Bu araştırma Florida&#39;nın Bankhead-Coley Takımı Bilim Grant (2BT03), Sağlık / Ulusal Kanser Enstitüsü (1R21CA164322-01) ve Moffitt Kanser Merkezi&#39;nin Takım Bilim Grant National Institutes Devlet tarafından finanse edildi. Bu eser, H. Lee Moffitt Kanser Merkezi ve Araştırma Enstitüsü, bir NCI belirlenen Kapsamlı Kanser Merkezi&#39;nde (P30-CA076292) de çeviriyle ilgili araştırma Çekirdek Tesisi tarafından kısmen desteklenmiştir. Birincil hücrelere Erişim Moffitt Kanser Merkezi&#39;nde Toplam Cancer Care Protokolü&#39;nün sayesinde mümkün olmuştur.

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	<li>Salmon, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+differential+sensitivity+of+human-tumor+stem+cells+to+anticancer+drugs.">Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs.</a> N Engl J Med. 298, 1321-1327 (1978).</li><li>Suggitt, M., Bibby, M. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+assay+for+chemosensitivity+in+multiple+myeloma.">A preclinical assay for chemosensitivity in multiple myeloma.</a> Cancer Res. 74, 56-67 (2014).</li><li>Misund, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measurement+of+drug+sensitivity+of+myeloma+cells+co-cultured+with+bone+marrow+stromal+cells.">A method for measurement of drug sensitivity of myeloma cells co-cultured with bone marrow stromal cells.</a> J Biomol Screen. 18, 637-646 (2013).</li><li>Ramasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+experimental+model+to+evaluate+the+concomitant+effect+of+drugs+on+the+tumour+microenvironment+and+cancer+cells.">Fluorescence-based experimental model to evaluate the concomitant effect of drugs on the tumour microenvironment and cancer cells.</a> Br J Haematol. 157, 564-579 (2012).</li><li>McMillin, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-specific+bioluminescence+platform+to+identify+stroma-induced+changes+to+anticancer+drug+activity.">Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity.</a> Nat Med. 16, 483-489 (2010).</li><li>Koh, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cells+for+microscopy+using+cytospin.">Preparation of cells for microscopy using cytospin.</a> Methods Enzymol. 533, 235-240 (2013).</li><li>Al-Quran, S. Z., Yang, L., Magill, J. M., Braylan, R. C., Douglas-Nikitin, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bone+marrow+plasma+cell+infiltrates+in+multiple+myeloma:+the+added+value+of+CD138+immunohistochemistry.">Assessment of bone marrow plasma cell infiltrates in multiple myeloma: the added value of CD138 immunohistochemistry.</a> Hum Pathol. 38, 1779-1787 (2007).</li><li>Najar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+promote+or+suppress+the+proliferation+of+T+lymphocytes+from+cord+blood+and+peripheral+blood:+the+importance+of+low+cell+ratio+and+role+of+interleukin-6.">Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6.</a> Cytotherapy. 11, 570-583 (2009).</li><li>Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+i+trialist.">Phase i trialist.</a> Lancet Oncol. 13, 236 (2012).</li></ol>

The authors have no conflict of interest to disclose.

Özet olarak, bu kemik iliği, bir yeniden birincil MM hücre ve ilaç ex vivo farmakodinamik kemosensitiviteyi ölçmek için güçlü ve yüksek kapasiteli bir yöntemdir. Bu protokol kapsamında kritik adımlar kuyuların uygun tohumlama ve yeterli (beklenen sonuçlar için bakınız Şekil 2) odaklama: MM ve stromal hücreler düzgün yayılı sağlamak, bu stromal hücreler, kuyunun dibine yapışık olan MM tüm Hücreler, MM hücreleri koyu halka çevrili aydınlık diskin yönü olan ay...

Bu yöntemin amacı, bu sonuçlar Kemoterapiye alt tespit etmek için kullanılabilir yeterli hassasiyetle, fizyolojik koşullara mümkün olduğunca yakın, ex vivo olarak ajanların bir panele birincil hücreler multipl miyeloma (MM), ilaç duyarlılığını karakterize etmektir tümör yükü ve içinde nüfusları sonuçta, klinik yanıtı tahmin etmek için tasarlanan hesaplama modelleri parameterize için. Hesaplamalı modelleri, kanser konak-terapi etkileşimleri gibi kompleks sistemler, analiz etmek güçlü araçlardır. Ancak, modeller sadece veri onları parameterize eskisi kadar iyi. Ne yazık ki, literatürde mevcut olan en veriler genellikle birbirini dışlayan deney koşullarında elde edilenler gibi, bu tür modeller parametrelerini elde etmek için, mevcut biçimde kullanılamaz. Böylece, yeni deneyler genellikle gerekli deneysel parametrelerin set elde etmek amacı ile ihtiyaç vardır. Çoğu canlılık deneyleri, ancak, yıkıcı ve inci vardırBize zaman noktalarında az sayıda, genellikle sadece birine sınırlı. Bu büyük ölçüde bu tür deneyler sistemin zamansal dinamikleri hakkında bilgi vermek için başarısız beri kemosensitivite tahlillerin kullanımı, hesaplama modelleri parameterize için yaşlılık ve sakatlık. Bu genellikle, böylece mevcut deneysel koşullar sayısının sınırlandırılması, numune başına birkaç milyona sınırlı ve biyopsi sonra kısa bir ömre sahip olan primer kanser hücrelerinin, özellikle de geçerlidir. Buna ek olarak, çoğu canlılık deneyleri ayrıca hücreleri bozan ve yapılabilir deneyler sayısını sınırlar, protokole ekstra çalışma ekler ölçümü, zamanında olmayan kanser (stroma) hücrelerinden kanser ayrılmasını gerektiren . 40 yıl kadar önce, somon ve ortak 1 tümör hücrelerinin duyarlılığında değerlendirilmesi için in vitro koloni oluşturma deneyi kendi ünlü önerdi. Ne yazık ki bu deney sayesinde çoklu mye az sayıda karışık başarı bulunduKontrol koşulları altında koloniler oluşturabilen edildi Loma (MM) hasta numuneleri: Bu, AA hücrelerin 0.1 ila% 0.001,% sadece küçük bir alt-grup replikasyon kabiliyetine sahip olduğu tahmin edilmiştir multipl miyelom kök hücreler 2 olarak adlandırılan edilir. Bu anlamlı daha yeni modeller 3, testin başarı oranını ve bir anda test edilebilir ilaçların sınırlı sayıda. (Standart ya da deneysel) MM ajanlar, dört yıl önce daha çoktur Bu sorun artık çok daha önemlidir. Ya bir hasta belirli bir ilaca &quot;hassas&quot; veya &quot;dirençli&quot; dir: Bu erken tahlillerin önemli bir sınırlama onların ikiye bölünmüş çıkışıdır. Hiçbir bilgi tümör nüfusun duyarlılık ve heterojen derecesine ilişkin sağlanmaktadır. Böylece, hızlı büyüyen dirençli hücrelerin küçük alt popülasyonlar olan hastalar tedaviye &quot;hassas&quot; olarak sınıflandırılır, ancak kısa bir süre nüks olur ve böylece değil bentedaviden Efit. Kanser hastalarının 4,5 genel sağkalım (OS) yanıt süresi önemi göz önüne alındığında, bu deneyler doğru sürekli OS tahmin etmek mümkün değildi nasıl açıktır. Amacıyla bu sınırlamaları aşmak için, ve uyuşturucu bir panele kişiselleştirilmiş klinik yanıtı tahmin ediyorum hastaya özgü hesaplama modelleri üretmek edebilmek için, biz multipl miyelom (MM) hücre hatlarının olmayan yıkıcı test ilaç duyarlılığı için bir yöntem geliştirdik ve hücre dışı matriks ve stroma 6 olmak üzere kemik iliği mikroçevresindeki bir ex vivo yeniden, birincil MM hücreleri. Bu deney, ancak, kimin boyutları ve maliyetli ekipman belirli bir parça bir defada yapılabilecek deneyler veya ilaç sayısını sınırlı belirli bir ticari mikroakışkan slayt, güvenerek sınırlama vardı. Burada anlatılan sistem, yüksek içine bu orijinal tahlil uzanırolmayan yıkıcı hücre canlılığını ölçmek için bir dijital görüntü analizi algoritmasına dayalı ilaçlar, in vitro tarama için Organotipik doz-yanıt platformu, -throughput. De 384 ya da 1536 oyuklu plakalarda her primer MM hücreleri, hücre-dışı matris ve hasta türevi stroma ve büyüme faktörleri de dahil olmak üzere kemik iliği mikro-3D rekonstrüksiyonu vardır. Canlı mikroskopi ve dijital görüntü analizi doz-yanıt yüzeyler üretmek için kullanılan farklı ilaç konsantrasyonları, hücre ölümü olayları tespit etmek için kullanılır. In vitro verilerden, bir matematiksel model hastanın tümör yükü içinde boyutu ve alt-popülasyonlarının kemosensitiviteyi tanımlar ve tümörü (klinik rejime 6 fizyolojik koşullarda ilaç (lar) nasıl tepki vereceğini simüle etmek için kullanılabilir Şekil 1). Bu platformun temel yenilikler şunlardır: İlaç konsantrasyonlarda başına gereken kanser hücrelerinin, (a), az sayıda (1,000-10,000rasyon); (B) anılan ilacı, fizyolojik koşullar etkinlik (hücre dışı matrisin, stroma hasta türevli büyüme faktörleri) değerlendirilmesi; (C) Sadece parlak bir alan görüntüleme beri canlılık belirteçleri 7 No toksisite, floresan 8 veya 9 biyoparlaklık ile hücreleri transfect böylece hiç gerek kullanılır; (D) Sürekli görüntüleme konsantrasyonu ve uygulama zamanı (farmakodinamik) &#39;in bir fonksiyonu olarak, ilaç etki sağlar; ve (e) in vitro ve hesaplamalı evrimsel modeller arasındaki entegrasyon, klinik sonucu 6 (Silva ve ark., hazırlık) tahmin etmek. Dijital görüntü analizi algoritması stromal hücrelerin kanser ayırt sağlar önemli bir faktördür altına onların farklı afinite de: dd hücreleri uygun ve stromal hücreler alt uygun ise, matris içinde süspansiyon halinde kalır yok iyi ve daha sonra streç. Bu ayrılık bile MM ve stroma a da oluşurAynı zamanda tohumlanmıştır ve inkübasyon O / N işlemi sırasında oluşur yeniden. plaka santrifüj aşağı iplik aşağıdaki tohumlama daha bu süreci hızlandırır. Stroma düşük bir profil ve koyu gölge (Şekil 2) korurken Sonuç olarak, MM hücreleri, karanlık bir halka ile çevrili yuvarlak parlak diskler gibi görüntüde görünür. Algoritması ayarlamaları gibi boyut ve şekil olarak diğer morfolojik özellikleri, dayalı ayrı kanser ve stroma ile yapılabilir rağmen Böylece, mevcut haliyle tahlil, yapışmayan kanser hücreleri için uygundur. Bu protokolde, (kemik iliğinden elde edilen mezenkimal stem hücreleri) BHYK ve HUVECler, kemik iliği interstisyel ve perivasküler niş temsil eden, hücre-dışı matrisi (kollajen ve temel zar matris) iki tip olarak ko-kültürler iki tip tarif sırasıyla. 384-plaka kullanarak, ilaç başına 5 konsantrasyonları ve iki çoğaltır, biz tes başardıkTek bir hastanın numune ile 31 farklı ilaçlara t kadar. Ek Şekil 1 bir robot pipet kullanarak optimum dağılımını temsil ederken 1,536 kuyucuğu bu sayı, 3 şu anda manuel hücre tohumlama için kullanılan düzen göstermektedir 127. Şekil olduğunu. Şekil 3&#39;ten tasarımda, her bir 384-oyuklu plaka 21 ilaç test 7 farklı ilaçlar, ya da bir hasta örneği 3 hasta numuneleri taşıyabilir. Her ilaç 5 konsantrasyonları ile temsil edilir, ve her bir durum çiftleri olarak tohumlanır. 4 kontrol kuyuları (ilaç yok) eklendi 7 ilaçlar (örneğin, kuyular 36-39, 126-129 ve 200-203) her biri için tohumlanır. Kullanılan ilaçların etkisini temin etmek için, insan miyeloma hücre kuşağı (örneğin, H929 ya da MM1.S), aynı zamanda tohumlandı ve (kuyular 76-89) kullanılan ilaçların her birinin en yüksek konsantrasyonda iki kopya halinde uyuşturulmuş. pozitif hücre hattı kontrolü onların doz yanıt eğrileri karşılaştırılabilir ac beri kullanılan ilaçlar, yeterli güce sahip olmalarını sağlarross deneyleri. Iki kuyu (kuyular 75 ve 90), görüntüleme sırasında tezgah üstü inkübatör olası sorunları tespit etmek için, başka bir deyişle, çevre koşulları için bir kontrol olarak, bir miyeloma hücre kuşağı ile tohumlanır. Kuyuların numaralandırma etme süresini azaltır ve ekipmanın aşınmasını azaltan mikroskop motorlu aşamasında tarafından katedilen mesafeyi azaltmak için zikzak bir yol izler.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EVOS FL AUTO</td>
			<td>AMG</td>
			<td>AMAFD1000 + AMC1000</td>
			<td>Digital microscope equipped with motorized stage and bench top incubator.</td>
		</tr>
		<tr>
			<td>384-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3683 SIGMA</td>
			<td>Corning CellBIND 384 well plates 
			384 well plate, polystyrene, CellBIND surface, sterile, clear flat bottom, black, w/lid</td>
		</tr>
		<tr>
			<td>1,536-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3832 ALDRICH</td>
			<td>Corning 1536 well plates, low base, surface treatment CellBIND, sterile</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-1440-02 SIGMA</td>
			<td>For in vitro isolation of lymphocytes from human peripheral blood.</td>
		</tr>
		<tr>
			<td>CD138 magnetic beads</td>
			<td>Miltenyi&#160;</td>
			<td>130-051-301</td>
			<td>Antibody conjugated magnetic beads for selection of CD138+ cells.</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi&#160;</td>
			<td>&#160;130-042-401</td>
			<td>Column for separation of cells.</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi&#160;</td>
			<td>130-042-302</td>
			<td>Magnet for separation column.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E6909 SIGMA</td>
			<td>ECM Gel from Engelbreth-Holm-Swarm murine sarcoma</td>
		</tr>
		<tr>
			<td>Getrex</td>
			<td>Life Technologies</td>
			<td>A1413201</td>
			<td>LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Life Technologies</td>
			<td>C-003-25P-B</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC )</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>GIBCO</td>
			<td>11875-093</td>
			<td>RPMI 1640 Medium + L-Glutamine + Phenol Red</td>
		</tr>
		<tr>
			<td>FBS Heat inactivated</td>
			<td>GIBCO</td>
			<td>10082-147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>3.1mg/mL Bovine collagen type I</td>
			<td>Advanced Biomatrix</td>
			<td>5005-B</td>
			<td>Bovine Collagen Solution, Type I, 3 mg/ml, 100 ml</td>
		</tr>
		<tr>
			<td>Precision XS</td>
			<td>BIOTEK</td>
			<td>PRC3841</td>
			<td>Robotic pipettor system</td>
		</tr>
	</tbody>
</table>

an organotypic high throughput system for characterization of drug sensitivity of primary multiple myeloma cells

Bu çalışmada biz, hasta kaynaklı multipl miyelom (MM) hücreler kemosensitiviteyi ve heterojenitesini tahmin etmek ex vivo ilaç duyarlılık testleri ve dijital görüntü analizi birleştiren yeni bir yaklaşım açıklar. Bu yaklaşım, hasta-Taze hücre dışı matrisin (kolajen ya da temel membran matrisi) ve stroma (dahil olmak üzere her kemik iliği mikro bir yeniden oluşan, çok oyuklu plakalara uygulanan mikroakışkan odaları içine, kemik iliği elde primer MM tohum hücreleri oluşur türetilmiş mezenkimal kök hücreleri) veya insandan türetilmiş endotel hücreleri (HUVECler). odaları, farklı ajanlar ve konsantrasyonları ile uyuşturulmuş olan ve bir dijital kamera ile donatılmış bir motorlu mikroskopta, parlak bir alan mikroskobu ile 96 saat süreyle sırayla görüntülenmiş. Dijital görüntü analiz yazılımı varlığı veya zar hareket yokluğu canlı ve ölü hücreleri algılar ve bir fonksiyon o kadar canlılığı değişim eğrileri üretirf ilaç konsantrasyonu ve maruz kalma süresi. Her ilaç, tümör nüfusun duyarlılığında parametrelerini yanı sıra, tümör heterojenite bir ölçüsü olarak, bu alt-popülasyonlarının sayısını belirlemek için bir hesaplama modeli kullanırlar. Bu hasta özel modeller daha sonra tedavi rejimleri taklit ve klinik yanıtı tahmin etmek için kullanılabilir.

Deney akışı kısaca Şekil 1&#39;de açıklanan tüm adımları başarıyla tamamlandığı takdirde, mikroskop ile elde edilen görüntüler Şekil 2&#39;ye eşdeğer olmalıdır. MM hücreleri açıkça parlak diskler ve BMSCs veya HUVECler zor görünür olmalıdır görülmelidir canlı ve de arka planda dağıtılır. Şekil 2A&#39;da görüldüğü gibi, DD hücreleri bir hastadan diğerine boyutu değişir, ancak genel olarak hücre çizgileri daha küçüktü...

Watch this Scientific Journal Video about An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells at JoVE.com

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Primer Multipl Miyelom Hücrelerinin İlaç Duyarlılık Karakterizasyonu için bir Organotipik Yüksek Verimli Sistemi

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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8.5K Görüntüleme.  H. Lee Moffitt Cancer Center and Research Institute. We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Video: An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Bir Organotipik Beyin Dilim ve kanser hücrelerinin 3D Sferoid ile Coculture Sistemi

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

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High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Nöroprotektif Stratejileri Tedavi Faktörleri teslimi için tasarlandı Erişkin Kök Hücre Yüksek Verimli Karakterizasyonu

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Chicken İnsan Multipl Miyelom Xenograftlarında Modeli Kurulması Tümör Büyüme, Invasion ve Anjiogenez Eğitim için

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

İlaç Tedavileri tümörün mikro incelenmesi ve Hedeflenen A Beyin Tümörü / Organotipik Dilim Co-kültür Sistemi

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin kaynaklı bağırsak Organoids şişme: Bir<em&gt; İn Vitro</em&gt; Deney Kistik Fibrozis Hastalarında İlaç Tepki değerlendirilmesi için

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor, büyük metabolitleri ve Lumacaftor Kistik fibrozis hastalarının biyolojik sıvı içinde klinik örnekler yüksek üretilen iş analizi için en iyi duruma getirilmiş LC-MS/MS yöntemi

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Dönem Placentae Fetal membranlar birincil insan Decidual hücre izolasyon

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Yüksek Verimli Bir Elektrokemilüminesans 7-Plex Testi Aynı Anda Tip 1 Diyabet ve Çoklu Otoimmün Hastalıklar İçin Tarama

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Yeni bir İnsan Organotipik Retina Kültürü Tekniğinin Karakterizasyonu

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...