作者没有确认。

1. 细胞的制备
<ol><li>在生物安全柜中使用无菌技术, 将2毫升培养基中的2万细胞放入6井板中的每一个井中。</li>
	<li>放置6井板在一个湿润的孵化器 (37 °c, 5% CO2) 为 24 h 允许细胞坚持板材。</li>
</ol>2. 制备用于治疗细胞的光敏剂
<ol><li>在培养基中准备0.5、1和2毫米的 5-阿拉溶液。如果胎牛血清 (血清) 是培养基中的一个成分, 则在培养基溶液中制备和治疗 5-孵化的细胞, 0.1% 的血清。11,12在这种情况下, SCC-13 细胞不需要血清, 因此 5-阿拉氨酸直接添加到角质的无血清培养基补充牛垂体提取物和表皮生长因子。</li>
	<li>吸入培养基, 用至少2毫升磷酸盐缓冲生理盐水 (PBS) 冲洗细胞。</li>
	<li>吸入 PBS 并将2毫升的0、0.5、1或2毫米的溶液放入单独的板材或水井中。</li>
	<li>在5至36摄氏度加热块上孵育0、0.5、1或2毫米 37-阿拉的细胞, 以防止细胞在使用铝箔孵化过程中受到光的照射。</li>
	<li>吸入 0, 0.5, 1 和2毫米 5-阿拉的解决方案, 并清洗细胞与2毫升的 PBS。</li>
	<li>吸入 PBS 并加入2毫升的培养基进行蓝光照射。</li>
</ol>3. 蓝光光疗法
<ol><li>在照射电池之前, 打开蓝光装置 (例如, 发光二极管、荧光灯或卤素灯) 并运行一个周期 (1000 秒) 以预热设备。蓝光装置的输出波长为 417 +/-5 nm。在处理前允许设备运行一个周期, 以确保蓝光波长和辐照度在整个 photoactivation 阶段是一致的。</li>
	<li>使用光度计测量细胞表面的辐照度。蓝色光辐照在细胞表面应该是10兆瓦/cm2。光和细胞之间的距离可能需要调整, 以达到10兆瓦/厘米2的辐照度取决于光源的强度。</li>
	<li>在蓝色光源下的黑色表面放置0、0.5、1和2毫米的治疗组。用蓝光照射细胞1000秒 (16 分钟和四十年代), 总剂量为 10 J/厘米2。在蓝光 photoactivation 之后, 细胞就可以进行分析了。</li>
</ol>4. 收集和染色
<ol><li>根据制造商的指导方针 (见材料表) 准备流量缓冲器。</li>
	<li>吸入培养基, 用2毫升的 PBS 冲洗细胞。</li>
	<li>添加1毫升0.25% 胰蛋白酶-EDTA, 并允许细胞分离约3至5分钟。细胞可以在显微镜下检查以确认细胞脱离。</li>
	<li>添加1毫升培养基 (或 PBS) 与10% 胎牛血清, 以停用胰蛋白酶。</li>
	<li>收集细胞悬浮到标记的5毫升流动管。</li>
	<li>在离心机中旋转5毫升的流动管, 在 201 x g处进行5分钟的抽吸溶液, 不去除细胞颗粒。</li>
	<li>添加200µL 的流缓冲器与共轭蛋白抗体 (1 µL 蛋白 v 每39µL 的流量缓冲区) 的每个样本。</li>
	<li>并用重悬细胞和地方在孵化器 (37 °c, 5% CO2) 为20分钟允许蛋白-V 捆绑。</li>
	<li>向每个样品添加3µL 7-反倾销。室温下孵育5分钟。</li>
</ol>5. 流量细胞分析
<ol><li>遵循流式细胞仪制造商的设置指南 (见材料表)。</li>
	<li>用流式细胞仪采集和分析样品。13
</li>
	<li>采用方差分析14对治疗和对照组进行统计学比较。将治疗组的方法与控制的平均值进行比较, 用 Dunnett 的试验进行后分析。</li>
</ol>

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DUSA/太阳制药提供了 5-阿拉和蓝光光设备。这份手稿由巴拉特·贾格迪奥博士的剩余资金支持。这些内容不代表美国退伍军人事务部或美国政府的意见。

30分钟孵化、1、2毫米 5-SCC-13 细胞凋亡显著增加, 其次为1000秒蓝光。这些结果与我们以前发表的研究相一致, 证明 5-孵化30分钟后蓝色光激活导致成纤维细胞在细胞凋亡12的百分比增加, 14。
该实验方法对 PDT 的研究具有优势和局限性。所描述的方法符合临床实践作为一个商业上可用的 PS 和光源使用。研究人员可以自定义不同细胞类型、PSs、光源和辐照参数的方法。然而, 这种方法有局限性, 因为这个协议是为在单层培养的黏附细胞设计的。在临床实践中, 组织结构或疾病病理学 (即底角化过度或纤维化) 的差异可能会降低 PS 吸收或光穿透15。因此, 治疗剂量和实验结果可能与临床实践没有直接的对应。以球形肿瘤模型和微流控芯片为方法, 对肿瘤微环境16、17、18进行了较严密的复制。球体有内部和外部细胞的利基, 可能差异纳入光敏和代表异构的肿瘤结构。其他研究人员使用微流控芯片筛选治疗条件和光敏的血管输送。然而, 对于不熟悉该技术的研究人员来说, 微流控芯片的开发或难于实现的成本可能很高。此外, 球体和微流控芯片可能无法反映光敏直接应用于肿瘤表面而无血管传播的皮肤癌的临床治疗。研究人员可能需要评估单层培养、球形肿瘤模型和微流控芯片在不同疾病系统中研究 PDT 的利弊。由于没有免疫细胞或细胞外基质, 因此不可能确定 PDT 如何影响复杂的细胞间相互作用。研究人员可能需要用动物模型和临床试验证实体外实验结果。在体外光动力实验中, 保持一致的孵化和光照参数, 对于提高检测的有效性具有重要意义。
已知温度改变了 PDT19的功效。一项研究表明, 细胞死亡, 5 的摄取, PP IX 的形成, 和细胞因子释放可能会加强孵化细胞与 5-阿拉在温度高达44摄氏度19。44摄氏度以上的潜伏期可能导致热诱导细胞死亡, 孵化温度低于20摄氏度可能不会导致显著的 5-阿拉的摄取和积累19。我们建议研究人员在恒温温度可调的加热块上执行 PS 孵化, 以控制潜在的混杂热效应。此外, 重要的是要孵化的光敏剂足够的时间, 以允许转化为 5-阿拉的 PP ix。在该协议中, SCC-13 细胞以 5-亚拉巴马州为30分钟的孵化, 成功诱导细胞凋亡。我们以前已经证明, 5-阿拉的10分钟孵化并没有显著增加成纤维细胞凋亡与未经处理的控制成纤维细胞11,14。PP-IX 开始积累在小鼠皮肤 5-阿拉孵化六分钟37摄氏度。因此, 经过十分钟的5的孵化, 可能没有足够的积累, PP IX 诱导细胞凋亡20。我们建议的最低潜伏期20至30分钟的 5-阿拉的基础上, 我们以前的研究11,14。研究人员可能需要根据实验室设置、感兴趣的光敏剂、细胞类型和临床指示来优化潜伏期。抗体滴定和优化实验可以执行的最佳结果使用制造商的指导方针。其他的兴趣分析可以执行后, 蓝色光 photoactivation 包括 dihydroethidium 流细胞定量的 ROS。14
在 photoactivation 阶段, 每单位表面积 (即辐照度) 和总辐照剂量 (即通量) 的光含量变化可能会影响 ROS 的形成和细胞凋亡21。通量、辐照度和时间的关系可以用以下等式描述:
剂量 (J/厘米2) = 辐照度 (W/厘米2) x 时间 (s)
由于剂量依赖于时间, 延长或缩短 photoactivation 阶段可能改变治疗效果。辐照度与光源和靶组织的距离平方成正比。因此, 如果光线太远, 可能没有足够的光能传递给目标组织来激发 PS. 或者, 辐照度可以通过反射周围表面的光线来增加。因此, 我们建议执行光辐照与放置在黑色表面的细胞培养板, 以防止光反射。研究人员可能获得商用或 FDA 批准的蓝光发光二极管和荧光器件用于 PDT 实验, 但这些器件可能有不同的功率输出和光场均匀性。每个实验前, 应使用波长特定的光度计测量光场在细胞表面的辐照度和均匀度, 以求得一致的结果。如果有必要, 可使用商用扩散器来提高场的均匀性。
总之, 我们描述了一种体外方法, 通过详述理论、实验方法、局限性、潜在缺陷和优化建议来调查 PDT。pdt 是一个有用的临床程序, 并在体外研究可以为新的 PSs 的发展, 协议的优化, 和新的迹象, pdt。

光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。联邦药物管理局 (FDA) 批准了 pdt 治疗日光脂溢性 (AK), 临床指南建议 pdt 作为治疗某些非黑色素瘤皮肤癌和寻常痤疮1,2。新出现的非皮肤病疗法包括胃肠道、前列腺癌和妇科癌症的治疗3。PDT 是一种有利的治疗方式, 因为它是低成本, 无创, 并与最小的不良事件和疤痕2。
在 PDT 的第一步, 应用 PS, 并允许积累 intracellularly2。在美国, 局部 5-氨基乙酰丙酸 (5-阿拉) 通常用于治疗 AKs。在孵化阶段, 5-亚拉巴马州通过在合并细胞中的血红素生物合成途径转化为原卟啉 ix (PP ix)3。癌症和癌前细胞减少了 ferrochelatase 活性, 这将原卟啉 ix (PP ix) 转化为最终产物, 血红素。结果, 与正常细胞4,5相比, 癌细胞积累了 PP IX。这种积累的 PP IX 在癌症和前癌细胞相对于周围组织允许 PDT 是一个有针对性的方法, 最小的不良事件。当氧接受来自 PP ix 的激发电子时, 光辐照会导致活性氧 (ROS) 的产生。PP-IX 在紫外光谱中具有峰值吸收, 在可见光谱中有较小的峰值, 包括蓝色和红光2。ROS 的形成可能最终导致细胞凋亡, 膜破坏, 线粒体损伤, 免疫调节, 角质细胞增殖, 胶原蛋白周转6,7,8,9,10。
PDT 是在二十世纪七十年代发展起来的, 是一种进化的治疗方式3。FDA 批准的治疗 AKs 建议 5-14–18 h 的皮肤孵化, 但1至 2 h 孵化通常用于临床实践2。在体外, 我们已经表明, 较短的15分钟 5-阿拉孵化可能会增加成纤维细胞凋亡相比, 未经处理的成纤维细胞11,12。此外, 研究了新的氯、酞菁和纳米粒子的 PSs, 以提高 PDT 功效3。在此, 我们提出了一种体外方法来研究 PDT 在黏附鳞状细胞癌细胞。在本协议中, SCC-13 细胞以0、0.5、1.0 和2毫米 5-阿拉的孵化为30分钟和光活化作用, 417 nm 蓝色光为 s。主要结果是凋亡和坏死, 蛋白和 7-aminoactinomycin D (7) 流式细胞术测量。该治疗协议旨在模拟 pdt, 并可调整以研究使用 pdt 与其他细胞系, 光敏, 孵化温度, 或 photoactivation 波长。可能需要准备额外的控制组, 包括无瑕, 单荧光染色, 阴性, 和积极的控制流式细胞术。对凋亡/坏死流式细胞术的理论、规程、实验设计和门控进行了全面的回顾, 可在别处找到13。

<table><tbody><tr><td>Keratinocyte serum free medium&amp;nbsp;</td><td>Thermofisher</td><td>17005042</td><td>Culture medium for SSC-13 cells</td></tr><tr><td>DMEM low glucose glutamax</td><td>Thermofisher</td><td>10567022</td><td>Possible culture medium for other cell types</td></tr><tr><td>6-well culture plates</td><td>Thermofisher</td><td>720083</td><td></td></tr><tr><td>PBS</td><td>Thermofisher</td><td>14190250</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Thermofisher</td><td>25200056</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermofisher</td><td>15250061</td><td>For cell counting and plating&amp;nbsp;</td></tr><tr><td>BLU-U light&amp;nbsp;</td><td>DUSA</td><td>Request from manufacturer</td><td>Other blue LED, flourescent, or halogen lights may used&amp;nbsp;</td></tr><tr><td>5-ALA</td><td>DUSA</td><td>Request from manufacturer</td><td></td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>C17032</td><td></td></tr><tr><td>FlowCellect Annexin Red Kit</td><td>Millipore Sigma&amp;nbsp;</td><td>FCCH100108&amp;nbsp;</td><td>Propidium iodide may replace 7-AAD. Comes with annexin-V, 7-AAD, and flow buffer</td></tr><tr><td>FACSCanto II</td><td>BD</td><td>Request from manufacturer</td><td>Any flow cytometer may be used&amp;nbsp;</td></tr><tr><td>Counting Chamber</td><td>Hausser</td><td>3120</td><td>For cell counting and plating&amp;nbsp;</td></tr><tr><td>FlowJo</td><td>FlowJo</td><td>Request from manufacturer</td><td>Flow cytometry analysis software</td></tr></tbody></table>

an in vitro approach to photodynamic therapy

光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。联邦药物管理局已批准 pdt 治疗日光脂溢性, 临床指南建议 pdt 作为治疗某些非黑色素瘤皮肤癌和寻常痤疮。PDT 是一种有利的治疗方式, 因为它是低成本, 无侵入性, 并与最小的不良事件和惊吓有关。在 PDT 的第一步, 应用 PS, 并允许积累 intracellularly。随后的光照射诱导活性氧的形成, 这可能最终导致细胞凋亡, 膜的破坏, 线粒体损伤, 免疫调节, 角质细胞增殖, 胶原蛋白的更替。在此, 我们提出了一种体外方法来研究 PDT 在黏附细胞系。该治疗协议旨在模拟 pdt, 并可调整以研究使用的 pdt 与各种细胞系, 光敏, 孵化温度, 或 photoactivation 波长。鳞状细胞癌细胞以0、0.5、1.0 和2毫米 5-氨基乙酰丙酸 (5-亚拉巴马州) 孵化, 30 分钟和光活化作用417纳米蓝光。以蛋白和 7-aminoactinomycin D 流式细胞术检测为主要结局指标为凋亡和坏死。在 5-阿拉三十分钟的孵化后, 细胞凋亡的剂量依赖性增加。在体外光动力实验中, 保持一致的孵化和光照参数, 对于提高检测的有效性具有重要意义。pdt 是一个有用的临床程序, 并在体外研究可以为新的 PSs 的发展, 协议的优化, 和新的迹象, pdt。

在 SCC-13 细胞孵化30分钟与 0, 0.5, 1, 2 毫米 5-阿拉和辐照 1000 s 蓝光, 有剂量依赖性增加的细胞凋亡。总凋亡 (平均标准误差平均值) 为3.94 秒 0.34, 7.90 @ 0.52, 23.86 @ 0.52, 和 38.33, 1.81 分钟后, 30, 0, 0.5, 1 毫米的孵化, 分别和2秒钟的蓝色光 (图 5)。我们用方差分析的方法比较了0、0.5、1和2毫米 5-阿拉的凋亡细胞中的平均百分率。1和2毫米 5-亚拉巴马州的细胞凋亡与0毫米 5-阿拉巴马治疗细胞相比显著增加。图 2A显示了具有代表性的流细胞浇口 (FSC) 与侧向散射 (SSC) 的 SCC-13 细胞孵化与 0, 0.5, 1, 2 毫米 5-阿拉巴马州。圆形浇口包围 SCC-13 细胞群。在这个实验中, 收集了7500个事件, SCC-13 细胞种群门捕获了至少90% 事件。图 2B显示了 y 轴上 x 轴和蛋白上的 7-反的代表图。细胞群被封闭成四象限 (Q1 到 Q4), 以区分凋亡细胞。Q1 (高蛋白, 低 7) 代表细胞凋亡早期。Q2 (高蛋白 v, 高 7) 代表细胞在晚期凋亡或坏死。Q3 (低蛋白, 高 7) 代表细胞正在坏死。Q4 (低蛋白, 低 7) 代表细胞不发生凋亡或坏死。为了计算细胞凋亡的百分比, 我们增加了 Q1 和 Q2 细胞的百分比。5-photoactivation 潜伏期长度、潜伏期温度或辐照剂量的不一致可能改变 PDT 疗效。图 3展示了一个代表性的蛋白和 7-细胞的流动, 当孵化温度变化 (即27, 36, 或42°c)。细胞凋亡的温度依赖性增加。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58190/58190fig1.jpg"> 图 1: 三十分钟孵化 5-亚拉巴马州后细胞凋亡的剂量依赖性增加.5-阿拉在36摄氏度孵化三十分钟后, SCC-13 细胞中1000秒的蓝光。条形表示每个治疗和控制组中蛋白阳性细胞的平均百分比。实验以三项技术进行。统计学意义由方差分析确定, p < 0.05 用星号 (*) 表示。误差线表示平均值的平均标准误差。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58190/58190fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58190/58190fig2.jpg"> 图 2: 代表性流式细胞仪地块.(A) 代表在0、0.5、1和2毫米 5-阿拉孵化细胞中的任意单位 (AU) 中有代表性的向前散射与侧散布式细胞仪地块。在这个实验中, 收集了7500个事件, SCC-13 细胞种群门捕获了90% 事件。(B) 代表蛋白与 7 -反流式细胞术在 AU 为 x 和 y 轴 0, 0.5, 1 和2毫米的孵化细胞。Q1: 早期凋亡细胞, Q2: 晚期凋亡/坏死细胞, Q3: 坏死细胞和 Q4: 存活细胞。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58190/58190fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58190/58190fig3.jpg"> 图 3: 在不同温度下孵化 5-阿拉可改变 PDT 功效.代表蛋白与 7 -反流式细胞仪在 AU 为 x 和 y 轴的 SCC-13 细胞孵化与0.5 毫米 5-亚拉巴马州在 27, 36 和42°c。Q1: 早期凋亡细胞, Q2: 晚期凋亡/坏死细胞, Q3: 坏死细胞和 Q4: 存活细胞。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58190/58190fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Vitro Approach to Photodynamic Therapy at JoVE.com

An In Vitro Approach to Photodynamic Therapy

光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。我们提出了一种体外pdt 协议, 旨在模拟 PDT, 可用于研究不同的 PS 孵化和光处理参数。

一种体外光动力治疗方法的研究

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

an-in-vitro-approach-to-photodynamic-therapy

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

An In Vitro Approach to Photodynamic Therapy

8.6K 次观看.  University of California, Davis. 光动力疗法 (PDT) 是一个医疗程序, 涉及孵化外部应用光敏剂 (PS) 后, 可见光 photoactivation 诱导细胞凋亡。我们提出了一种体外pdt 协议, 旨在模拟 PDT, 可用于研究不同的 PS 孵化和光处理参数。

视频: An In Vitro Approach to Photodynamic Therapy

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

科研

医学

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

化学

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

<meta charset="utf8"></head><body>5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

癌症研究

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

生物工程

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

生物化学

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

Source: Chang, J. E., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a>&#160;J. Vis. Exp.&#160;(2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

一种体外光动力治疗方法的研究

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一个系统来研究肿瘤休眠和转移性增长

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5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

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从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子

体外光毒性测定:一种基于 PDT 的方法，用于评估光敏剂在肺癌细胞中的光毒性潜力

一种体外光动力治疗方法的研究

摘要

探索更多视频

一个系统来研究肿瘤休眠和转移性增长

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

光动力疗法治疗骨肉瘤细胞的细胞毒功效

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

光控生物活性化合物的体外和体内评价 - 癌症光药理学的潜在候选药物

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

体外光毒性测定:一种基于 PDT 的方法，用于评估光敏剂在肺癌细胞中的光毒性潜力

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子