The authors would like to thank Baden-W&#252;rttemberg Stiftung for their financial support in the "Bioinspired Material Synthesis" framework (BioMatS-14).

1. Hydrogel Preparation
<ol>
	<li>Mix 200 mg of BSA with 1 mL of deionized H2O to create 20% (w/v) BSA stock (stock solution A).</li>
	<li>Mix 165 &#181;L of THPC solution (134 mg/mL) with 4.835 mL of deionized water to create THPC stock solution (stock solution B).</li>
	<li>Weigh 1 mg of KCSSGKSRGDS (1,111.1 g/mol) peptide (or an equivalent cell-adhesive peptide) and dilute it in 100 &#181;L of sterile H2O to obtain a 10 mg/mL solution (stock solution C). 
	NOTE: This step is optional and o...

<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. 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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=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. 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The authors declare that they have no competing financial interests.

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 are materials that form insoluble 3D networks capable of binding large amounts of water. Such materials can provide excellent environmental conditions for living cells. Currently, there is increasing interest in the generation of three-dimensional hydrogel structures and in the development of processes to tailor their chemical and physical properties. Once this is achieved, a template for the growth of cells and the manipulation of cellular behavior can be generated1,2,3,4. These 3D structures not only create a more natural and realistic environment than conventional two-dimensional approaches, but they also reveal new possibilities for the growth of stem cells or tumor models5. Different materials possess a range of characteristics that mainly depend upon the pore size of the gel6. The pores play a crucial role in cell culture applications, tissue engineering, and the directed growth of stem cells. For example, oxygen and nutrients diffuse through the matrix, and adequate amounts must be able to reach the cells7. On the other hand, harmful metabolites must be removed as quickly as possible, and sufficient space for cell growth must be available7. Consequently, the properties of the material, and thus the pore size, severely influence the potential benefit and possible applications of the matrix. Depending upon the properties of the material, different cell-growth processes can occur in 3D cell culture, including the formation of neuronal structures; the growth and differentiation of skin or bone cells; and the directed growth of special stem cell lines, like hepatocytes or fibroblasts2,3,8,9,10,11. Another crucial point influencing the possible application of a material is its stability towards external stimuli12. For example, the hydrogel must maintain its mechanical integrity in cell culture media or the human body.
In recent years, research on 3D cell culture hydrogels intensified, and many studies were carried out to resolve the 3D architectures of the systems13. Hydrogels composed of chemically synthesized components are most commonly investigated because they can be easily synthesized and chemically modified and they exhibit high stability (see Zhu et al., 2011 for a review)5. However, proteins have many beneficial properties: as so-called &#34;precision polymers,&#34; they are biocompatible; they have a defined length; they are relatively easy to modify; and they have a large number of target sites14,15. In this regard, highly specific, innovative structures can be generated for application in many fields. In this study, a protein-based hydrogel16 was used to demonstrate the ability of well-established methods to influence the 3D architecture of the material. Furthermore, the capability of and applicability to pore generation was also investigated.
Many different techniques are available to modify 3D structures, including both simple methods and sophisticated, highly specialized techniques from different fields of material science. A widespread technique is the use of electrospinning to generate well-defined structures17. Charged fibers are pulled from a solution by an electric field and then solidify upon exposure to oxygen. In this way, fibers in the range of several nanometers up to several microns can be produced. Additional techniques to tune the size, structure, and distribution of the pores within the matrix are soft lithography, photolithography, hydrodynamic focusing, electro-spraying, and bio-printing18,19,20. A significant drawback of these techniques is their dependency upon specific, expensive equipment and special chemicals or materials. Furthermore, experience with these techniques is often not directly transferrable to protein-based materials, and many of the chemicals and methods are not cell compatible.
On the other hand, many techniques do not rely upon special equipment, making them easier and cheaper to apply and to reproduce. A widespread method for structure manipulation is solvent casting21,22,23. Particles are added prior to the polymerization reaction and are distributed homogenously to saturate the solution. After the polymerization, a change of conditions, such as a dilution or a pH change, leads to the solvation of the particles, while the pores remain within the material. The chemicals used in these techniques, such as salt, sugar, paraffin, gelatin, and chalk, are cheap and readily available. In freeze-drying, swollen hydrogels are frozen. The subsequent sublimation of the liquid phases under a vacuum is then performed23,24,25. Water sublimation from the network is gentle enough to maintain the specific 3D structures of the material. In gas foaming, a solution is streamed with a gas while the polymerization takes place, leaving pores within the gel21. The size and distribution of the pores can be adjusted depending upon the gas stream.
To form the protein hydrogel, BSA is reacted with tetrakis (hydroxymethyl) phosphonium chloride (THPC) in a Mannich-type reaction to allow for the formation of covalent bonds between primary amines and the hydroxy groups of the four-armed linker molecule26. Possible harmful intermediates are removed by excessive washing of the material after the reaction occurs.
This study demonstrates the possibility of treating a BSA-based material with different techniques to manipulate and tailor the size of the pores. Each of the techniques can be used in any laboratory worldwide, as no special equipment is necessary. In addition, different parameters, such as swelling ratio, enzymatic degradability, pH stability, and temperature sensitivity, were examined and compared to each other, especially respect to the influence of the different techniques on the generation of 3D architectures. Finally, the materials were functionalized with cell-adhesive peptides to investigate the possible application of the materials to cell culture. Two different model cell lines were used: A549 and 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>
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			<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>
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			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
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			<td height="20" style="height:20px;">Tubes 1,5 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
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			<td>Sarsted Ag und Co.</td>
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			<td height="20" style="height:20px;">CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td>
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easy manipulation of architectures in protein-based hydrogels for cell culture applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (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

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

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.

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Research

JoVE Journal

Biochemistry

6.6K visualizações.  Ulm University. 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 mill...