Name: production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d
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Description: Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

Forfatterne takker T. Palmer og H. Babu (Stanford Neurokirurgi) for at give murine NPCs. vektor kunst i figur 4 blev anvendt og tilpasset fra Servier medicinsk Art under Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/ licenses/by/3.0/legalcode). En del af dette arbejde blev udført på den Stanford Nano delte faciliteter (SNSF), støttet af National Science Foundation under award ECCS-1542152. N.A.S. anerkender støtte fra National Institute of General Medical Sciences af National Institutes of Health (32GM 008412). C.M.M. anerkender støtte fra en NIH NRSA præ ph.d.-stipendium (F31 EB020502) og Siebel lærde Program. S.C.H. anerkender støtte fra National Institutes of Health (U19 AI116484 og R21 EB018407), National Science Foundation (DMR 1508006) og California Institute for regenerativ medicin (RT3-07948). Denne forskning har modtaget støtte fra den europæiske Alliance for regenerativ rehabilitering forskning & uddannelse (AR3T), som understøttes af Eunice Kennedy Shriver National Institute of Child Health og menneskelige udvikling (NICHD), Statens Institut for Neurologiske lidelser og slagtilfælde (NINDS) og Statens Institut for biomedicinsk Imaging og bioteknologi (NIBIB) af de nationale kontorer i sundhed under Award nummer P2CHD086843. Indholdet er udelukkende ansvarlig for forfattere og repræsenterer ikke nødvendigvis synspunkter af National Institutes of Health.

1. ELP udtryk protokol
<ol><li>Dag 1: Voksende starter koloni
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<li>Forberede ampicillin og chloramphenicol agar plader ved autoklavering 25 g Luria, bouillon og 15 g agar per 1 L ultrarent vand. Når løsningen er afkølet til ~ 60 ° C, der tilsættes 1 mL ampicillin stock (100 mg/mL i ultrarent vand) og 1 mL af chloramphenicol stock (34 mg/mL i 70% ethanol) til 1 L af agar løsning for endelige 100 µg/mL og 34 µg/mL , henholdsvis. Overføre 20 mL af endelige løsning til 10 cm petriskåle med ...

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A., Mrksich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+Stem+Cell+Fate+by+Controlling+the+Affinity+and+Density+of+Ligand-Receptor+Interactions+at+the+Biomaterials+Interface.">Directing Stem Cell Fate by Controlling the Affinity and Density of Ligand-Receptor Interactions at the Biomaterials Interface.</a> Angewandte Chemie International Edition. 51 (20), 4891-4895 (2012).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Hydrogel+Substrates+with+Tunable+Mechanical+Properties.">Preparation of Hydrogel Substrates with Tunable Mechanical Properties.</a> Current Protocols in Cell Biology. , 10.16.1-10.16.16 (2010).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. 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E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+recombinant+proteins+by+fusion+with+thermally-responsive+polypeptides.">Purification of recombinant proteins by fusion with thermally-responsive polypeptides.</a> Nature Biotechnology. 17 (11), 1112-1115 (1999).</li><li>Aladini, F., Araman, C., Becker, C. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+characterization+of+elastin-like+polypeptides+(ELPs)+with+variable+guest+residues.">Chemical synthesis and characterization of elastin-like polypeptides (ELPs) with variable guest residues.</a> Journal of Peptide Science. 22 (5), 334-342 (2016).</li><li>McMillan, R. A., Caran, K. L., Apkarian, R. P., Conticello, V. 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Rekombinante protein proteinekspression og -oprensning er et kraftfuldt værktøj til at syntetisere biomaterialer med høj reproducerbarhed. Grund stort set fremkomsten af kommercialiseret molekylære kloning, kan brugerdefinerede rekombinant plasmider købes fra flere leverandører, hvilket reducerer tid til at arbejde med materialer som ELPs. På samme måde, plasmider kan bestilles direkte fra den oprindelige lab, når den oprindelige arbejde blev støttet af en føderal kontrakt og den fremtidige arbejde bliver til ...

I det forløbne århundrede, har todimensionale (2D) vævskultur udviklet sig til en integreret værktøjssæt til at studere grundlæggende celle biologi i vitro. Derudover har de relativt billige og enkle protokoller for 2D cellekultur ført til dens vedtagelse på tværs af mange biologiske og medicinske discipliner. Dog har tidligere forskning vist, at traditionel 2D platforme kan føre til resultater at afvige markant fra de indsamlede i vivo, forårsager kostbar tid og finansiering spildt for klinisk orienteret forskning1,2, 3. Vi og andre hypotesen om, at denne uoverensstemmelse kan tilskrives manglen på native biokemiske og biofysiske stikord til cellerne kulturperler på 2D overflader, som kan være nødvendige for optimal spredning og modning af forskellige celletyper.
For at løse disse begrænsninger og hjælp bro over kløften mellem 2D har i in vitro og i vivo undersøgelser, forskerne udviklet tre-dimensionelle (3D) hydrogel platforme for celle-indkapsling1,4,5 ,6. Hydrogels er ideelle materialer til at sammenfatte den endogene mikromiljø ekstracellulære matrix (ECM) i vivo på grund af deres væv-lignende mekaniske egenskaber og vand-hævede struktur, der giver mulighed for hurtig transport af næringsstoffer og signalering faktorer7,8. Derudover kan 3D hydrogels være designet til at have uafhængig kontrol over de mekaniske og biokemiske egenskaber ved skafottet. Både matrix mekanik9,10,11,12 og celle-klæbende ligander13,14,15 er velkendt at påvirke celle adfærd i vitro og in vivo. Således, 3D hydrogels med afstemmelige egenskaber tilbyder en platform for at studere årsagssammenhænge mellem celler og deres mikromiljø. Kriterier for en ideel 3D hydrogel matrix omfatter enkle, ikke-cytotoksiske celle-indkapsling samt uafhængige tunability af fysiologisk relevante mekaniske egenskaber og efterligner af indfødte celle-dobbeltklæbende motiver.
Både syntetisk (fx., polyethylenglycol, polylactic syre, poly (glykolsyre)) og naturligt afledt (fx., natriumalginat, kollagen, Matrigel) hydrogels har fordele over 2D i vitro kultur platforme; men de har også betydelige mangler, som begrænser deres anvendelighed. Første, mange syntetisk og naturligt afledt platforme kræver barske crosslinking betingelser, som kan være potentielt giftige for pattedyrceller, hvilket fører til nedsat celle levedygtighed7. Derudover mange syntetiske platforme mangler native bioactivity og skal være functionalized gennem sekundære kemiske reaktioner, som kan tilføje øget omkostningerne og kompleksiteten16. Endelig, mens naturligt afledt materialer indeholder typisk iboende bio-aktive domæner, de er ofte plaget af høj batch til batch variationer og ofte er begrænset til danner relativt svage geler7,17.
Rekombinante protein engineering præsenterer en unik værktøjssæt til materialedesign af så eksplicit kontrol over protein sekvens, og dermed de potentielle mekaniske og biokemiske egenskaber af den endelige hydrogel stillads18. Derudover ved at udnytte den velkendte biologiske maskiner af Escherichia coli (E. coli) til at udtrykke proteiner, kan materialer produceres omkostningseffektivt og konsekvent med begrænset inter - og intra-batch variabilitet. Elastin-lignende protein (ELP) præsenteres her har tre manipuleret domæner: (1) en T7 og His6 tag, der giver mulighed for mærkning fluorescently mærkede antistoffer, (2) en 'elastin-lignende' region giver elastisk mekaniske egenskaber og giver mulighed for kemiske Crosslinking, og (3) en 'bio-aktive' region, der koder for celle-dobbeltklæbende motiver.
Vores elastin-lignende region er baseret på den kanoniske (Val-Pro-Gly-Xaa-Gly)5 elastin sekvens hvor fire af 'Xaa' aminosyre websteder er isoleucin (Ile), men kunne være designet til at være nogen aminosyre undtagen prolin. Denne sekvens forlener rekombinant ELPs med lavere kritisk løsning temperatur (LCST) adfærd, der kan udnyttes til simpel rensning efter udtryk via termisk cykling19,20. Denne LCST egenskab kan indstilles til termisk samlede ved forskellige temperaturer ved at ændre gæst 'Xaa' rester21,22.
Her, er 'Xaa' holdningen på en af de fem elastin-lignende gentagelser blevet erstattet med Amin-præsenterer lysin (Lys) amino syre, som er udnyttet til hydrogel crosslinking. Vores tidligere arbejde har vist ikke-cytotoksiske og robust crosslinking via reaktion med Amin-reaktive crosslinker tetrakis (hydroxymethyl) phosphonium chlorid (THPC)23. Af varierende samlede protein indhold og crosslinker koncentration er vi i stand til at producere hydrogels, der kan indstilles til at spænde over en fysiologisk relevante stivhed vifte (~0.5-50 kPa)9,23,24. Ud over tuning mekaniske egenskaber, celle vedhæftning inden for hydrogel resultaterne fra integration af canonical celle-klæbende domæner i rygraden i ELP protein. For eksempel, indarbejdelse af den udvidede fibronektin-afledte 'RGDS' aminosyresekvens giver mulighed for celle vedhæftning og konformationelle fleksibilitet, mens den scrambled, bindende 'RDGS' variant begrænser celle-matrix vedhæftning24. Af modulerende forholdet mellem celle-klæbende til ikke-klæbende proteiner samt den samlede proteinkoncentration, er vi i stand til effektivt at producere hydrogels, der spænder over en bred vifte af ligand koncentration. Resultantly, har vi udviklet en hydrogel platform med afkoblede biokemiske og biofysiske egenskaber, som kan indstilles uafhængigt for optimal 3D kultur af forskellige celletyper.
Matrix stivhed og klæbende ligand tunability har rekombinante hydrogels kapacitet til at design specifikke materialet nedbrydning profiler, som er nødvendige for celle spredning, spredning, og migration inden for en 3D sammenhæng4 , 9. denne nedbrydning, der ydes af celle sekretion af proteaser, der specifikt målretter enten udvidet 'RGDS'9 eller elastin-lignende sekvens25. ELP hydrogels har også vist sig at støtte de efterfølgende biokemiske analyser, der er nødvendige for at studere cellernes levedygtighed og funktion, herunder immuncytokemi samt DNA/RNA/protein udvinding for kvantitative bakgear transskription-polymerase kædereaktion (qRT-PCR) og Western blot9. ELP varianter har også været brugt i en række i vivo modeller og er kendt for at være veltolereret af immunsystemet26.
Taget under ét, ELP som en væsentlig platform for celle-indkapsling undersøgelser kan prale af en lang række fordele i forhold til syntetisk eller naturligt afledt materiale platforme, som ofte ikke har samme grad af biokemiske og biofysiske tunability og reproducerbarhed. Derudover ELPS enkle og ikke-cytotoksiske brug med en lang række celletyper (fx., chick dorsalrodsganglier14,24, murine neurale stamfader celler9, humane mesenkymale stamceller celler27, kvæg neonatal chondrocytter28, menneskelige endothelial celler29,30) giver mulighed for en mere fysiologisk relevante model af den endogene 3D ECM sammenlignet med 2D cellekultur. Heri, præsenterer vi en protokol for udtryk for recombinantly udvundet, ELPs til brug som en afstemmelige hydrogel platform for 3D cell indkapsling. Yderligere præsenterer vi metoden for down-stream fluorescerende mærkning og konfokal mikroskopi af indkapslede celler.

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Elastin-Like Protein Expression and Purification</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm Petri Dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">FB0875713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td>RICCA Chemical</td>
			<td style="width:123px;">2546.70-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">A3920-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP1760-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">9002-18-0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21(DE3)pLysS Competent Cells</td>
			<td>Invitrogen</td>
			<td style="width:123px;">C606003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloramphenicol</td>
			<td style="width:260px;">Amresco</td>
			<td style="width:123px;">0230-100G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA disodium salt, dihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">O2793-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">A451-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP1755-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Broth</td>
			<td style="width:260px;">EMD Millipore</td>
			<td>1.10285.5007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>VWR</td>
			<td style="width:123px;">52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>MP Biomedicals</td>
			<td style="width:123px;">195381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">S 8045-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Filter Unit (0.22 &#956;m)</td>
			<td>Millipore</td>
			<td style="width:123px;">SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terrific Broth</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:123px;">71754-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Encapsulation in 3D ELP Hydrogels</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#956;m syringe filters</td>
			<td style="width:260px;">Millipore</td>
			<td style="width:123px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 mm thick silicone sheet</td>
			<td>Electron Microscopy Science</td>
			<td style="width:123px;">70338-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well tissue culture plates&#160;</td>
			<td>Corning</td>
			<td style="width:123px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (2 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (4 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-34</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch (5 mm)</td>
			<td>Integra Miltex</td>
			<td style="width:123px;">33-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)&#160;</td>
			<td>Corning</td>
			<td style="width:123px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">No. 1 12 mm glass coverslips</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:123px;">12-545-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">404861-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5% Tryspin/EDTA</td>
			<td>Thermo Fisher&#160;</td>
			<td style="width:123px;">15400054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Immunocytochemistry of Cells in 3D ELP Hydrogels</td>
			<td style="width:260px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% (w/v) Paraformaldehyde (PFA)</td>
			<td style="width:260px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">15701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:260px;">Roche</td>
			<td style="width:123px;">3116956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Molecular Probes</td>
			<td style="width:123px;">D1306&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey Serum</td>
			<td style="width:260px;">Lampire Biological Labs</td>
			<td style="width:123px;">7332100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse Secondary Antibody (AF488)</td>
			<td style="width:260px;">Molecular Probes</td>
			<td style="width:123px;">A-11017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit Secondary Antibody (AF546)</td>
			<td style="width:260px;">Molecular Probes</td>
			<td style="width:123px;">A-11071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum</td>
			<td>Gibco</td>
			<td style="width:123px;">16210-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Mouse Nestin Primary Antibody</td>
			<td style="width:260px;">BD Pharmingen</td>
			<td style="width:123px;">556309</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Sox2 Primary Antibody</td>
			<td style="width:260px;">Cell Signaling Technology</td>
			<td style="width:123px;">23064S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Nail Polish</td>
			<td style="width:260px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">72180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">X100-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield Hardset Mounting Medium&#160;</td>
			<td style="width:260px;">Vector Labs</td>
			<td style="width:123px;">H-1400&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d

Todimensionale (2D) vævskultur teknikker har været afgørende for vores forståelse af grundlæggende cellebiologi. Men, traditionelle 2D vævskultur systemer mangler en tredimensional (3D) matrix, hvilket resulterer i en betydelig afbryde mellem resultater indsamlet in vitro- og in vivo. For at løse denne begrænsning, har forskere udviklet 3D hydrogel vævskultur platforme, der kan efterligne i vivo celle mikromiljø biokemiske og biofysiske egenskaber. Denne forskning har motiveret behovet for at udvikle materiale platforme, der understøtter 3D celle indkapsling og downstream biokemiske assays. Rekombinante protein engineering tilbyder en unik værktøjssæt til 3D hydrogel materiel design og udvikling ved at give mulighed for den specifikke kontrol af protein sekvens og derfor i forlængelse heraf resulterende potentielle mekaniske og biokemiske egenskaber matrix. Vi præsenterer her, en protokol for at udtrykke recombinantly afledt elastin-lignende protein (ELP), som kan bruges til form hydrogels med selvstændigt afstemmelige mekaniske egenskaber og celle-klæbende ligand koncentration. Præsenterer vi yderligere en metode for celle indkapsling i ELP hydrogels og efterfølgende immunfluorescent farvning af indlejrede celler for downstream analyse og kvantificering.

De ELPs bruges i denne protokol består af fem regioner: en T7 tag, His6 tag, enterokinase (EK) kavalergang site, en bio-aktive region og en elastin-lignende region (figur 1). Koderne T7 og His6 giver mulighed for nem identifikation gennem standard vestlige skamplet teknikker. Indførelsen af EK kavalergang site giver mulighed for den enzymatiske fjernelse af tag-regionen, hvis det er nødvendigt. Den bio-aktive region koder for udvidet, fibronektin-afledte c...

Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Rekombinante protein-manipuleret hydrogels er fordelagtige for 3D cellekultur, som de giver mulighed for komplet tunability af polymer rygrad og derfor celle mikromiljø. Her, beskriver vi processen med rekombinant elastin-lignende protein oprensning og dens anvendelse i 3D hydrogel celle indkapsling.

Produktionen af Elastin-lignende Protein Hydrogels til indkapsling og immunfarvning af celler i 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

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