从BBSRC资金体谅（批准号BB H011293 / 1）。

概观第1章描述的pH值敏感的纳米传感器和纳米传感器响应利用荧光光谱和它们的大小用扫描电镜pH值表征的准备。 第2节描述的静电聚合物支架和它们的形态和大小用扫描电镜表征的准备。部2还描述了自我报告的支架这是电纺支架与夹杂物的pH值敏感的纳米传感器的制备方法。所得到的自我报告的支架的特征在于通过SEM以评估是否在电纺纤维的任何形态学变化已经发生。从掺入纳米传感器产生的自我报告的支架的荧光共聚焦显微镜进行评估。 第3节因静电脚手架和自我报告脚手架描述细胞的培养。该支架在preparat先消毒离子为其支架进行评估，以确定消毒和细胞的培养是否会影响自我报告脚手架的荧光能力的细胞培养以下。该协议还介绍了纳米传感器交付给那些因静电支架培养的细胞。 Lysotracker染料被用来确保纳米传感器没有被考虑到的细胞的酸性区室。 1。制备及对pH值敏感的溶胶 - 凝胶纳米传感器分析 1.1制备的pH响应溶胶 - 凝胶纳米传感器注意：该制剂应在通风橱中进行。 <ol><li>制备荧光团为通过溶解5将pH纳米传感器中使用 - （和6） - 羧基荧光素，琥珀酰亚胺酯（FAM-SE）（1.5毫克）的二甲基甲酰胺（DMF）中（1毫升）在圆底烧瓶中，6 - 羧基四甲基罗丹明，琥珀酰亚胺酯（TAMRA-SE）（1.5毫克）的DMF（1毫升）在另一个圆底烧瓶中。添加3 -氨基丙基三乙氧基硅烷（APTES）（1.5毫升）到每个培养瓶中，并在干燥的氮气氛下（21℃）下搅拌24小时，在黑暗中，即包裹样品中金属箔。 </li><li>上述两种染料（250微升）添加到乙醇（6毫升）和氢氧化铵（30重量％，4毫升）包含在一个圆底烧瓶中的混合物中，并搅拌1小时（21℃）。 </li><li>慢慢加入正硅酸四乙酯（TEOS）（0.5毫升）到混合物中，继续再搅拌2小时。该混合物会变成混浊。纳米传感器可以通过旋转蒸发来收集。纳米传感器可以在4℃以备将来使用而保存在玻璃小瓶中。 注意：应在通风橱中或在称量它们的平衡应该密封，例如在一个Weighsafe的情况下进行的纳米传感器的进一步的处理在其干燥形式。 </li></ol> 1.2纳米传感器响应pH值<ol><li>准备索伦森的磷酸盐缓冲溶液从pH值5.5-8.0由MIXI吴特定的磷酸二氢钠（0.2M）和磷酸氢二钠（0.2M）股票方案的比率。用pH计检查最终的pH值。使用氢氧化钠（NaOH）（4 M）或盐酸（HCl）（2 M）使任何轻微调整pH值。 </li><li>通过首先固定装有一个涡旋混合3分钟，然后浸没在容器中的超声发生器的纳米传感器溶液的容器悬浮在pH缓冲液（5毫克/毫升）将pH纳米传感器，直到溶液变浑浊（约5分钟）。重复这些步骤，充分悬浮于溶液中的纳米传感器。 </li><li>将第一溶液为光反应杯并使用488 nm的激发波长为5 - （和6） - 羧基荧光素（FAM）和568纳米的6 - 羧基四甲基罗丹明（TAMRA）。收集发射波长在500-530纳米的FAM和558-580 nm的使用TAMRA荧光光谱仪。 </li><li>改变在光学比色皿的解决方案，使得pH值可以在不同的pH下观察到，并继续Çollecting的发射光谱。 </li><li>计算在每个pH值产生的，产生的校正曲线的发射最大值之比。 </li></ol> 1.3扫描电子显微镜的纳米传感器<ol><li>将纳米传感器的样品到一个碳涂覆的电子显微镜存根和溅射涂层具有氩气气氛下，金为5分钟。 </li><li>通过扫描电子显微镜（SEM）观察。在成像调节工作距离，电压和放大倍数，以减少电子的充电（ 图1）。 </li></ol> 2。 PLGA支架的制备与分析 2.1静电纺丝PLGA支架<ol><li>在通风橱中溶解聚在二氯甲烷与吡啶甲酸（乳酸 - 共 - 乙醇酸）（PLGA）（DCM）（20％（W / W））（PF）（1％（W / W））。将溶液到10ml注射器18G的钝针填充和牢固地贴合到注射泵。 </li><li>距离针尖20 cm的距离20厘米x 15厘米铝制收集板。 </li><li>附加的高电压电源的电极，以将注射器和地球到铝集电板的一角。 注意：应注意高电压用于处理任何静电纺丝设备之前， 即始终把电力供应关闭。 </li><li>使用3.5米/小时的恒定流速在12千伏2小时递送解决方案。使用这些条件的电纺丝将给予约60微米的支架的深度。 </li><li>离开该支架在通风橱中放置24小时，以允许溶剂残留挥发。 </li></ol> 2.2静电对pH值敏感的PLGA支架<ol><li>准备在2.1节，但与添加纳米传感器的聚合物溶液（5毫克/毫升）的修改说明纳入对pH值敏感的纳米传感器支架。暂停在PLGA溶液中的纳米传感器与超声波的协助下（约5分钟）前<html>要放置到一个10毫升注射器。 </li></ol> 2.3 SEM PLGA支架和pH值响应脚手架<ol><li>将PLGA支架或pH响应性支架材料的样品上的碳涂覆的电子显微镜存根并进行如下描述的纳米传感器的扫描电镜（ 图1）。 </li></ol> 2.4校准pH值响应脚手架<ol><li>将pH值反应支架的样品放入35毫米培养板，沉浸在适当的缓冲溶液。 </li><li>在共聚焦显微镜使用488 nm的氩激光激发FAM和568 nm的氪激光器的纳米传感器内激发TAMRA。观察pH值敏感支架的荧光发射的500-530纳米的FAM和558-580纳米的TAMRA。 （ 图2）。使用顺序扫描，以避免收集激发波长。 </li></ol> 3。在PLGA支架和pH响应PLGA支架细胞培养ntent“&gt;注：以下应在细胞培养柜中进行。 3.1制备PLGA支架的细胞培养<ol><li>切PLGA或pH响应PLGA支架成2厘米2件。 </li><li>通过用紫外（UV）光在8厘米15分钟每一侧的距离照射消毒支架。 </li><li>传送支架到12孔培养板中，放置一个钢环上顶。加青霉素/链霉素溶液（5％V / V）在磷酸盐缓冲盐水（PBS）的内侧和钢环以外，孵育过夜（37℃，5％CO 2）。注意钢圈于英国诺丁汉大学制造，并具有以下尺寸：3 cm外直径1厘米内直径1厘米深。 </li><li>除去消毒液和洗涤用PBS。 </li><li>加细胞培养基到内侧（500微升）和外侧（500微升）的钢环，地点在培养箱中（37℃，5％CO 2的</子&gt;）而制备细胞悬浮液。 </li><li>制备细胞悬浮液，并用台盼蓝排除法进行细胞计数。 </li><li>建立细胞悬浮液以达到1×10 6个细胞/ ml的细胞数。 </li><li>从钢板环的内侧和外侧除去介质。 </li><li>替换上的钢圈（1米）的外预热的细胞培养基。 </li><li>加入300μL的细胞悬液到钢圈的内侧 - 岩板向前和向后轻轻散发细胞。 </li><li>将细胞培养板放入培养箱中（37℃，5％CO2） -细胞附着应该发生在24小时内，但是继续培养，只要实验要求-刷新媒体每2-3天。 </li></ol> 3.2纳米传感器传递到细胞在PLGA支架培养<ol><li>如3.1节所述的种子细胞到PLGA支架。在这项研究中的空白PLGA支架（含有多种不宁纳米传感器）是成像时使用，由于荧光发射的重叠。 </li><li>将细胞接种到支架恒温箱（37℃，5％CO 2）72小时，以使细胞在整个纳米传感器，以提供之前的支架生 ​​长和迁移。 </li><li>用PBS（37℃，5％CO2）1小时前，纳米传感器交付洗细胞和媒体的改变从标准培养基抗生素和无血清培养基，培养。注意：标准培养基为3T3细胞包括补充有胎牛血清（10％（V / V）），L-谷氨酰胺溶液（2毫摩尔），青霉素/链霉素的Dulbecco改良的Eagle培养基（1％（V / V）） 。 </li><li>发病期间短暂超声处理的纳米传感器（5毫克）用的Optimem（50微升）来创建溶液A制备纳米传感器 - 脂质体复合物</li><li>通过将脂质体2000（5微升）到的Optimem（45微升）创建B溶液，在室温下孵育5分钟。 </li><li>添加解决方案A至溶液B，​​在室温下孵育20分钟以形成溶液C这是纳米传感器 - 脂质体复合物。 </li><li>加入溶液C（100微升）对细胞进行培养（37℃，5％CO 2）为3小时。 </li><li>从培养箱中，抽吸介质除去细胞接种支架，加入PBS（1ml）中，以允许活细胞可视化通过共聚焦显微镜之前，​​用PBS洗两次。注意：用PBS在这个协议使得酚红指示剂存在于细胞培养基中，因为正在执行校准在不同的pH没有引起自发荧光和也。然而，可用于细胞培养的较长期分析酚红培养基中。 </li><li>沉浸细胞接种到支架的不同pH值的缓冲液，并监控与细胞相关的纳米传感器的响应特性。 </li><li>监视的pHi通过确定从FAM和TAMRA的荧光强度的比率通过使用共聚焦显微镜（FIGU观察荧光再3）。 </li></ol>哺乳动物细胞内3.3纳米传感器位置<ol><li>培养细胞用含有仅3.2节所描述的FAM染料（这是因为TAMRA的荧光发射是在同一光谱区为LysoTracker红色）的纳米传感器。在1.1节，但与遗漏，并准备加入TAMRA的介绍准备这些纳米传感器。 </li><li>随着纳米传感器交货周期稀释LysoTracker红原液至50nm。 </li><li>添加稀释的溶液（2微升）到细胞后PLGA支架培养物的等分试样。 </li><li>孵育细胞LysoTracker红1小时（37℃，5％CO2）。 </li><li>以下潜伏期移除介质和改变介质的PBS（pH 7.4）中（2ml）中，准备使用共聚焦显微镜观察前，用PBS（pH 7.4）中洗细胞两次。注：可以用来代替PBS酚红的自由媒体。 </li><li>加入核染色绘制直线Q5到PBS中，使最终浓度为5微米，并孵育细胞3分钟（37℃，5％CO 2）。这是没有必要以下的潜伏期与DRAQ5改变介质。 </li><li>使用488nm的氩激光激发FAM只有纳米传感器，一个568 nm的氪激光激发LysoTracker红色和633 nm的氦/氖激光激发DRAQ5生色团，并观察荧光在500-530纳米，波长580-600，650 -750 nm处（ 图3）。使用顺序扫描，以避免收集激发波长。 </li></ol>

<ol>
	<li>Takimoto, Y., Dixit, V., Arthur, M., Gitnick, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+liver+tissue+formation+in+rats+using+a+novel+collagen-polypropylene+scaffold.">De novo liver tissue formation in rats using a novel collagen-polypropylene scaffold.</a> Cell Transplantation. 12 (4), 413-421 (2003).</li><li>Sales, V. L., Engelmayr, G. C., 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=Protein+precoating+of+elastomeric+tissue-engineering+scaffolds+increased+cellularity,+enhanced+extracellular+matrix+protein+production,+and+differentially+regulated+the+phenotypes+of+circulating+endothelial+progenitor+cells.">Protein precoating of elastomeric tissue-engineering scaffolds increased cellularity, enhanced extracellular matrix protein production, and differentially regulated the phenotypes of circulating endothelial progenitor cells.</a> Circulation. 116 (11), I55-I63 (2007).</li><li>Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+scaffold+design+for+tissue+engineering.">Porous scaffold design for tissue engineering.</a> Nature Materials. 4 (7), 518-524 (2005).</li><li>Li, D., Xia, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+nanofibers:+Reinventing+the+wheel?.">Electrospinning of nanofibers: Reinventing the wheel?.</a> Advanced Materials. 16 (14), 1151-1170 (2004).</li><li>Sill, T. J., von Recum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro+spinning:+Applications+in+drug+delivery+and+tissue+engineering.">Electro spinning: Applications in drug delivery and tissue engineering.</a> Biomaterials. 29 (13), 1989-2006 (2008).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+polymeric+nanofibers+for+tissue+engineering+applications:+A+review.">Electrospinning of polymeric nanofibers for tissue engineering applications: A review.</a> Tissue Engineering. 12 (5), 1197-1211 (2006).</li><li>Sawyer, N. B. E., Worrall, L. 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=In+situ+monitoring+of+3D+in+vitro+cell+aggregation+using+an+optical+imaging+system.">In situ monitoring of 3D in vitro cell aggregation using an optical imaging system.</a> Biotechnology and Bioengineering. 100 (1), 159-167 (2008).</li><li>Dan, L., Chua, C. K., Leong, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Response+to+Interstitial+Flow:+A+State-of-the-Art+Review.">Fibroblast Response to Interstitial Flow: A State-of-the-Art Review.</a> Biotechnology and Bioengineering. 107 (1), 1-10 (2010).</li><li>Pancrazio, J. J., Wang, F., Kelley, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+tools+for+tissue+engineering.">Enabling tools for tissue engineering.</a> Biosensors &amp; Bioelectronics. 22 (12), 2803-2811 (2007).</li><li>You, Y., Lee, S. W., Youk, J. H., Min, B. M., Lee, S. J., Park, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+degradation+behaviour+of+non-porous+ultra-fine+poly(glycolic+acid)/poly(L-lactic+acid)+fibres+and+porous+ultra-fine+poly(glycolic+acid)+fibres.">In vitro degradation behaviour of non-porous ultra-fine poly(glycolic acid)/poly(L-lactic acid) fibres and porous ultra-fine poly(glycolic acid) fibres.</a> Polymer Degradation and Stability. 90 (3), 441-448 (2005).</li><li>Dong, Y. X., Liao, S., Ramakrishna, S., Chan, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+degradation+behaviors+of+electrospun+PGA,+PLGA+and+P(LLA-CL)+nanofibers+cultured+with/without+cell+culture.">Distinctive degradation behaviors of electrospun PGA, PLGA and P(LLA-CL) nanofibers cultured with/without cell culture.</a> Multi-Functional Materials and Structures. 47 - 50, 1327-1330 (2008).</li><li>Xu, Y. H., Sun, J. J., Mathew, G., Jeevarajan, A. S., Anderson, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+glucose+monitoring+and+control+in+a+rotating+wall+perfused+bioreactor.">Continuous glucose monitoring and control in a rotating wall perfused bioreactor.</a> Biotechnology and Bioengineering. 87 (4), 473-477 (2004).</li><li>Harrington, H. C., Rose, F. R. A. J., Reinwald, Y., Buttery, L. D. K., Ghaemmaghami, A. M., Aylott, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+PLGA+fibre+sheets+incorporating+fluorescent+nanosensors:+self-reporting+scaffolds+for+application+in+tissue+engineering.">Electrospun PLGA fibre sheets incorporating fluorescent nanosensors: self-reporting scaffolds for application in tissue engineering.</a> Analytical Methods. 5 (1), (2013).</li><li>Wang, X. Y., Drew, C., Lee, S. H., Senecal, K. J., Kumar, J., Sarnuelson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+membranes+for+highly+sensitive+optical+sensors.">Electrospun nanofibrous membranes for highly sensitive optical sensors.</a> Nano Letters. 2 (11), 1273-1275 (2002).</li><li>Yang, Y., Yiu, H. H. P., El Haj, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-line+fluorescent+monitoring+of+the+degradation+of+polymeric+scaffolds+for+tissue+engineering.">On-line fluorescent monitoring of the degradation of polymeric scaffolds for tissue engineering.</a> Analyst. 130 (11), 1502-1506 (2005).</li><li>Blackwood, K. A., McKean, R., 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=Development+of+biodegradable+electrospun+scaffolds+for+dermal+replacement.">Development of biodegradable electrospun scaffolds for dermal replacement.</a> Biomaterials. 29 (21), 3091-3104 (2008).</li><li>Bashur, C. A., Dahlgren, L. A., Goldstein, A. S. <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+fiber+diameter+and+orientation+on+fibroblast+morphology+and+proliferation+on+electrospun+poly(D,L-lactic-co-glycolic+acid)+meshes.">Effect of fiber diameter and orientation on fibroblast morphology and proliferation on electrospun poly(D,L-lactic-co-glycolic acid) meshes.</a> Biomaterials. 27 (33), 5681-5688 (2006).</li><li>Li, W. J., Laurencin, C. T., Caterson, E. J., Tuan, R. S., Ko, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+structure:+A+novel+scaffold+for+tissue+engineering.">Electrospun nanofibrous structure: A novel scaffold for tissue engineering.</a> Journal of Biomedical Materials Research. 60 (4), 613-621 (2002).</li><li>Sung, H. J., Meredith, C., Johnson, C., Galis, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+scaffold+degradation+rate+on+three-dimensional+cell+growth+and+angiogenesis.">The effect of scaffold degradation rate on three-dimensional cell growth and angiogenesis.</a> Biomaterials. 25 (26), 5735-5742 (2004).</li></ol>

作者宣称，他们有没有竞争的财务权益。

组织工程立志创建生物替代品，可以用来作为在体内像体外组织模型和组织替代疗法进行修复，替换，维持或提高特定组织或器官的功能。合成的替代物已经使用了很多年，以取代或组织抢修，但这些往往是由于与宿主组织和/或感染，最终导致拒绝或进一步修正手术融合不佳失败。生成植入前活组织，在实验室可以解决实现全面整合的问题，并减少需要翻修手术。然而，这个目标要...

在组织工程中的一个关键的策略是使用生物相容性的材料来制作支架，其形态类似于组织，它要替代，也能够支持细胞生长和功能1,2。脚手架允许细胞附着和增殖但允许在整个三维细胞结构的空隙细胞迁移提供机械支撑。该支架还必须考虑到的细胞营养物质传输，而不是抑制代谢废物清除3。 静电纺丝已经成为一种很有前途的方法，能够支持细胞生长4-6聚合物支架的制造。生产的非织造电纺纤维是适合于细胞生长，因为它们往往是多孔的，并允许在整个三维细胞构建体7的间隙中的细胞-细胞相互作用，以及细胞迁移。它吨期间监测细胞存活力是重要他培养期间，确保细胞活力保持在整个三维构建体。例如，培养条件，如氧和pH值需要小心控制，因为在分析物的浓度梯度可以在三维结构内的存在。生物反应器或灌注系统可受聘于间质流体内条件，因此增加营养转移和代谢废物清除8模仿。这种系统是否正在确保恒定的微环境条件的问题可以通过评估在实时细胞微环境加以解决。 这可以实时监测关键微环境指标包括：温度，细胞培养基的化学成分，溶解的氧和二氧化碳的浓度，pH和湿度。这些指标中，温度可最容易使用的探针原位监测。方法监测通常在余下的上市指标沃尔沃去除等分取样，因此干扰了细胞培养和增加污染的风险。连续，实时的方法正在追捧。目前的监测方法通常依赖于物理上探测细胞结构，如pH监测仪或氧探头的仪器。然而，这些侵入性的方法可以破坏细胞结构，扰乱正在进行的实验。无创监测的三维结构中的分析物浓度可以使各种环境因素，如营养枯竭9实时监控。这将使营养供给的评估，以结构内更深的区域，并确定是否代谢废物得到有效10,11去除。试图解决侵袭的问题的系统一般涉及使用已灌注腔的传递培养基同时通过培养容器和外部传感器来监控pH值，氧气和葡萄糖12。该再是在发展中国家，可以直接集成到培养容器中，不需要除去的等分试样进行采样的，因此会在原位监测传感器提供越来越大的兴趣。 为解决现场和非侵入性的监测，我们已经把分析物响应纳米传感器为静电支架，产生自我报告的支架13微环境条件等缺点。即通过监测荧光活性充当传感装置的支架已预先制备的，其中所述感测装置是要么通过电或通过使用分析物响应性染料被加入到聚合物中之前，形成支架14,15的所产生的实际的聚合骨架。然而，这些传感装置必须给由从其他分析物可能的干扰错误的光输出的潜力。使用比例传感装置的su通道的那些在所描述的方案制备持有以消除这些可能的不利影响，并提供特定于所讨论的分析物的响应的可能性。 这里介绍的静电支架乃从合成的共聚物的聚（乳酸-共-乙醇酸）（PLGA），由于选择具有食品和药物管理局（FDA）批准的，由于其可生物降解和生物相容特性和轨道支持各种细胞类型16-18的生长和功能的记录。所制备的比例分析物响应纳米传感器的响应pH值。纳米传感器包括两个荧光染料成生物相容的溶胶 - 凝胶基质中，其中一种染料，FAM是响应于pH和其他，TAMRA作为内标，因为它是不响应的pH值。另外两个FAM和TAMRA的荧光可以单独进行分析，因为它们不显著重叠。确定所述荧光EMI的比例两种染料的特定波长裂变给出的pH响应独立于其它环境条件。自报支架可以让pH值的重复评估， 原位和实时而不破坏开发的3D模型。我们已经证明，这些支架能够支持细胞附着和增殖，并保持响应到有问题的分析。的酸性副产物的工程化构建体的动力学保持充分研究，因此使用对pH值敏感的支架可以大大促进这些研究19。此外，使用了自行申报组织工程支架的应用提供了机会，以充分了解，监控和优化的三维模型的组织结构在体外生长，无创性和实时性。 对pH值敏感的纳米传感器也已交付给成纤维细胞在静电PLGA scaffol培养的细胞内环境DS。荧光发射的从染料的比率被用来监测和pHi值相比，自我报告的支架结合的pH纳米传感器。纳米传感器的细胞在3D环境中培养的递送可以使深部以非破坏性的方式构造内的监测的分析物浓度的影响。因此，纳米传感器可能是一个可行的成像工具，非破坏性评估整个3D细胞行为的构造允许长期分析。筛选三维结构中的单个细胞的分析物浓度可以确保它们得到足够的营养物和氧的浓度。监测过程参数可有助于标准化的技术的发展为氧气和营养物质的有效质量传输。纳米传感器对细胞内环境和纳米传感器为高分子支架结合的交付可以合并，允许在3D常量评估细胞活力以及支架的性能在组织生长过程ructs。这可能会导致这些结构增加了知识和进步的生物学相关的组织替代品的制造。

<table border="0" cellpadding="0" cellspacing="0" width="481">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Ethanol</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">32221</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Anhydrous dimethylformamide (DMF)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">270547</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Ammonium hydroxide 50% (v/v) aqueous solution</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:137px;">35574</td>
			<td style="width:79px;">diluted to 30% (v/v) with pure water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">TEOS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">13190-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">3-Aminopropyltriethoxysilane (APTES)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">A3648</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">5-(and-6)-carboxyfluorescein, succinimidyl ester (FAM-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1311</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">6-carboxytetramethylrhodamine, succinimidyl ester (TAMRA-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1171</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate monobasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate dibasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-0876</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">NaOH</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S8045</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Trypsin/EDTA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T4174</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">P0781</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">PBS</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D8537</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DMEM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D6046</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">FBS</td>
			<td style="width:71px;">Source Bioscience</td>
			<td style="width:137px;">Batch-213-101992</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">L-Glutamine</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">G7513</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Lipofectamine 2000</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11668-019</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Optimem</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11058-021</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">LysoTracker Red</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">L-7528</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Draq5</td>
			<td style="width:71px;">Biostatus Ltd</td>
			<td style="width:137px;">DR50050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Nitrogen gas</td>
			<td style="width:71px;">BOC</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DCM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">320269</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">TFA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T6508</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:195px;">Confocal microscope</td>
			<td style="width:71px;">Leica TCS-SP</td>
			<td style="width:137px;"></td>
			<td style="width:79px;">equipped with argon and krypton lasers and a 63X 0.9NA water immersion lens</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">UV light</td>
			<td style="width:71px;">UVLS28 UVP, USA</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Stirrer plate</td>
			<td style="width:71px;">SB161-3 Jencons-PLS</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">pH meter</td>
			<td style="width:71px;">Jenway model 3510</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Rotary Evaporator</td>
			<td style="width:71px;">Buchi Rotary Evaporator R200</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Centrifuge (nanosensors)</td>
			<td style="width:71px;">Hermle Z300</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:195px;">Centrifuge (cell culture)</td>
			<td style="width:71px;">Thermo Scientific Heraeus Biofuge Primo</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Vortex</td>
			<td style="width:71px;">Whirlimixer Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Ultrasonicator</td>
			<td style="width:71px;">FB11021 Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Aluminum sheet</td>
			<td style="width:71px;">Nottingham University</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">35mm cell culture plate</td>
			<td style="width:71px;">Iwaki</td>
			<td style="width:137px;">3000035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">10 ml syringe</td>
			<td style="width:71px;">Becton Dickenson</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">3T3 Fibroblast cells</td>
			<td style="width:71px;">European Collection of Cell Cultures</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">PLGA</td>
			<td style="width:71px;">Lakeshore Biomaterials</td>
			<td>7525 DLG 7E</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pyridinium formate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>P8535</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Trypan blue</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>T8154</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium phosphate monobasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium phosphate dibasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S5136</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HCl</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>320331</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

self-reporting scaffolds for 3-dimensional cell culture

培养细胞在三维上合适的支架材料被认为是更好的模拟体内微环境和提高细胞间的相互作用。所得到的三维蜂窝结构往往可以更相关的研究的分子事件和细胞间的相互作用比研究中的2D类似的实验。在整个支架创建具有高细胞活性的有效三维培养的培养条件，如氧和pH值必须仔细控制在分析物的浓度梯度可以在整个三维结构存在。这里我们描述了制备生物相容的pH响应性溶胶 - 凝胶纳米传感器和它们掺入到聚（乳酸 - 共 - 乙醇酸）（PLGA）电纺支架以及它们的用于哺乳动物细胞的培养以后的制剂的方法。将pH响应性支架材料可以用作工具，以一个三维蜂窝结构内确定微环境的pH值。此外，我们详细的pH响应nanose交付nsors哺乳动物细胞，其增长主要由电纺PLGA支架支撑的细胞内环境。对pH值敏感的纳米传感器的细胞质位置可以利用正在进行的实验过程中监测细胞内pH（PHI）。

利用SEM，其中纳米传感器成像的人口进行测定，发现具有纳米尺寸在240-470纳米（ 图1A）的范围内制得的pH值敏感的纳米传感器的尺寸分布进行了表征。实现一个狭窄的，合理的小直径与使用施托贝尔法制备纳米粒子是一致的。已经发现，纳米粒子的合成过程中使用碱性pH环境即纳米颗粒制备使用施托贝尔方法，允许良好控制的大小的，而使用酸性条件制剂生产中颗粒大小广泛分...

Watch this Scientific Journal Video about Self-reporting Scaffolds for 3-Dimensional Cell Culture at JoVE.com

Self-reporting Scaffolds for 3-Dimensional Cell Culture

生物相容的pH响应性溶胶 - 凝胶纳米传感器可并入聚（乳酸 - 共 - 乙醇酸）（PLGA）电纺支架。所产生的自我报告的支架可用于在微环境条件原位监测，同时在所述支架中培养细胞。这是有益的，因为在三维细胞构建体可以在实时而不干扰试验来监测。

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

self-reporting-scaffolds-for-3-dimensional-cell-culture

Research

JoVE Journal

Bioengineering

13.1K 次观看.  University of Nottingham. 生物相容的pH响应性溶胶 - 凝胶纳米传感器可并入聚（乳酸 - 共 - 乙醇酸）（PLGA）电纺支架。所产生的自我报告的支架可用于在微环境条件原位监测，同时在所述支架中培养细胞。这是有益的，因为在三维细胞构建体可以在实时而不干扰试验来监测。 

视频: Self-reporting Scaffolds for 3-Dimensional Cell Culture

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

科研

生物工程

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...