作者要感谢巴登-符腾堡基金会的财政支持，"仿生材料合成"框架 (BioMatS-14)。

1.水凝胶制备
<ol>
	<li>用去离子 H2O 打造 20%(w/v) BSA 股票 （股票解决方案 A） 1 毫升混合 200 毫克的牛血清白蛋白。</li>
	<li>用去离子水，创建 THPC 4.835 毫升混合 165 微升 THPC 解决方案 （134 毫克/毫升） 的股票 （股票解决方案 B） 解。</li>
	<li>体重 1 毫克的 KCSSGKSRGDS (1,111.1 g/mol) 肽 （或等效的细胞粘附肽） 和稀释在 100 μ L 的无菌 H2O 获得 10 毫克/毫升的解决方案 （股票 C）。 注: 此步骤是可选的只需要包括如果水凝胶是基于细胞培养中的应用。</li>
	<li>底部的 96 孔板中删除并替换可移动的塑料包装。 注意: 对于板用在这里，底部可以很容易被删除所到的每口井; 底部施加压力薄塑料底只会掉出来。</li>
	<li>牛血清白蛋白原液的混合 100 微升 （A） 和 100 μ L THPC 的股票解 (B) (可选: 4 微升股票解 C 官能化、 细胞粘附凝胶的) 在 96 孔板获得 200 微升的水凝胶。吹打向上和向下至少 5 次，保证均匀的凝胶聚合后混合组件。</li>
	<li>96 孔板置于室温 (RT) 约 10 分钟，直到所有水凝胶进行了正确的聚合。</li>
	<li>仔细地，慢慢地从板的底部去除塑料包装。</li>
	<li>按出使用一张小邮票 96 孔板水凝胶并将它们转移到无菌的 PBS，pH 7.4 1.5 毫升管。 注: 水凝胶具有圆柱状，直径约 4 毫米和 8 毫米的高度。</li>
	<li>存储在磷酸盐缓冲液 (PBS) 在 4 ° C 到几个月的水凝胶。</li>
</ol>
2.冷冻水凝胶
<ol>
	<li>1.5 毫升管装满 500 µ L 的无菌去离子 H2O 和取消上限。将水凝胶转移到 1.5 毫升反应管使用一把铲子。</li>
	<li>包好 1.5 毫升管与石蜡膜在至少三种层的每个小瓶紧密相关。使用一根针穿透小孔的膜，使管中的气体释放。</li>
	<li>请继续执行以下步骤之一:
	<ol>
		<li>将小瓶转移到液态氮解决方案 5 分钟，以保证水和水凝胶完全冻结。从液氮中取出后立即, 将小瓶转移到冷冻干燥机，防止材料解冻。 注意: 此过程导致毛孔大约 10-15 微米的半径。</li>
		<li>把玻璃瓶放在-20 ° C 隔夜慢慢冻结的水凝胶。立即删除后从-20 ° C，将小瓶转移到冷冻干燥机，防止材料解冻。 注意: 此过程导致毛孔大约半径 50-60 微米。</li>
	</ol>
	</li>
	<li>后 24 小时 （和完全蒸发的水和周围的水凝胶），解冻材料由从冷冻干燥机中移除它。 注: 孔隙大小可以用共焦激光扫描显微镜 （步骤 5，"水凝胶可视化"） 分析。</li>
</ol>
3.颗粒浸出
<ol>
	<li>准备水凝胶在 1.1-1.5 的步骤中所述。</li>
	<li>直接混合组件后, 添加 NaCl 直到饱和发生 （36 毫克/毫升）。添加盐，直到盐的晶体可以看作在溶液中是白色沉淀。</li>
	<li>转移到振动筛的 96 孔板，摇动直到聚合发生 （约 10 分钟）。水凝胶从删除板，1.7-1.8 步骤中所述。</li>
	<li>孵育水凝胶在室温 (20 rpm) 钻井液振动筛的无菌水中洗脱所有盐从水凝胶模板至少 24 小时。直到几个月在 PBS 为存储在 4 ° C 的水凝胶。</li>
</ol>
4.通道形成
<ol>
	<li>在步骤 1 中所述制备水凝胶。使用一把钳子的解决方案中移除水凝胶、 删除多余的水与高度吸水纸，和放在一块干冰。</li>
	<li>冻结的水凝胶为 30 s 和仔细地从块中删除它。不损坏水凝胶;仔细地用一把铲子刮掉块。转移到 1.5 毫升管中的水凝胶和干它一夜之间在 37 ° c。</li>
</ol>
5.水凝胶可视化
<ol>
	<li>备罗丹明 B 原液稀释 1 毫克的罗丹明 B 在 10 毫升的 PBS。直到达到 0.001 毫克/毫升浓度稀释在 PBS 中的罗丹明原液法制备连续稀释 (稀释因子: 100)</li>
	<li>从存储解决方案 （例如，从步骤 2.4.） 中移除水凝胶和转移到 1.5 毫升管与 1 毫升的 0.01 毫克/毫升罗丹明 B 解决方案。染色水凝胶一夜之间在罗丹明 B 溶液在室温下。</li>
	<li>第二天，转移至 10 毫升的 PBS (pH 7.4) 和至少 3 小时洗要可视化的水凝胶。</li>
	<li>好转移到 µ 幻灯片 8 水凝胶和用 PBS 盖住它。</li>
	<li>小切出的水凝胶，并在使用刀片 （约 0.5 毫米的高度）。</li>
	<li>使用激光共聚焦显微镜，可视化的水凝胶在波长为 514 nm (目的: 教统会计划-Neofluar 40 x / 1.30 油 DIC M27，平面扫描模式: 514 nm 和缩放: 1 X)。</li>
</ol>
6.细胞文化可行性
<ol>
	<li>无菌过滤所有股票解决方案 （A、 B 和 C） 0.45 微米的过滤层。</li>
	<li>制备水凝胶在 1.1-1.4，包括细胞粘附肽在水凝胶聚合之前的步骤中所述。</li>
	<li>上述各组分混合后立即移液器混合物 µ 幻灯片 8 成很顺利，直到底部完全覆盖。 注: 此步骤必须执行快速、 直接混合组件后, 作为聚合在幻灯片中的几分钟内发生。</li>
	<li>粘附细胞培养及通道获得单个细胞悬浮液中的标准细胞培养技术。转移细胞提前预热、 无菌 DMEM 细胞培养液中辅以胎牛血清 (FBS，10%(w/v))、 青霉素-链霉素 （1%(w/v)) 和非必需氨基酸溶液 (MEM，1%(w/v))。</li>
	<li>与 Neubauer 计数室和仔细吸管 200 微升细胞 (2 x 105细胞/厘米2) 水凝胶表面所需数目的单元格进行计数。</li>
	<li>涵盖 µ 幻灯片 8 井盖，把它转移到孵化器 （37 ° C，5%CO2）。孵育至少 4 h 在 37 ° c。</li>
	<li>后的细胞附着至少 4 小时，洗两次带 200 µ L 无菌细胞培养 PBS 的单元格。</li>
	<li>与 200 微升的 3.7%(v/v) 在室温下 10 分钟的甲醛固定的细胞和两次用 PBS 洗 （~ 200 μ）。 注: 穿适当的个人防护设备时处理甲醛。</li>
	<li>通透细胞带 200 微升 0.1%Triton X 5 分钟洗两次用 PBS 的单元格。</li>
	<li>与鬼笔罗丹细胞染色由 195 µ L 的 PBS 混合 5 µ L 的甲醇股票并将其添加到单元格在室温染色细胞 20 分钟在黑暗中。两次用 PBS 洗涤。</li>
	<li>探讨细胞粘附特性利用共聚焦显微镜在 514 nm (目的: 教统会计划-Neofluar 40 x / 1.30 油 DIC M27，平面扫描模式: 514 nm 和缩放: 1 X)。使用适当的软件图像进行分析 （见表的材料）。</li>
</ol>
7.水凝胶性质
<ol>
	<li>膨胀率。
	<ol>
		<li>至少一天完全干凝胶在 37 ° C。</li>
		<li>衡量每个水凝胶，并注意确切的重量。</li>
		<li>2 毫升反应管装满 1.5 毫升的 PBS。</li>
		<li>转移到此 2 毫升反应管的水凝胶，并让它完全沉浸在 PBS。</li>
		<li>在 PBS 在室温下达到平衡与 PBS 中至少两天离开水凝胶。</li>
		<li>三天后，从解决方案中移除水凝胶和干燥用纸巾要从水凝胶表面除去多余的水分。</li>
		<li>权衡的水凝胶。</li>
		<li>计算的溶胀比使用下面的公式: 
		<img alt="Equation" src="/files/ftp_upload/55813/55813eq1.jpg"> 干凝胶 （第 7.1.2 步。） 重量和 Ws Wd在哪里是湿凝胶 （步骤 7.1.7） 的重量。乘以 100 要膨胀的比率 %。</li>
	</ol>
	</li>
	<li>ph 值和温度稳定性。
	<ol>
		<li>转移到 15 毫升反应管中 5 毫升的 PBS。</li>
		<li>用氢氧化钠和盐酸.带至适当的温度 （例如， RT，37 ° C 或 80 ° C） 解决方案调整到适当的 ph 值 (例如， pH 2，7 或 10) 解决方案。</li>
		<li>转让是要追究到适当的解决方案及其稳定性的水凝胶。如有必要，请调整 ph 值。 注: 所有水凝胶应该完全肿起来了在这一点 （见的溶胀比），防止由于肿胀的材料重量的正确解释。</li>
		<li>在特定的时间间隔后, 从解决方案 （例如，每个小时至 2 天） 中移除水凝胶，用高吸水的纸巾，除去多余的水分，并权衡它干。</li>
	</ol>
	</li>
	<li>酶促降解。
	<ol>
		<li>准备股票酶溶液 300 U 胰蛋白酶、 胃蛋白酶，按照制造商的说明。</li>
		<li>转移到适当的解决方案 （例如，从步骤 1.8） 水凝胶。 注: 所有水凝胶应该完全肿起来了在这一点上，防止体重的不正确解释。</li>
		<li>经过一定的时间间隔，从解决方案 （例如，每个小时） 中移除水凝胶，用高吸水的纸巾，除去多余的水分，并权衡它干。</li>
	</ol>
	</li>
</ol>

<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>

作者宣称，他们有没有经济利益的竞争。

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 the network, thus rendering the material useless or simply inapplicable for the desired application12. Proteins possess a known potential for application in hydrogels due to their defined structure, their intrinsic biocompatibility (because they are derived from natural sources), and their many available modification sites (e.g., reactive amino-, hydroxyl- or carboxyl- groups). However, many protein-based hydrogels lack sufficient mechanical stability compared to their synthetic counterparts16. Because the introduction of large 3D architecture may lead to the complete structural disintegration of a hydrogel, the feasibility of methods to tune 3D structures must be critically evaluated to obtain the desired material properties and to achieve the full potential of the protein material. This was one reason for the evaluation of the freeze-drying and particle leaching processes and of the effect those procedures had on the resulting properties of the protein hydrogels in this study.
A critical point while working with hydrogels is the visualization of the materials on the macro-scale. Quite often, electron microscopy-based methods are used to examine the 3D structure of a hydrogel28. While doing this, it is important to consider that many procedures used to prepare the sample for electron microscopy might interact with the material and significantly alter its properties and integrity (e.g., the freezing of the sample). To overcome this problem, confocal laser scanning microscopy was used here to evaluate the produced 3D architectures and the size and distribution of the pores within the hydrogels. As opposed to electron microscopy, only a simple staining step in the native hydrated environment of the swollen state of the material is necessary to analyze the micro-scale structure of the matrix.
Freeze-drying is a very well-established method in soft matter research and biochemistry. It allows for the production of pores of a certain size and regular distribution throughout the matrix due to ice crystal formation in the network. By applying very low pressure (i.e., ~0.05 mbar) and temperatures below 0 &#176;C, the ice sublimates from the material, leaving pores behind without further disintegrating the surrounding network. In the swollen state, pores of about 10 to 70 &#181;m were detected in the template (Figure 1), with a narrow size distribution for smaller pores and a wider distribution for larger pores. Those sizes are within a suitable range for most cell culture-related applications, like hematopoietic stem cell incorporation3 and the ingrowth of osteoids and fibroblasts21. However, this method also has certain limitations. For some applications, the creation of larger pores might be beneficial22. The size limit that could be achieved with freeze drying was approximately 70 &#181;m in this material, but larger pores have already been developed by different groups21.
The dried hydrogels must be handled with care; due to their fragile structure, they tend to break apart when slight pressure is applied to the dried material. The critical step in the preparation of the material is the polymerization. Both components must be mixed properly and quickly to guarantee the production of a uniform hydrogel. If the hydrogels are not mixed immediately, a partial polymerization might occur, leading to an uneven pore distribution within the matrix.
Another technique that is well-established for the modification of synthetic polymer materials is the so-called particle leaching. Solid but dispersible particles are incorporated into the material prior to the gel-sol transition and are later eliminated from the polymerized material by changing external conditions, leading to the solubilization of the particles13,29. Paraffin and gelatin can be melted by increasing the temperature29, while crystalline particles, like sugars and salts, are dissolved by changing the buffer composition or dilution13. For this protein-based material, the radii of the pores could be changed 10-fold within the 3D matrix. Untreated salt crystals created the largest pores, with radii about 50 to 70 &#181;m (Figure 2). One limitation of this method is the maximum size of the pores: above 70 &#181;m, a considerable population of the pores exhibited fractures in their backbones, thus destabilizing the matrix. To overcome this problem, salt crystals were ground with a mortar and pestle, and significantly smaller pores, without damaged structural backbones, were produced. To improve the method, the crystals can be sieved to achieve a specific size3. Another critical step is the saturation of the solution: the hydrogels must be completely liquid when adding the salt. If the hydrogel is already partly polymerized, uneven pores will appear in the mesh, or the gel will break. If too little salt is added, the solution might be too diluted, causing no pores appear. To overcome this problem, a test series must be made prior to the experiment to find the exact amount of salt that is necessary for saturation.
Channels within a material can be useful for certain applications that require scaffold material (e.g., the ingrowth of axons into a template)11, but they are often difficult to produce in soft materials due to the lack of stability and rigidity. We established a process that includes the use of a block of solid carbon dioxide to produce linear-oriented channels within the protein hydrogel, with a length of several hundred microns and a radius of approximately 20 &#181;m (Figure 3). During their formation, the time that the hydrogel remains on the block of dry ice is essential and must be evaluated individually for each material. Short freezing times do not lead to the formation of structures, while longer freezing times break the network. When removing the material, damage to the hydrogel can be avoided by pressing a spatula against the block of ice and scratching some of the ice off the block along with the hydrogel.
All procedures influence the structure of the material and may thus severely reduce the beneficial properties of the material. To address this effect, different basic gel parameters were evaluated. The swelling ratio is one of the most important features with respect to the use of a hydrogel in cell culture, as it directly correlates with the free water content, an essential factor for the growth and survival of cells within a 3D template. All treated hydrogels were dried and brought to swelling equilibrium prior to the measurements. The swelling ratios for the different methods varied from 534% to 1,153% (Table 1). The free water content is crucial to prevent the accumulation of harmful intermediates in the environment of the cell. At the same time, nutrients, growth factors, and oxygen should be able to diffuse rapidly throughout the gel to fulfill the demands of a growing and dividing cell7. With both freeze-drying and salt leaching, a high water content could be achieved for the protein-based macroporous hydrogels. Furthermore, the swelling ratio has a significant influence on the differentiation of stem cells within a matrix and can be further improved by incorporating PEG into the material8. This is easy to do in protein hydrogels with a variety of target sites.
Many hydrogels are intended for use in biotechnological fields, such as cell culture2, tissue engineering30, or transplantation medicine31. The material must deal with harsh external conditions, including temperature or pH changes and the presence of degradation agents, like proteases or catalytic active substances. Especially in materials intended for use in the human body, such as coatings for medical devices32 or injectable hydrogels33, the systems might mutually interact with the host system, thus influencing their stability and possibly limiting their use. As a variety of methods to tune 3D architecture severely influence the material stability, all procedures used in this study were investigated for their influence on the mechanical and chemical properties of the hydrogel. The reaction of the BSA protein hydrogel mainly depends upon the applied stimuli. For example, a temperature increase from RT to 37 &#176;C or a pH shift into the acidic range did not alter the protein stability, while a shift to basic conditions reduced the residual weight after a week (Table 1). More importantly, none of the procedures used here influenced the stability of the matrix in comparison to one another (Table 1).
For many applications, a cleavable backbone is desirable. Most protein hydrogels are biodegradable. However, many hydrogels require extended decomposing times, as agents must often work from the outside to the inside of the material. This further leads to uneven degradation patterns, resulting in the complete breakdown of the network after a certain, undefined time. Untreated BSA hydrogel was degraded after several days, with the complete breakdown and disintegration of the network after a certain time. Hydrogels treated with freeze-drying or particle leaching approaches degraded in a predictable manner within hours when in the presence of enzymes, as shown in Figure 4. For macroporous hydrogels, enzymes can freely diffuse throughout the matrix and cleave the backbone of the whole template, leading to an degradation and predictable degradation times. The degradation time was found to be dependent upon the sizes of the pores within the material. For example, hydrogels frozen at -20 &#176;C, with an average pore radius of 60 &#181;m, could be degraded to half of their weight within 3.2 h using 300 U of pepsin. Hydrogels frozen at -196 &#176;C, with a significantly smaller average pore radius of 10 &#181;m, had a half-life of 6.5 h at the same pepsin concentration (Table 1).
A major concern for cell culture applications is the adhesion potential of the matrix. Many materials facilitate adhesion due to their inherent adhesion properties, while other systems must be provided with proper cellular adhesion through the incorporation of cell-adhesive structures. One prominent example of guaranteed cell adhesion is a cell-adhesive peptide, with RGD one of the most prominent and well-characterized34. Protein-based materials are normally easy to modify due to the vast amount of accessible target sites within the template. To prove the general feasibility of this BSA-based protein hydrogel for cell culture applications, the material was functionalized with a cell-adhesive RGD peptide. To facilitate the process, a specific KCSSGKSRGDS peptide was designed with a primary amine in the lysine moiety, which can be targeted by the crosslinking agent during polymerization. A549 and MCF7 cells were grown on the surface of the protein hydrogel and showed distinctive adhesion after 24 h of growth, with high viability16. The growth of the cells and the strong adhesion, which could not be reversed by washing with PBS, show the viability of the cells on the matrix and the general feasibility of the produced material for cell culture application. The direct incorporation of the peptide is both an advantage and a limitation of this system: on the one hand, it simplifies the procedure and eliminates the need for an additional reaction step. On the other hand, many different cell-adhesive peptides have been developed and used over the last decade5, and some of them bear primary amines in their structure. Those amines will readily react with the hydroxy groups of the linker molecule, thus potentially disabling the adhesion properties of the peptide. The peptide must be chosen according to this system. Another option is to add the peptide of choice after the polymerization. This way, it can be linked to different moieties in the protein backbone, whichever is best for the desired application.
For further use in 3D cell culture, the system must be optimized and adapted for each specific cell line. By changing the protein or crosslinker concentration, the properties can already be tuned, especially the mechanical properties of the material. The presented methods offer a range of tools for the manipulation of hydrogels, with no need for special equipment and with low costs. The techniques maintain the general properties of the protein-based hydrogel, making them useful for the initial evaluation of 3D cell culture applications.

水凝胶是水的形成不溶性的 3D 网络能够结合大量的材料。这种材料可以提供优良的环境条件，活细胞。目前，那里越来越感兴趣，在三维水凝胶结构的生成和发展的过程来定制他们的化学和物理特性。一旦完成，该细胞的生长和操纵细胞行为的模板可以生成的1，2，，34。这些 3D 结构不仅创造一个更为自然和现实的环境，比传统的二维方法，但他们也揭示新的可能性，为干细胞的生长或肿瘤模型5。不同的材料拥有一系列的特性主要取决于孔隙大小的凝胶6。毛孔发挥关键作用，细胞培养应用程序、 组织工程和干细胞的定向的生长。例如，通过矩阵，弥漫的氧气和营养物质和适当数额必须能够达到细胞7。另一方面，必须尽快，去除有害代谢物和足够的空间用于细胞生长必须可用7。因此，材料特性和，因此孔隙大小，严重影响矩阵的潜在利益和可能的应用程序。取决于材料的性能，不同的细胞生长过程可以发生在三维细胞培养，包括形成的神经元结构;生长和分化的皮肤或骨细胞;和特殊的干细胞，如肝细胞或成纤维细胞2，3，8，9，，1011定向的生长。影响材料的可能应用的另一个关键点是对外部刺激12其稳定性。例如，水凝胶必须保持其在细胞培养基或人体的机械完整性。
近年来，研究三维单元文化水凝胶加剧，并进行了许多研究解决系统13的 3D 建筑。由化学合成的成分组成的水凝胶最常用因为他们可以很容易合成和化学改性和他们表现出较高的稳定性进行了研究 （见朱等人，2011 年为审查）5。然而，蛋白质有许多有益的特性: 如所谓的"精密聚合物"，他们都是生物相容性;他们有一个已定义的长度;它们是相对容易的修改;和他们有大量的目标站点14，15。在这方面，特异性高、 创新结构可以生成在许多领域中的应用。在此研究中，基于蛋白质的凝胶16用于演示的既定方法影响材料的 3D 架构的能力。此外，还研究了适用于孔隙水的产生和能力。
有许多的不同技术，可用来修改三维结构，包括方法简单、 复杂、 高度专业化的技术，从不同科学领域的材料。广泛的技术是利用静电纺丝技术来生成定义良好的结构17。带电的纤维由电场拉扯从解决方案，然后凝固暴露在氧气。这种方式，可以产生几个纳米到几个微米范围内的纤维。额外的技术调整大小、 结构和分布的毛孔内矩阵是软光刻技术、 光刻、 水动力聚焦、 电喷涂、 和生物印刷18，，1920。这些技术的一个重大缺点是他们对具体的、 昂贵的设备和特殊的化学物质或材料的依赖。此外，这些技术的经验往往不是直接转移到蛋白质为基础的材料，和许多的化学品和方法不是兼容的单元格。
另一方面，许多技术不要依赖特殊的设备，使他们更容易和便宜适用，并重现。结构操作的普遍做法是溶液浇铸21，，2223。粒子在聚合反应之前添加和均匀分布，饱和溶液。在聚合反应后, 变化的条件，如稀释或 ph 值的变化，导致溶剂化物的粒子，而毛孔留在材料内。在这些技术，如盐、 糖、 石蜡、 明胶和粉笔，所使用的化学品是廉价和容易获得。在冷冻干燥，肿水凝胶被冻结。真空条件下液相的后续升华是然后执行23，，2425。从网络的水升华是足够温和，保持材料的特定三维结构。在气体发泡，解决方案是用气体分流聚合反应发生，留下毛孔内凝胶21时。取决于气体流可以调整的大小和分布的毛孔。
形成蛋白质凝胶，与四羟甲基氯化磷 (THPC) 在曼尼希反应，以便形成共价键伯胺和链接器四武装分子26的羟基之间的反应是牛血清白蛋白。反应发生后通过过度清洗的材料移除可能有害的中间体。
这项研究表明治疗不同的技术来操纵和裁缝毛孔的大小基于 BSA 的材料的可能性。每个技术可以用作在任何实验室全世界，没有特别的设备是必要的。此外，不同的参数，如溶胀率、 酶促降解性、 pH 稳定性和温度敏感性、 被检测，并比较彼此，尤其是尊重对不同技术的影响上的 3D 架构生成。最后，材料是羧基与细胞粘附肽调查材料对细胞培养中的可能应用。使用了两种不同的模型细胞系: A549 和 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

水凝胶被公认的有前途的材料，因为它们能够提供高度水合的细胞环境细胞文化申请。领域的 3D 模板正上升由于潜在的相似之处的那些材料到天然细胞外基质。基于蛋白质的凝胶是特别有前途，因为他们很容易能官能化，可以实现定义的结构可调的理化性质。然而，大孔 3D 模板单元格文化应用程序使用天然材料的生产，常常受到其较弱的力学性能相比，这些合成材料。在这里，不同的方法进行计算以产生大孔牛血清白蛋白 (BSA)-基于水凝胶系统，与在半径 10 至 70 微米范围内可调的孔径大小。此外，建立了一种方法来生成渠道在这几个几百微米长的蛋白质基础材料。不同的方法来产生毛孔，以及孔隙大小对材料的性能如膨胀比、 ph 值、 温度稳定性和酶降解行为的影响进行分析。利用共焦激光扫描显微镜对凝胶本机、 肿州考察了孔径大小。细胞培养应用的可行性进行了评价使用蛋白系统和两个模型细胞株的细胞粘附 RGD 多肽修饰: 人类乳腺癌细胞 (A549) 和 adenocarcinomic 肺泡基底上皮细胞 (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 microscopy. This is because the sample preparation (i.e., drying) process often changes the hydrogel properties. To overcome this problem, the material samples here were analyzed using confocal microscopy, a method that allows for the characterization of the material in its native, water-swollen state. Rhodamine B can bind to the hydrogel backbone via electrostatic interaction and facilitates the visualization of the material. To investigate the feasibility of a freeze-drying approach to modify this protein-based system, hydrogels were subsequently polymerized and frozen at 196 &#176;C in liquid nitrogen and at -20 &#176;C. The results show a clear influence of the freezing temperature on the resulting pore sizes. At -196 &#176;C, small pores with a radius of about 10 &#181;m and a narrow size distribution were produced. On the other hand, freezing at higher temperatures leads to the production of materials with much larger pores, whose radii are between 50 and 70 &#181;m, as seen in Figure 1. The slow freezing process leads to the formation of larger ice crystals, which results in bigger pores within the material after the sublimation of the ice.
Another technique for pore generation, which is well-described for synthetic materials, is particle leaching. Solid particles like paraffin, gelatin, salt, or another solid-state material, are incorporated into the hydrogel prior to solidification. They are then eluted from the solid hydrogel by changing external conditions, such as temperature, Ph, or buffer composition by dilution. The BSA-based hydrogel was polymerized in the presence of high salt concentrations. After the sol-to-gel transition, salt crystals were removed from the material by adding an excess of water to dilute the crystals. This was followed by the confocal analysis of the resulting materials. The size of the salt crystals was changed by grinding NaCl with a mortar and pestle. As shown in Figure 2, the pore size can be tremendously changed using this method. Depending upon the crystals used (untreated or ground for a certain time), pore sizes from 10 to 70 &#181;m could be produced. Appropriately sized pores are essential for many applications that feature the incorporation of cells into a hydrogel. However, more sophisticated structures might be needed when the creation of more complex structures is desired (e.g., to provide structural guidance for neuronal cells along a gradient). Complex architectures can often only be produced with special equipment (e.g., spin coating, electrospinning, lithographic, or bioprinting techniques). However, the production of linear channels can be realized with a simple freeze-drying approach and the use of dry ice. The channels produced have radii of about 20-30 &#181;m and lengths of several hundreds of micrometers, as shown in Figure 3.
Another crucial feature of hydrogels is the biodegradability of the template matrix, both in vivo and in vitro1. Protein hydrogels offer the advantage of being biodegradable. However, many systems lack fast degradation due to the restricted target sites within the material. Proteolytic agents must work their way from the outside of a material to the inside, resulting in very slow degradation times. In contrast, macroporous hydrogels are normally well-diffusible. This allows enzymes to target protein structures throughout the whole template simultaneously, which greatly reduces degradation times. For the presented protein-based hydrogel, the proteolytic degradation time depends mainly on the pore sizes within the matrix. A representative degradation kinetic is shown in Figure 4. Pore generation reduces the time from several days to a few hours for trypsin and pepsin degradation, as shown in Table 1.
The main benefit of hydrogels is the high water content within the materials, which is desirable to mimic the extracellular matrix27. The swelling ratio represents the amount of water a gel can absorb and hold after drying and is a valid indicator of the free water content of a macroporous hydrogel. Furthermore, diffusion depends strongly upon the free water content and is required for the transport of nutrients towards the cells and the removal of toxic metabolites. The swelling ratio correlates directly with the pore size within the material, as shown in Table 1. By changing the pore size using the demonstrated methods, it is possible to alter the diffusion behavior in the template and thus to influence cell fate by manipulating their feed.
The stability of a hydrogel to external stimuli, such as pH and temperature changes, severely limits or expands the possible applications of a system. Particularly for application in a cell culture-related area, resistance to changes in the external parameters is essential, as cells and cell culture matrix mutually influence their properties and behavior (e.g., the acidification of the medium might lead to hydrogel degradation, and hydrogel degradation leads to cell release or death)22. To demonstrate the feasibility of the methods described here for pore generation, all hydrogel stabilities were determined for increasing temperatures (37 &#176;C and 80 &#176;C) and changing pH values (pH 2, 7, and 10). The residual weights of the hydrogels, which were treated using different methods, are summarized in Table 1. In conclusion, the presented protein-based hydrogels are stable over a wide range of conditions, while the residual weights of the hydrogels at high temperatures only reduce gradually. For the swelling ratio, it is important to consider that a partial unfolding might occur at higher temperatures, even if most BSA molecules should be held in place by the four-armed linker.
Whenever a macroporous hydrogel is intended for use in cell culture, the adhesion properties of the material play a crucial role in the individual application. Some materials bear inherent adhesion properties (e.g., heparin, short, extracellular matrix-derived peptide structures, or fibronectin)5. For those that do not have these features, the possibility to efficiently modify the material is desirable in order to introduce cell adhesion properties. One major advantage of protein hydrogels is the presence of a variety of accessible functional groups on the surface, which can be targeted by specific reactive linker molecules. Another option is the incorporation of cell adhesive peptides during polymerization, which can further facilitate the handling of the material and the production process, as only a single step is necessary to produce cell-adhesive materials. By using a four-armed, amine-reactive crosslinking agent26, the cell adhesion mediating peptide (in this case, RGD) can be directly co-polymerized during hydrogel formation. This procedure results in the proper adhesion of the cells, as shown in Figure 5. Two model cell lines, human breast cancer cells (A549, CCL-185) and adenocarcinomic human alveolar basal epithelial cells (MCF 7, HTB-22) were used to investigate the general feasibility of using this modified hydrogel in 3D cell culture. Both cell lines showed very good adhesion potentials on the modified hydrogels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55813/55813fig1.jpg" /> 
Figure 1: Freeze-drying the hydrogels. After hydrogel polymerization, the gels were transferred to -196 &#176;C (liquid nitrogen) or -20 &#176;C. The material was stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The size and distribution of the pores within the material were analyzed with imaging software and were plotted for the different temperatures. On the right side, representative pictures of the materials frozen at (A) -196 &#176;C and (B) -20 &#176;C are shown. The error bars represent the standard deviation. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55813/55813fig2.jpg" /> 
Figure 2: Hydrogel particle leaching. Salt crystals are ground with a mortar and pestle for (A) 10 min, (B) 2 min, (C) 1 min, and (D) 0 min. Both hydrogel components are mixed, and the solution is saturated with salt crystals. After the polymerization of the materials, salt is eluted from the material by diluting the template in large amounts of deionized water. The material is stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The size and distribution of the pores within the material were analyzed with imaging software and plotted for the different grinding times. Images A to D show the pores within representative hydrogels, where salt crystals of varying sizes were used. The error bars represent the standard deviation. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55813/55813fig3.jpg" /> 
Figure 3: Channel formation. The polymerized hydrogels were transferred to a block of dry ice for 30 s. This was followed by the sublimation of the ice crystals from the structure at 0.05 mbar and -85 &#176;C. The material was stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The channels were several hundred microns long and had diameters of about 20 &#181;m. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55813/55813fig4.jpg" /> 
Figure 4: Enzymatic degradation. Representative degradation pattern of a macroporous hydrogel treated using a salt-leaching approach with ground salt crystals (10 min). Degradation took place in solution with 300 U trypsin and pepsin and was compared to an untreated hydrogel in PBS by measuring the residual weight of the material every hour. The error bars represent the standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55813/55813fig5.jpg" /> 
Figure 5: Cellular behavior on hydrogels. To investigate the potential of the material in cell culture, hydrogels were functionalized with a cell-adhesive RGD peptide. The hydrogels were polymerized into a &#181;-slide 8 well and seeded with 2 x 105/cm2 A549 and MCF7 cells. The cells were allowed to grow and adhere to the material for 24 h. After fixation with 3.7% formaldehyde and permeabilization with 0.1% Triton X-100, the cells were stained with cell-specific phalloidin-rhodamine and were visualized with inverted confocal laser scanning microscopy at a wavelength of 514 nm. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Salt leaching</td>
			<td rowspan="2">Salt leaching (grinded salt, 10 min)</td>
			<td>Frozen at&#160;</td>
			<td>Frozen at&#160;</td>
			<td rowspan="2">Gradient freezing</td>
			<td rowspan="2">Non-porous hydrogels</td>
		</tr>
		<tr>
			<td>-20&#176;C</td>
			<td>-196&#176;C</td>
		</tr>
		<tr>
			<td>pH 2 [%]</td>
			<td>95.3 &#177; 3.8</td>
			<td>96.2 &#177; 3.3</td>
			<td>94.4 &#177; 3.4</td>
			<td>96.2 &#177; 5.2</td>
			<td>89.7 &#177; 4.3</td>
			<td>69.5 &#177; 4.4</td>
		</tr>
		<tr>
			<td>pH 7 [%]</td>
			<td>95.6 &#177; 2.3</td>
			<td>94.4 &#177; 4.2</td>
			<td>95.3 &#177; 5.6</td>
			<td>94.2 &#177; 3.2</td>
			<td>94.1 &#177; 3.2</td>
			<td>98.2 &#177; 1.6</td>
		</tr>
		<tr>
			<td>pH 10 [%]</td>
			<td>82.1 &#177; 4.4</td>
			<td>86.1 &#177; 3.2</td>
			<td>76.3 &#177; 5.5</td>
			<td>83.2 &#177; 4.3</td>
			<td>84.2 &#177; 4.5</td>
			<td>42.3 &#177; 4.1</td>
		</tr>
		<tr>
			<td>RT [%]</td>
			<td>97.4 &#177; 4.4</td>
			<td>95.4 &#177; 0.42</td>
			<td>91.3 &#177; 2.2</td>
			<td>94.3 &#177; 4.1</td>
			<td>97.1 &#177; 1.9</td>
			<td>99.1 &#177; 2.2</td>
		</tr>
		<tr>
			<td>37&#176;C [%]</td>
			<td>95.3 &#177; 4.2</td>
			<td>97.4 &#177; 0.4</td>
			<td>93.2 &#177; 3.3</td>
			<td>96.2 &#177; 1.9</td>
			<td>95.3 &#177; 4.3</td>
			<td>98.3 &#177; 3.4</td>
		</tr>
		<tr>
			<td>80&#176;C [%]</td>
			<td>81.2 &#177; 4.4</td>
			<td>83.6 &#177; 4.5</td>
			<td>84.2 &#177; 4.9</td>
			<td>83.5 &#177; 3.4</td>
			<td>91.4 &#177; 8.1</td>
			<td>70.2 &#177; 6.2</td>
		</tr>
		<tr>
			<td>Swelling ratio [%]</td>
			<td>1153 &#177; 110</td>
			<td>534 &#177; 45</td>
			<td>1312&#160; &#177; 91</td>
			<td>834 &#177; 78&#160;</td>
			<td>823 &#177; 163</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin [h]</td>
			<td>4.5 &#177; 0.23</td>
			<td>7.4 &#177; 0.29</td>
			<td>3.2 &#177; 0.21</td>
			<td>6.5 &#177; 0.13</td>
			<td>4.2 &#177; 0.13</td>
			<td>55.0 &#177; 2.48</td>
		</tr>
		<tr>
			<td>Pepsin [h]</td>
			<td>3.1 &#177; 0.19</td>
			<td>4.0 &#177; 0.22</td>
			<td>2.4 &#177; 0.13</td>
			<td>3.8 &#177; 0.14</td>
			<td>3.5 &#177; 0.19</td>
			<td>46.5 &#177; 3.02</td>
		</tr>
	</tbody>
</table>
Table 1: Hydrogel properties. To investigate the influence of the pore formation methods used here (top line), different types of hydrogels were investigated for their pH and temperature stabilities, swelling ratio, and enzymatic degradation pattern (left column). For pH values of 2, 7, and 10 and temperatures of RT, 37 &#176;C, and 80 &#176;C, the average residual weights of the hydrogels after 7 days are displayed as percentages. For enzymatic degradation, the half-lives (h) of the materials in 300 U trypsin and pepsin are shown. For the swelling ratios, the water uptakes based upon the dried gels are shown are percentages.

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

不同的方法来操作基于蛋白质的凝胶中的三维结构对材料性能在这里评价。大孔网络一种细胞粘附肽，羧基化，它们在细胞培养的可行性评估使用两种不同的模型细胞株。

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

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

easy-manipulation-architectures-protein-based-hydrogels-for-cell

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.7K 次观看.  Ulm University. 不同的方法来操作基于蛋白质的凝胶中的三维结构对材料性能在这里评价。大孔网络一种细胞粘附肽，羧基化，它们在细胞培养的可行性评估使用两种不同的模型细胞株。

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

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

定量应用同质MALDI样品的制备

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

科研

生物化学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

磁性镊子 quadruplexes 单分子操作

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

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

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

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

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

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

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

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

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

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

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

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

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

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

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

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

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

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

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

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

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

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

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.