Name: Författare i fokus: Undersöker angiogenes genom utmaningar och innovationer inom analysutveckling
Uploaded: 2024-05-31T14:50:00
Description: Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

Författarna tackar Sophie Krüger och Gabriele Prinz för deras utmärkta tekniska support. Vi tackar Sebastian Maier för att ha utvecklat pluginet ImageJ för att kvantifiera sfäroidskott och Lighthouse Core Facility, Zentrum für Translationale Zellforschung (ZTZ), Department of Medicine I, Universitetssjukhuset Freiburg för användningen av IncuCyte-systemet. Grafiken skapades med biorender.com. Detta arbete stöddes av Deutsche Forschungsgemeinschaft [Bu3135/3-1 + Bu3135/3-2 till F.B.], Medizinische Fakultät der Albert-Ludwigs- Universität Freiburg [Berta-Ottenstein-Program för kliniska forskare och avancerade kliniska forskare till F.B.], Else Kröner-Fresenius-Stiftung [2021_EKEA.80 till F.B.], det tyska cancerkonsortiet [CORTEX fellowship for Clinician Scientists till J.R.] och Volker Homann Stiftung [till J.N.+F.B.] och "Freunde der Universitäts-Augenklinik Freiburg e.V." [till P.L.]

1. Cellodling av HUVEC
OBS: Utför alla följande steg under sterila arbetsförhållanden (steril arbetsbänk).
<ol><li>Expanderande HUVEC:er<ol><li>För båda angiogenesanalyserna, använd poolade humana navelvensendotelceller (HUVEC) eller humana mikrovaskulära endotelceller (HMVECs).</li>
		<li>Odla cellerna tills de når 90 % sammanflöde i en obelagd T75-kolv med endotelcellstillväxtmedium (EGM) innan du utför en delning. Utför mediumbyte 3 gånger i veckan. OBS:...

Studie av angiogenes med hjälp av skrapsårmigration och sfäroidgroningsanalyser

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+wound+healing.">Angiogenesis in wound healing.</a> J Investig Dermatol Symp Proc. 5 (1), 40-46 (2000).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+angiogenesis+in+tumor+growth+and+metastasis.">Role of angiogenesis in tumor growth and metastasis.</a> Semin Oncol. 29, 15-18 (2002).</li><li>Crawford, T. N., Alfaro, D. V., Kerrison, J. B., Jablon, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy+and+angiogenesis.">Diabetic retinopathy and angiogenesis.</a> Curr Diabetes Rev. 5 (1), 8-13 (2009).</li><li>Ng, E. W., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+angiogenesis,+the+underlying+disorder+in+neovascular+age-related+macular+degeneration.">Targeting angiogenesis, the underlying disorder in neovascular age-related macular degeneration.</a> Can J Ophthalmol. 40 (3), 352-368 (2005).</li><li>Blanco, R., Gerhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegf+and+notch+in+tip+and+stalk+cell+selection.">Vegf and notch in tip and stalk cell selection.</a> Cold Spring Harb Perspect Med. 3 (1), 006569 (2013).</li><li>Hellstr&#246;m, M., Kal&#233;n, M., Lindahl, P., Abramsson, A., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+PDGF-B+and+PDGFR-&#946;+in+recruitment+of+vascular+smooth+muscle+cells+and+pericytes+during+embryonic+blood+vessel+formation+in+the+mouse.">Role of PDGF-B and PDGFR-&#946; in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.</a> Development. 126 (14), 3047-3055 (1999).</li><li>Nowak-Sliwinska, 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=Consensus+guidelines+for+the+use+and+interpretation+of+angiogenesis+assays.">Consensus guidelines for the use and interpretation of angiogenesis assays.</a> Angiogenesis. 21 (3), 425-532 (2018).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+forces+in+fibrillar+extracellular+matrices+control+directional+capillary+sprouting.">Tensional forces in fibrillar extracellular matrices control directional capillary sprouting.</a> J Cell Sci. 112, 3249-3258 (1999).</li><li>Rapp, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+and+3D+in+vitro+angiogenesis+assays+highlight+different+aspects+of+angiogenesis.">2D and 3D in vitro angiogenesis assays highlight different aspects of angiogenesis.</a> Biochim Biophys Acta Mol Basis Dis. 1870 (3), 167028 (2024).</li><li>Rapp, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+signaling+induced+by+the+IL-6+family+of+cytokines+modulates+angiogenesis.">STAT3 signaling induced by the IL-6 family of cytokines modulates angiogenesis.</a> J Cell Sci. 136 (1), 260182 (2023).</li><li>Heiss, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+spheroids+as+a+versatile+tool+to+study+angiogenesis+in+vitro.">Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro.</a> FASEB J. 29 (7), 3076-3084 (2015).</li><li>Rapp, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncostatin+m+reduces+pathological+neovascularization+in+the+retina+through+m&#252;ller+cell+activation.">Oncostatin m reduces pathological neovascularization in the retina through m&#252;ller cell activation.</a> Invest Ophthalmol Vis Sci. 65 (1), 22 (2024).</li><li>Fagotto, F., Gumbiner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+contact-dependent+signaling.">Cell contact-dependent signaling.</a> Dev Biol. 180 (2), 445-454 (1996).</li><li>Rapp, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressing+bias+in+manual+segmentation+of+spheroid+sprouting+assays+with+U-Net.">Addressing bias in manual segmentation of spheroid sprouting assays with U-Net.</a> Mol Vis. 29, 197-205 (2023).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J Exp Med. 115 (3), 453-466 (1962).</li><li>Guy, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+cell+invasion+and+migration+process:+A+comparison+of+the+video+microscope-based+scratch+wound+assay+and+the+Boyden+chamber+assay.">Evaluation of the cell invasion and migration process: A comparison of the video microscope-based scratch wound assay and the Boyden chamber assay.</a> J Vis Exp. (129), e56337 (2017).</li><li>Decicco-Skinner, K. L., 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=Endothelial+cell+tube+formation+assay+for+the+in+vitro+study+of+angiogenesis.">Endothelial cell tube formation assay for the in vitro study of angiogenesis.</a> J Vis Exp. (91), e51312 (2014).</li><li>Vernon, R. B., Angello, J. C., Iruela-Arispe, M. L., Lane, T. F., Sage, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+of+basement+membrane+matrices+by+cellular+traction+promotes+the+formation+of+cellular+networks+in+vitro.">Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro.</a> Lab Invest. 66 (5), 536-547 (1992).</li><li>Craig, L. E., Spelman, J. P., Strandberg, J. D., Zink, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+from+diverse+tissues+exhibit+differences+in+growth+and+morphology.">Endothelial cells from diverse tissues exhibit differences in growth and morphology.</a> Microvasc Res. 55 (1), 65-76 (1998).</li><li>M&#252;ller, A. M., Hermanns, M. I., Cronen, C., Kirkpatrick, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+adhesion+molecule+expression+in+cultured+human+macro-and+microvascular+endothelial+cells.">Comparative study of adhesion molecule expression in cultured human macro-and microvascular endothelial cells.</a> Exp Mol Pathol. 73 (3), 171-180 (2002).</li><li>Chiew, G. G. Y., Wei, N., Sultania, S., Lim, S., Luo, K. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+three-dimensional+co-culture+of+cancer+cells+and+endothelial+cells:+A+model+system+for+dual+analysis+of+tumor+growth+and+angiogenesis.">Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis.</a> Biotechnol Bioeng. 114 (8), 1865-1877 (2017).</li><li>Smith, L. 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I denna rapport presenterade vi ett spektrum av tekniker med funktionella och molekylära avläsningar för att studera angiogenes in vitro. 
Migrationsanalysen representerar en väletablerad teknik som används inom alla områden av våtlaboratoriearbete. Vi valde den kommersiellt tillgängliga metoden för avbildning av levande celler för att dra nytta av formatet med 96 brunnar som lämpar sig för screening och dos-responsexperiment, den standardiserade och reproducerbara sårst...

Angiogenes, som hänvisar till bildandet av nya blodkärl från redan existerande1, är en avgörande process under fysiologisk utveckling och patologiska tillstånd. Det är oumbärligt för att ge energi till mycket metaboliskt aktiva vävnader som näthinnan2 eller det utvecklande centrala nervsystemet3 och under läkning av skadad vävnad4. Onormal angiogenes är å andra sidan grunden för många sjukdomar. Solida tumörer, såsom kolorektal cancer eller icke-småcellig lungcancer, underlättar deras tillväxt och den nödvändiga energiförsörjningen genom att främja angiogenes5. Förutom cancer är neovaskulära sjukdomar i ögat som diabetisk retinopati eller åldersrelaterad makuladegeneration, som är de främsta orsakerna till blindhet i den utvecklade världen, ett resultat av avvikande kärltillväxt 6,7. En detaljerad förståelse av den underliggande patomekanismen är avgörande för att förstå hur fysiologisk angiogenes kan förbättras, till exempel vid sårläkning, samtidigt som man bättre kontrollerar patologiska tillstånd som vasoproliferativa ögonsjukdomar.
På cellulär nivå aktiveras vaskulära endotelceller av olika signalmolekyler i angiogenes, vilket initierar cellproliferation och migration8. Cellerna organiserar sig därefter i en hierarki, med icke-prolifererande spetsceller som bildar filopodier i framkanten av den utvecklande kärlgrenen8. Längs med följer snabbt växande stjälkceller spetscellerna, vilket bidrar till bildandet av det framväxande kärlet. Därefter rekryteras andra celltyper, såsom pericyter eller glatta muskelceller, för att ytterligare stabilisera den begynnande gren9.
För att utforska molekylära processer på vaskulär endotelcellsnivå har många in vitro-protokoll utvecklats och nyligen granskats10. Dessa analyser kan vanligtvis delas in i två kategorier: mer förenklade men skalbara 2D-metoder och mer avancerade 3D-protokoll. I ett nyligen genomfört projekt genomförde vi en omfattande jämförande analys mellan 2D-skrapsårmigrationsanalysen och 3D-sfäroidgroningsanalysen11 för att bedöma omfattningen av deras skillnader och deras förmåga att modellera olika aspekter av angiogenes12. 
Även om båda erbjuder fördelarna med att vara tillförlitliga och lätta att implementera, på molekylär nivå, var 3D-sfäroidgroningsanalysen gynnsam när det gällde att ta itu med viktiga aspekter av angiogenes jämfört med in vivo-data, såsom metaboliska omkopplare eller cell-matrisinteraktioner. Eftersom in vitro-analyser av angiogenes används för att utvärdera den angiomodulerande potentialen hos signalvägar13 och för att screena för terapeutiska medel, är det viktigt att in vitro-resultat kan överföras till in vivo-miljöer. Dessutom är möjligheten för omics-baserade analyser på RNA- och proteinnivå för att karakterisera de molekylära förändringarna som svar på riktad modulering av angiogena processer under kontrollerade förhållanden fortfarande en viktig fördel jämfört med in vivo-miljöer 14,15.
I den här publikationen presenterar vi viktiga analyser för att utforska angiogenesrelaterade frågor genom användning av en levande cellavbildningsanalys och en sfäroidspirande analys, inklusive efterföljande molekylära analyser som RNA-sekvensering för transkriptomisk analys och immunhistokemi på proteinnivå.

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		<tr>
			<td>10x Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M0650</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-(2-Hydroxyethyl)1-piperazinyl)-ethan-sulfons&#228;ure</td>
			<td>PAN-Biotec</td>
			<td>P05-01100</td>
			<td>HEPES</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated AffiniPure F(ab)&#8216;2-Fragment</td>
			<td>Jackson IR&#160;</td>
			<td>115-606-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Vert. A1</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CapturePro 2.10.0.1</td>
			<td>JENOTIK Optical Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type 1 rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche&#160;</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td>EBM</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Serva</td>
			<td>11290.02</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Bio&#38;SELL</td>
			<td>S 0615</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells, pooled</td>
			<td>Lonza</td>
			<td>C2519A</td>
			<td>HUVEC</td>
		</tr>
		<tr>
			<td>IncuCyte ImageLock 96-well plates&#160;</td>
			<td>Sartorius</td>
			<td>4379</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte S3 Live-Cell Analysis System</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methocel</td>
			<td>Sigma</td>
			<td>m-0512</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Endothelial Cell Medium with 10% FBS</td>
			<td>PB-MH-100-4090-GFP</td>
			<td>PELOBiotech</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Carl Roth</td>
			<td>P031.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-Fluorescein Isothiocyanate Labeled (0.5 mg/mL Methanol)</td>
			<td>Sigma-Aldrich</td>
			<td>P5282-.1MG</td>
			<td>Phalloidin-FITC</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Thermo Fisher Scientfic</td>
			<td>14190-094</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Primary Human Retinal Microvascular Endothelial Cells</td>
			<td>Cell Systems</td>
			<td>ACBRI 181</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Glass Antifade Mountant with NucBlue</td>
			<td>Invitrogen by ThermoFisher Scientific</td>
			<td>2260939</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>QIAGEN</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human Vascular Endothelial Growth Factor</td>
			<td>PeproTech</td>
			<td>100-20</td>
			<td>VEGF</td>
		</tr>
		<tr>
			<td>Squared petri dish</td>
			<td>Greiner</td>
			<td>688102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>PAN-Biotec</td>
			<td>P10-024100</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF-R2 (monoclonal)</td>
			<td>ThermoFisher Scientific Inc.</td>
			<td>B.309.4</td>
			<td></td>
		</tr>
		<tr>
			<td>WoundMaker</td>
			<td>Sartorius</td>
			<td>4493</td>
			<td></td>
		</tr>
	</tbody>
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investigating angiogenesis on a functional and molecular level by leveraging the scratch wound migration assay and the spheroid sprouting assay

Angiogenes spelar en avgörande roll i både fysiologiska och patologiska processer i kroppen, inklusive tumörtillväxt eller neovaskulär ögonsjukdom. En detaljerad förståelse av de underliggande molekylära mekanismerna och tillförlitliga screeningmodeller är avgörande för att effektivt rikta in sig på sjukdomar och utveckla nya behandlingsalternativ. Flera in vitro-analyser har utvecklats för att modellera angiogenes och dra nytta av de möjligheter som en kontrollerad miljö ger för att belysa angiogena drivkrafter på molekylär nivå och screena för terapeutiska mål.
Denna studie presenterar arbetsflöden för att undersöka angiogenes in vitro med hjälp av humana endotelceller i navelvenen (HUVEC). Vi beskriver en skrapsårmigrationsanalys med hjälp av ett levande cellavbildningssystem som mäter endotelcellsmigration i en 2D-miljö och sfäroidgroddanalysen som bedömer endotelcellsgroning i en 3D-miljö som tillhandahålls av en kollagenmatris. Dessutom skisserar vi strategier för provberedning för att möjliggöra ytterligare molekylära analyser såsom transkriptomik, särskilt i 3D-miljö, inklusive RNA-extraktion samt immunocytokemi. Sammantaget erbjuder detta ramverk forskare en pålitlig och mångsidig verktygslåda för att fullfölja sina vetenskapliga undersökningar i in vitro angiogenesanalyser.

Angiogenesis, which refers to the formation of new blood vessels from pre-existing ones1, is a crucial process during physiological development and pathologic conditions. It is indispensable for providing energy to highly metabolically active tissues such as the retina2 or the developing central nervous system3 and during the healing of damaged tissue4. Abnormal angiogenesis, on the other hand, is the basis for numerous diseases. Solid tumors, such as colorectal cancer or non-small cell lung cancer, facilitate their growth and the necessary energy supply by promoting angiogenesis5. Apart from cancer, neovascular diseases of the eye like diabetic retinopathy or age-related macular degeneration, which represent leading causes of blindness in the developed world, result from aberrant vessel growth6,7. A detailed understanding of the underlying pathomechanism is crucial to comprehend how physiological angiogenesis can be enhanced, for instance, in wound healing while better controlling pathological conditions such as vasoproliferative eye diseases.
On a cellular level, vascular endothelial cells are activated by various signaling molecules in angiogenesis, initiating cell proliferation and migration8. The cells subsequently organize into a hierarchy, with non-proliferating tip cells forming filopodia at the leading edge of the developing vessel branch8. Alongside, fast-proliferating stalk cells trail the tip cells, contributing to the formation of the emerging vessel. Subsequently, other cell types, such as pericytes or smooth muscle cells, are recruited to further stabilize the nascent branch9.
To explore molecular processes at the vascular endothelial cell level, numerous in vitro protocols have been developed and recently reviewed10. These assays typically fall into two categories: more simplistic but scalable 2D approaches and more elaborate 3D protocols. In a recent project, we conducted a comprehensive comparative analysis between the 2D scratch wound migration assay and the 3D spheroid sprouting assay11 to assess the extent of their differences and their ability to model various aspects of angiogenesis12. 
While both offer the advantages of being reliable and easily implementable, on a molecular level, the 3D spheroid sprouting assay was favorable in addressing key aspects of angiogenesis compared to in vivo data, such as metabolic switches or cell-matrix interactions. Since in vitro angiogenesis assays are used to evaluate the angiomodulatory potential of signaling pathways13 and to screen for therapeutic agents, transferability of in vitro results to in vivo settings is essential. Furthermore, the opportunity for omics-based analyses on the RNA and protein levels to characterize the molecular changes in response to targeted modulation of angiogenic processes under controlled conditions remains an important benefit compared to in vivo settings14,15.
In this publication, we present key assays for exploring angiogenesis-related questions through the utilization of a live-cell imaging migration assay and a spheroid sprouting assay, including subsequent molecular analyses like RNA sequencing for transcriptomic analysis and immunohistochemistry on the protein level.

Författare i fokus: Undersöker angiogenes genom utmaningar och innovationer inom analysutveckling

Undersöka angiogenes på en funktionell och molekylär nivå genom att utnyttja Scratch Wound Migration Assay och Spheroid Spiring Assay

Angiogenesis is a hallmark process involved in embryology and in disease development, including tumor growth and vasoproliferative eye disease. In this project, we aim to present a comprehensive framework of in vitro techniques that can be used to study angiogenesis on a molecular level in a very controlled setting, and to screen for potential therapeutic options. Translating in vitro to in vivo and ultimately to the clinical setting is challenging due to the risk of false positive or false negative results.Selecting the right angiogenesis assay is therefore crucial and knowing its limitations. This study aims to offer guidelines in how to interpret and how to establish two common angiogenesis assays. Recently, we compared a 2D scratch wound migration assay with a 3D spheroid sprouting assay.While the 2D assay is easily scalable, the 3D assay captures angiogenesis in greater detail, including tip stalk cell formation, matrix interaction, and the glycolytic switch. This data offer a scientists guidelines on which assay to choose next in their projects. Our laboratory will continue to characterize key aspects of the presented in vitro assays by directly comparing them to in vivo settings in order to facilitate translations of in vitro results to clinical applications.

För migrationsanalysen är det avgörande att noggrant undersöka de bilder som tagits vid tidpunkten t = 0 timmar för att säkerställa att närvaron av ett fullt utvecklat cellmonolager detekteras korrekt av systemet (figur 1B). Dessutom bör det bekräftas att den repiga kanten är klar och rak (figur 1B). Det cellfria området bör vara i stort sett fritt från skräp. I slutet av analysen bör en grupp som stimulerats med till exempel 25 ng/ml VEGF som en...

Denna studie presenterar den tvådimensionella (2D) skrapsårmigrationsanalysen och den tredimensionella (3D) sfäroidgroningsanalysen, tillsammans med deras respektive nedströmsanalysmetoder, inklusive RNA-extraktion och immunocytokemi, som lämpliga analyser för att studera angiogenes in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

investigating-angiogenesis-on-functional-molecular-level-leveraging

Research

JoVE Journal

Medicine

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Scratch Wound Migration Assay of Human Umbilical Vein Endothelial Cells (HUVECs) for Investigating Angiogenesis In Vitro

To begin, seed 100 microliters of HUVEC cells into the wells of a 96 well plate. Place the plate in a cell culture incubator at 37 degrees Celsius under 5%carbon dioxide for six hours to facilitate cell settling. Next, replace the medium in each well with 100 microliters of endothelial cell growth basal medium, containing 2%FBS, and incubate again.The following day, position the plate in a 96 well wound maker tool. Press down the lever of the device to generate a scratch. Carefully lift off the lid of the wound maker tool before releasing the lever to prevent double scratching.Wash the wells two times with 200 microliters of FBS-supplemented endothelial cell growth basal medium. After confirming total debris removal, pipette 100 microliters of the prepared stimulation or control solutions into each well. Use a live cell imaging microscope to acquire images every hour for 24 hours.The positive control showed successful migration in the original scratched area. Technical issues were seen as patchy cell growth, double scratching, or waves in the scratch borders. A 25%increase in relative wound distance was considered optimal.Incorrect separation of the positive and negative controls indicated low dynamic range.

Skrapsårmigrationsanalys av humana navelvensendotelceller (HUVEC) för undersökning av angiogenes in vitro

Fixing and Blocking 2D Human Umbilical Vein Endothelial Cell (HUVEC) Cultures for Immunohistochemical Analysis

To begin, add 30 milliliters of 5%potassium hydroxide in methanol to a 50 milliliter tube. Incubate 100 cover slips with a diameter of 12 millimeters in the tube for 30 minutes under constant agitation. Next, wash the cover slips in demineralized water for 30 minutes.Once washing is complete, transfer the cover slips into 70%isopropyl solution for storage. To coat the cover slips, first lean them individually against the wall of a square Petri dish. When the isopropyl alcohol has evaporated, transfer them into a 24 well plate.Pipette one milliliter of collagen in PBS into each well and incubate. Next, wash the cover slips about 4 to 5 times for five minutes each with one milliliter of PBS. Pipette cultured HUVECs into the wells with the coated cover slips.After culturing the cells for a desired length of time, wash the cells in one milliliter of PBS for five minutes. Fix the cells in one milliliter of 2%paraformaldehyde for 20 minutes at room temperature. Now, rinse the cells in one milliliter of PBS for 3 to 4 wash cycles of five minutes each.Finally, pipette one milliliter of blocking buffer into the well plate before staining.

Fixering och blockering av 2D humana navelvensendotelcellskulturer (HUVEC) för immunhistokemisk analys

Spheroid Sprouting Assay of Human Umbilical Vein Endothelial Cells (HUVECs) for Investigating Angiogenesis In Vitro 

To begin, add HUVECs into a 10 milliliter Falcon tube containing eight milliliters of endothelial growth medium. Add two milliliters of METHOCEL stock solution into the tube and shake well. Next, transfer 25 microliters of the mixture onto the inverted inner surface of a large, square Petri dish.Gently rotate the lid by 180 degrees and place it on the bottom of a cell culture vessel and incubate overnight. The next day, add 2.3 milliliters of rat tail collagen Type I to a vial containing 0.28 milliliters of 10X Medium 199. Keep this mixture on ice and mix until it is evenly yellow.Titrate the mixture against sodium hydroxide until it turns orange. Then, add 50 microliters of HEPES buffer to the final product. Collect the hanging cultured cells into a 50 milliliter Falcon tube with 20 milliliters of PBS, then centrifuge.After discarding the supernatant, add 0.1 milliliters of FBS and 0.4 milliliters of endothelial basal medium to the spheroid pellet. Gently tap the tube to resuspend the pellet. Now, pipette two milliliters of METHOCEL stock solution and mix well.Then, add two milliliters of the prepared collagen mixture to the resuspended spheroids. Dispense 0.5 milliliters of the resulting mixture into the wells of a 24-well plate and incubate. Next, pipette 100 microliters of diluted recombinant human vascular endothelial growth factor into the wells of the plate.Incubate the cell culture at 37 degrees Celsius under 5%carbon dioxide supplementation for 12 hours. The next day, with an inverted microscope, capture the images of the spheroids that are not in contact with the well rim, with each other, or show signs of damage. The spheroids stimulated with only basal medium or recombinant human vascular endothelial growth factor were clearly distinguishable.Spheroids in low-quality gels appeared to drop to the ground and dispersed. The negative control showed a moderate baseline sprouting rate while the human vascular endothelial growth factor doubled or tripled the relative sprouting length. The number of sprouts showed a similar dynamic range.

Sfäroidgroendeanalys av humana navelvensendotelceller (HUVEC) för undersökning av angiogenes in vitro 

Immunocytochemistry of Human Umbilical Vein Endothelial Cell (HUVEC) Spheroids in a 3D Collagen Matrix for Investigating Angiogenesis In Vitro

To begin, fix the sprouted spheroid HUVEC gels in one milliliter of 4%paraformaldehyde for one hour. Wash the gels gently in one milliliter of PBS about three to four times. Detach the gels fully from the surface of each well of the plate, then transfer them into new 24 well plates.Add one milliliter of blocking solution to the well plate, then incubate on an orbital shaker for one hour at room temperature. After incubation, pipette 500 microliters of the primary antibody solution into the wells before incubation. The next day, wash the gels gently with PBS for five wash cycles of five minutes each.Add 500 microliters of the corresponding secondary antibody diluted with phalloidin-FITC in blocking buffer. Incubate the plate overnight at four degrees celsius on an orbital shaker. After washing the gels in PBS, transfer them onto microscope slides.Add two drops of DAPI-containing mounting medium onto a cover slip and place it over each gel. Seal the dried gels with nail polish. Place them in the dark before storing at four degrees Celsius until further analysis.