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 gerne takke Baden-Württemberg Stiftung for deres finansielle støtte i forbindelse med "Bioinspired materiale syntese" (BioMatS-14).

1. Hydrogel forberedelse
<ol>
	<li>Bland 200 mg af BSA med 1 mL deioniseret vand H2O oprette 20% (w/v) BSA lager (stock opløsning A).</li>
	<li>Mix 165 µL af THPC løsning (134 mg/mL) med 4.835 mL deioniseret vand til at oprette THPC lager løsning (stamopløsning B).</li>
	<li>Vejer 1 mg KCSSGKSRGDS (1,111.1 g/mol) peptid (eller en tilsvarende celle-klæbende peptid) og fortynde det i 100 µL sterilt H2O at opnå en 10 mg/mL opløsning (stamopløsning C). Bemærk: Dette trin er valgfrit, og ...

<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...

Hydrogels er materialer, der danner uopløselige 3D netværk habil bindende store mængder vand. Sådanne materialer kan give gode miljømæssige betingelser for levende celler. I øjeblikket, der stigende interesse i generation af tre-dimensionelle hydrogel strukturer og i udvikling af processer til at skræddersy deres kemiske og fysiske egenskaber. Når dette er opnået, kan en skabelon for væksten af celler og manipulation af cellulære adfærd være genereret1,2,3,4. Disse 3D strukturer ikke kun skaber en mere naturlig og realistisk miljø end konventionelle to-dimensionelle tilgange, men de også afslører nye muligheder for vækst af stamceller eller tumor modeller5. Forskellige materialer har en række karakteristika, der hovedsagelig afhænger af porestørrelse gel6. Porerne spiller en afgørende rolle i celle kultur applikationer, vævsmanipulering og den styret vækst af stamceller. For eksempel, ilt og næringsstoffer diffuse gennem matrixen, og tilstrækkelige mængder skal kunne nå celler7. På den anden side skadelige metabolitter skal fjernes så hurtigt som muligt, og tilstrækkelig plads til cellevækst skal være tilgængelig7. Derfor påvirke egenskaber af materialet, og dermed porestørrelse, alvorligt de potentielle fordele og mulige anvendelser af matrixen. Afhængig af materialets egenskaber, kan forskellige cellevækst processer forekomme i 3D cellekultur, herunder dannelsen af neuronal strukturer; vækst og differentiering af huden eller knogle celler; og styret væksten af særlige stamcellelinjer, som hepatocytter eller fibroblaster2,3,8,9,10,11. Et andet afgørende punkt påvirke en eventuel anvendelse af et materiale, er dens stabilitet mod eksterne stimuli12. For eksempel, skal hydrogel opretholde dens mekaniske integritet i celle Kulturmedier eller den menneskelige krop.
I de seneste år, forskning på 3D celle kultur hydrogels intensiveres, og mange undersøgelser blev udført for at løse de 3D arkitekturer systemer13. Hydrogels består af kemisk syntetiseret komponenter er hyppigst undersøgt, fordi de kan nemt syntetiseret og kemisk modificerede og de udviser høj stabilitet (Se Zhu et al., 2011 for en anmeldelse)5. Proteiner har dog mange gavnlige egenskaber: som såkaldte "præcision polymerer," de er biokompatible; de har en defineret længde; de er forholdsvis nemme at ændre; og de har et stort antal mål websteder14,15. I denne forbindelse kan meget specifikke, nyskabende strukturer genereres til anvendelse inden for mange områder. I denne undersøgelse, blev en protein-baseret hydrogel16 brugt til at påvise veletablerede metoder evne til at påvirke den 3D arkitektur af materialet. Derudover blev kapacitet og anvendelighed til pore generation også undersøgt.
Mange forskellige teknikker er til rådighed til at ændre 3D strukturer, herunder både enkle metoder og sofistikeret, højt specialiserede teknikker fra forskellige områder af materiallære. En udbredt teknik er brugen af electrospinning til at generere veldefinerede strukturer17. Ladede fibre er trukket fra en løsning af et elektrisk felt og derefter størkne ved udsættelse for ilt. På denne måde kan blive produceret fibre i rækken af adskillige nanometer op til flere mikron. Supplerende teknikker til at tune størrelse, struktur og fordeling af porer i matrixen er bløde litografi, fotolitografi, hydrodynamiske fokusering, electro-sprøjtning, og bio-udskrivning18,19,20. En væsentlig ulempe af disse teknikker er deres afhængighed på specifikke, dyrt udstyr og specielle kemikalier eller materialer. Desuden erfaring med disse teknikker er ofte ikke direkte overføres til materialer baseret på protein, og mange af kemikalier og metoder er ikke celle kompatibel.
På den anden side stole mange teknikker ikke på specialudstyr, hvilket gør dem lettere og billigere at anvende og til at reproducere. En udbredt metode til struktur manipulation er solvent støbning21,22,23. Partikler er tilføjet før polymerisation reaktion og distribueres homogen måde for at mætte løsningen. Efter polymerisering medfører en ændring af betingelser, såsom en fortynding eller en ændring i pH, solvation af partiklerne, medens porerne i materialet. Kemikalier, der anvendes i disse teknikker, såsom salt, sukker, paraffin, gelatine og kridt, er billige og let tilgængelige. I frysetørring, er hævede hydrogels frosset. Den efterfølgende sublimering af flydende faser under en vakuum er derefter udført23,24,25. Vand sublimation fra netværket er blide nok til at opretholde de specifikke 3D strukturer af materialet. I gas skummende, der en løsning streames med en gas mens polymerisation finder sted, forlader porerne i gelen21. Størrelse og fordeling af porer kan justeres afhængigt af gasstrømmen.
For at danne protein hydrogel, reagerede BSA med tetrakis (hydroxymethyl) phosphonium chlorid (THPC) i en Mannich-type reaktion at muliggøre dannelsen af kovalente bindinger mellem primære aminer og hydroxy grupper af fire bevæbnede linker molekyle26. Mulige skadelige mellemprodukter er fjernet af overdreven vask af materialet efter reaktionen opstår.
Denne undersøgelse viser muligheden for behandling af en BSA-baseret materiale med forskellige teknikker til at manipulere og tilpasse størrelsen af porerne. Hver af teknikkerne, der kan bruges i ethvert laboratorium på verdensplan, som ingen særlige udstyr er nødvendig. Derudover respekt forskellige parametre, såsom hævelse ratio, enzymatisk nedbrydelighed, pH stabilitet og temperatur følsomhed, er undersøgt og sammenlignet med hinanden, især for indflydelse fra de forskellige teknikker på generation af 3D arkitekturer. Endelig, materialerne blev functionalized med celle-klæbende peptider til at undersøge den mulige anvendelse af materialer til cellekultur. To forskellige model cellelinjer blev anvendt: A549 og 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

Hydrogels er anerkendt som lovende materialer til celle kultur ansøgninger på grund af deres evne til at yde meget hydreret celle miljøer. Inden for 3D skabeloner stigende på grund af den potentielle ligheden af disse materialer til de naturlige ekstracellulære matrix. Protein-baserede hydrogels er særligt lovende, fordi de let kan blive functionalized og kan opnå definerede strukturer med justerbar fysisk-kemiske egenskaber. Men produktion af kombineret makroporøs 3D skabeloner til celle kultur applikationer ved hjælp af naturlige materialer er ofte begrænset af deres svagere mekaniske egenskaber i forhold til de af syntetiske materialer. Her, forskellige metoder blev evalueret for at producere kombineret makroporøs bovint serumalbumin (BSA)-baserede hydrogel systemer, med justerbar pore størrelser i rækken af 10-70 µm i radius. Derudover blev en metode til at generere kanaler i dette protein-baseret materiale, der er flere hundrede mikron lange etableret. De forskellige metoder til at producere porer samt påvirkning af porestørrelse materialeegenskaber såsom hævelse ratio, pH, temperatur stabilitet og Enzymatisk nedbrydning adfærd, blev analyseret. Pore størrelser blev undersøgt i den indfødte, hævede tilstand af hydrogels ved hjælp af Konfokal laser scanning mikroskopi. Muligheden for celle kultur programmer blev evalueret ved hjælp af en celle-klæbende M.H.T. peptid ændring af ordningen for protein og to model cellelinjer: humane brystkræftceller (A549) og adenocarcinomic menneskelige alveolær basal 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

Forskellige metoder til at manipulere tredimensionalt arkitektur i protein-baserede hydrogels evalueres her med hensyn til materialeegenskaber. Kombineret makroporøs netværk er functionalized med en celle-klæbende peptid, og deres gennemførlighed i cellekultur evalueres ved hjælp af to forskellige model cellelinjer.

Let Manipulation af arkitekturer i Protein-baseret Hydrogels til celle 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

Udarbejdelse af Homogene MALDI Prøver til Kvantitative applikationer

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

Enkelt-molekyle Manipulation af G-quadruplexes af magnetiske pincet

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

Et Fluorogenic peptid kavalergang Assay til at screene for proteolytiske aktivitet: ansøgninger om 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

Undersøgelse af metabolismen af seks systemiske insekticider i en nyetableret celle affjedrings kultur afledt af te (Camellia sinensis L.) Blade

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

Cellekultur på silicium nitrid membraner og Cryopreparation for Synchrotron røntgen fluorescens Nano-analyse

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 analyse af cellulære lipidom af Saccharomyces Cerevisiae Ved hjælp af flydende kromatografi kombineret med tandem massespektrometri

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

Tværgående 2000 af ældre ris(Oryza sativa L.) Kerner til scanning elektronmikroskopi imaging ved hjælp af pipette tips som immobilisering Support

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

Kvantitative metabolomics af saccharomyces Cerevisiae Ved hjælp af flydende kromatografi kombineret med tandem massespektrometri

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 fluorescensbaseret endonukleasekoblet DNA-methyleringsassay til screening for DNA-methyltransferasehæmmere

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