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

Författarna vill tacka Baden-Württemberg Stiftung för deras finansiella stöd inom ramen för ”Bioinspired Material syntes” (BioMatS-14).

1. Hydrogel förberedelse
<ol>
	<li>Blanda 200 mg av BSA med 1 mL avjoniserat H2O skapa 20% (w/v) BSA lager (lager lösning A).</li>
	<li>Blanda 165 µL THPC lösning (134 mg/mL) med 4.835 mL avjoniserat vatten för att skapa THPC stamlösning (stamlösning B).</li>
	<li>Väger 1 mg KCSSGKSRGDS (1,111.1 g/mol) peptid (eller en motsvarande cell-lim-peptid) och späd det i 100 µL sterilt H2O få en 10 mg/mL lösning (stamlösning C). Obs: Detta steg är valfritt och behöver bara inkluderas om h...

<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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomimetic+hydrogel+based+on+methacrylated+dextran-graft-lysine+and+gelatin+for+3D+smooth+muscle+cell+culture.">A biomimetic hydrogel based on methacrylated dextran-graft-lysine and gelatin for 3D smooth muscle cell culture.</a> Biomaterials. 31 (6), 1158-1170 (2010).</li><li>Raic, A., R&#246;dling, L., Kalbacher, H., Lee-Thedieck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+macroporous+PEG+hydrogels+as+3D+scaffolds+for+the+multiplication+of+human+hematopoietic+stem+and+progenitor+cells.">Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells.</a> Biomaterials. 35 (3), 929-940 (2014).</li><li>Wong Po Foo, C. T. S., Lee, J. 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. V., Zuo, P., Nie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+3D+cell+culture:+potential+application+for+tissue-based+bioassays.">Microfluidic 3D cell culture: potential application for tissue-based bioassays.</a> Bioanalysis. 4 (12), 1509-1525 (2012).</li><li>Park, H., Guo, X., 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+Swelling+Ratio+of+Injectable+Hydrogel+Composites+on+Chondrogenic+Differentiation+of+Encapsulated+Rabbit+Marrow+Mesenchymal+Stem+Cells+In+Vitro.">Effect of Swelling Ratio of Injectable Hydrogel Composites on Chondrogenic Differentiation of Encapsulated Rabbit Marrow Mesenchymal Stem Cells In Vitro.</a> Biomacromolecules. 10 (3), 541-546 (2010).</li><li>Stokols, S., Tuszynski, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fabrication+and+characterization+of+linearly+oriented+nerve+guidance+scaffolds+for+spinal+cord+injury.">The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury.</a> Biomaterials. 25 (27), 5839-5846 (2004).</li><li>Sung, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Impact+of+2D+and+3D+Fibroblast+Cultures+on+In+Vitro+Breast+Cancer+Models.">Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models.</a> PLoS One. 8 (10), 1-13 (2013).</li><li>Tsai, E. C., Dalton, P. D., Shoichet, M. S., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+hydrogel+guidance+channels+facilitate+regeneration+of+adult+rat+brainstem+motor+axons+after+complete+spinal+cord+transection.">Synthetic hydrogel guidance channels facilitate regeneration of adult rat brainstem motor axons after complete spinal cord transection.</a> J Neurotrauma. 21 (6), 789-804 (2004).</li><li>Shoichet, M. S., Li, R. H., White, M. L., Winn, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+hydrogels+used+in+cell+encapsulation:+An+in+vitro+comparison+of+alginate+and+agarose.">Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose.</a> Biotechnol Bioeng. 50 (4), 374-381 (1996).</li><li>Chiu, Y. -. C., Kocag&#246;z, S., Larson, J. C., Brey, E. M. <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+physical+and+mechanical+properties+of+porous+poly+(ethylene+glycol)-co-(L-lactic+acid)+hydrogels+during+degradation.">Evaluation of physical and mechanical properties of porous poly (ethylene glycol)-co-(L-lactic acid) hydrogels during degradation.</a> PloS One. 8 (4), e60728 (2013).</li><li>Jonker, A. M., L&#246;wik, D. W. P. M., van Hest, J. C. 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. A. <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+cell+culture+matrices:+state+of+the+art.">Three-dimensional cell culture matrices: state of the art.</a> Tissue Eng Part B Rev. 14 (1), 61-86 (2008).</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=Evaluation+of+methods+for+pore+generation+and+their+influence+on+physio-chemical+properties+of+a+protein+based+hydroge.">Evaluation of methods for pore generation and their influence on physio-chemical properties of a protein based hydroge.</a> Biotech Rep. 12, 6-12 (2016).</li><li>Raja, S. T. K., Thiruselvi, T., Mandal, A. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+transformation+and+physical+properties+of+a+hydrogel-forming+peptide+studied+by+NMR,+transmission+electron+microscopy,+and+dynamic+rheometer.">Structural transformation and physical properties of a hydrogel-forming peptide studied by NMR, transmission electron microscopy, and dynamic rheometer.</a> Biophys J. 103 (5), 979-988 (2012).</li><li>Draghi, L., Resta, S., Pirozzolo, M. G., Tanzi, 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=Microspheres+leaching+for+scaffold+porosity+control.">Microspheres leaching for scaffold porosity control.</a> J Mater Sci. 16 (12), 1093-1097 (2005).</li><li>Whang, 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=Engineering+Bone+Regeneration+with+Bioabsorbable+Scaffolds+with+Novel+Microarchitecture.">Engineering Bone Regeneration with Bioabsorbable Scaffolds with Novel Microarchitecture.</a> Tissue Eng. 5 (1), 35-51 (1999).</li><li>Ziv, 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=A+tunable+silk-alginate+hydrogel+scaffold+for+stem+cell+culture+and+transplantation.">A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation.</a> Biomaterials. 35 (12), 3736-3743 (2014).</li><li>Butruk-Raszeja, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Athrombogenic+hydrogel+coatings+for+medical+devices--Examination+of+biological+properties.">Athrombogenic hydrogel coatings for medical devices--Examination of biological properties.</a> Colloid Surface B. 130, 192-198 (2015).</li><li>L&#252;, S., Li, B., Ni, B., Sun, Z., Liu, M., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+injectable+hydrogel+for+three-dimensional+cell+culture:+chondroitin+sulfate+bioconjugated+with+poly(N-isopropylacrylamide)+synthesized+by+RAFT+polymerization.">Thermoresponsive injectable hydrogel for three-dimensional cell culture: chondroitin sulfate bioconjugated with poly(N-isopropylacrylamide) synthesized by RAFT polymerization.</a> Soft Matter. 7 (22), 10763 (2011).</li><li>Ruoslahti, E., Pierschbacher, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+in+cell+adhesion:+RGD+and+integrins.">New perspectives in cell adhesion: RGD and integrins.</a> Science. 238 (4826), 491-497 (1987).</li></ol>

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...

Hydrogeler är material som bildar olösliga 3D nätverk kan bindande stora mängder vatten. Sådant material kan ge utmärkta miljöförhållanden för levande celler. För närvarande, det finns ett ökat intresse i generationen av tredimensionella hydrogel strukturer och i utveckling av processer för att skräddarsy deras kemiska och fysikaliska egenskaper. När detta är uppnått, kan en mall för tillväxten av celler och manipulering av cellulära beteende vara genererade1,2,3,4. Dessa 3D-strukturer inte bara skapa en mer naturlig och realistisk miljö än konventionella tvådimensionell metoder, men de också avslöja nya möjligheter för tillväxt av stamceller eller tumör modeller5. Olika material besitter en rad egenskaper som främst är beroende av porstorlek av gel6. Porerna har en avgörande roll i cell kultur program, vävnadsteknik och riktad tillväxt av stamceller. Till exempel syre och näringsämnen diffunderar genom matrisen och tillräckliga mängder måste kunna nå de celler7. Å andra skadliga metaboliter måste tas bort så snabbt som möjligt och tillräckligt med utrymme för cellernas tillväxt måste vara tillgängliga7. Följaktligen, egenskaperna för materialet, och därmed porstorlek, allvarligt påverka de potentiella nytta och möjliga tillämpningarna av matrisen. Beroende på egenskaperna hos materialet, kan olika cell-tillväxt processer uppstå i 3D cellodling, däribland bildandet av neuronala strukturer. tillväxt och differentiering av celler i huden eller ben; och riktad tillväxt av särskilda stamcellslinjer, som hepatocyter eller fibroblaster2,3,8,9,10,11. En annan avgörande punkt påverkar en eventuell tillämpning av ett material är dess stabilitet mot yttre stimuli12. Till exempel måste hydrogel bibehålla sin mekanisk integritet i cell kulturmassmedia eller den mänskliga kroppen.
Under senare år har forskning om 3D cell kultur hydrogels intensifieras, och många studier genomfördes till lösa de 3D arkitekturerna av system13. Hydrogeler består av kemiskt syntetiserade komponenter undersöks oftast eftersom de kan enkelt syntetiseras och kemiskt modifierade och de uppvisar hög stabilitet (se Zhu et al., 2011 för en granskning)5. Proteiner har dock många välgörande egenskaper: som so-called ”precision polymers”, de är biokompatibla; de har en definierad längd; de är relativt lätta att ändra; och de har ett stort antal mål platser14,15. I detta avseende kan mycket specifika, innovativa strukturer genereras för tillämpning inom många områden. I denna studie användes en protein-baserade hydrogel16 att demonstrera förmågan av väletablerade metoder att påverka materialet 3D arkitektur. Dessutom undersöktes också den förmåga och tillämplighet till pore generation.
Det finns många olika tekniker att ändra 3D-strukturer, inklusive både enkla metoder och sofistikerade, högt specialiserade tekniker från olika områden inom materialvetenskap. En utbredd teknik är användningen av electrospinning att generera väl definierade strukturer17. Laddade fibrer dras från en lösning av ett elektriskt fält och sedan stelna vid exponering för syre. På detta sätt kan fibrer i spänna av flera nanometer upp till flera mikrometer produceras. Ytterligare tekniker att justera storlek, struktur och fördelning av porerna inom matrisen är mjuk litografi, photolithography, hydrodynamisk fokusering, electro-sprutning och bio-utskrift18,19,20. En betydande nackdel med dessa tekniker är deras beroendeställning specifika, dyr utrustning och särskilda kemikalier eller material. Dessutom erfarenhet med dessa tekniker är ofta inte direkt överföras till protein-baserade material, och många av de kemikalier och metoder är inte cell kompatibel.
Å andra sidan, lita många tekniker inte på särskild utrustning, göra dem enklare och billigare att tillämpa och att reproducera. En utbredd metod för struktur manipulation är lösningsmedel gjutning21,22,23. Partiklar läggs före polymerisation reaktionen och distribueras likartad för att mätta lösningen. Efter polymerisation leder en förändring av villkor, såsom en utspädning eller en pH förändring, till utläggning av partiklarna, medan porerna förblir inom materialet. Kemikalier som används i dessa tekniker, såsom salt, socker, paraffin, gelatin och krita, är billiga och lätt tillgängliga. I frystorkning, är svullna hydrogeler frysta. Den efterföljande sublimering av de flytande faserna under en vakuum är sedan utförs23,24,25. Vatten sublimering från nätverket är skonsam nog att bibehålla särskilda 3D-strukturer av materialet. I gasar skumbildning, direktuppspelas en lösning med en gas medan polymerisation sker, lämnar porer inom gel21. Storleken och fördelningen av porerna kan justeras beroende på gasströmmen.
För att bilda den protein hydrogel, är BSA reagerade med tetrakis (hydroximetyl) phosphonium chloride (THPC) i en Mannich-typ reaktion att möjliggöra bildandet av kovalenta bindningar mellan primära aminer och hydroxy grupper av fyra beväpnade linker molekyl26. Möjligt skadligt intermediärer avlägsnas genom överdriven tvättning av materialet efter reaktionen inträffar.
Denna studie visar möjligheten att behandla en BSA-baserat material med olika tekniker för att manipulera och anpassa storleken på porerna. Var och en av teknikerna som kan användas i alla laboratorier över hela världen, som ingen särskild utrustning är nödvändiga. Dessutom olika parametrar, såsom svullnad baserat, enzymatisk nedbrytbarhet, pH stabiliteten och temperatur känslighet, undersökts och förhållande till varandra, särskilt med avseende på påverkan av de olika teknikerna på generationen av 3D arkitekturer. Slutligen, material var functionalized med cell-självhäftande peptider för att undersöka en eventuell tillämpning av material till cellkultur. Två olika modell cellinjer användes: A549 och MCF7.

<table border="0" cellpadding="0" cellspacing="0" width="1102">
	<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">THPC</td>
			<td>Sigma Aldrich</td>
			<td>404861-100ML</td>
			<td>Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.1 % Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td>Slightly toxic</td>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#181; slide 8 well</td>
			<td>Ibidi GmbH</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCSSGKSRGDS peptide</td>
			<td>UPEP Ulm</td>
			<td>Custom sysnthesis</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<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

Hydrogeler redovisas som lovande material för cell kultur tillämpningar på grund av deras förmåga att tillhandahålla mycket hydrerad cell miljöer. Fältet för 3D mallar ökar på grund av den potentiella likheten mellan dessa material till naturliga extracellulärmatrix. Protein-baserade hydrogeler är särskilt lovande eftersom de kan enkelt vara functionalized och kan uppnå definierade strukturer med justerbar fysikalisk-kemiska egenskaper. Produktionen av får 3D mallar för cell kultur program som använder naturliga material begränsas dock ofta av deras svagare mekaniska egenskaper jämfört med de av syntetiska material. Här, olika metoder utvärderades för att producera avdunsta bovint serumalbumin (BSA)-baserade hydrogel system, med en justerbar porstorlek i intervallet 10 till 70 µm i radie. Dessutom inrättades en metod att generera kanaler i detta protein-baserade material som flera hundra mikrometer lång. De olika metoderna att producera porer, liksom påverkan av porstorlek på materialets egenskaper såsom svullnad baserat, pH, temperaturstabilitet och enzymatiska nedbrytningen beteende, analyserades. Porstorlek undersöktes i den infödda, svullna delstaten den hydrogels använder confocal microscopy för laserscanning. Genomförbarheten för cell kultur program utvärderades med hjälp av en cell-självhäftande RGD peptid modifiering av protein och två modell cellinjer: mänskliga bröstcancer cancerceller (A549) och adenocarcinomic human alveolär basala 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

Olika metoder för att manipulera tredimensionella arkitektur i protein-baserade hydrogels utvärderas här avseende materialegenskaper. Avdunsta nätverken är functionalized med en cell-lim-peptid och deras genomförbarhet i cellkultur utvärderas med hjälp av två olika modell cell linjer.

Enkelt att använda arkitekturer i Protein-baserade hydrogeler för Cell kultur applikationer

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

The overall goal of this methodology is to create macroporous hydrogels with varying pore sizes for cell culture applications. This method can help answer key questions in the field of 3D cell culture such as the principle feasibility of a novel hydrogel material for 3D cell culture applications. The main advantage of this methodology is that it offers a range of techniques to optimize and adjust a novel material to the requirements of different types of cells.To begin, dilute one milligram of the RGD peptide or an equivalent cell adhesive peptide in 100 microliters of sterile water to obtain the 10 milligram per milliliter solution. Next, remove the bottom of a 96 well plate and replace it with removal plastic wrap. Mix 100 microliters of BSA stock solution and 100 microliters of THPC stock solution in the 96 well plate to obtain 200 microliters of the hydrogel.Mix the components by pipetting up and down at least five times to guarantee a uniform hydrogel after polymerization. Place the 96 well plate at room temperature for about 10 minutes until all hydrogels are properly polymerized. Following polymerization, carefully and slowly remove the plastic wrap from the bottom of the plate.Press the hydrogels out of the 96 well plate using a small stab and transfer them to 1.5 milliliter tubes with sterile PBS pH 7.4. Store the hydrogels in phosphate buffered saline at four degrees Celsius for up to several months. To freeze-dry the hydrogels, first fill 1.5 milliliter tubes with 500 microliters of sterile deionized water and remove the caps.Transfer the hydrogels to the 1.5 milliliter reaction tubes using forceps. Wrap the 1.5 milliliter tubes tightly with paraffin film using at least three layers for each vial. Use a needle to pierce small holes in the film to enable gas release from the tubes.To guarantee that the water and hydrogel completely freeze, transfer the vial to liquid nitrogen solution for five minutes. Immediately after removal from the liquid nitrogen, transfer the vials to the freeze-dryer to prevent the material from thawing. As an alternative freezing step, keep the vials at minus 20 degrees Celsius overnight to slowly freeze the hydrogels.Immediately after removal from minus 20 degrees Celsius, transfer the vials to the freeze-dryer to prevent the material from thawing. After 24 hours and the complete evaporation of the water in and around the hydrogel, thaw the material by removing it from the freeze-dryer. For particle leaching, mix the components of the hydrogel as before.Following mixing, immediately add sodium chloride until saturation occurs. Add salt until salt crystals can be seen in the solution as a white precipitate. Pore sizes can be altered by grinding the salt crystals prior to use.Transfer the 96 well plate to a shaker and shake until polymerization takes place. After about 10 minutes, remove the hydrogels from the plate as before. Incubate the hydrogels for at least 24 hours at room temperature in sterile water on a rotator to elute all salt from the hydrogel template.Store the hydrogels at four degrees Celsius in PBS for up to several months. For gentle formation, prepare hydrogels as described. Then remove a hydrogel from solution using a pincer.Remove the excess water with highly absorbent paper and place it on top of a block of dry ice. Freeze the hydrogel for 30 seconds and carefully remove it from the block. Do not damage the hydrogel.Carefully use a spatula to scrape it off the block. Transfer the hydrogel to a 1.5 milliliter tube and dry it overnight at 37 degrees Celsius. Prepare a Rhodamine B stock solution as well as serial dilutions as described in the text protocol.Remove the hydrogel from the stored solution and transfer it to a 1.5 milliliter tube with one milliliter of 0.01 milligram per milliliter Rhodamine B solution. Stain the hydrogel overnight in Rhodamine B solution at room temperature. The next day, transfer the hydrogel that is to be visualized to 10 milliliters of PBS and wash for at least three hours.Cut small slices out of the hydrogel using a blade. Then transfer the hydrogel onto an eight well microscopic slide and cover it with PBS. Using a confocal laser scanning microscope, visualize the hydrogel at a wavelength of 514 nanometers.Sterile filter all stock solutions with a 0.45 micron filter. Begin preparation of the hydrogel and include the cell adhesive peptide prior to hydrogel polymerization. After mixing the components, immediately pipette the mixture into an eight well microscopic slide until the bottom is completely covered.Transfer the cells into pre-warmed sterile DMEM cell culture medium supplemented with fetal bovine serum, penicillin streptomycin, and nonessential amino acid solution. After counting the cells with a Neubauer counting chamber, carefully pipette 200 microliters of the desired number of cells onto the hydrogel surface. Cover the eight well microscopic slide with a lid and transfer it to an incubator.After at least four hours of cellular attachment, wash the cells twice with 200 microliters of sterile cell culture PBS. Next, fix the cells with 200 microliters of 3.7%formaldehyde for 10 minutes at room temperature and wash twice with PBS. Permeabilize the cells with 200 microliters of 0.1%Triton X for five minutes before washing twice more with PBS.Stain the cells with Toluidine Rhodamine by mixing five microliters of methanolic stock with 195 microliters of PBS and adding it to the cells at room temperature. Stain the cells for 20 minutes in the dark before washing twice with PBS. Investigate the cell adhesion properties using a confocal microscope at 514 nanometers.Analyze the hydrogel properties as described in the text protocol. As shown here, the polymerized and stained hydrogels have no distinctive architectures. After freeze-drying the hydrogels at different temperatures, the average size of the pores within the hydrogel can be extended up to 60 microns.Salt leaching results in uniform networks with pore sizes of about 10 microns. The size can be further adjusted by grinding salt crystals prior to use. The use of a gradient freezing approach on a block of dry ice leads to the formation of channels with a diameter of about 20 microns and a length of several hundreds of microns.Prior to functionalization with the cell adhesive moiety, cells cannot adhere to the surface of the hydrogel and show a round morphology. Only after providing the material with the cell adhesive peptide, the cells find focal adhesion points which enable a proper adhesion of the cell onto a material. This is crucial for use of novel materials in 3D cell culture applications.After watching this video, you should have a good understanding of how to manipulate a hydrogel's architecture with simple methods. This method can be applied for most novel hydrogel systems to learn about the use in cell culture and to optimize the hydrogel's architecture depending on the intended use.

Research

JoVE Journal

Biochemistry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

Framställning av Homogena MALDI Prover för kvantitativa Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Singel-molekyl Manipulation av G-quadruplexes av magnetisk pincett

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

En Fluorogenic peptid klyvning analysen till skärmen för den proteolytiska aktiviteten: program för coronavirus spike protein aktivering

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

Studie om metabolismen av sex systemiska insekticider i en nyetablerad cell SUS pension kultur härledd från Tea (Camellia sinensis L.) Lämnar

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Cell odling på Kiselnitridmembran och Kryopreparation för Synchrotron röntgenfluorescens Nanoanalys

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Kvantitativ analys av cellulär lipidom e av Saccharomyces Cerevisiae med hjälp av flytande kromatografi tillsammans med Tandem Mass Spectrometri

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Tvärgående sektionering av moget ris (Oryza sativa L.) Kärnor för skanning av elektronmikroskopiavbildning med pipettespetsar som immobiliseringsstöd

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Kvantitativ metabolomik av sackaromyces Cerevisiae med flytande kromatografi i kombination med tandemmasspektrometri

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

Kontinuerlig fluorescensbaserad endonukleaskopplad DNA-metyleringsanalys för att screena för DNA-metyltransferashämmare

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...