فإن الكتاب أن نعترف مؤسسة بيكر و &quot;النساء في زمالة العلوم&quot; مؤسسة الملكي للأطفال بدعم من Zerina Lokmic. أندرو G. Elefanty وإدوارد G. ستانلي هي NHMRC كبار زملاء الأبحاث. وأيد العمل في المختبرات من قبل NHMRC والخلايا الجذعية أستراليا.

<ol>
	<li>Baluk, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+specialized+junctions+between+endothelial+cells+of+lymphatic+vessels.">Functionally specialized junctions between endothelial cells of lymphatic vessels.</a> J Exp Med. 204 (10), 2349-2362 (2007).</li><li>Yao, L. C., Baluk, P., Srinivasan, R. S., Oliver, G., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+button-like+junctions+in+the+endothelium+of+airway+lymphatics+in+development+and+inflammation.">Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.</a> Am J Pathol. 180 (6), 2561-2575 (2012).</li><li>Zawieja, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+physiology+of+lymphatics.">Contractile physiology of lymphatics.</a> Lymphat Res Biol. 7 (2), 87-96 (2009).</li><li>Witte, M. 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=Structure+function+relationships+in+the+lymphatic+system+and+implications+for+cancer+biology.">Structure function relationships in the lymphatic system and implications for cancer biology.</a> Cancer Metastasis Rev. 25 (2), 159-184 (2006).</li><li>Andrade, M., Jacomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+human+lymphatic+system.">Anatomy of the human lymphatic system.</a> Cancer Treat Res. 135, 55-77 (2007).</li><li>Kumar, V., Abbas, A. K., Aster, J. C., Ch Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cotran Pathologic Basis of Disease. 43-78. 2, (2009).</li><li>Shayan, R., Achen, M. G., Stacker, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+vessels+in+cancer+metastasis:+bridging+the+gaps.">Lymphatic vessels in cancer metastasis: bridging the gaps.</a> Carcinogenesis. 27 (9), 1729-1738 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mulliken &amp; Young's Vascular Anomalies Hemangiomas and. , 1059-1076 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mulliken &amp; Young's Vascular Anomalies Hemangiomas. 14, 562-594 (2013).</li><li>Bellini, C., Hennekam, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+disorders+of+primary+malfunctioning+of+the+lymphatic+system.">Clinical disorders of primary malfunctioning of the lymphatic system.</a> Adv Anat Embryol Cell Biol. 214, 187-204 (2014).</li><li>Schook, C. 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=Primary+lymphedema:+clinical+features+and+management+in+138+pediatric+patients.">Primary lymphedema: clinical features and management in 138 pediatric patients.</a> Plast Reconstr Surg. 127 (6), 2419-2431 (2011).</li><li>Brouillard, P., Boon, L., Vikkula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+lymphatic+anomalies.">Genetics of lymphatic anomalies.</a> J Clin Invest. 124 (3), 898-904 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=for+the+Study+of+Vascular+Anomalies+(ISSVA)+classification.">for the Study of Vascular Anomalies (ISSVA) classification.</a> Available from: http://www.issva.org/classification.com. , (2014).</li><li>Croteau, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposiform+lymphangiomatosis:+a+distinct+aggressive+lymphatic+anomaly.">Kaposiform lymphangiomatosis: a distinct aggressive lymphatic anomaly.</a> J Pediatr. 164 (2), 383-388 (2014).</li><li>Lokmic, Z., 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+lymphatic+malformation+endothelial+cells,+their+in+vitro+characterization+and+in+vivo+survival+in+a+mouse+xenograft+model.">Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model.</a> Angiogenesis. 17 (1), 1-15 (2014).</li><li>Smith, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+malformations.">Lymphatic malformations.</a> Lymphat Res Biol. 2 (1), 25-31 (2004).</li><li>Chen, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similar+histologic+features+and+immunohistochemical+staining+in+microcystic+and+macrocystic+lymphatic+malformations.">Similar histologic features and immunohistochemical staining in microcystic and macrocystic lymphatic malformations.</a> Lymphat Res Biol. 7 (2), 75-80 (2009).</li><li>Norgall, 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=Elevated+expression+of+VEGFR-3+in+lymphatic+endothelial+cells+from+lymphangiomas.">Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.</a> BMC cancer. 7 (2), 75-80 (2007).</li><li>Garzon, M. C., Huang, J. T., Enjolras, O., Frieden, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+malformations.">Vascular malformations.</a> Part I. J Am Acad Dermatol. 56 (3), 353-370 (2007).</li><li>Osborn, A., Dickie, P., Adams, D., Dickie, 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> 20th International workshop on Vascular Anomalies. ISSVA. , (2014).</li><li>Baluk, P., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+microscopic+imaging+of+lymphangiogenesis+and+angiogenesis.">Markers for microscopic imaging of lymphangiogenesis and angiogenesis.</a> Ann N Y Acad Sci. 1131, 1-12 (2008).</li><li>Lokmic, Z., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+microcirculation.+Tissue+engineering.">Engineering the microcirculation. Tissue engineering.</a> Part B, Reviews. 14 (1), 87-103 (2008).</li><li>Lokmic, Z., Mitchell, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+and+stereological+assessment+of+blood+and+lymphatic+vessels.">Visualisation and stereological assessment of blood and lymphatic vessels.</a> Histol Histopathol. 26 (6), 781-796 (2011).</li><li>Hagerling, R., 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=A+novel+multistep+mechanism+for+initial+lymphangiogenesis+in+mouse+embryos+based+on+ultramicroscopy.">A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy.</a> EMBO J. 32 (5), 629-644 (2013).</li><li>Pollmann, C., Hagerling, R., Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+lymphatic+vessel+development,+growth,+and+function.">Visualization of lymphatic vessel development, growth, and function.</a> Adv Anat Embryol Cell Biol. 214, 167-186 (2014).</li><li>Breiteneder-Geleff, 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=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> Am J Pathol. 154 (2), 385-394 (1999).</li><li>Kaipainen, 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=Expression+of+the+fms-like+tyrosine+kinase+4+gene+becomes+restricted+to+lymphatic+endothelium+during+development.">Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.</a> Proc Natl Acad Sci U S A. 92 (8), 3566-3570 (1995).</li><li>Schacht, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T1alpha/podoplanin+deficiency+disrupts+normal+lymphatic+vasculature+formation+and+causes+lymphedema.">T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.</a> EMBO J. 22 (14), 3546-3556 (2003).</li><li>Kriehuber, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+dermal+lymphatic+and+blood+endothelial+cells+reveal+stable+and+functionally+specialized+cell+lineages.">Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.</a> J Exp Med. 194 (6), 797-808 (2001).</li><li>Florez-Vargas, 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=Comparative+analysis+of+D2-40+and+LYVE-1+immunostaiing+in+lymphatic+malformations.">Comparative analysis of D2-40 and LYVE-1 immunostaiing in lymphatic malformations.</a> Lymphology. 41 (3), 103-110 (2008).</li><li>Makinen, 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=PDZ+interaction+site+in+ephrinB2+is+required+for+the+remodeling+of+lymphatic+vasculature.">PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.</a> Genes Dev. 19 (3), 397-410 (2005).</li><li>Connell, F. 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=Analysis+of+coding+regions+of+VEGFR3+and+VEGFRC+in+Milroy+disese+and+other+primary+lymphoedemas.">Analysis of coding regions of VEGFR3 and VEGFRC in Milroy disese and other primary lymphoedemas.</a> Hum Genet. 124 (6), 625-631 (2009).</li></ol>

The authors have nothing to disclose.

موظفين المدنيين المحليين تلعب دورا هاما في الحفاظ على توازن السوائل، والاستجابة المناعية لمستضدات الخارجية وامتصاص ونقل بعض المواد الغذائية. يمكن أن يتأثر توازن LEC من خلال عمليات المرض مثل الالتهابات البكتيرية والورم ورم خبيث، ولكن يمكن أن موظفين المدنيين المحليي?...

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies (Gibco)</td>
			<td>11965-092</td>
			<td>Used to collect tissue samples from the operating theatre.</td>
		</tr>
		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>EGM-2 MV Bullet Kit contains 500 ml of EBM-2 media and human EGF, hydrocortisone, gentamycin (GA-1000), fetal bovine serum (FBS), VEGF, human FGF-b, R3-IGF-1 and ascorbic acid.</td>
		</tr>
		<tr>
			<td>VEGF-C</td>
			<td>R&#38;D Systems</td>
			<td>2179-VC-025</td>
			<td>Complete endothelial cell media is supplemented with 50 ng/mL VEGF-C</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution (100x)</td>
			<td>Life Technologies (Gibco)</td>
			<td>15240-062</td>
			<td>This solution contains 10,000U/ml of penicillin, 10,000 mg/ml of streptomycin and 25 mg/ml of Fungizone antimycotic. Use at 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Used at 10 mg/ml concentrations. It can be re-used for up to 1 month if kept at 4 degrees and maintained sterile. Use 7 mL to coat 150 cm2 flask.</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell dissociation reagent</td>
			<td>Life Technologies (Gibco)</td>
			<td>A11105-01</td>
			<td>StemPro&#174;Accutase&#174; is used at 0.05 ml per cm2 to cover the entire surface area of the flask. Using lesser volumes may result in incomplete cell detachment.</td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue dye</td>
			<td>Life Technologies (Gibco)</td>
			<td>15250-061</td>
			<td>Used as a viability stain.</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche Applied Biosciences</td>
			<td>4942078001</td>
			<td>Used at 0.04%</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Worthington Lab</td>
			<td>4176</td>
			<td>Used at 0.25%</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche Applied Biosciences</td>
			<td>11284932001</td>
			<td>Used at 0.01%</td>
		</tr>
		<tr>
			<td>70 mm nylon cell strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated VEGFR-3 clone 9D9F9, clone WM59</td>
			<td>BioLegend</td>
			<td>356204</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated CD34, clone 581</td>
			<td>BioLegend</td>
			<td>343516</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti human CD31, clone WM59</td>
			<td>BioLegend</td>
			<td>303116,</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated rat anti human PODOPLANIN, clone NC-80.</td>
			<td>BioLegend</td>
			<td>337006</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>PE-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400112</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400126</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400120</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated mouse IgG2a, k isotype, clone RATK2758</td>
			<td>BioLegend</td>
			<td>400525</td>
			<td>Used at 1:200 dilution</td>
		</tr>
	</tbody>
</table>

isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting

اضطرابات الجهاز اللمفاوي مثل وذمة لمفية الأولية، والتشوهات اللمفاوية وأورام اللمفاوية هي الحالات النادرة التي تسبب اعتلال كبير ولكن لا يعرف الكثير عن علم الأحياء الخاصة بهم. ان عزل خلايا نقي للغاية الإنسان اللمفاوية البطانية (موظفين المدنيين المحليين) من الأنسجة المريضة والسليمة تسهيل الدراسات البطانة اللمفاوية عند مستويات الوراثية، الجزيئية والخلوية. ومن المتوقع أن هذه التحقيقات قد تكشف عن أهداف لعلاجات جديدة يمكن أن تغير الإدارة السريرية لهذه الشروط. ويرد بروتوكول يصف عزل القلفة البشرية موظفين المدنيين المحليين وتشوه اللمفاوي الخلايا البطانية اللمفاوية (LM موظفين المدنيين المحليين). للحصول على ومفروم واحدة الأنسجة تعليق خلية وتعامل إنزيمي استخدام dispase الثاني وكولاجيناز II. ثم وصفت مما أدى تعليق خلية واحدة مع الأجسام المضادة لمجموعة من التمايز (CD) علامات CD34، CD31، الأوعية الدموية غشائي عامل نمو 3 (VEGFR-3) وPODOPLANIN. STAIتم فرز خلايا قابلة للحياة نيد على فارز خلية تنشيط fluorescently (FACS) لفصل CD34 CD31 منخفضة نقاط البيع VEGFR-3 نقاط البيع PODOPLANIN نقاط البيع LM LEC السكان من البطانية وغيرها من الخلايا غير البطانية. وLM موظفين المدنيين المحليين مرتبة كانت مثقف وتوسعت في قوارير فبرونيكتين المغلفة للاستخدام مزيد من التجارب.

بعد الهضم الأنسجة الأولي، بعد 24 ساعة في الثقافة العينات غير المجزأ، متميزة مستعمرات الخلايا البطانية ويمكن ملاحظة (الشكل 2A) جنبا إلى جنب مع خلايا تشبه الخلايا الليفية وخلايا العضلات الملساء. وبعد الفرز وبعد 24 ساعة في الثقافة الخلية، CD34 CD31 منخفضة نقاط الب?...

Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

والهدف من هذا البروتوكول هو عزل الخلايا البطانية المبطنة اللمفاوية تشوه اللمفاوي السفن وغلفة مثل الكيس البشرية باستخدام الخلايا مضان تنشيط الفرز (FACS). زراعة الخلايا اللاحقة والتوسع في هذه الخلايا تسمح مستوى جديد من التطور التجريبي للدراسات الجينية، البروتين والفنية وتمايز الخلايا.

عزل خلايا الإنسان اللمفاوي البطانية التي كتبها متعدد المعلمة تنشيط الإسفار الفرز خلية

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

isolation-human-lymphatic-endothelial-cells-multi-parameter

Research

JoVE Journal

Medicine

14.2K Views.  The Royal Children&#146;s Hospital.  والهدف من هذا البروتوكول هو عزل الخلايا البطانية المبطنة اللمفاوية تشوه اللمفاوي السفن وغلفة مثل الكيس البشرية باستخدام الخلايا مضان تنشيط الفرز (FACS). زراعة الخلايا اللاحقة والتوسع في هذه الخلايا تسمح مستوى جديد من التطور التجريبي للدراس...

Video: Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

في الوقت الحقيقي المعاوقة الكهربائية تقنية تستند إلى قياس غزو أحادي الطبقة الخلية البطانية من قبل خلايا السرطان

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

العزلة، وتوصيف المقارن التمييز بين الخلايا الجذعية البشرية الأسنان اللب المستمدة من الأسنان الدائمة باستخدام طريقتين مختلفتين

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

موت الخلايا المرتبطة الانقسام الشاذ التي لاحظها التصوير متحد البؤر في الخلايا السرطانية لايف

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

العزلة والثقافة، والتصوير من الجنين البنكرياس الإنسان الكتل الخليوي

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

دراسة سرطان البنكرياس الجذعية خصائص الخلايا لتطوير استراتيجيات علاجية جديدة

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

العزلة وثقافة توسيع ورم محددة الخلايا البطانية

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

العزلة والثقافة من الخلايا البطانية الأولية من الشرايين والأوردة الكلاب

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

عزل الخلايا البطانية السلف من المتطوعين الأصحاء وإمكاناتهم الكثيرة تأثر عينات المصل بعد جراحة القلب

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

إعداد و<em&gt; في المختبر</em&gt; توصيف ممغنط مير تعديل الخلايا البطانية

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.