Name: bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation
Uploaded: 2018-07-02T14:30:00
Description: Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

Tao Jiang tak Kina stipendium Rådet (201403170354) og McGill Engineering ph.d.-bedømmelse (90025) for deres stipendium midler. Jose G. Munguia-Lopez tak CONACYT (250279, 290936 og 291168) og FRQNT (258421) for deres stipendium midler. Salvador Flores-Torres tak CONACYT for deres stipendium midler (751540). Joseph M. Kinsella tak National Science og Engineering Research Council, den canadiske Foundation for Innovation, Townshend-Malene Stevnsbo Family Foundation og McGill University for deres finansiering. Vi vil gerne takke Allen Ehrlicher for at lade os bruge hans rheometer, Dan Nicolau for at lade os bruge hans Konfokal mikroskop, og Morag Park for at give os adgang til fluorescently mærket cellelinjer.

1. forberedelse af materialer, Hydrogel og celle kultur materialer
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Materiale og løsning forberedelse
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<li>Vask og tør 250 mL og 100 mL glas bægre, magnetiske omrørere, spatler, 10 mL patroner, 25 G cylindrisk dyser med (en længde på 0,5 i) og en indre diameter på 250 µm. Sterilisere materialer ved autoklavering dem ved 121 ° C/15 min./1 atm. holde materialer under sterile forhold indtil brug. Bemærk: Henvise til Bordet af materialer til leverandøroplysninger...

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	<li>Cui, X., Hartanto, Y., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+multicellular+spheroids+formation.">Advances in multicellular spheroids formation.</a> Journal of the Royal Society Interface. 14 (127), (2017).</li><li>Yip, D., Cho, C. 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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=Rapid+casting+of+patterned+vascular+networks+for+perfusable+engineered+three-dimensional+tissues.">Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.</a> Nature Materials. 11 (9), 768-774 (2012).</li><li>Jia, W., 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=Direct+3D+bioprinting+of+perfusable+vascular+constructs+using+a+blend+bioink.">Direct 3D bioprinting of perfusable vascular constructs using a blend bioink.</a> Biomaterials. 106, 58-68 (2016).</li><li>Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A., Lewis, J. 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H., Zhao, J. J., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+for+cancer+research.">Bioprinting for cancer research.</a> Trends in Biotechnology. 33 (9), 504-513 (2015).</li><li>Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+and+prototyping+of+tissues+and+scaffolds.">Printing and prototyping of tissues and scaffolds.</a> Science. 338 (6109), 921-926 (2012).</li><li>Nair, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cell+viability+during+bioprinting+processes.">Characterization of cell viability during bioprinting processes.</a> Biotechnology Journal. 4 (8), 1168-1177 (2009).</li><li>Costa, E. 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=3D+tumor+spheroids:+an+overview+on+the+tools+and+techniques+used+for+their+analysis.">3D tumor spheroids: an overview on the tools and techniques used for their analysis.</a> Biotechnology Advances. 34 (8), 1427-1441 (2016).</li><li>Zhao, 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=Three-dimensional+printing+of+Hela+cells+for+cervical+tumor+model+in+vitro.">Three-dimensional printing of Hela cells for cervical tumor model in vitro.</a> Biofabrication. 6 (3), 035001 (2014).</li><li>Ling, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting-based+high-throughput+fabrication+of+three-dimensional+MCF-7+human+breast+cancer+cellular+spheroids.">Bioprinting-based high-throughput fabrication of three-dimensional MCF-7 human breast cancer cellular spheroids.</a> Engineering. 1 (2), 269-274 (2015).</li><li>Liang, 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=A+cell-instructive+hydrogel+to+regulate+malignancy+of+3D+tumor+spheroids+with+matrix+rigidity.">A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity.</a> Biomaterials. 32 (35), 9308-9315 (2011).</li><li>Szot, C. S., Buchanan, C. F., Freeman, J. W., Rylander, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+in+vitro+bioengineered+tumors+based+on+collagen+I+hydrogels.">3D in vitro bioengineered tumors based on collagen I hydrogels.</a> Biomaterials. 32 (31), 7905-7912 (2011).</li><li>Carey, S. P., Kraning-Rush, C. M., Williams, R. M., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+control+of+invasive+tumor+cell+behavior+by+extracellular+matrix+microarchitecture.">Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture.</a> Biomaterials. 33 (16), 4157-4165 (2012).</li><li>Hospodiuk, M., Dey, M., Sosnoski, D., Ozbolat, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioink:+a+comprehensive+review+on+bioprintable+materials.">The bioink: a comprehensive review on bioprintable materials.</a> Biotechnology Advances. 35 (2), 217-239 (2017).</li><li>Caliari, S. R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+hydrogels+for+cell+culture.">A practical guide to hydrogels for cell culture.</a> Nature Methods. 13 (5), 405-414 (2016).</li><li>Bhutani, U., Laha, A., Mitra, K., Majumdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+alginate+and+gelatin+hydrogels:+viscosity+effect+on+hydrophobic+drug+release.">Sodium alginate and gelatin hydrogels: viscosity effect on hydrophobic drug release.</a> Materials Letters. 164, 76-79 (2016).</li><li>Biswal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mechanical+and+electrical+behavior+of+gelatin+hydrogels+on+drug+release+and+cell+proliferation.">Effect of mechanical and electrical behavior of gelatin hydrogels on drug release and cell proliferation.</a> Journal of the Mechanical Behavior of Biomedical Materials. 53, 174-186 (2016).</li><li>Rowley, J. A., Madlambayan, G., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+hydrogels+as+synthetic+extracellular+matrix+materials.">Alginate hydrogels as synthetic extracellular matrix materials.</a> Biomaterials. 20 (1), 45-53 (1999).</li><li>Djabourov, M., Leblond, J., Papon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelation+of+aqueous+gelatin+solutions.+I.+Structural+investigation.">Gelation of aqueous gelatin solutions. I. Structural investigation.</a> Journal de Physique (France). 49 (2), 319-332 (1988).</li><li>Djabourov, M., Leblond, J., Papon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelation+of+aqueous+gelatin+solutions.+II.+Rheology+of+the+sol-gel+transition.">Gelation of aqueous gelatin solutions. II. Rheology of the sol-gel transition.</a> Journal de Physique (France). 49 (2), 333-343 (1988).</li><li>Coussot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rheometry of Pastes, Suspensions, and Granular Materials: Applications in Industry and Environment. , (2005).</li><li>Ouyang, L., Yao, R., Zhao, Y., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bioink+properties+on+printability+and+cell+viability+for+3D+bioplotting+of+embryonic+stem+cells.">Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.</a> Biofabrication. 8 (3), 035020 (2016).</li><li>Michon, C., Cuvelier, G., Launay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+dependence+of+the+critical+viscoelastic+properties+of+gelatin+at+the+gel+point.">Concentration dependence of the critical viscoelastic properties of gelatin at the gel point.</a> Rheologica Acta Rheologica Acta: An International Journal of Rheology. 32 (1), 94-103 (1993).</li><li>Mouser, V. 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=Yield+stress+determines+bioprintability+of+hydrogels+based+on+gelatin-methacryloyl+and+gellan+gum+for+cartilage+bioprinting.">Yield stress determines bioprintability of hydrogels based on gelatin-methacryloyl and gellan gum for cartilage bioprinting.</a> Biofabrication. 8 (3), 035003 (2016).</li><li>Benbow, J. J., Oxley, E. W., Bridgwater, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extrusion+mechanics+of+pastes-the+influence+of+paste+formulation+on+extrusion+parameters.">The extrusion mechanics of pastes-the influence of paste formulation on extrusion parameters.</a> Chemical Engineering Science. 42 (9), 2151-2162 (1987).</li><li>Bingham, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluidity and plasticity. , (1922).</li><li>Horrobin, D. J., Nedderman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+entry+pressure+drops+in+paste+extrusion.">Die entry pressure drops in paste extrusion.</a> Chemical Engineering Science. 53 (18), 3215-3225 (1998).</li><li>Soman, 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=Cancer+cell+migration+within+3D+layer-by-layer+microfabricated+photocrosslinked+PEG+scaffolds+with+tunable+stiffness.">Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness.</a> Biomaterials. 33 (29), 7064-7070 (2012).</li><li>Asghar, W., 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=Engineering+cancer+microenvironments+for+in+vitro+3-D+tumor+models.">Engineering cancer microenvironments for in vitro 3-D tumor models.</a> Materials Today. 18 (10), 539-553 (2015).</li><li>Lin, R. 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Celle-belæsset strukturer kan blive kompromitteret, hvis forurening (biologiske eller kemiske) opstår på ethvert tidspunkt i processen. Normalt, biologisk forurening er set efter to eller tre dage af kulturen som en farve ændring i kultur medier eller bioprinted struktur. Derfor, sterilisation (fysisk og kemisk desinfektion) er et afgørende skridt for alle de celle-relaterede processer. Bemærkelsesværdigt, autoklavering gelatine ændrer sin geldannende egenskaber, som gjorde det gel langsommere i de forsøg, vi ha...

Selvom 2D cellekultur er meget udbredt i kræftforskning, findes begrænsninger som celler dyrkes i et éncellelag format med en ensartet koncentrationen af næringsstoffer og ilt. Disse kulturer mangler vigtige celle-celle og celle-matrix interaktioner i native tumor mikromiljø (TME). Derfor, disse modeller dårligt sammenfatte fysiologiske forhold, hvilket resulterer i afvigende celle adfærd, herunder unaturlige morfologier, uregelmæssige receptor organisation, membran polarisering og unormale Gen-ekspression, blandt andet betingelser1,2,3,4. På den anden side tilbyder 3D cellekultur, hvor celler er udvidet i en målekolbe plads som aggregater, spheroids eller organoids, en alternativ teknik for at skabe mere præcise in vitro- miljøer for at studere grundlæggende cellebiologi og fysiologi. 3D celle kultur modeller kan også fremme celle-ECM interaktioner, der er kritiske fysiologiske egenskaber af indfødte TME in vitro-1,4,5. Den nye 3D bioprinting-teknologi giver muligheder for at bygge modeller, der efterligner den heterogene TME.
3D bioprinting er afledt af rapid prototyping og giver mulighed for fremstilling af 3D mikrostrukturer, der er i stand til at efterligne nogle af kompleksiteten af levende væv prøver6,7. De nuværende bioprinting metoder omfatter inkjet, ekstrudering og laser-assisteret udskrivning8. Blandt dem giver metoden ekstrudering heterogenitet skal kontrolleres inden for de trykte matricer af præcis positionering forskellige typer af materialer på forskellige oprindelige placeringer. Det er derfor, den bedste fremgangsmåde til at fabrikere heterogene in vitro- modeller som omfatter flere typer af celler eller matricer. Ekstrudering bioprinting har held været anvendt til at opbygge auricular formet stilladser9, vaskulære strukturer10,11,12, og huden væv13, hvilket resulterer i høje udskrivning troskab og celle levedygtighed. Teknologien er også udstyret med alsidigt materiale valg, evnen til at deponere materialer med celler indlejret med en kendt tæthed, og høj reproducerbarhed14,15,16,17 . Naturlige og syntetiske hydrogels er ofte brugt som bioinks for 3D bioprinting på grund af deres biokompatibilitet, bioactivity og deres hydrofile netværk, der kan blive manipuleret til at strukturelt ligner ECM7,18 ,19,20,21,22,23. Hydrogels er også fordelagtigt, da de kan indeholde klæbende websteder for celler, strukturelle elementer, permeabilitet for næringsstoffer og luftarter, og de relevante mekaniske egenskaber til at fremme celle udvikling24. For eksempel tilbyder kollagen hydrogels integrin anchorage websteder, at celler kan bruge til at knytte til matrixen. Gelatine, denatureret kollagen, bevarer lignende celle vedhæftning websteder. Derimod calciumalginat bioinert men giver mekaniske integritet ved at danne krydsbindinger med divalent ioner25,26,27,28.
I dette arbejde udviklet vi et komposit hydrogel som en bioink, bestående af natriumalginat og gelatine, med ligheder til den mikroskopiske arkitektur i en native tumor stroma. Brystkræftceller og fibroblaster var indlejret i hydrogels og udskrives via en ekstrudering-baserede bioprinter for at oprette en 3D-model, der efterligner i vivo mikromiljø. Manipuleret 3D miljø tillader kræftceller til at danne flercellede tumor spheroids (MCTS) med en høj rentabilitet i lange perioder af cellekultur (> 30 dage). Denne protokol viser metoder for syntese sammensatte hydrogels, kendetegner materialer mikrostruktur og trykproces, bioprinting cellular heterogene modeller, og observere dannelsen af MCTS. Disse metoder kan anvendes til andre bioinks i ekstrudering bioprinting samt om forskellige designs heterogent væv modeller med potentielle anvendelser i stof screening, celle migration assays og undersøgelser, der fokuserer på grundlæggende celle fysiologiske funktioner.

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Sodium alginate</td>
			<td style="width:213px;">FMC BioPolymer</td>
			<td style="width:173px;">CAS-No: 9005-38-3</td>
			<td style="width:167px;">Protanal LF 10/60 FT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Gelatin</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td style="width:173px;">G9391</td>
			<td>Type B gelatin from bovine skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Dubelcco&#39;s phosphate buffered saline (DPBS 1X)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:173px;">LS14190136</td>
			<td>1&#215;, w/o calcium, w/o magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Magnetic hotplate</td>
			<td style="width:213px;">Corning&#160;</td>
			<td style="width:173px;">N/A</td>
			<td>Stirrer/hot plate model PC-420</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">50 mL centrifuge tubes</td>
			<td style="width:213px;">Corning</td>
			<td style="width:173px;">352098</td>
			<td>Falcon&#174; 50mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Centrifuge</td>
			<td style="width:213px;">GMI</td>
			<td style="width:173px;">N/A</td>
			<td>Sorvall RT6000D, GMI, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride anhydrous</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">MilliQ water</td>
			<td style="width:213px;">Millipore</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Millipore 0.22 &#181;m filters</td>
			<td style="width:213px;">Millipore</td>
			<td style="width:173px;">SLGS033SB</td>
			<td>Millex-GS Syringe Filter Unit, 0.22&#160;&#181;m, mixed cellulose esters, 33&#160;mm, ethylene oxide sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Oscillation rheometer MCR 302</td>
			<td style="width:213px;">Anton Paar</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Rheometer measuring tool CP25</td>
			<td style="width:213px;">Anton Paar</td>
			<td style="width:173px;">79038</td>
			<td>Conical plate geometry for rheometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">RheoCompass</td>
			<td style="width:213px;">Anton Paar</td>
			<td style="width:173px;">N/A</td>
			<td>Software controlling rheometer MCR 302</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Scanning electron microscope</td>
			<td style="width:213px;">Hitachi</td>
			<td style="width:173px;">N/A</td>
			<td>SEM, Hitachi SU-3500 Variable Pressure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Paraformaldehyde, 96%, extra pure</td>
			<td style="width:213px;">Acros Organics</td>
			<td style="width:173px;">416785000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Dulbecco modified eagle medium (DMEM)</td>
			<td style="width:213px;">Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic/Antimycotic solution (100X) stabilized</td>
			<td style="width:213px;">Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Wisent Bioproducts</td>
			<td style="width:173px;">080-150</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:372px;">Cell culture T-75 flasks</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td style="width:173px;">CLS430641</td>
			<td>75 cm2 TC-Treated surface treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D bioprinter BioScaffolder 3.1</td>
			<td style="width:213px;">GeSiM</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeSim software</td>
			<td style="width:213px;">GeSiM</td>
			<td style="width:173px;">N/A</td>
			<td>Software controlling BioScaffolder 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc cartridge UV resist</td>
			<td>EFD Nordson</td>
			<td>7012126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">End cap</td>
			<td>EFD Nordson</td>
			<td>7014472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tip cap</td>
			<td>EFD Nordson</td>
			<td>7014469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piston&#160;</td>
			<td>EFD Nordson</td>
			<td>7012182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless nozzle G25</td>
			<td>EFD Nordson</td>
			<td>7018345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Water bath</td>
			<td style="width:213px;">VWR</td>
			<td style="width:173px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Agarose</td>
			<td style="width:213px;">Sigma-Aldrich</td>
			<td style="width:173px;">A9539</td>
			<td>Bioreagent, for molecular biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Costar 6-well plates&#160;</td>
			<td style="width:213px;">Corning</td>
			<td style="width:173px;">3516</td>
			<td>TC-Treated Multiple Well Plates, Individually Wrapped, Sterile&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Confocal spinning disk inverted microscope</td>
			<td style="width:213px;">Olympus Life Science</td>
			<td style="width:173px;">N/A</td>
			<td>Olympus IX83</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">MTS assay kit</td>
			<td style="width:213px;">Promega</td>
			<td style="width:173px;">G3582</td>
			<td>CellTiter 96&#174; AQueous&#160;One Solution Cell Proliferation Assay&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Live/Dead viability cytotoxicity kit</td>
			<td>Molecular Probes,ThermoFisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin 0.25/EDTA 1X</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Corning 96-well plate</td>
			<td style="width:213px;">Corning</td>
			<td style="width:173px;">3595</td>
			<td>Clear Flat Bottom Polystyrene TC-Treated Microplate, Individually Wrapped, with Low Evaporation Lid, Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Autoclave Tuttnauer</td>
			<td style="width:213px;">Heidolph Brinkmann</td>
			<td style="width:173px;">N/A</td>
			<td>Heidolph Tuttnauer 2540E Autoclave Sterilizer Electronic Model with 4 Stainless Steel Trays, 23L Capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Trypan blue</td>
			<td style="width:213px;">Invitrogen&#160;</td>
			<td style="width:173px;">T10282</td>
			<td>0.4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Ethanol</td>
			<td style="width:213px;">Commercial Alcohols</td>
			<td style="width:173px;">P016EA95</td>
			<td>Greenfield Speciality Alcohols</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:372px;">CO2 Incubator</td>
			<td style="width:213px;">Panasonic</td>
			<td style="width:173px;">N/A</td>
			<td>MCO 19AIC-PA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Lyophilizer&#160;</td>
			<td style="width:213px;">SP Scientific</td>
			<td style="width:173px;">N/A</td>
			<td>Virtis Sentry 2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">SolidWorks</td>
			<td style="width:213px;">Dassault Systems</td>
			<td style="width:173px;">N/A</td>
			<td>A CAD software used to build demostrative propeller-like model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">MATLAB</td>
			<td style="width:213px;">The MathWorks</td>
			<td style="width:173px;">N/A</td>
			<td>A programming software used to generate G-code for BioScaffolder 3.1</td>
		</tr>
	</tbody>
</table>

bioprintable alginate/gelatin hydrogel 3d in vitro model systems induce cell spheroid formation

Den cellulære, biokemiske og biofysiske heterogenitet af indfødte tumor mikromiljø er ikke sammenfattet af voksende udødeliggjort kræft cellelinjer ved hjælp af konventionelle todimensionale (2D) cellekultur. Disse udfordringer kan overvindes ved hjælp af bioprinting teknikker til at bygge heterogene tredimensionale (3D) tumor modeller hvor forskellige typer af celler er integreret. Calciumalginat og gelatine er to af de mest almindelige biomaterialer ansat i bioprinting på grund af deres biokompatibilitet, Biomimetik og mekaniske egenskaber. Ved at kombinere de to polymerer, opnåede vi en bioprintable sammensatte hydrogel med ligheder til den mikroskopiske arkitektur i en native tumor stroma. Vi studerede trykproces sammensatte hydrogel via reologi og opnået den optimale udskrivning vindue. Brystkræftceller og fibroblaster var indlejret i hydrogels og udskrives til at danne en 3D-model efterligne i vivo mikromiljø. Bioprinted heterogen model opnår en høj rentabilitet for langsigtet cellekultur (> 30 dage) og fremmer den samlesæt af brystkræftceller i flercellede tumor spheroids (MCTS). Vi observerede migration og interaktion af kræft-associerede fibroblastceller (cafïr) med MCTS i denne model. Ved hjælp af bioprinted celle kultur platforme som fælles kultur systemer, tilbyder det et unikt værktøj til at studere afhængighed af tumordannelse stroma sammensætning. Denne teknik har en høj overførselshastighed, lave omkostninger og høj reproducerbarhed, og det kan også give en alternativ model til konventionelle éncellelag cellekulturer og animalske tumor modeller at studere kræft biologi.

Temperatur feje viser en tydelig forskel på A3G7 forløber på 25 ° C og 37 ° C. Forstadiet er flydende ved 37 ° C og har en kompleks viskositet af 1938.1 ± 84,0 mPa x s, som er valideret af en større G "over G'. Da temperaturen falder, gennemgår forløberen fysiske gellation på grund af den spontane fysiske indvikling af gelatine molekyler i en tri-helix dannelse29,30. Begge G' og G "øge og konvergerer på 30.6 ° C, med...

Watch this Scientific Journal Video about Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation at JoVE.com

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Vi udviklede en heterogen bryst kræft model bestående af udødeliggjort tumor og fibroblast celler indlejret i en bioprintable calciumalginat/gelatine bioink. Modellen sammenfatter i vivo tumor mikromiljø og letter dannelsen af flercellede tumor spheroids, giver indsigt i de mekanismer, der driver tumordannelse.

Bioprintable calciumalginat/gelatine Hydrogel 3D In Vitro modelsystemer fremkalde celle klumpformet dannelse

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

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Bioengineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Øjenirritation Test (EIT) for Hazard Identifikation af Eye Irriterer Kemikalier hjælp rekonstrueret human Cornea-lignende Epithelial (RHCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Modeling Kræft i æggestokkene flercellede Sfæroide Behavior i en dynamisk 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

en<em&gt; In vitro</em&gt; Orgel Kultur Model af muse diskusprolaps

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Forbedret 3D Hydrogel kulturer af primære Glial celler for In Vitro modellering af Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

3D trykt porøse Cellulose Nanocomposite Hydrogel stilladser

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

En menneskelig 3D ekstracellulær matrix-Adipocyte kultur model for at studere matrix-celle metaboliske Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Protokoller af 3D Bioprinting af gelatine Methacryloyl hydrogel baserede Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Brug af multilayered Hydrogel Bioink i tredimensionel bioprint til homogen cellefordeling

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Avancerede 3D-levermodeller til in vitro genotoksicitetstest efter langvarig nanomaterialeeksponering

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

3D-udskrivning af in vitro hydrogelmikrobærere ved vekslende viskøs inertial kraftstråle

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...