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

Universidad de Cartagena UDEC

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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

生物学

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

癌症研究

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.

Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo ...

使用 3D 声学组装装置可扩展生成均匀的单元球体

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

作者聚焦:用于高质量 3D 细胞球状体培养的无支架声学组装方法的开发 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

用于细胞球体批量生产的三维声学装配装置

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...

自我报告的支架3维细胞培养

摘要

探索更多视频

此视频中的章节

一个简单的悬滴细胞生成三维球体文化协议

芯片尺寸支架的三维细胞培养的微细

监测三D培养中的癌细胞入侵和T细胞毒性

使用胶原基支架的神经母细胞瘤三维体外仿生模型

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

三维肽支架中哺乳动物细胞的培养

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

用于细胞球体批量生产的三维声学装配装置

用于细胞球体批量生产的三维声学装配装置

自我报告的支架3维细胞培养

摘要

探索更多视频

此视频中的章节

一个简单的悬滴细胞生成三维球体文化协议

芯片尺寸支架的三维细胞培养的微细

监测三D培养中的癌细胞入侵和T细胞毒性

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

三维肽支架中哺乳动物细胞的培养

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数

用于细胞球体批量生产的三维声学装配装置

用于细胞球体批量生产的三维声学装配装置

使用胶原基支架的神经母细胞瘤三维体外仿生模型