The authors wish to acknowledge and thank those additional research personnel that contributed to the development of the described protocol and its isolation of ASCs, including: Dr. H. Peter Lorenz, MD, Dr. Hiroshi Muzuno, MD, Dr. Jerry Huang, MD, Dr. Adam Katz, MD, Dr. William Futrell, MD, Dr. Rong Zhang, DDS, PhD, Dr. Larissa Rodriguez, MD, Dr. Zeni Alfonso, PhD, and Dr. John Fraser, PhD. The results presented were funded, in part, by research grants from the National Institutes of Health, including the NIAMS and NIDCR Institutes.

<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>

The authors of this manuscript are co-inventors on a patent owned by The Regents of the University of California and licensed to Cytori Therapeutics.

Adipose tissue for the isolation of ASCs can come in many forms: from solid pieces of tissue obtained through resection or lipoplasty to smaller pieces obtained through either syringe extraction or suction-assisted lipoplasty (i.e. liposuction ). Whether more SVF cells (and thereby ASCs) can be obtained from resected or aspirated adipose samples is unclear as conflicting studies have been presented 16, 17. It is possible that either form of adipose tissue is more than suitable for the isolation of SVF...

In 2001, a putative population of multipotent stem cells from adipose tissue was described in the journal Tissue Engineering 1. These cells were given the name Processed Lipoaspirate or PLA cells due to their derivation from processed lipoaspirate tissue obtained through cosmetic surgery. The isolation method described in this article was based on existing enzymatic strategies for the isolation of the stromal vascular fraction (SVF) from adipose tissue 2. The SVF has been defined as a minimally processed population of red blood cells, fibroblasts, endothelial cells, smooth muscle cells, pericytes and pre-adipocytes that have yet to adhere to a tissue culture substrate 2, 3. Culturing of this SVF over time is proposed to eliminate many of these contaminating cell populations and result in an adherent, fibroblastic population. These fibroblasts have been identified in the literature for the last 40 years as being pre-adipocytes. However, our research group demonstrated that these cells possessed mesodermal multipotency and renamed the adherent SVF population as PLA cells. Subsequent studies by numerous other research groups have added to this potential, suggesting both endodermal and ectodermal potentials (for review see 4). Since that time, numerous additional terms for these cells have appeared in the literature. In order to provide some type of consensus, the term Adipose-derived Stem Cells or ASCs was adopted at the 2nd annual IFATS conference. As such, the term ASC will be used in this article. 
The protocol described in this article is a relatively simple procedure that requires standard laboratory equipment and uses simple reagents such as phospho-buffered saline, standard tissue culture media reagents and collagenase. It can produce large numbers of ASCs depending on the amount of starting adipose tissue volume and subsequent culture time. However, the processing of such a large amount of adipose tissue can present some physical issues that can be mitigated to a degree using this protocol. Furthermore, this protocol does require sterile tissue culture facilities and approved biosafety hoods, thereby necessitating the use of an approved tissue culture facility. This requirement can also decrease the utility of the ASC population in clinical applications unless they are isolated in good manufacturing practice (GMP)-approved facilities designed for the isolation and expansion of materials for clinical use. As an alternative, automated systems that can isolate ASCs in a closed system in the operating theatre would avoid this key issue and allow for the immediate use of ASCs without any need for subsequent in vitro expansion. To date, there are six automated systems that are available commercially for the isolation of cells from human tissue. These systems may make it possible to isolate a significant number of ASCs from large amounts of adipose tissue immediately following its harvest. These ASCs could then be reintroduced into the patient for a variety of regenerative purposes without the patient ever having to leave the operating room. In addition to this protocol describing the manual isolation of ASCs, a protocol for automated isolation of ASCs using the Celution System is also given in a companion article.

<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

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

The protocol outline above describes a manual, enzymatic method for the isolation of an SVF from a large volume lipoaspirate sample. Within this SVF are numerous cell populations, including the ASC. Numerous studies propose that culturing this SVF under standard tissue culture conditions will select for an adherent fibroblast population likely to be composed mainly of the ASC type. Consistent with this, we have shown, using flow cytometry and immunofluorescence, that cultured SVF pellets become essentially free of the ...

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

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

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

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41.1K Views.  Cytori Therapeutics Inc. In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

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

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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.

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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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.

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 of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

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.

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&#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.

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 of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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.

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