作者は認めるしたいを含む記載されているプロトコルとASCのその単離の発展に貢献し、それらの追加の研究人材、感謝博士H.ピーター·ローレンツ博士、博士宏Muzuno、MD、博士ジェリー黄、MDを博士アダム·カッツ、MD、博士はウィリアム·フトレル、MD、博士は栄チャン、DDS、PhDは、博士ラリッサロドリゲス、MD、博士はゼニアルフォンソ博士は、博士ジョン·フレイザー博士。提示された結果は、NIAMSおよびNIDCR研究所などの国立衛生研究所から研究助成金によって部分的に資金を供給されました。

<ol>
	<li>Zuk, P. A., 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=Multi-lineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multi-lineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Engineering. 7 (2), 211-226 (2001).</li><li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+isolated+fat+cells.">Metabolism of isolated fat cells.</a> J. Biol. Chem. 239, 375-380 (1964).</li><li>Poznanski, W. J., Waheed, I., Human Van, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fat+cell+precursors.+Morphologic+and+metabolic+differentiation+in+culture.">fat cell precursors. Morphologic and metabolic differentiation in culture.</a> Lab Invest. 29 (5), 570-576 (1973).</li><li>Zuk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+Stem+Cells+in+Tissue+Regeneration:+A+Review.">Adipose-derived Stem Cells in Tissue Regeneration: A Review.</a> ISRN Stem Cells. , (2012).</li><li>Zuk, P. A., 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=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Mol. Biol. Cell. 13, 4279-4295 (2002).</li><li>Mitchell, J. B., 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=Immunophenotype+of+human+adipose-derived+cells:+temporal+changes+in+stromal-associated+and+stem+cell-associated+markers.">Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.</a> Stem Cells. 24 (2), 376-385 (2006).</li><li>Oedayrajsingh-Varma, M. J., 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=Phenotypical+and+functional+characterization+of+freshly+isolated+adipose+tissue-derived+stem+cells.">Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells.</a> Stem Cells Dev. 16 (1), 91-104 (2007).</li><li>Yoshimura, K., 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=Characterization+of+freshly+isolated+and+cultured+cells+derived+from+the+fatty+and+fluid+portions+of+liposuction+aspirates.">Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates.</a> J Cell Physiol. 208 (1), 64-76 (2006).</li><li>Zannettino, A. C., 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=Multipotential+human+adipose-derived+stromal+stem+cells+exhibit+a+perivascular+phenotype+in+vitro+and+in+vivo.">Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo.</a> J Cell Physiol. 214 (2), 413-421 (2008).</li><li>Chung, M. 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=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li><li>Li, 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=Adipogenic+potential+of+adipose+stem+cell+subpopulations.">Adipogenic potential of adipose stem cell subpopulations.</a> Plast Reconstr Surg. 128 (3), 663-672 (2011).</li><li>Rada, T., Reis, R. L., Gomes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stem+cells+subpopulations+isolated+from+human+adipose+tissue+exhibit+different+chondrogenic+and+osteogenic+differentiation+potential.">Distinct stem cells subpopulations isolated from human adipose tissue exhibit different chondrogenic and osteogenic differentiation potential.</a> Stem Cell Rev. 7 (1), 64-76 (2011).</li><li>Heydarkhan-Hagvall, S., 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=Human+Adipose+Stem+Cells:+A+Potential+Cell+Source+for+Cardiovascular+Tissue+Engineering.">Human Adipose Stem Cells: A Potential Cell Source for Cardiovascular Tissue Engineering.</a> Cells Tissues Organs. 187 (4), 263-274 (2008).</li><li>Jack, G. S., 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=Processed+lipoaspirate+cells+for+tissue+engineering+of+the+lower+urinary+tract:+implications+for+the+treatment+of+stress+urinary+incontinence+and+bladder+reconstruction.">Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction.</a> J Urol. 174 (5), 2041-2045 (2005).</li><li>Banas, A., 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=Rapid+hepatic+fate+specification+of+adipose-derived+stem+cells+and+their+therapeutic+potential+for+liver+failure.">Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure.</a> J Gastroenterol Hepatol. 24 (1), 70-77 (2009).</li><li>Schreml, S., 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=Harvesting+human+adipose+tissue-derived+adult+stem+cells:+resection+versus+liposuction.">Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction.</a> Cytotherapy. 11 (7), 947-957 (2009).</li><li>Oedayrajsingh-Varma, M. J., 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=Adipose+tissue-derived+mesenchymal+stem+cell+yield+and+growth+characteristics+are+affected+by+the+tissue-harvesting+procedure.">Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.</a> Cytotherapy. 8 (2), 166-177 (2006).</li><li>Ahmad, J., Eaves, F. F., Rohrich, R. J., Kenkel, J. 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+American+Society+for+Aesthetic+Plastic+Surgery+(ASAPS)+survey:+current+trends+in+liposuction.">The American Society for Aesthetic Plastic Surgery (ASAPS) survey: current trends in liposuction.</a> Aesthet Surg J. 31 (2), 214-224 (2011).</li><li>Tierney, E. P., Kouba, D. J., Hanke, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+of+tumescent+and+laser-assisted+liposuction:+review+of+the+literature.">Safety of tumescent and laser-assisted liposuction: review of the literature.</a> J Drugs Dermatol. 10 (12), 1363-1369 (2012).</li><li>Mojallal, A., Auxenfans, C., Lequeux, C., Braye, F., Damour, O. <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+negative+pressure+when+harvesting+adipose+tissue+on+cell+yield+of+the+stromal-vascular+fraction.">Influence of negative pressure when harvesting adipose tissue on cell yield of the stromal-vascular fraction.</a> Biomed Mater Eng. 18 (4-5), 193-197 (2008).</li><li>Matsumoto, D., 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=Influences+of+preservation+at+various+temperatures+on+liposuction+aspirates.">Influences of preservation at various temperatures on liposuction aspirates.</a> Plast Reconstr Surg. 120 (6), 1510-1517 (2007).</li><li>Francis, M. P., Sachs, P. C., Elmore, L. W., Holt, 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=Isolating+adipose-derived+mesenchymal+stem+cells+from+lipoaspirate+blood+and+saline+fraction.">Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction.</a> Organogenesis. 6 (1), 11-14 (2010).</li><li>Boquest, A. C., Shahdadfar, A., Brinchmann, J. E., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stromal+stem+cells+from+human+adipose+tissue.">Isolation of stromal stem cells from human adipose tissue.</a> Methods Mol Biol. 325, 35-46 (2006).</li><li>Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+isolation,+expansion+and+differentiation.">Adipose-derived stem cells: isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Dubois, S. G., 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=Isolation+of+human+adipose-derived+stem+cells+from+biopsies+and+liposuction+specimens.">Isolation of human adipose-derived stem cells from biopsies and liposuction specimens.</a> Methods Mol Biol. 449, 69-79 (2008).</li><li>Mosna, F., Sensebe, L., Krampera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow+and+adipose+tissue+mesenchymal+stem+cells:+a+user's+guide.">Human bone marrow and adipose tissue mesenchymal stem cells: a user's guide.</a> Stem Cells Dev. 19 (10), 1449-1470 (2010).</li><li>Zachar, V., Rasmussen, J. G., Fink, T. <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+growth+of+adipose+tissue-derived+stem+cells.">Isolation and growth of adipose tissue-derived stem cells.</a> Methods Mol Biol. 698, 37-49 (2011).</li><li>Williams, S. K., McKenney, S., Jarrell, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+lot+selection+and+purification+for+adipose+tissue+digestion.">Collagenase lot selection and purification for adipose tissue digestion.</a> Cell Transplant. 4 (3), 281-289 (1995).</li><li>Wang, H., Van Blitterswijk, C. A., Bertrand-De Haas, M., Schuurman, A. H., Lamme, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzymatic+isolation+of+fibroblasts+for+the+creation+of+autologous+skin+substitutes.">Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.</a> In Vitro Cell Dev Biol Anim. 40 (8-9), 268-277 (2004).</li><li>Pilgaard, L., Lund, P., Rasmussen, J. G., Fink, T., Zachar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+highly+defined+proteases+for+the+isolation+of+adipose+tissue-derived+stem+cells.">Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells.</a> Regen Med. 3 (5), 705-715 (2008).</li><li>Kurita, 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=Influences+of+centrifugation+on+cells+and+tissues+in+liposuction+aspirates:+optimized+centrifugation+for+lipotransfer+and+cell+isolation.">Influences of centrifugation on cells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer and cell isolation.</a> Plast Reconstr Surg. 121 (3), 1033-1041 (2008).</li><li>Poloni, A., 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=Human+dedifferentiated+adipocytes+show+similar+properties+to+bone+marrow-derived+mesenchymal+stem+cells.">Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.</a> Stem Cells. 30 (5), 965-974 (2012).</li><li>D'Andrea, 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=Large-scale+production+of+human+adipose+tissue+from+stem+cells:+a+new+tool+for+regenerative+medicine+and+tissue+banking.">Large-scale production of human adipose tissue from stem cells: a new tool for regenerative medicine and tissue banking.</a> Tissue Eng Part C Methods. 14 (3), 233-242 (2008).</li><li>Tallone, 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=Adult+human+adipose+tissue+contains+several+types+of+multipotent+cells.">Adult human adipose tissue contains several types of multipotent cells.</a> J Cardiovasc Transl Res. 4 (2), 200-210 (2011).</li><li>De Francesco, 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=Human+CD34/CD90+ASCs+are+capable+of+growing+as+sphere+clusters,+producing+high+levels+of+VEGF+and+forming+capillaries.">Human CD34/CD90 ASCs are capable of growing as sphere clusters, producing high levels of VEGF and forming capillaries.</a> PLoS One. 4 (8), e6537 (2009).</li><li>Haasters, 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=Morphological+and+immunocytochemical+characteristics+indicate+the+yield+of+early+progenitors+and+represent+a+quality+control+for+human+mesenchymal+stem+cell+culturing.">Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing.</a> J Anat. 214 (5), 759-767 (2009).</li></ol>

この原稿の著者は、カリフォルニア大学とサイトリピューにライセンスが所有する特許の共同発明者である。

ASCの分離のための脂肪組織は、さまざまな形で来ることができる：注射器抽出または吸引補助脂肪形成術（ すなわち脂肪吸引）のいずれかによって得られた小さな断片に切除または脂肪形成術によって得られた組織の固体片から。競合する研究は16,17提示されているように、複数のSVF細胞（ASCは、それによって）が切除または脂肪吸引サンプルから得ることができるかどうか?...

2001年には、脂肪組織から多能性幹細胞の集団は、推定上のジャーナル·ティッシュ·エンジニアリング1に記載されている。これらの細胞は、美容整形によって得られた処理脂肪吸引組織からの派生のために名前を処理脂肪吸引またはPLA細胞を与えられた。この資料に記載されている分離方法は、脂肪組織2から間質血管画分（SVF）を単離するための既存の酵素の戦略に基づいていた。 SVFは、組織培養基質2、3に付着するには至っていない赤血球、線維芽細胞、内皮細胞、平滑筋細胞、周皮細胞および前脂肪細胞の最小限の加工集団として定義されている。経時このSVFの培養は、これらの混入細胞集団の多くを排除し、付着性、線維芽細胞集団を生じることが提案されている。これらの線維芽細胞は事前にされていると、最後の40年間の文献で確認されている脂肪細胞。しかし、我々の研究グループでは、これらの細胞は中胚葉多分化を保有し、PLA細胞として付着したSVFの人口と改名することを実証した。他の多くの研究グループによるその後の研究では、（レビュー4を参照）、内胚葉及び外胚葉の両方電位を示唆し、この潜在的に追加した。その時以来、これらの細胞のための多数の追加の用語が文献に登場している。コンセンサスいくつかのタイプを提供するために、長期的な脂肪由来幹細胞またはASCを、2 回目の年次IFATS会議で採択された。このように、長期的なASCが、この記事で使用されます。 この資料に記載されたプロトコルは、標準的な実験室装置を必要とし、そのようなリン酸緩衝化生理食塩水、標準的な組織培養培地試薬およびコラゲナーゼなどの単純な試薬を使用して比較的簡単な手順である。それは、脂肪組織量およびその後のcと出発の量に応じてASCの多数を生成することができるulture時間。しかし、脂肪組織のような大量の処理は、このプロトコルを使用してある程度まで軽減することができるいくつかの物理的な問題を提示することができる。さらに、このプロトコルは、無菌の組織培養施設を必要としませんし、それによって承認された組織培養施設の使用を必要と、バイオセーフティフードを承認した。これらは適正製造基準分離と臨床使用のための材料の拡大のために設計された（GMP）が承認した施設で隔離されていない限り、この要件には、臨床応用におけるASCの人口の有用性を減少させることができます。別の方法として、手術室に閉鎖系でのASCを単離することができる自動化されたシステムは、この重要な問題を回避するとその後のin vitroでの拡大を必要とせずにASCのすぐに使用可能にする。今日まで、ヒト組織からの細胞の単離のために市販の6つの自動化システムがある。これらのシステムは、単離することを可能にすることができるすぐに収穫後の脂肪組織に大量のからASCのかなりの数。これらのASCは、患者がこれまでに手術室を離れることなく、回生、様々な目的のために患者に再導入することができた。 ASCの手動分離を記述し、このプロトコルに加えて、セリューション·システムを使用したASCの自動化された単離するためのプロトコルも、関連記事に記載されています。

<table border="1">
	<tbody>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Reagent</td>
		</tr>
		<tr>
			<td>
				DMEM (Dulbecco&#39;s Modification of Eagle&#39;s Medium)</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				10-013-CV</td>
			<td>
				with 4.5 g/ml glucose, L-glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>
				Penicillin/Streptomycin</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				30-002-CI</td>
			<td>
				10,000 IU/ml penicillin/10,000 &mu;g/ml streptomycin</td>
		</tr>
		<tr>
			<td>
				Amphotericin B</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				30-003-CF</td>
			<td>
				250 &mu;g/ml amphotericin B</td>
		</tr>
		<tr>
			<td>
				10X PBS (Phospho-buffered Saline)</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				25-053-CI</td>
			<td>
				without calcium, without magnesium</td>
		</tr>
		<tr>
			<td>
				Trypsin/EDTA</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				20-031-CV</td>
			<td>
				0.25 % trypsin/2.21mM EDTA</td>
		</tr>
		<tr>
			<td>
				Collagenase type IA (from Clostridium histolyticum)</td>
			<td>
				Sigma</td>
			<td>
				C2674</td>
			<td>
				crude preparation; &lt;125 collagen digestion units/mg solid</td>
		</tr>
		<tr>
			<td>
				FBS (Fetal Bovine Serum) heat inactivated</td>
			<td>
				Gemini Bioproducts</td>
			<td>
				100106</td>
			<td>
				USDA source, heat inactivated</td>
		</tr>
		<tr>
			<td>
				10 ml serological pipettes</td>
			<td>
				Genesee Scientific</td>
			<td>
				12-104</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				25 ml serological pipettes</td>
			<td>
				Genesee Scientific</td>
			<td>
				12-106</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				50 ml polypropylene centrifuge tubes</td>
			<td>
				Genesee Scientific</td>
			<td>
				21-106</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				100 mm tissue culture dishes</td>
			<td>
				Genesee Scientific</td>
			<td>
				25-202</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				150 mm tissue culture dishes</td>
			<td>
				Genesee Scientific</td>
			<td>
				25-203</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				500 ml Stericup Filter Units</td>
			<td>
				Millipore</td>
			<td>
				SCGPU05RE</td>
			<td>
				PES membrane, 0.22 &mu;m pore</td>
		</tr>
		<tr>
			<td>
				Cell strainers</td>
			<td>
				FisherBrand</td>
			<td>
				22-363-549</td>
			<td>
				100 &mu;m nylon mesh</td>
		</tr>
		<tr>
			<td>
				dexamethasone - water soluble</td>
			<td>
				Sigma</td>
			<td>
				D-2915</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				L-ascorbic-acid 2 phosphate</td>
			<td>
				Sigma</td>
			<td>
				A-8960</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				&beta;-glycerophosphate disodium salt</td>
			<td>
				Sigma</td>
			<td>
				G-9422</td>
			<td>
				also known as glycerophosphate</td>
		</tr>
		<tr>
			<td>
				insulin</td>
			<td>
				Sigma</td>
			<td>
				I-6634</td>
			<td>
				made from bovine pancreas</td>
		</tr>
		<tr>
			<td>
				indomethacin</td>
			<td>
				Sigma</td>
			<td>
				I-7378</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				apo-transferrin</td>
			<td>
				Sigma</td>
			<td>
				T-4382</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				TGF&beta;1</td>
			<td>
				R&amp;D Systems</td>
			<td>
				240-B-002</td>
			<td>
				recombinant human</td>
		</tr>
		<tr>
			<td>
				Oil Red O</td>
			<td>
				Sigma</td>
			<td>
				O-0625</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Alcian Blue</td>
			<td>
				Sigma</td>
			<td>
				A-5268</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Silver nitrate</td>
			<td>
				Sigma</td>
			<td>
				S-0319</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Hydrochloric acid</td>
			<td>
				Fisher Scientific</td>
			<td>
				A144</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Paraformaldehyde</td>
			<td>
				Fisher Scientific</td>
			<td>
				30525-89-4</td>
			<td>
				supplied as a 16 % stock</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				[header]</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Equipment Needed</td>
		</tr>
		<tr>
			<td>
				Class II A/B Biosafety hood</td>
			<td>
				Thermo Scientific</td>
			<td>
				&nbsp;</td>
			<td>
				ensure hood has vacuum lines for aspiration</td>
		</tr>
		<tr>
			<td>
				Benchtop centrifuge</td>
			<td>
				Hermle Labnet</td>
			<td>
				Z383</td>
			<td>
				Swing-out rotor for 50 ml tubes required, capable of 1200 x g</td>
		</tr>
		<tr>
			<td>
				Water bath</td>
			<td>
				Fisher Scientific Isotemp</td>
			<td>
				S52602Q</td>
			<td>
				5-10L capacity, capable of 37 C</td>
		</tr>
		<tr>
			<td>
				Automated Pipette Aids</td>
			<td>
				Drummond Pipette Aid XL</td>
			<td>
				4-000-105</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				CO2 Incubator</td>
			<td>
				Thermo Scientific</td>
			<td>
				Forma 310</td>
			<td>
				direct heat or water jacketed</td>
		</tr>
	</tbody>
</table>

	&nbsp;

manual isolation of adipose-derived stem cells from human lipoaspirates

2001年には、カリフォルニア大学ロサンゼルス校の研究者は、彼らが最初に処理脂肪吸引細胞またはPLA細胞と呼ばれる脂肪吸引脂肪組織からの成体幹細胞の新たな集団の単離を記述した。それ以来、これらの幹細胞は、脂肪由来幹細胞またはASCをと改名され、幹細胞研究と再生医療の分野で最も人気のある成体幹細胞集団の1になるために行っている。数千件もの記事になりました骨再生、末梢神経修復および心血管工学など、再生様々な動物モデルにおいてASCの使用を記載している。最近の記事クリニックでのASCの用途の無数を記述するために始めている。この記事に示されているプロトコルは、手動および酵素的に化粧品の手順から得た脂肪吸引物、大量のからのASCを分離するための基本的な手順を説明します。このプロトコルは、簡単またはaccommodまでスケールアップすることができます脂肪吸引の量を食べ、abdominoplastiesや他の同様の手順で得られた脂肪組織からのASCを隔離するために適合させることができる。

上記のプロトコルの概要については、マニュアル、大容量の脂肪吸引サンプルからSVFを単離するための酵素法を説明しています。このSVF内にASCを含む多数の細胞集団である。多くの研究は、標準的な組織培養条件下でこのSVFを培養するASCの種類を主成分とする可能性が付着した線維芽細胞集団を選択することを提案する。これと一致して、我々は、培養されたSVFペレットが主要な混入細胞型?...

Watch this Scientific Journal Video about Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates at JoVE.com

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

2001年には、UCLAの研究者は、成体幹細胞の集団の単離を説明した脂肪組織から、脂肪由来幹細胞またはASCをと呼ばれる。この記事では、コラゲナーゼを使用して手動、酵素消化プロトコルを使用して脂肪吸引物からASCの分離の概要を説明します。

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

manual-isolation-adipose-derived-stem-cells-from-human

Research

JoVE Journal

Biology

40.9K ビュー.  Cytori Therapeutics Inc.  2001年には、UCLAの研究者は、成体幹細胞の集団の単離を説明した脂肪組織から、脂肪由来幹細胞またはASCをと呼ばれる。この記事では、コラゲナーゼを使用して手動、酵素消化プロトコルを使用して脂肪吸引物からASCの分離の概要を説明します。 

ビデオ: Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

生物学

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

ヒト膵臓癌異種移植片由来の幹細胞の分離

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

アカゲザル脂肪由来幹細胞の単離・増殖・分化

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

培養ヒト間葉系幹細胞からの細胞外小胞の単離、特性評価、および治療応用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

脂肪組織由来幹細胞の単離と特性評価

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

著者スポットライト:Sprague Dawleyラットの脂肪組織に由来する間葉系幹細胞の単離と同定  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

スプレイグドーリーラットの脂肪組織由来間葉系幹細胞の単離と同定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...