Name: easy manipulation of architectures in protein-based hydrogels for cell culture applications
Uploaded: 2017-08-04T11:00:00
Description: Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Forfatterne vil gjerne takke Baden-Württemberg Stiftung for deres økonomiske støtte i "Bioinspired materiale syntese" framework (BioMatS-14).

1. Hydrogel forberedelse
<ol>
	<li>Bland 200 mg BSA med 1 mL av deionisert H2O lage 20% (w/v) BSA lager (lagerløsning A).</li>
	<li>Mix 165 µL THPC løsning (134 mg/mL) med 4.835 mL deionisert vann for å lage THPC lagerløsning (lagerløsning B).</li>
	<li>Veie 1 mg av KCSSGKSRGDS (1,111.1 g/mol) peptid (eller en tilsvarende celle-klebende peptid) og fortynne den i 100 µL av sterile H2O å få en 10 mg/mL løsning (lagerløsning C). Merk: Dette trinnet er valgfritt, og trenger bare å være...

<ol>
	<li>Geckil, H., Xu, F., Zhang, X., Moon, S., Demirki, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hydrogels+as+extracellular+matrix+mimics.">Engineering hydrogels as extracellular matrix mimics.</a> Nanomedicine. 5 (3), 469-484 (2011).</li><li>Liu, Y., Chan-Park, M. 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S., Mulyasasmita, W., Parisi-Amon, A., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-component+protein-engineered+physical+hydrogels+for+cell+encapsulation.">Two-component protein-engineered physical hydrogels for cell encapsulation.</a> Proc Nat Acad Sci USA. 106 (52), 22067-22072 (2009).</li><li>Zhu, J., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+properties+of+hydrogel+tissue-engineering+scaffolds.">Design properties of hydrogel tissue-engineering scaffolds.</a> Expert Rev Med Devic. 8 (5), 607-626 (2011).</li><li>Samaryk, 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=Versatile+Approach+to+Develop+Porous+Hydrogels+with+a+Regular+Pore+Distribution+and+Investigation+of+their+Physicomechanical+Properties.">Versatile Approach to Develop Porous Hydrogels with a Regular Pore Distribution and Investigation of their Physicomechanical Properties.</a> J Appl Polym Sci. 114 (4), 2204-2212 (2009).</li><li>Li, X. J., Valadez, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-+and+Protein-Based+Hydrogels.">Peptide- and Protein-Based Hydrogels.</a> Chem Mater. 24 (5), 759-766 (2012).</li><li>Bodenberger, N., 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=Beyond+bread+and+beer:+whole+cell+protein+extracts+from+baker's+yeast+as+a+bulk+source+for+3D+cell+culture+matrices.">Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.</a> Appl Microbiol Biot. 101 (5), 1-11 (2016).</li><li>Bodenberger, N., Paul, P., Kubiczek, D., Walther, P., Gottschalk, K. E., Rosenau, F. <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+cheap+and+easy+to+handle+protein+hydrogel+for+3D+cell+culture+applications+a+high+stability+matrix+with+tunable+elasticity+and+cell+adhesion+properties.">A novel cheap and easy to handle protein hydrogel for 3D cell culture applications a high stability matrix with tunable elasticity and cell adhesion properties.</a> Chem Sel. 1 (7), 1353-1360 (2016).</li><li>Agarwal, S., Wendorff, J. H., Greiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrospinning+technique+for+biomedical+applications.">Use of electrospinning technique for biomedical applications.</a> Polymer. 49 (26), 5603-5621 (2008).</li><li>Hong, J., deMello, A. J., Jayasinghe, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-electrospraying+and+droplet-based+microfluidics:+control+of+cell+numbers+within+living+residues.">Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues.</a> Biome Mater. 5 (2), 21001 (2010).</li><li>Jayasinghe, S. N., Irvine, S., McEwan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+electrospinning+highly+concentrated+cellular+suspensions+containing+primary+living+organisms+into+cell-bearing+threads+and+scaffolds.">Cell electrospinning highly concentrated cellular suspensions containing primary living organisms into cell-bearing threads and scaffolds.</a> Nanomedicine. 2 (4), 555-567 (2007).</li><li>Selimovi&#263;, &. #. 3. 5. 2. ;., Oh, J., Bae, H., Dokmeci, M., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+strategies+for+generating+cell-encapsulating+hydrogels.">Microscale strategies for generating cell-encapsulating hydrogels.</a> Polymers. 4 (3), 1554-1579 (2012).</li><li>Annabi, N., Nichol, J. 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=Controlling+the+porosity+and+microarchitecture+of+hydrogels+for+tissue+engineering.">Controlling the porosity and microarchitecture of hydrogels for tissue engineering.</a> Tissue Eng Part B Rev. 16 (4), 371-383 (2010).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. 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B., Gnanamani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+and+redox+sensitive+albumin+hydrogel:+A+self-derived+biomaterial.">pH and redox sensitive albumin hydrogel: A self-derived biomaterial.</a> Sci Rep. 5, 15977 (2015).</li><li>Hennink, W. E., van Nostrum, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+crosslinking+methods+to+design+hydrogels.">Novel crosslinking methods to design hydrogels.</a> Adv Drug Deliver Rev. 64, 223-236 (2012).</li><li>Chung, C., Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrakis(hydroxymethyl)+phosphonium+chloride+as+a+covalent+cross-linking+agent+for+cell+encapsulation+within+protein-based+hydrogels.">Tetrakis(hydroxymethyl) phosphonium chloride as a covalent cross-linking agent for cell encapsulation within protein-based hydrogels.</a> Biomacromolecules. 13 (12), 3912-3916 (2008).</li><li>Cal&#243;, E., Khutoryanskiy, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+hydrogels:+A+review+of+patents+and+commercial+products.">Biomedical applications of hydrogels: A review of patents and commercial products.</a> Eur Polym J. 65, 252-267 (2015).</li><li>Huang, H., Herrera, A. I., Luo, Z., Prakash, O., Sun, X. 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The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of th...

Hydrogels er materialer som uløselig 3D nettverk kan binde store mengder vann. Slike materialer kan gi fremragende miljøforhold for levende celler. Foreløpig er det økende interesse i generasjonen av tredimensjonale hydrogel strukturer og utvikling av prosesser å skreddersy sine kjemiske og fysiske egenskaper. Når dette er oppnådd, kan en mal for veksten av celler og manipulering av mobilnettet atferd være generert1,2,3,4. Disse 3D strukturer ikke bare skape et mer naturlig og realistisk miljø enn konvensjonelle todimensjonal tilnærminger, men de også avsløre nye muligheter for vekst av stamceller eller svulst modeller5. Ulike materialer har en rekke egenskaper som hovedsakelig avhenger porestørrelse av gel6. Porene spille en avgjørende rolle i celle kultur programmer, tissue engineering og regissert veksten av stamceller. For eksempel oksygen og næringsstoffer diffus gjennom matrisen og tilstrekkelig mengder må kunne nå celler7. På den annen side, skadelige metabolitter må fjernes så raskt som mulig, og nok plass til cellevekst må være tilgjengelig7. Følgelig påvirke egenskapene til materialet, og dermed porestørrelse, alvorlig den potensielle fordel og mulige anvendelser av matrix. Avhengig av egenskapene til materialet, kan ulike cellevekst prosesser oppstå i 3D cellekultur, inkludert dannelsen av neuronal strukturer; vekst og differensiering av huden eller bein celler. og regissert veksten av spesielle stamcelleforskningen linjer, som hepatocytter eller fibroblaster2,3,8,9,10,11. En annen avgjørende punktet påvirker mulig anvendelse av et materiale er dens stabilitet mot ytre stimuli12. For eksempel må hydrogel opprettholde integriteten mekanisk i celle kultur medier eller menneskekroppen.
De siste årene, forskning på 3D celle kultur hydrogels intensivert, og mange studier var gjennomført for å løse de 3D arkitekturene systemer13. Hydrogels består av kjemisk syntetisert komponenter er oftest undersøkt fordi de kan lett syntetisk og kjemisk endret og de viser høy stabilitet (se Zhu et al., 2011 for en gjennomgang)5. Men proteiner har mange fordelaktige egenskaper: som såkalte "presisjon polymerer," de er biokompatible; de har en definert lengde; de er relativt lett å endre; og de har et stort antall mål nettsteder14,15. I denne forbindelse, kan svært spesiell strukturer genereres for programmet på mange felt. I denne studien, ble en protein-basert hydrogel16 brukt til å demonstrere veletablerte metoder å påvirke 3D arkitektur av materialet. Videre ble evnen og anvendbarhet pore generasjon også undersøkt.
Mange ulike teknikker er tilgjengelig endre 3D strukturer, inkludert både enkle metoder og sofistikert, høyt spesialiserte teknikker fra ulike felt av materielle vitenskap. En utbredt teknikken er bruk av electrospinning å generere veldefinerte strukturer17. Ladet fibre trekkes fra en løsning av et elektrisk felt og deretter stivne ved kontakt med oksygen. På denne måten kan fiber i området flere nanometer til flere mikrometer produseres. Flere teknikker for å justere størrelsen, struktur og distribusjon av porene innenfor matrix er myk litografi, klima og jordsmonn, etter fokus, elektro-sprøyting og bio-utskrift18,19,20. En betydelig ulempe av disse teknikkene er avhengigheten på spesifikke, dyrt utstyr og spesielle kjemikalier eller materialer. Videre erfaring med disse teknikkene er ofte ikke direkte overføres til protein-basert materiale, og mange av kjemikalier og metodene er ikke cellen kompatibel.
På den annen side, stol mange teknikker ikke på spesialutstyr, noe som gjør dem enklere og billigere å bruke og å reprodusere. En utbredt metode for struktur manipulasjon er solvent avstøpning21,22,23. Partikler legges før polymerisasjon reaksjonen og distribueres homogenously for å mette løsningen. Etter polymerisasjon fører en endring av forhold, som en fortynning eller en pH endring, til solvation av partikler, mens porene forblir i materialet. Kjemikaliene som brukes i disse teknikkene, som salt, sukker, parafin, gelatin og kritt, er billig og lett tilgjengelig. I Frysetørring, er hovne hydrogels frosset. Etterfølgende sublimering av flytende faser under et vakuum er utført23,24,25. Vann sublimering fra nettverket er skånsom nok å opprettholde spesifikke 3D strukturer av materialet. I gass skummende, streames en løsning med en gass mens polymerisasjon foregår, forlater porene i gel21. Størrelse og distribusjon av porene kan justeres avhengig gasstrømmen.
For å danne den protein hydrogel, er BSA reagert med tetrakis (hydroxymethyl) phosphonium chloride (THPC) i en Mannich-type reaksjon å tillate dannelsen av kovalente bindinger mellom primære aminer og de hydroxy gruppene fire bevæpnede koblingsfunksjonalitet molekyl26. Mulig skadelig mellomprodukter fjernes ved overdreven vask av materialet etter reaksjonen oppstår.
Denne studien viser muligheten for behandling av en BSA-basert materiale med ulike teknikker for å manipulere og tilpasse størrelsen på porene. Hver av teknikkene kan brukes i alle laboratorium verden over, som ingen spesielt utstyr er nødvendig. I tillegg hensyn ulike parametre, slik som hevelse forholdet, enzymatisk nedbrytbarhet, pH stabilitet og temperatur følsomhet, undersøkt og forhold til hverandre, spesielt til påvirkning av ulike teknikker på generering av 3D arkitekturer. Endelig var materialer functionalized med celle-klebende peptider å undersøke mulig bruk av materialet til cellekultur. To forskjellige modell linjer ble brukt: A549 og MCF7.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:479px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:253px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">10010023</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (high glucose)</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Nonessential Amino Acid Solution</td>
			<td>Sigma Aldrich</td>
			<td>M7145-100ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin EDTA 0.05 % Phenol Red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>&#214;lfabrik Schmidt</td>
			<td>2133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Carl Roth&#160;</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumin Fraction V</td>
			<td>Carl Roth&#160;</td>
			<td>3854.2</td>
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		<tr height="20">
			<td height="20" style="height:20px;">THPC</td>
			<td>Sigma Aldrich</td>
			<td>404861-100ML</td>
			<td>Toxic</td>
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			<td height="20" style="height:20px;">0.1 % Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td>Slightly toxic</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin-rhodamine&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3.7 % Formaldehyde&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>F8775-25ML</td>
			<td>Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rhodamine B</td>
			<td>Sigma Aldrich</td>
			<td>81-88-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filtropur S 0.2,&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>2 83.1826.001&#160;&#160;&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">&#181; slide 8 well</td>
			<td>Ibidi GmbH</td>
			<td>80826</td>
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		<tr height="20">
			<td height="20" style="height:20px;">KCSSGKSRGDS peptide</td>
			<td>UPEP Ulm</td>
			<td>Custom sysnthesis</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>Carl Roth&#160;</td>
			<td>K928.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 5 ml Polysterene Round-Bottom Tube&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubes 50 ml&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubes 1,5 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,690,001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubes 2 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,691</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td>
			<td>Greiner</td>
			<td>655073</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FreezeDryer Epsilon 1-6D,&#160;</td>
			<td>Christ, Osterode am Harz, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Laser Scanning Microscope&#160;</td>
			<td>Carl Zeiss AG, Oberkochen, Germany</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484</td>
			<td>Carl Zeiss AG, Oberkochen, Germany</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7</td>
			<td>GSA GmbH</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

easy manipulation of architectures in protein-based hydrogels for cell culture applications

Hydrogels gjenkjennes som lovende materialer for celle kultur programmer evne til å gi svært hydratiserte celle miljøer. Feltet 3D maler er økende på grunn av potensielle likheten av disse materialene til naturlige ekstracellulær matrix. Protein-basert hydrogels er særlig lovende fordi de kan lett bli functionalized og kan oppnå definerte strukturer med justerbar mekanisk-egenskaper. Men er produksjon av macroporous 3D maler for celle kultur programmer ved hjelp av naturlige materialer ofte begrenset etter svakere mekaniske egenskaper sammenlignet med de av syntetiske materialer. Her metoder ble vurdert for å produsere macroporous bovin serum albumin (BSA)-baserte hydrogel systemer, med justerbar pore størrelser mellom 10-70 µm i radius. Videre, en metode for å generere kanaler i denne protein-basert materiale som er flere hundre mikron lange ble etablert. De forskjellige metodene for å produsere porene, samt påvirker porestørrelse materialegenskaper som hevelse forholdet, pH, temperatur stabilitet og enzymatisk degradering atferd, ble analysert. Pore størrelser ble undersøkt i hydrogels med AC confocal mikroskopi for laserskanning opprinnelig, hovne staten. Muligheten for celle kultur programmer ble vurdert en celle-klebende RGD peptid endring av protein systemet og to modell linjer: menneskelige brystkreft celler (A549) og adenocarcinomic menneskelig alveolar basale epitelceller (MCF7).

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron micr...

Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Forskjellige metoder for å manipulere tredimensjonale arkitektur i protein-basert hydrogels er evaluere her materialegenskaper. Macroporous-nettverk er functionalized med en celle-klebende peptid og deres gjennomførbarhet i cellekultur evaluert med to forskjellige modell linjer.

Enkel manipulering av arkitekturer i Protein-basert Hydrogels for celle kultur programmer

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Research

JoVE Journal

Biochemistry

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