Bu çalışma Eğitim, Gençlik ve Spor (LO1304) Çek Bakanlığı ve Çek Cumhuriyeti Teknolojik Ajansı (TE01020028) hibe tarafından desteklenmiştir. Yazarlar çevresel kapak fotoğraf çekerken Dr. Lakshman Varanasi teşekkür etmek istiyorum.

Agaroz kaplı plakalar hazırlanması 1. <ol><li> düşük erime noktalı agaroz 0.75 g tartılır ve serumsuz ml McCoy&#39;un 5A ortamı (ya da fenol kırmızısı olmadan) 100 ekleyin. Bir mikrodalga çözüm ısıtın ve tamamen agaroz çözmek için her 1-2 dakikada bir girdap. sterilize çözüm Otoklav. </li><li> yaklaşık 70 ° C&#39;ye kadar agaroz otoklava soğutun ve bir laminer akış kutusu, vakumla süzülerek 500 mL, 0.22 um&#39;lik bir filtre üst yoluyla filtre. bir kerede tüm çözüm kullanarak değilse küçük hacimlere% 0.75 filtrelenmiş agaroz çözeltisi (FAS) alikotu. soğuk oda ya da 4 hafta boyunca 4 ° C&#39;de buzdolabında Bu hazır kullanım agaroz çözelti aseptik saklayın. </li><li> bir akış kutusu steril fosfat-tamponlu tuzlu su (PBS) ile daha sonra% 70 etanol (EtOH) ile, bir Combi reaktifi dağıtıcı ve birinci kaset plastikli veya metal uçlu dağıtma kaseti ile küçük bir tüp takın. </li><li> Kullanımdan önce, FAS I depolanmış bir kısım ısı<html> n mikrodalga bunu eritmek için. Başbakan Agaroz çözüm ve ceket 384 gözlü, doku kültürü (TC) ile dağıtım kaset FAS 15 uL mikropleytin &quot;özel&quot; ile tedavi edilen. plaka agaroz hücrelerin ekim önce 15-20 dakika soğumasını bekleyin. Soğuk bir odada ya da 4 ° C&#39;de ve doğrudan ışıktan bir polietilen torbaya sarılmış aseptik agaroz kaplı plakalar, saklayın. Not: Stok solüsyonu agaroz konsantrasyonunda bir değişikliği önlemek için% 0.75 FAS tekrarlanan ısıtma kaçının. Yukarıda sözü edilen koşullar altında depolandığında Agaroz kaplı plakalar 2 haftaya kadar saklanabilir. FAS plakalarının kaplanması esnasında ısıtma gerektirmez. </li><li> kaset ipuçları ve tüplerde kalan agaroz kaldırmak için 70-80 ° C steril su ile macun ile dağıtım kaset temizleyin. </li></ol> 2. Hücre Kültürü ve MCTS oluşumu <ol><li> Kültür, insan kolorektal karsinoma HCT116 hücreleri, daha önce tarif edildiği gibilass = &quot;xref&quot;&gt; 8. </li><li> agaroz kaplı, 384 gözlü, soğuk depolama mikro TC ile muamele edilmiş ve istenen sayıda çıkarın ve 15 dakika süre ile oda sıcaklığında (RT) kadar dengeye getirin. </li><li> % 70 EtOH ve steril PBS ile bir standart tüp dağıtım kaseti emişli hücre tohumlama için Kombi reaktif dağıtıcı hazırlayın. Gerekli mcL tohumlama hacmi ve dağıtıcı manuel ayar düğmelerini kullanarak ortama dağıtım hızını ayarlayın. Not: tüm hücre ekim işlemi, steril koşullar altında, bir laminar akış kutusunda gerçekleştirilmektedir. </li><li> bir rekombinant hücre ayrışma enzim kullanılarak doku kültürü şişesi yapışkan hücreleri ayırmak. Tam büyüme ortamında 50 ul oyuk başına 2.5 x 10 4 hücre / mL&#39;lik bir yoğunlukta, tohum hücreleri ile steril bir beherde bir hücre süspansiyonu stok yapmak. Birden fazla 384 WP tohum zaman, kabın dibine yerleşmesini önlemek için, bir manyetik karıştırıcı kullanılarak hücrelerin karıştırın. </li><li> izin vermekPlakalar oda sıcaklığında 30 dakika dinlendirilir ve daha sonra 4 x g&#39;de 15 dakika boyunca santrifüj. </li><li> Bu arada, bir buharlaştırma düşürücü çevresel kapak alır ve onu kısa kenarlara steril H 8 mL 2, O ya da% 5 dimetil sülfoksit (DMSO) ile doldurun 5 ml pipet (Şekil 1A) ile (sol ve sağ). İlk olarak, yavaş yavaş yukarı ve aşağı pipet süpürülerek sol taraftaki çukur içine sıvı dolum 4 mL dağıtmak. Sağ taraftaki çukur ile bu adımı yineleyin. NOT: Sıvı kapağının merkezinde birleştirme ve gaz değişimi (Şekil 1A) için bir boşluk bırakmaz yan olukları eklenen emin olun. Kapağın dış ve daha sonra 384 gözlü TC levhaları dış kuyu içine H H2O sızma 2 O sonuçlar aşırı miktarda eklenmesi. </li><li> (Şekil 2A, steril H2O ile 384 gözlü TC levhaları sıvı haznesini ve sıvı dolgulu çevresel kapak düzenli levha kapaklar yerine). </li><li> % 95 nem,% 5 CO2 ve% 20 O2 ile 37 ° C, döner bir inkübatörde tabak yerleştirin ve hücreler, 4 gün boyunca MCTSes halinde toplamak için izin verir. aniden düşmesini nem seviyesini önlemek için sonraki günlerde çok uzun süre inkübatör kapıyı açmaktan kaçının. </li></ol> Bir Robotik Sistemi Kullanılarak 3. Orta Değişim <ol><li> MCTS oluşumundan sonra 4. günde, otomatik bir mikro yıkama dağıtıcı kullanılarak göz başına önceden ısıtılmış ortam 30 uL ekleyin. MCTSes ek olarak 3 gün boyunca büyümeye izin verin. onlar deney için istediğiniz boyutu ulaşmak kadar, düzenli olarak her 3 günde bir orta yerine. </li><li> Her bir ortamdaki bir tanımlanan hacmi aspire ampirik yıkama manifoldunun Z -height ayarlayın. Aspire 30 kuyu başına orta ul taze, önceden ısıtılmış orta 30 uL ile değiştirin. NOT: yıkama manifoldu aşağı hareket hangi dağıtım hızı ve hız ayarlamaen düşük oranda kuyuların içine kuyularda türbülans en aza indirmek için. </li></ol> MCTS 4. Yüksek içerik Görüntüleme ve Yarı-otomatik Görüntü Analizi <ol><li> Resim 4X hava objektif kullanarak bir yüksek içeriği otomatik görüntüleme sisteminde MCTS (NA 16). </li><li> Numarası ve aralık z -stacks ve oyuk başına bütün bir MCTS yakalamak için deney için istediğiniz gibi piksel binning ayarlayın 11 ms maruz kalma süresi ve 4 x 4&#39;e binning ayarlayın. </li><li> programlama dilinde yazılmış bir in-house yordamını kullanarak &quot;, benioku&quot; bölümünde açıklandığı gibi, resim kademeli olarak işleyin. NOT: .m kodu ve .txt benioku dosyaları ek kod dosyaları (Şekil 1B) olarak mevcuttur. Rutin tedbirler 2D görüntüleri MCTS alanı, majör ve minör eksenler, çevre ve sağlamlık. </li></ol><img alt="Şekil 1" src="/files/ftp_upload/55403/55403fig1.jpg" /> Şekil 1:Çevresel kapak hazırlanması ve yarı otomatik rutin bir akış şemasıdır. Doğru (sol) ve fazla miktarda sıvı dolgu (sağ) bir miktarda (burada,% 5 DMSO) ile dolu bir buharlaştırma düşürücü çevresel kapağın (A) görüntü. ok sıvı eklemek için kısa kenar çukur gösterir. Yıldız işareti DMSO doğru hacminin eklenmesinden sonra kapağın ortasında boşluk gösterir. (B) yarı otomatik görüntü analizi rutin yer alan adımları gösteren bir iş akışı. <a href="http://ecsource.jove.com/files/ftp_upload/55403/55403fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Filtreli Agaroz ile 384 Kuyu TC Plakaları Kaplama LOT standart bir uygulama kaplamak için agaroz ve / veya dağıtma birimi gerekir plakaları, agaroz 6 jelleşmenin önlenmesi için ısıtıldı tutulması için% 1-1.5, düşük-erime noktalı agarozda kullanmaktır. çapı 0.2 ila 0.4 mm arasında değişen küçük bir boru delikler sıvı dağıtma kasetleri kullanılarak birden fazla tabak hazırlarken agaroz jel potansiyel bir sorun değildir. Bu dağıtma sıras...

tümörlerde kanser hücreleri fizyolojik hücre dışı matris ve etkileşim hücreleri ile çevrili bir kompleks 3 boyutlu (3D) bir yapı içerisinde düzenlenir. Yaklaşık dokularda bütün hücreler 3 boyutlu bir ortamda bulunan tümör özellikleri taklit nitro tümör modellerinde daha fazla fizyolojik olarak uygun olan ihtiyaç çok 3D kültür teknikleri, 1, 2, 3 geliştirilmesi ile sonuçlanmıştır. Bu modeller artık 3D 2 terapötik metastazı ve hücre yanıtına tümör mikro rolü çalışmak için temel araştırma araçları haline gelmektedir. Ayrıca, 2-boyutlu (2B) hücre kültürleri 4 ile karşılaştırıldığında, 3 boyutlu modeller hücre içi sinyal yollar etkileyen tümör stromanın etkileşimleri, daha iyi anlaşılması için izin verir. kanser hücre hatlarının çok hücreli tümör sferoidler (MCTSes) sık sık 3D cep c kullanılanin vivo tümörlerin göreli yakınlığı nedeniyle ulture modelleri. Kullanılan çeşitli teknikler dışında, agaroz ile kaplanmış plakalar MCTS nesil sıvı katlamalı tekniği (lot) talebi optimizasyonu ve hedef doğrulama 5, 6, 7, 8, 9, 10, 11 için büyük ilgi görmüştür. Bu LOT 6, 7 kullanılarak MCTS kültürlerde bileşik kütüphaneler pilot ekranları çalıştırmak için başarılı başardık son çalışmalar bellidir. Ancak, orta buharlaşma kaynaklı düzensiz kaybına MCTS morfolojisi ve büyüme iyi-to-kuyu değişkenlik mikrotitre levhaları kullanılarak LOT (milletvekilleri) eşlik ortak engelli bulunmaktadır. Sonuç olarak, üniform olmayan MCTSes oluşumu önemini ve alaka ödünFarmakolojik Testler 8, 12, 13 veri. birimleri dağıtma otomatik sıvı kullanırken uyarlık konularına ek olarak, LOT-tabanlı yüksek verimli deneyleri etkileyen başka pratik bir sorun milletvekilleri kaplama agaroz ile. Besleme ünitesi agaroz jelleşmenin önlenmesi için ısıtıldı tutulabilir, ancak tevzi kaset ve boru tıkanma robotik sistemler 6 için potansiyel bir husustur. Bu zorlukların bazılarının üstesinden gelmek için, biz son zamanlarda MCTS kültürü 8 LOT birkaç değişiklik geliştirmişlerdir. Bu modifikasyonlar özellikle yaygın yüksek verimli tarama laboratuvarlarında bulunan araçları kullanarak milletvekilleri gelen düzensiz orta kaybını önlemek için olası yolları dayanmaktadır. 3 arasında muntazam boyutlu ve tekrarlanabilir MCTSes üretilmesi için modifiye edilmiş çok bir ayrıntılı işlem84-kuyu plakaları (İP) sunulmuştur. Eser özellikle, enine kesit alanının ölçümü için net bir şekilde tanımlanmış bir sınır yoktur, kısmen parçalanmış, ilaçla işleme tabi tutulmuş MCTSes olarak, MCTS boyutunun değerlendirilmesi için bir yarı-otomatik rutin sunulur.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Low-melting</td>
		</tr>
		<tr>
			<td>McCOY&#39;s 5A Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M8403</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;rapid&#8221; Filtermax filter</td>
			<td>TPP</td>
			<td>99505</td>
			<td>0.22 &#956;m, 500 mL</td>
		</tr>
		<tr>
			<td>Multidrop&#8482; Combi Reagent Dispenser&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Tube Dispensing cassette&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24073295</td>
			<td>Metal tip&#160;</td>
		</tr>
		<tr>
			<td>384-well TC plate&#160;</td>
			<td>PerkinElmer</td>
			<td>6057308</td>
			<td>Plate type- CellCarrier</td>
		</tr>
		<tr>
			<td>Standard Tube Dispensing Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>24072670</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroClime Environmental Lid</td>
			<td>Labcyte</td>
			<td>LLS-0310</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Incubator (SteriStore )</td>
			<td>HighRes Biosolutions</td>
			<td>23641</td>
			<td>Serial No.: D00384</td>
		</tr>
		<tr>
			<td>Microplate Washer Dispenser&#160;</td>
			<td>BioTek</td>
			<td>Unspecified</td>
			<td>Model: EL406&#160;</td>
		</tr>
		<tr>
			<td>High-Content Imaging System (CellVoyager )</td>
			<td>Yokogawa Electric Corporation</td>
			<td>Unspecified</td>
			<td>Model: CV7000</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>New England Biolabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE&#8482; Express recombinant cell dissociation reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604021</td>
			<td>Phenol red free</td>
		</tr>
	</tbody>
</table>

evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

Yakından in vivo koşullarda taklit Tümör modelleri potansiyel anti-kanser ilaçların taranması için ilaç keşfi ve geliştirilmesi giderek daha popüler hale gelmektedir. Çok hücreli tümör sferoidler (MCTSes) etkili bir talebi optimizasyonu ve hedef doğrulama için in vitro bir model bunları uygun hale getirir, katı tümörlerin fizyolojik koşulları taklit eder. MCTS kültürü için çeşitli tekniklerin dışında, agaroz üzerinde sıvı bindirme yöntemi MCTS üretimi için en ucuz yöntemlerden biridir. Bununla birlikte, yüksek verimli tarama için sıvı kaplama kullanarak MCTS kültürleri güvenilir aktarım agaroz ile mikrotiter plakaları (MAS) kaplanması ve kuyu boyunca tek MCTS formasyonunun irreproducibility dahil kısıtlamaları bir dizi tarafından tehlikeye. Milletvekilleri ilaç için tüm plaka kullanımını engelleyen, plaka dış ortamdaki aşırı buharlaşma sonucu etkileri kenar önemli yatkındırtestleri. Bu el yazması üniforma MCTS formasyonu ölçeklenebilirlik ve tekrarlanabilirlik arttırmak için sıvı bindirme tekniği detaylı teknik geliştirmeler sağlar. ilaç tedavisi sunulmuştur sonra Ayrıca, MCTS değerlendirilmesi için basit, yarı otomatik ve evrensel olarak uygulanabilir yazılım aracı ile ilgili detaylar sunuyor.

Özellikle periferik kuyulardan orta düzensiz kaybı, küçük kültür hacimleri ile milletvekilleri sık karşılaşılan bir sorundur. Bu tür iyi kontrol sıcaklık / nemlendirme sistemleri ve buharlaştırma azaltıcı MP&#39;lerin ve plaka kapaklı kuluçka esas olarak geliştirilmiş kültür koşulları, kuyu 8 boyunca ortam önemli kaybını azaltır. nispi bir buharlaşma ölçmek için, Orange G (RG) eşit hacimleri her bir kuyucuğa ilave edildi ve 3 ...

Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

mikrotiter plakaları orta düzensiz kaybı üniforma çok hücreli tümör sfero oluşumu tekrarlanabilirlik etkiler. Kültür şartlarının iyileştirilmesi küremsi oluşumu tekrarlanabilirlik ve sıvı üst tabaka tekniği ile küremsi bazlı deneylerin sonuçları geliştirmek önemli bir orta kaybını azaltmak.

Buharlaşma azaltıcı Kültür Durumu Mikrotiter Tabaklar Multisellüler Sfero Oluşumunun Tekrarlanabilirlik artırır

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

evaporation-reducing-culture-condition-increases-reproducibility

Research

JoVE Journal

Cancer Research

6.9K Görüntüleme.  Palacky University in Olomouc.  mikrotiter plakaları orta düzensiz kaybı üniforma çok hücreli tümör sfero oluşumu tekrarlanabilirlik etkiler. Kültür şartlarının iyileştirilmesi küremsi oluşumu tekrarlanabilirlik ve sıvı üst tabaka tekniği ile küremsi bazlı deneylerin sonuçları geliştirmek önemli bir orta kaybını azaltmak. 

Video: Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

İşlevsel Genomik Tarama kullanılması Kanser Hücre Sfero Kültürleri Potansiyel Yeni İlaç Hedefleri Belirlemek İçin

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Kanser Araştırmaları

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

ΓH2AX ve 53BP1 oluşumu ve DNA çift iplikli kırılmalara tamiri analiz etmek için ayirt mikroskobu

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Baş ve boyun Skuamöz hücreli karsinom için bir küresel tabanlı 3D hücre kültür modelinde test terapi

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

Sözde Hemangioblastoma tahlil çimlenme küresel kullanarak neovaskülarizasyon Vitro performansını değerlendirmek için kapsamlı bir yordam

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

3D Organotypic Melanom küresel Cilt modeli

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Antikanser antikor etkinliğinin Antineoplastic uyuşturucu tarafından kullanılmasının muhtemelen: antikor-uyuşturucu Synergism kombinasyon Dizin denklem kullanılarak tespiti

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Kimlik, histolojik karakterizasyonu ve için fare Vitro 3D küresel kültür modelleri prostat lob diseksiyonu

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Bir küresel öldürme tahlil araba T hücreleri tarafından

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Üç boyutlu Küroid Istilası tahlil tam ınsan tümör tabanlı matris

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Hücre viability ve ölüm kanser hücrelerinin 3D Küroid kültürlerde değerlendirilmesi

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...