込まれるようになった骨芽細胞前駆細胞からの最終分化細胞である。それらは骨細胞集団の中で最も豊富で最も長生きする細胞です。それらは裂孔内に存在し、樹状突起が小管と呼ばれるチャネルを通って伸びる特徴的な星状形態を有し、周囲の環境および骨表面との通信および代謝交換の広範なネットワークを形成する2。骨細胞は、骨リモデリングにおける骨芽細胞および破骨細胞の両方の役割を振り付け、それらは機械的負荷3への適応を付与する主要な機械感覚細胞であり、リン酸恒常性4 および骨基質石灰化5に関与し、ラクノカナリキュラー系とともに、遠方組織6にシグナル伝達する内分泌器官として作用する。
骨細胞は石灰化マトリックス内に位置するため、アクセスが制限され、単離が困難になり、in vitro調査が妨げられます。最初の単離方法の1つは、後にPHEX(X染色体上のエンドペプチダーゼと相同性を有するPHosphate調節遺伝子)8の鳥変異体であることが知られる骨細胞特異的モノクローナル抗体(OB7.3)7を使用してニワトリ頭蓋冠から単離された骨細胞について説明しました。他の研究者は、ラット9またはマウス10の長骨の逐次消化を使用して、骨細胞の純度が約70%であると報告されている骨細胞に富む画分を取得しました9。この手順の制限には、骨細胞以外の他の細胞タイプで汚染された培養物の純度が最適ではないこと、および骨細胞が分裂する能力を失ったため、骨細胞が培養中の他の細胞によって潜在的に増殖する可能性があることが含まれます。これらの課題は、長期的な文化の使いやすさを制限します。
これらの制限を克服するために、異なる骨細胞細胞株が開発された。MLO-Y4細胞株11およびMLO-A5細胞株12は、特に初期段階の骨細胞の研究に有用な最も広く研究されている細胞株である。しかし、それらは成熟骨細胞マーカーである低レベルのスクレロスチンとFGF2313を発現するため、成熟骨細胞シグナル伝達の研究にはあまり有用ではありません。IDG−SW3 14およびOcy45415を含む他の細胞株は、高レベルのスクレロスチンおよびFGF23を発現し、後期骨細胞期の研究において有用である。細胞株は有用な研究ツールであることが証明されています。それにもかかわらず、それらは初代細胞の生物学を完全に表していないため、制限なしに来るわけではありません。異なる細胞株は骨細胞成熟スペクトルの異なる発生段階を表し、細胞株は初代骨細胞の不均一性を表すことができません16,17。
初代骨細胞の純粋な培養物を得るために、研究者らは、8 kb dmp1プロモーターを使用して骨細胞における緑色蛍光タンパク質(GFP)発現を促進するcreマウスモデルを利用しました18,19。Paicらによる二重トランスジェニックマウス(pOB-Col 2.3-GFP-シアンおよびDMP1-GFP-topaz)19およびNakashimaらによるdmp1-topazトランスジェニックマウス20が、骨細胞集団を取得するために使用されている。その中で彼らは、初代骨細胞の培養物を取得するためにGFPを発現する骨細胞の逐次消化およびFACSを採用した19,20。tdTomatoタンパク質を活性化する10 kb dmp1レポーターマウスAi9のcreの方向は、骨髄内の骨細胞、骨芽細胞、筋肉、および細胞に存在することが示されました。8 kb dmp1プロモーターは同じ発現パターンを有していたが、骨芽細胞および骨髄細胞の一部のみがタンパク質を発現しており、これは8 kb dmp1プロモーターがより特異的であることを示している21。それにもかかわらず、8 kb dmp1プロモーターを使用して得られた結果は慎重に解釈する必要があり、得られた集団が十分に高い純度であることを確認するために、骨細胞と骨芽細胞特異的マーカーを使用して遺伝子発現プロファイルを日常的に実行する必要があります。
破骨細胞マーカーOSCARとDcstampは、造血非枯渇骨細胞集団と枯渇骨細胞集団で見出され、この発見により、著者らは、8 kb dmp1-topaz新生児頭蓋冠の分画とGFPソーティングから得られた消化物が造血細胞で汚染されていると結論付けました。GFP陽性造血細胞はGFP陽性間葉系細胞(骨細胞)よりもGFP強度が適度に低いため、造血細胞による汚染はGFPソートゲートを締めることで軽減できたはずです22。
in vitroで骨細胞を研究する方法は、骨細胞生物学に関する最近の豊富な情報に貢献しています。しかしながら、骨細胞の単離は、依然として労働集約的で時間のかかる手順であり、細胞収率は低い。コラゲナーゼおよびEDTAを用いた骨消化の記載された方法は、しばしば画分8から23まで、骨細胞の生存率が課税される数時間を要する。研究者らは、細胞選別に画分(2〜20)を使用したことを報告しており20、骨細胞に関連する遺伝子と骨芽細胞に関連する遺伝子の発現プロファイルが、純粋な骨細胞集団の単離の成功を確認することを示しました20。この記事では、フラクション(2〜2)を取得するプロセスについて説明し、フラクション1から8までの各フラクションからの骨細胞の収量を比較して、各フラクションの骨細胞の戻りを決定します。また、新生dmp1-topazマウス頭蓋冠の解剖とコラゲナーゼとエチレンジアミン四酢酸(EDTA)を用いた頭蓋骨消化、およびFACS用細胞の調製についても説明します。

<table>
	<tbody>
		<tr>
			<td>BD FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Data aquisition and analysis</td>
		</tr>
		<tr>
			<td>BD Falcon Tube</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>12 x 75 mm Tube with Cell Strainer Cap, 35 &#956;m nylon mesh.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Wako, Osaka, Japan</td>
			<td>034-22363</td>
			<td>0.2% (w/v), crude collagenase mix sourced from C. histolyticum.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo, Kumamoto, Japan</td>
			<td></td>
			<td>5mM EDTA prepared with 0.1% BSA</td>
		</tr>
		<tr>
			<td>FACSAriaTM II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation buffer</td>
			<td></td>
			<td></td>
			<td>70mM NaCl, 10mM NaHCO, 60mM sorbitol, 3mM K2HPO4, 1mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES</td>
		</tr>
		<tr>
			<td>Millex Sterile Filter Unit</td>
			<td>Merck Millipore, Ireland</td>
			<td>SLGV033RS</td>
			<td>0.22&#956;m</td>
		</tr>
		<tr>
			<td>Nylon cell strainer</td>
			<td>FALCON, NY, USA</td>
			<td></td>
			<td>40&#956;m</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies, NY, USA</td>
			<td></td>
			<td>0.5% x10. diluted to x1 in PBS</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Wako, Osaka, Japan</td>
			<td></td>
			<td>Containing 10% fetal bovine serum, 100 IU/ml penicillin G, and 100 &#956;g/ml streptomycin</td>
		</tr>
	</tbody>
</table>

obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes

骨細胞は、かつては機械的負荷を感知する舞台裏の機能を与えられて骨の受動的な常駐であると考えられていましたが、今では脚光を浴びており、細胞外マトリックスを積極的に修飾し、それを囲むラクノカナキュラー系で内分泌器官を形成し、離れた部位にメッセージを送信するなど、複数の主要な機能を持っていることが示されています。初代骨細胞を単離して骨細胞様細胞株までin vitroで骨細胞を試験することが可能になった方法のおかげで、骨細胞は現在、構造と機能に関する大きな関心と知識の急増を経験しています。骨細胞の生物学や他の分子成分との相互作用の多くの側面はまだ発見されていません。このプロトコルでは、骨細胞で緑色蛍光タンパク質を発現するdmp1-topaz新生児マウス頭蓋冠からの初代骨細胞の効率的な単離について、細胞分画とその後のFACSによる初代骨細胞の培養の取得によって詳細に説明します。

このプロトコルの目的は、dmp1-topaz新生児マウス頭蓋冠から初代骨細胞の培養物を得るプロセスを実証することです コラゲナーゼを使用してコラーゲンマトリックスを分解し、EDTAを使用してカルシウムキレート化を行う分画プロセスを通じて、その後、FACS用に細胞を調製して骨細胞を他の細胞集団から分離します。
新生児マウス頭蓋冠から初代骨細胞を得るための方法?...

Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

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

このプロトコルでは、蛍光活性化細胞選別(FACS)のための骨細胞調製に加えて、新生児dmp1-topazマウス頭蓋冠の解剖と、細胞消化と分画による緑色蛍光タンパク質を発現する骨細胞の単離について説明します。

GFP発現骨細胞のマウス頭蓋骨分画による初代骨細胞の取得

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 ...

obtaining-primary-osteocytes-through-murine-calvarial-fractionation

Research

JoVE Journal

Biology

2.0K ビュー.  Tohoku University Graduate School of Dentistry. このプロトコルでは、蛍光活性化細胞選別(FACS)のための骨細胞調製に加えて、新生児dmp1-topazマウス頭蓋冠の解剖と、細胞消化と分画による緑色蛍光タンパク質を発現する骨細胞の単離について説明します。

ビデオ: Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

アフリカツメガエル女性から卵子を入手

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

生物学

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

アフリカツメガエル卵抽出物の調製と分画

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

キメラゼブラフィッシュ胚における網膜と脳の開発のライブイメージングのためのGFP発現割球の移植

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

初代マウス骨芽細胞から骨細胞培養物を入手するレチノイン酸のアプリケーション

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.