Bu araştırma Kanser Araştırma Ulusal Sağlık Enstitüleri İntramural Araştırma Programı, Ulusal Kanser Enstitüsü, Merkezi tarafından desteklenmiştir.

Not: Aşağıdaki protokol NUR-PIT kullanarak belirli hücreleri ortadan kaldırmak için gereken adımları açıklar. Kontroller ve NUR-PIT ve hücre canlılığı ile ilgili diğer ayrıntıları başka bir yerde 18 bulunabilir. Monoklonal antikorlara IR700 1. Konjugasyon (mAb) <ol><li> 2-5 mg / 0.1 M Na 2 HPO 4 (pH 8.6) çözeltisi içinde ml&#39;de ilgi mAb hazırlayın. </li><li> Mikrosantrifüj tüpü içinde 0.1 M Na 2 HPO 4 çözeltisi (pH 8.6) içinde 10 mM IR700 30.8 nmol olan mAb 6.8 nmol karıştırın ve alüminyum folyo ile kaplı 1 saat için oda sıcaklığında inkübe edilir. </li><li> iki kez 15 ml PBS ile PD-10 kolonu (Malzeme / Ekipman bkz: İçindekiler) yıkayın. Adım 1.2 numuneyi yükleyin. </li><li> Üreticinin talimatlarına uygun olarak, PBS ile akıtılarak PD-10 kolonu ile bir karışım saflaştınlır. Not: Burada, elüsyon IR700 band rengi boyunca oldu. 300 ul örnek, PBS elüsyon fraksiyonu 4.4 ml, tipik olarak 2,5 ila ml&#39;dir. </li><li> belirlemekBir spektrofotometre 9 595 nm&#39;de emilimin ölçülmesiyle, Coomassie boyama ile protein konsantrasyonu. Her mAb molekülüne 9 konjuge florofor moleküllerinin sayısını onaylamak için 689 nm&#39;de absorpsiyon ile IR700 konsantrasyonunu belirleyin. Not: bir spektrofotometre ile 1 MAB&#39;deki IR700 moleküllerinin optimal konjügasyon sayısını belirlemek için önemlidir. Genel olarak, 1 mAb molekül içinde 3 çevresinde IR700 molekülleri, in vitro ve in vivo çalışmaları en uygunudur. HPLC ve SDS-PAGE yöntemleri mAb ile IR700 bağlı olup olmadıklarını teyit etmek için kullanılabilir. </li><li> protein konsantrasyonu belirlendikten sonra 4 ° C&#39;de saklayın. </li></ol> Karışık 3D Hücre Kültürü 2. Hazırlık (spheroid Karışık) <ol><li> damla plakaları asılı levha rezervuar kısmına steril su (yaklaşık 1 ml) uygulayın. </li><li> A431-luc-GFP hücreleri ve 3T3-RFP hücrelerini - çeşitli ilgi hücre tiplerinin oranları hazırlayın -Kültür ortamı, 50 ul içerisinde süspansiyon haline toplam 5000 hücreleri ihtiva eden her bir örnek ile. Not: kültür ortamı daki hücre türlerinin oranı, hücre tipine bağlı olarak vb 100: 100, 10: 100, 25: 100, 50: 1 hazırlayın. </li><li> 37 ° C ve% 5 karbon dioksit nemlendirilmiş bir inkübatör içinde 96 gözenekli asılı bırakma plakasında 5-7 gün karışımı inkübe edin. 2 günde kültür ortamı değiştirin. Not: hücreleri içeren damlalar olarak, 3D tam küremsi yapmak hafifçe plaka işlemek için 3D şekiller oluşturan önce kolayca düşmek. </li><li> 40X büyütme - 10X bir ters aydınlık mikroskop kullanılarak küremsi morfolojisi ve boyutunu dikkate alınmalıdır. Not: Bu hücre tipleri ve asılı damla büyüklüğüne bağlı olmasına rağmen, sfero çapı de damla plaka asılı 96&#39;da kuluçkalamadan 7 gün sonra yaklaşık 400-600 um olduğundan emin olun. </li></ol> Karışık 3D Hücre Kültürü için İn Vitro NUR-PIT 3. <ol><li> ha ortamı değiştirmek10 ug / ml antikor-photoabsorber konjügatı (APC) ihtiva eden ortama açılan levhalar donanımının değiştirilmesi, ve 37 ° C&#39;de ve% 5 karbon dioksit nemlendirilmiş bir inkübatörde 6 saat inkübe edilir. </li><li> 6 saat kuluçkalamadan sonra, taze kültür ortamı (fenol kırmızısı Free) ile iki kez sfero yıkayın. ucu kesilmiş hafifçe steril bir 200 ul pipet kullanarak 100 ul taze fenol kırmızısı ücretsiz kültür ortamı ile bir cam tabanlı 50 mm çanak sfero aktarın. Her yemeğin bir sfero yerleştirin. </li><li> morfoloji değişikliği tespit etmek için ters aydınlık mikroskop ile sfero dikkate alınmalıdır. Aşağıdaki filtre ayarları ile floresan mikroskop kullanmak optik gazetecilere (örneğin, GFP ve RFP) gözlemlemek için: GFP - 469 nm uyarma filtresi ve 525 nm emisyon filtresi; RFP - 559 nm uyarma filtresi ve 630 nm emisyon filtresi. </li><li> ışınlanması için cam tabanlı tabak üzerinde ışık yayan diyot (LED) koyun. Not: küremsi NIR maruz kalabilirmikroskop veya laminer kaputu ya yanar. <ol><li> Bir optik güç ölçer 9 NIR ışık güç yoğunluğunu ölçün. Bu ölçüme göre, 2 J / cm2 de 670 nm 710 dalga boylarında ışık yayan ışık yayan diyotlar (LED&#39;ler) üzerinden NIR ışık saçmak. Not: LED ışık yaklaşık 5 inç maksimum derinliği nüfuz edebilir. NIR PIT sitotoksik etkileri bağımsız olarak güç yoğunluğu ve maruz kalma 23 süresi, sadece belirli bir enerji bağlıdır. </li></ol></li><li> Işık verilmesinden sonra taze kültür ortamı 50 ul yeni Asılı damla plakasına sfero aktarmak ve 37 ° C&#39;de ve% 5 karbon dioksit, bir nemlendirilmiş inkübatörde 1 gün süreyle inkübe edilir. </li><li> Yavaşça kesilmiş ucu ile steril 200 ul pipet kullanarak taze kültür ortamı (fenol kırmızısı ücretsiz), 100 ul bir cam tabanlı çanak sfero aktarın. NUR-PIT kullanarak fi sonra bir floresan mikroskop 1 gün sfero gözlemlemekltresi ayarları adım 3.3 tarif. <ol><li> Veya bir floresan mikroskobu (Şekil 2B) 14,18,22 kullanılarak sitoplazmik GFP floresans kaybı ile 2 ug / ml&#39;lik bir son konsantrasyonda ortamına propidyum iyodür eklenerek ölü hücreleri algılar. </li></ol></li><li> Hedef hücreler tamamen ortadan değilse tekrarlayın 3,1-3,6 adımları. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Bu NIR PIT kullanarak hedef olmayan hücrelere zarar vermeden karışık bir 3D hücre kültüründen spesifik hücre eliminasyonu için bir yöntem ortaya koymaktadır. Şimdiye kadar, hiçbir pratik hücre eleme yöntemi doku sağlandıktan sonra veya transplantasyon sonrasında bulunmamaktadır. Böylece, NIR-PIT bunu gerçekleştirmek için gelecek vadeden bir yöntemdir. APC &#39;ler mAb kendisi benzer farmakokinetiği göstermektedir, çünkü bu teknik, aynı zamanda, in vivo 18,22 <e...

Diğer hücrelere zarar vermeden özel hücrelerin ortadan kaldırılması özellikle kurulmuş doku olarak, son derece zordur ve doku mühendisliği alanında bir hücre eleme yöntemi için acil bir ihtiyaç vardır. 3 - Günümüzde rejeneratif tıp alanında, embriyonik kök hücreleri (ES), pluripotent kök hücrelerini (PSC), ya da uyarılmış pluripotent kök hücre (iPS) kullanarak doku kültürleri umut verici malzemeler 1 bulunmaktadır. Bu doku rejenerasyonu umut verici olmasına rağmen, istenmeyen hücreleri ile kirlenme önemli bir husustur. Ayrıca, transplantasyon 4,5 sonra tümorijenisite &#39;nin güvenlik endişesi var. Birçok çalışmalar bu konular üzerinde duruldu olmasına rağmen özellikle rejeneratif tıp 6, belirli hücreleri ortadan kaldırmak için - 8, hiçbir pratik bir yöntem geliştirilmiştir. Yakın Kızılötesi photoimmunotherapy (NIR PIT) bir antikor-photoabsorber conjugat göre bir tedavi yöntemie (APC). Bir APC hücreye spesifik monoklonal antikor (mAb) ve photoabsorber, IR700 oluşur. IR700 bir hidrofilik silika ftalosiyanin türevi ve tek başına 9 ile fototoksisite indüklemez. IR700 kovalent lisin molekülü yan zinciri üzerinde amid artıkları ile antikora konjuge edilir. APC hücre membran hedef molekülüne bağlanması ve daha sonra 690 nm &#39;de, NIR ışığa maruz kaldıktan sonra yaklaşık anında hücre nekrozunu indükler. 14 - NUR-ışık, hücre ölümüne 9 yol açan hücresel membran rüptürü maruz kalma sırasında. 21 - NIR PIT anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mezotelin, anti-GPC3 ve anti-CEA 15 de dahil olmak üzere birden fazla antikor ya da antikor fragmanları, etkili olduğu kanıtlanmıştır. Bu nedenle, NIR PIT hedef moleküller, çok çeşitli karşı kullanılabilir. Ayrıca, NIR-PIT NIR ligh kısıtlayarak belirli bölgelerde seçici tedavi sağlayan bir iyi-kontrollü tedavit ışınlama 18,22. Burada, karışık 3D kültürlerden gelen NIR PIT kullanarak belirli hücre eleme bir yöntem mevcut.

<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.

optik olarak EGFR aşın NIR PIT, A431 hücre hattı, etkin olabilmesi için, genetik olarak GFP ve lusiferaz (A431-Luc-GFP) ifade etmek için modifiye edilmiştir. NIR PIT olmayan bir hedef olarak Balb / 3T3 hücre çizgisi optik RFP (3T3-RFP) ifade etmek için modifiye edilmiştir. APC panitumumab-IR700 (pan-IR700) sentezlendi. Hücreleri (A431-luc-GFP ve 3T3-RFP) çeşitli oranlarda oluşmuştur Karışık sferoidler, bu protokol (Şekil 1) &#39;e göre imal edildi. T...

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).

Bir Yakın Kızılötesi Photoimmunotherapy Tekniği Karışık 3D Kültür seçici hücre Eliminasyon

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

Research

JoVE Journal

Bioengineering

7.8K Görüntüleme.  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). 

Video: 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.

Bir Tümör Sferoid Modelinde Antikora Bağımlı Hücresel Sitotoksisitenin Ölçülmesi: İlaç Keşfi Uygulaması

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

Kanser Araştırmaları

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.

2D Hücre Kültürü Modelinde Verteporfin Sitotoksisitesini Arttırmak için Şirket İçi İnşa Edilmiş ve Işık Yayan Diyot Tabanlı Fotodinamik Terapi Cihazı

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 ...

Biyokimya

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.

Fotokontrollü Biyolojik Olarak Aktif Bileşiklerin İn Vitro ve İn Vito Değerlendirilmesi - Kanser Fotofarmakolojisi için Potansiyel İlaç Adayları

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

Kimya

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, where success depends on the distribution of specific immune cell subpopulations. Over the past decade, multiple sophisticated technologies have been introduced to achieve detailed resolution of the TME using two-dimensional sections from either formalin-fixed, paraffin-embedded (FFPE) or fixed-frozen tissues. While these thin sections are easier to procure and analyze, they lack the three-dimensional architecture needed to reliably and comprehensively characterize a tumor. To address this limitation, a tissue mounting and imaging technique was developed to enable the three-dimensional analysis of tumor lesions in their native in vivo state. This protocol outlines the procurement of human tumor tissue, the mounting of samples on custom-printed platforms, and staining procedures for post-fixation samples. The multiplexed immunofluorescence technique, IBEX (Iterative Bleaching Extends Multiplexity), was adapted to characterize the three-dimensional TME with up to 15 markers for tumor, immune, and stromal cells using commercially available antibodies. Imaging depths of up to 100 &#181;m were achieved using an inverted white-light laser confocal microscope with a custom-printed imaging adaptor and commercial glass-bottom dishes to ensure optimal tissue orientation. This protocol highlights the potential of the IBEX method to expand multiplexed immunofluorescence studies, providing a more comprehensive understanding of TME composition.

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

Yinelemeli Beyazlatma Genişletilmiş Çokluk Yaklaşımı kullanılarak tümör taşıyan dokunun üç boyutlu görüntülenmesi

The spatial heterogeneity of the tumor microenvironment (TME) is a critical determinant of therapeutic response, particularly for immune-oncology agents, ...

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.

Manyetik Hipertermi ve Pothototermal Kombinasyon Kanser Tedavisi için Manyetik, Akustik ve Optik-Üçlü Duyarlı Mikrobubbles

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 ...

Biyomühendislik

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.

Fotodinamik Tedavi bir Vitro yaklaşım

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

Tıp

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.

Plevral Yayılım Fare Modelleri için Yakın Kızılötesi Fotoimmünoterapi

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

Candida glabrata'da Minyon Koloni Oluşumu ve İlaç Direncinin Analizi

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.

Yazar Spotlight: Antifungal Araştırmalar için İlaca Dirençli Candida'da Minyon Kolonileri İndüklemek İçin Yeni Bir Yaklaşım Olarak Fotodinamik Terapi

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.

Rose Bengal Aracılı Fotodinamik Terapi ile Candida glabrate'de Minyon Kolonilerin İndüksiyonu

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

Bir Yakın Kızılötesi Photoimmunotherapy Tekniği Karışık 3D Kültür seçici hücre Eliminasyon

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Bir Tümör Sferoid Modelinde Antikora Bağımlı Hücresel Sitotoksisitenin Ölçülmesi: İlaç Keşfi Uygulaması

2D Hücre Kültürü Modelinde Verteporfin Sitotoksisitesini Arttırmak için Şirket İçi İnşa Edilmiş ve Işık Yayan Diyot Tabanlı Fotodinamik Terapi Cihazı

Fotokontrollü Biyolojik Olarak Aktif Bileşiklerin İn Vitro ve İn Vito Değerlendirilmesi - Kanser Fotofarmakolojisi için Potansiyel İlaç Adayları

Yinelemeli Beyazlatma Genişletilmiş Çokluk Yaklaşımı kullanılarak tümör taşıyan dokunun üç boyutlu görüntülenmesi

Manyetik Hipertermi ve Pothototermal Kombinasyon Kanser Tedavisi için Manyetik, Akustik ve Optik-Üçlü Duyarlı Mikrobubbles

Fotodinamik Tedavi bir Vitro yaklaşım

Plevral Yayılım Fare Modelleri için Yakın Kızılötesi Fotoimmünoterapi

Rose Bengal Aracılı Fotodinamik Terapi ile Candida glabrate'de Minyon Kolonilerin İndüksiyonu

Rose Bengal Aracılı Fotodinamik Terapi ile Candida glabrate'de Minyon Kolonilerin İndüksiyonu

Rose Bengal Aracılı Fotodinamik Terapi ile Candida glabrate'de Minyon Kolonilerin İndüksiyonu