Name: selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique
Uploaded: 2016-03-14T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

这项研究是由美国国立卫生研究院院内研究计划，国家癌症研究所，癌症研究中心的支持。

注意:以下协议描述必要的步骤来消除使用NIR-PIT特定细胞。控制和有关NIR-PIT和细胞活力等细节可以在其他地方18中找到。 1.共轭IR700，以单克隆抗体（MAB） <ol><li>在2-5毫克/毫升的0.1M 的 Na 2 HPO 4（pH为8.6）的溶液制备的兴趣的mAb。 </li><li>混合mAb的6.8纳摩尔用10mM IR700 30.8纳摩尔在0.1M 的 Na 2 HPO 4在微量离心管中液（pH 8.6），并在室温孵育1小时，用铝箔覆盖。 </li><li>用15ml的PBS洗涤PD-10柱（参见材料/设备的数据表），两次。从步骤1.2装载样品。 </li><li>根据制造商的说明通过PBS洗脱纯化经由PD-10柱的混合物。 注:在这里，洗脱沿IR700的带颜色。对于300微升样品，PBS洗脱分数毫升〜4.4毫升通常为2.5之间。 </li><li>确定蛋白质浓度用考马斯染色，通过测量在595nm处的吸收用分光光度计9。确定IR700与吸收的浓度在689纳米到确认缀合每种mAb分子9荧光团分子的数目。 注意:确定在1单抗IR700分子的最佳偶联次数用分光光度计是很重要的。通常，在1单抗分子约3 IR700分子是最适用于在体外和体内的工作。 HPLC和SDS-PAGE方法可以用于确认单抗和IR700是否可能会或没有。 </li><li>测定蛋白质浓度后储存于4℃。 </li></ol> 2.准备混合3D细胞培养（混合球体） <ol><li>应用无菌水（约1ml）中成悬滴板的板储部分。 </li><li>制备的细胞类型的利益的各种比例 - A431-LUC-GFP细胞和3T3-RFP细胞 -用含有5,000个细胞总数，悬浮于50μl培养基的每一个样品。 注意:根据细胞类型的培养基中的细胞类型的兴趣的比率100 等 :准备1:100，10:100; 25:100，50。 </li><li>在37℃和5％二氧化碳孵育在96孔悬滴板5-7天的混合物在加湿培养箱中。改变培养基，每2天。注:为了使3D球体准确，轻拿轻放盘，如含有细胞滴形成3D形状之前，容易脱落。 </li><li> 40X放大率 - 使用倒置亮视野显微镜在10倍观察球体的形态和尺寸。注意:尽管这取决于细胞类型和悬滴的大小，确保该球体的直径后，在96孵育7天后的孔悬滴板周围400-600微米。 </li></ol> 3. 在体外 NIR-PIT混合3D细胞培养<ol><li>更改HA媒体nging滴板，以10微克/毫升抗体 - 光吸收剂结合物（APC）含有介质，并且在37℃和5％二氧化碳的潮湿培养箱孵育6小时。 </li><li>后6小时温育后，用新鲜的培养基（无酚红）洗球体的两倍。轻轻球体用100微升新鲜无酚红培养基用无菌200微升枪头，转移到玻璃底50毫米菜尖切断。放在每道菜1球体。 </li><li>观察球体用倒置明显微镜检测形态的变化。观察光学记者（如 GFP和RFP）使用具有以下过滤器设置荧光显微镜:GFP - 469 nm激发过滤器和525 nm发射滤波器; RFP - 559 nm的激发滤光片和630nm的发射滤光器。 </li><li>放置照射玻璃底培养皿上方的发光二极管（LED）。 注:球体​​可以接触到近红外光无论是在显微镜或在层流罩。 <ol><li>测量近红外光的功率密度与光功率计9。根据该测定，通过发光二极管（LED），其在在2J / cm 2的670至710纳米的波长的发射光照射近红外光。 注:LED灯可以穿透大约5英寸最大深度。不管功率密度和曝光23的持续时间的近红外PIT的细胞毒性作用是只依赖于给定的能量。 </li></ol></li><li>照射后，转移球体到一个新的悬滴板用50μl新鲜培养基，并在37℃和5％二氧化碳的潮湿培养箱孵育1天。 </li><li>轻轻球体用100微升使用带切断尖端无菌200微升枪头，新鲜培养基（无酚红）的转移到玻璃底菜。观察与NIR-PIT使用网络连接后，荧光显微镜1天球体在步骤3.3说明过滤器设置。 <ol><li>通过，或通过使用荧光显微镜（ 图2B）14,18,22细胞质的GFP荧光的损失以2微克/毫升的最终浓度加入碘化丙啶至媒体检测死细胞。 </li></ol></li><li>如果靶细胞没有完全消除重复步骤3.1-3.6。 </li></ol>

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作者什么都没有透露。

我们证明特定细胞消除从混合三维细胞培养物的方法，而不使用近红外-PIT损害对非靶细胞。到目前为止，存在一旦组织被建立或细胞消除无实际方法移植后。因此，NIR-PIT是实现这个很有前途的方法。这种技术也可在体内 18,22利用，因为的APCs显示出类似的药代动力学为单克隆抗体本身。靶细胞类型可适于与各种的APC。各种抗体或抗体片段，其中包括抗EGFR，抗PSMA，抗间皮素和抗CEA已经用作的APC 15 - 21。此外，改变NIR照射的区域可以最小化处理的区域。这里，以萤光素酶活性的定量和荧光对靶细胞，特定的细胞完全消除了确认。 NIR-PIT是一种基于光治疗，因而临界点为t他的近红外光源和目标之间的距离，由于光能量并根据一平方反比定律（步骤3.4），因此细胞坏死减少。使三维球体确切地说，板应处理并轻轻孵育越好。含滴细胞很容易被破坏，或形成3D形状面前有点惊世骇俗脱落。 NIR-PIT有一些独特的优势。首先，NIR-PIT的APCs展示出相似静脉内药代动力学给父母的非共轭的抗体，由于IR700的亲水性，导致高度特异性结合到靶细胞和最小非目标累积。因此，即使在组织移植后，NIR-PIT可通过在APC随后感兴趣近红外区域的曝光的静脉注射治疗的靶细胞。第二，近红外光可以穿透更深入组织比紫外线或可见光，因此，近红外PIT可以治疗不仅表面而且ENTI再通过暴露于NIR表面处的移植组织的厚度。最后，近红外PIT可反复使用，以消除靶细胞而没有限制。当靶细胞在细胞表面表达18靶分子足够的副本，此方法非常有用。 不过，也有这种技术有一些限制。首先，在APC的渗透性被限制，因为它是一个大的分子。为了克服这个问题，反复NIR-PIT治疗或抗体片段可以利用18,19,22。另一个限制是光线穿透。虽然近红外光可正确地穿透体外培养，翻译到体内治疗需要适于的方法，如消化内镜，支气管镜，或直接胸腔镜与光导纤维移动光源更靠近目标区域。 NIR-PIT的进一步修改将允许在本地和规范使用这种方法的IFIC细胞消灭在许多领域，如免疫调节或肿瘤微环境中24 - 26。

消除特定细胞而不损害其他细胞是非常困难的，特别是在建立组织，并且存在用于在组织工程领域中的细胞消除方法的迫切需要。现今在再生医学领域中，使用胚胎干细胞（ES），多能干细胞（的PSCs），或诱导的多能干细胞（IPS）的组织培养物是有前途的材料1 - 3。 虽然这个组织再生是有前途的，具有不需要的细胞污染是一个大问题。此外，有移植4,5-之后致瘤性的安全问题。虽然许多研究都集中在这些问题上，以消除特定细胞，尤其是在再生医学6 - 8，没有实际的方法已被开发出来。 近红外photoimmunotherapy（NIR-PIT）是一种基于抗体 - 光吸收剂conjugat的处理E（APC）。一个APC由细胞特异性单克隆抗体（mAb）和光吸收剂，IR700的。 IR700是亲水性二氧化硅的酞菁衍生物和本身并不9诱发光毒性。 IR700是通过赖氨酸分子的侧链酰胺残基共价结合到抗体上。 APC的结合细胞膜上的靶分子，然后诱导暴露于NIR光在690纳米的近后立即细胞坏死。在暴露于近红外光，细胞膜破裂，导致细胞死亡9 - 14。 NIR-PIT已被证明是具有多个抗体或抗体片段，其中包括抗EGFR，抗HER2，抗PSMA，抗CD25，抗间皮素，抗GPC3，和抗CEA 15有效- 21。因此，近红外PIT可对多种靶分子使用。此外，近红外PIT是良好控制的治疗，通过限制近红外二极管灯允许特定区域的选择性治疗ŧ照射18,22。 这里，我们目前采用NIR-PIT从混合3D文化特异性细胞清除的方法。

<table><tbody><tr><td>IRDye 700DX Ester Infrared Dye</td><td>LI-COR Bioscience (Lincoln, NE, USA)</td><td>929-70011</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH (St. Louis, MO, USA)</td><td>S9763</td><td></td></tr><tr><td>Sephadex G25 column (PD-10)&amp;nbsp;</td><td>GE Healthcare (Piscataway, NJ, USA)</td><td>17-0851-01</td><td></td></tr><tr><td>Coomassie (bradford) Plus protein assay</td><td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td><td>PI-23200</td><td></td></tr><tr><td>Perfecta3D 96-Well hanging Drop Plates</td><td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td><td>HDP1096-8</td><td></td></tr><tr><td>Optical power meter</td><td>Thorlabs (Newton, NJ, USA)</td><td>PM100</td><td></td></tr><tr><td>LED: L690-66-60</td><td>Marubeni America Co. (Santa Clara, CA, USA)</td><td>L690-66-60</td><td></td></tr><tr><td>Vectibix (panitumumab)</td><td>Amgen (Thousand Oaks, CA, USA)</td><td></td><td></td></tr><tr><td>35&amp;nbsp;mm glass bottom dish, dish size 35&amp;nbsp;mm, well size 10&amp;nbsp;mm</td><td>Cellvis (Mountain View, CA, USA)</td><td>D35-10-0-N</td><td></td></tr></tbody></table>

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.

以光学监控NIR-PIT中，A431细胞系，其过表达EGFR的效果，被遗传修饰以也表达GFP和荧光素酶（A431-LUC-GFP）。作为近红外PIT的一个非目标，所述的Balb / 3T3细胞系的光学修饰以表达RFP（3T3-RFP）。 APC的，帕尼单抗-IR700（泛IR700），合成。混合球状体，其中分别组成细胞（A431-LUC-GFP和3T3-RFP）的各种比例的是按照此协议（ 图1）制成。反复NIR-PIT与泛IR700（参见图2A方案）的孵化执行。从这种混合三维培养靶细胞消除达到与荧光和荧光素酶的活动（ 图2B，C）进行监测。另一方面，非靶细胞持续增长。 <img alt="图1" src="/files/ftp_upload/53633/53633fig1.jpg" /> <str翁&gt;图1.混合球状体的各种比例的代表性图像。混合球状体的代表性图像A431-LUC-GFP细胞和在第7天3T3-RFP细胞各种比例被示出。比例尺= 200微米。这个数字已经从16修改。 <a href="https://www.jove.com/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53633/53633fig2.jpg" /> 在三维细胞球体图2.靶细胞消除（A431-LUC-GFP和3T3-RFP细胞混合球体）。（A）的近红外-PIT（2J / cm 2）的方案中示出。 （B）的重复NIR-PIT完全消除，没有损害非靶细胞（3T3-RFP）靶细胞（A431-LUC-GFP）中的混合三维球体。栏= 200微米。荧光素酶的活动（C）定量（RLU比）靶细胞（每组10球体）的证明完全消除。这个数字已经从18修改。数据表示为平均值±SEM <a href="https://www.jove.com/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

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

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 ...

selective-cell-elimination-from-mixed-3d-culture-using-near-infrared

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K 次观看.  National Cancer Institute. 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). 

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

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

科研

癌症研究

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 ...

化学

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 ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

生物工程

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

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 ...

医学

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

近红外光免疫疗法为小鼠模型的胸膜传播

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

分析光滑念珠菌的小集落形成和耐药性

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

作者聚焦:光动力疗法作为在耐药 念珠菌 中诱导娇小菌落用于抗真菌研究的新方法

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

通过 Rose Bengal 介导的光动力疗法诱导念珠菌中的小菌落

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

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

摘要

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近红外光免疫疗法为小鼠模型的胸膜传播

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量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

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

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