Dr. Rountree acknowledges current support from the Children's Miracle Network, National Institute of Health, K08DK080928 and R03DK088013, and the American Cancer Society Research Scholar Award, MGO-11651. Dr. Rountree acknowledges that this procedure was initially developed and refined while funded by the Pediatric Scientist Development Program (NICHD Grant Award K12-HD00850).

<ol>
	<li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD133-expressing+murine+liver+oval+cell+population+with+bilineage+potential.">A CD133-expressing murine liver oval cell population with bilineage potential.</a> Stem Cells. 25, 2419-2429 (2007).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++liver+cancer+stem+cells+from+methionine+adenosyl+transferase+1A-deficient+mice+demonstrate+resistance+to+transforming+growth+factor+(TGF)-beta-induced+apoptosis.">CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis.</a> Hepatology. 49, 1277-1286 (2009).</li><li>Rountree, C. B., Ding, W., He, L., Stiles, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+CD133-expressing+liver+cancer+stem+cells+in+liver-specific+phosphatase+and+tensin+homolog+deleted+on+chromosome+10-deleted+mice.">Expansion of CD133-expressing liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on chromosome 10-deleted mice.</a> Stem Cells. 27, 290-299 (2009).</li><li>Rountree, C. B., Senadheera, S., Mato, J. M., Crooks, G. M., Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+liver+cancer+stem+cells+during+aging+in+methionine+adenosyltransferase+1A-deficient+mice.">Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice.</a> Hepatology. 47, 1288-1297 (2008).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> J. Cell. Physiol. 213, 286-300 (2007).</li><li>Fausto, N., Campbell, J. S., Riehle, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> Hepatology. 43, 45-53 (2006).</li><li>Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+Stem+Cells.+III.+Emergent+themes+of+liver+stem+cell+biology:+niche,+quiescence,+self-renewal,+and+plasticity.">Gastrointestinal Stem Cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 290, 189-193 (2006).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+liver+repopulating+capacity+of+murine+oval+cells.">The origin and liver repopulating capacity of murine oval cells.</a> Proc Natl Acad Sci U. S. A. 100, 11881-11888 (2003).</li><li>Greenbaum, L. E., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stem+cells+in+liver+repair+and+fibrosis.">The role of stem cells in liver repair and fibrosis.</a> Int. J. Biochem. Cell Biol. 43, 222-229 (2011).</li><li>Choi, S. S., Omenetti, A., Syn, W. K., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Hedgehog+signaling+in+fibrogenic+liver+repair.">The role of Hedgehog signaling in fibrogenic liver repair.</a> Int. J. Biochem. Cell. Biol. 43, 238-244 (2011).</li><li>Khurana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scopolamine+treatment+and+muscarinic+receptor+subtype-3+gene+ablation+augment+azoxymethane-induced+murine+liver+injury.">Scopolamine treatment and muscarinic receptor subtype-3 gene ablation augment azoxymethane-induced murine liver injury.</a> J. Pharmacol. Exp. Ther. 333, 639-649 (2010).</li><li>Sell, S., Leffert, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+cancer+stem+cells.">Liver cancer stem cells.</a> J. Clin. Oncol. 26, 2800-2805 (2008).</li><li>Lee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+prognostic+subtype+of+human+hepatocellular+carcinoma+derived+from+hepatic+progenitor+cells.">A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.</a> Nat. Med. 12, 410-416 (2006).</li><li>Sell, S., Dunsford, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+stem+cell+origin+of+hepatocellular+carcinoma+and+cholangiocarcinoma.">Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma.</a> Am. J. Pathol. 134, 1347-1363 (1989).</li><li>Amin, R., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+tgf-Beta+in+hepatic+carcinogenesis.">Liver stem cells and tgf-Beta in hepatic carcinogenesis.</a> Gastrointest Cancer Res. 2, 27-30 (2008).</li><li>Tang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor/stem+cells+give+rise+to+liver+cancer+due+to+aberrant+TGF-beta+and+IL-6+signaling.">Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.</a> Proc Natl Acad Sci. 105, 2445-2450 (2008).</li><li>Kitisin, K., Pishvaian, M. J., Johnson, L. B., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+molecular+signaling+pathways+in+hepatocellular+carcinoma.">Liver stem cells and molecular signaling pathways in hepatocellular carcinoma.</a> Gastrointest Cancer Res. 1, 13-21 (2007).</li><li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+fails+to+differentiate+into+liver+epithelium+during+murine+development+and+regeneration.">Bone marrow fails to differentiate into liver epithelium during murine development and regeneration.</a> Hepatology. 45, 1250-1260 (2007).</li><li>Shimano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+oval+cells+have+the+side+population+phenotype+defined+by+expression+of+ATP-binding+cassette+transporter+ABCG2/BCRP1.">Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1.</a> Am J Pathol. 163, 3-9 (2003).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+isolation+and+clonal+identification+of+self-renewing+bipotent+hepatic+progenitor+cells+in+adult+mouse+liver.">Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.</a> Hepatology. , (2008).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+and+characterization+of+self-renewing+pluripotent+stem+cells+in+the+developing+liver.">Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.</a> J Cell Biol. 156, 173-184 (2002).</li><li>Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpCAM-positive+hepatocellular+carcinoma+cells+are+tumor-initiating+cells+with+stem/progenitor+cell+features.">EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.</a> Gastroenterology. 136, 1012-1024 (2009).</li><li>Yamashita, T., Budhu, A., Forgues, M., Wang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hepatic+stem+cell+marker+EpCAM+by+Wnt-beta-catenin+signaling+in+hepatocellular+carcinoma.">Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma.</a> Cancer Res. 67, 10831-10839 (2007).</li><li>Furuyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver,+exocrine+pancreas+and+intestine.">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43, 34-41 (2011).</li><li>Kordes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++hepatic+stellate+cells+are+progenitor+cells.">CD133+ hepatic stellate cells are progenitor cells.</a> Biochem Biophys Res Commun. 352, 410-417 (2007).</li><li>Berg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Growth+Factor+10+is+critical+for+liver+growth+during+embryogenesis+and+controls+of+hepatoblast+survival+via+b-catenin+activation.">Fibroblast Growth Factor 10 is critical for liver growth during embryogenesis and controls of hepatoblast survival via b-catenin activation.</a> Hepatology. , (2007).</li><li>Turner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatic+stem+cell+and+maturational+liver+lineage+biology.">Human hepatic stem cell and maturational liver lineage biology.</a> Hepatology. 53, 1035-1045 (2011).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Galicia, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+hepatic+tumor+progenitor+cells+in+Pten-null+mice+requires+liver+injury+and+is+reversed+by+loss+of+AKT2.">Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.</a> Gastroenterology. 139, 2170-2182 (2010).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+of+murine+liver+tumor+cells+promotes+invasion.">Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion.</a> Hepatology. 52, 945-953 (2010).</li></ol>

Dr. Rountree receives research support from Bayer Pharmaceutics. Drs. Ding and Crooks and Ms. Dang and Ms. VanKirk have no disclosures to report.

Unlike the hematopoietic system, in which hematopoietic stem cells are responsible for maintaining a cellular differentiation system that replacesnormal physiologic turn-over of leukocytes, red blood cells, and platelets, liver stem cell, or adult liver progenitor cells, do not participate in normal liver homeostasis.5,6 After acute liver injury or partial hepatectomy, hepatocytes, as differentiated liver epithelium, undergo several rounds of proliferation to replace the lost liver mass.5,6Only during chronic injury are liver stem cells observed to proliferate.1,5-11 These adult, organ specific stem cells are proposed to differentiate into both hepatocytes and cholangiocytes.1,5-8Interestingly, the vast majority of liver cancer develops on the background of chronic injury, and thus liver stem cells are also hypothesized to be the precursors for a subset of liver cancer.2-4,12-17
One of the major challenges to stem cell work in the liver is isolation of rare populations of cells for functional analysis. For example, the vast majority of cells in the liver are hepatocytes, which are significantly larger than cholangiocytes and other smaller non-parenchymal cells. By breaking the whole liver into cell compartments (large hepatocytes - parenchymal fraction and smaller cells - non-parenchymal fraction), and further selecting for CD45 negative cells (non-hematopoietic cells), we are able to enrich the starting population of stem cells by over 600-fold.1,18-21Of note, we are by no means indicating that the CD133+ population is a 100% pure stem cell population, but clearly represents a heterogeneous population of cells, with different lineage and repopulation potential. One of the major limitations of the field is definitions of stem cells and progenitor cells. We use the term "stem cell" more broadly in this work, but in strict definition, CD133+ non-parenchymal cells represent a bi-lineage progenitor population. Given the state of current stem and progenitor research in the liver, this report does provide a starting place for investigators who are interested in this field. As new markers emerge, such as EpCAM,22,23 or transcription factors, such as Sox9,24 they can be incorporated. For example, we have found a fairly high rate of overlap between EpCAM+ cells and CD133+ cells.
In this report, we detail a process for stem cell isolation from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after isolation.1,3,4 The specific goal of this process is to isolate a relatively pure population of liver stem cells that can be clonally expanded in vitro. 
The primary limitation to this procedure is that the majority of cells isolated will not be viable after flow cytometry (see Trouble Shooting section). This is the result of the hours required to prepare the cells, and the numerous procedures required to refine the population prior to isolation. If the liver is digested in single cell suspension for immediate FACS analysis, the size difference between hepatocytes and other non-parenchymal cells will make effective FACS gate creation impossible. If the liver non-parenchymal cells are utilized, without elimination of hematopoietic cells, there is a risk that a significant number of CD133+ cells may be of hematopoietic origin may contaminate the fraction. Furthermore, by processing the cells through the Miltenyi filter, only single cells and very small clusters of cells are collected. This ensures that the sample will not clog the FACS intake needle.
Alternatives for this procedure include modification for any alternative cell surface marker, such as CD49f, EpCAM, or combination of markers, such as CD133+EpCAM+A second published report utilizes a density gradient to isolate liver stem cells from a non-parenchymal fraction. This procedure requires an Ultra-centrifuge (8000 x g), adds significant time to the procedure, and in our experience, significantly reduced pre-FACS cellular yield and post-FACS viability.8
Future experiments include a broader range of gene expression analysis, including fetal liver genes, such as HNF3, HNF4&alpha;, and &alpha;fp.In addition, Western blot analysis and immunocytochemistry of expressed proteins can be utilized to confirm RT-PCR results of cells in culture.
One issue to note is recent work that identified CD133 expression on hepatic stellate cells.25 We now routinely screen our samples for markers of stellate cells (see Trouble Shooting section), and have not identified significant contamination in our fractions. This may be related to different techniques of liver digestion and cell isolation.2,3,26
Based on the fact that the majority of cells will not be viable immediately after FACS isolation, we recommend that the cells be plated, either as bulk CD133+ cells or single cells, prior to use in animals. 5-7 days in vitro will significantly improve results of tumor analysis. In addition, given the rigors of single cell isolation, this should only be conducted once colonies can be expanded from bulk CD133+ isolated cells. A more thorough discussion of the various culture conditions and scaffold proteins which may be utilized to culture liver stem and progenitor cells has been well characterized by Lola Reid. This work provides alternative conditions and modifications, which investigators may incorporate into their research program once basic isolation techniques are mastered.27,28 Dr. Reid's work also provides a more detailed analysis of the lineage biology and maturation between hepatic stem cells and committed progenitors. 
In terms of in vivo tumor analysis, we have had success using freshly isolated cells and cells from clonally expanded CD133+ cells in vitro, primarily using 1x106 cells. We have focused ontwo genetic strains of chronic liver injury, the MAT1a-/- and liver specific Pten-/- mice, and we have utilized both nude mice and wild-type mice as hosts for tumor growth. In our experience, CD133+ liver stem cells will only form tumors if they are isolated from significant liver injury models that are pre-malignant. Note that the tumors formed from CD133+ cells general have both hepatocellular carcinoma and cholangiocarcinoma features, suggesting a stem cell or progenitor cell origin to the tumors.2-4,29
Follow-up work after tumors are documented includes standard pathologic analysis of tumor tissue (H&amp;E staining) and immunohistochemical staining. In addition, tumors can be minced and digested for FACS analysis or re-culture.2-4,30
In conclusion, we have detailed a procedure for the isolation, expansion, and basic characterization of CD133+ liver stem cells and CD133+ cancer stem cells.
Trouble Shooting: 
CD45 contamination: 
For Step 3, if there is contamination of CD45+ cells, which can be assessed by adding a CD45-FITC Ab prior to FACS analysis and isolation, check to ensure that the CD45 microbead antibody is not expired and that the filtrate was collected only while the filter was in the magnetic holder. Any filtrate collected while the filter is not in the magnetic holder will contain CD45+ cells. 
Low cell number: 
For the uninjured liver, the total number of CD133+ non-parenchymal isolated may be less than 10,000. These cells are rare in the quiescent liver. For a chronic injury model, such as the DDC 0.1% diet, the number will increase greatly to 100,000 cells. If the total cell number is significantly below these numbers, one issue to consider is the FACS Ab staining. Check to ensure that the CD133 Ab is not expired, as poor staining will result in a poor yield. Also, we recommend performing a FACS analysis of liver non-parenchymal cells to determine the relative population prior to attempting FACS isolation. 
Low cell viability after FACS: 
One of the issues of viability may be related to how the cells are processed and over what time. Ideally, the entire cell isolation procedure, steps 1-4, should be conducted without any delays between steps and completed in the same day. Any significant delay between steps 1-4 will greatly reduce the cell viability. A second issue related to cell viability may be related to the sheath pressure used for FACS isolation. We recommend using a lower sheath pressure. Lastly, once the cells are isolated, they should be immediately plated, as any significant storage on ice after sorting will also reduce viability. 
CD133+ non-parenchymal heterogeneity: 
Recent reports indicate that hepatic stellate cells may also have CD133 expression and have some plasticity.25 Therefore, in addition to verifying bi-potency genes with Albumin and Krt19, additional verification can include genes associated with stellate cells, such as glial fibrillary acidic protein in quiescent stellate cells and alpha-smooth muscle actin and desmin in activated stellate cells.3,4,26 In addition, the CD133+ population represents a broader progenitor population. The addition of a second marker, such as EpCAM, may help to refine the population further, and limit heterogeneity.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Invitrogen</td>
			<td>10565-018</td>
			<td>With phenol red</td>
		</tr>
		<tr>
			<td>CD45 microbeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepatocyte Growth Factor	</td>
			<td>Sigma</td>
			<td>	H1404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor		</td>
			<td>Sigma</td>
			<td>E4127</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase			</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td>1 gram</td>
		</tr>
		<tr>
			<td>Collagenase D		</td>
			<td>Roche</td>
			<td>1088874</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pronase		</td>
			<td>Roche</td>
			<td>0165921</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 micron mesh strainer		</td>
			<td>Fisher</td>
			<td>352350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Omniscript RT			</td>
			<td>Quaigen</td>
			<td>205111</td>
			<td>50 reactions</td>
		</tr>
		<tr>
			<td>HotStarTaq</td>
			<td>Quaigen</td>
			<td>203203</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Miltenyi LD column</td>
			<td>Miltenyi</td>
			<td>130-042-901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CD133-PE FACS Ab		</td>
			<td>eBioscience</td>
			<td>12-1331-82</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin coated plates			</td>
			<td>BD</td>
			<td>354410</td>
			<td>96 well</td>
		</tr>
		<tr>
			<td>Trypsin 0.05% EDTA			</td>
			<td>Invitrogen</td>
			<td>25300-354</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit	</td>
			<td>Quaigen	</td>
			<td>74004	</td>
			<td>50 columns for 5x105 cells or less</td>
		</tr>
		<tr>
			<td>Pharm Lyse			</td>
			<td>BD</td>
			<td>555899</td>
			<td>10X concentration</td>
		</tr>
	</tbody>
</table>

isolation of cd133+ liver stem cells for clonal expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Watch this Scientific Journal Video about Isolation of CD133+ Liver Stem Cells for Clonal Expansion at JoVE.com

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

クローン性増殖のためのCD133 +肝幹細胞の単離

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

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22.3K ビュー.  Pennsylvania State College of Medicine. Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

ビデオ: Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

造血幹細胞の分離と移植（造血幹細胞）

生物学

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.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

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.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

マウス唾液腺幹細胞の単離

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

初代ヒト肝細胞と非実質肝細胞の対応する主な集団の単離のためのプロトコル

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...