Cette recherche a été financée par l&#39;État de Bankhead-Coley Science Team Grant de la Floride (2BT03), les National Institutes of / Institut national du cancer de la Santé (1R21CA164322-01) et Science Team Grant Moffitt Cancer Center. Ce travail a été financé en partie par le Fonds pour la recherche translationnelle de base au H. Lee Moffitt Cancer Center &amp; Research Institute, un Comprehensive Cancer Center NCI désigné (P30-CA076292). L&#39;accès aux cellules primaires a été rendue possible grâce au Protocole de Total Care Cancer au Moffitt Cancer Center.

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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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50+years+of+preclinical+anticancer+drug+screening:+empirical+to+target-driven+approaches.">50 years of preclinical anticancer drug screening: empirical to target-driven approaches.</a> Clin Cancer Res. 11, 971-981 (2005).</li><li>Kirshner, J., 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+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112, 2935-2945 (2008).</li><li>Durie, B. G., Jacobson, J., Barlogie, B., Crowley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnitude+of+response+with+myeloma+frontline+therapy+does+not+predict+outcome:+importance+of+time+to+progression+in+southwest+oncology+group+chemotherapy+trials.">Magnitude of response with myeloma frontline therapy does not predict outcome: importance of time to progression in southwest oncology group chemotherapy trials.</a> J Clin Oncol. 22, 1857-1863 (2004).</li><li>Harousseau, J. L., Attal, M., Avet-Loiseau, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+complete+response+in+multiple+myeloma.">The role of complete response in multiple myeloma.</a> Blood. 114, 3139-3146 (2009).</li><li>Khin, Z. 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.

En résumé, ceci est une méthode de débit puissant et haute de quantifier chimiosensibilité des cellules de MM primaires et ex vivo pharmacodynamique de médicaments dans une reconstitution de la moelle osseuse. Les étapes critiques au sein de ce protocole sont le bon ensemencement des puits et en se concentrant adéquate (voir Figure 2 pour les résultats escomptés): veiller à ce que les cellules de MM et stromales sont uniformément réparties, que les cellules stromales sont adhérent...

Le but de cette méthode est de caractériser la sensibilité aux médicaments du myélome multiple (MM) des cellules primaires pour un panneau d&#39;agents ex vivo, aussi près que possible des conditions physiologiques, et avec suffisamment de précision que ces résultats peuvent être utilisés pour identifier des sous-chimiorésistance populations au sein de la charge tumorale et, finalement, à paramétrer des modèles de calcul visant à estimer la réponse clinique. Les modèles informatiques sont des outils puissants pour analyser les systèmes complexes, tels que le cancer interactions hôte-thérapie. Cependant, les modèles sont seulement aussi bon que les données utilisées pour les paramétrer. Malheureusement, la plupart des données disponibles dans la littérature ne peuvent pas être utilisés dans sa forme actuelle pour paramétrer ces modèles, car ils sont souvent obtenus dans des conditions expérimentales mutuellement exclusifs. Ainsi, de nouvelles expériences sont souvent nécessaires dans le but d&#39;obtenir l&#39;ensemble des paramètres expérimentaux nécessaires. La plupart des essais de viabilité, cependant, sont destructrices et enous limités à un petit nombre de points dans le temps, souvent un seul. Cette handicape grandement l&#39;utilisation de tests de chimiosensibilité de paramétrer des modèles de calcul, puisque de telles expériences ne parviennent pas à fournir des informations sur la dynamique temporelle du système. Cela est particulièrement vrai avec les cellules cancéreuses primaires, qui sont souvent limitées à quelques millions par échantillon, et ont une courte durée de vie après la biopsie, limitant ainsi le nombre de conditions expérimentales disponibles. En outre, la plupart des essais de viabilité nécessitent la séparation du cancer des cellules non cancéreuses (stroma) au moment de la quantification, qui ajoute un surcroît de travail pour le protocole, perturbe en outre les cellules, et limite le nombre d&#39;expériences qui peuvent être effectuées . Il ya 40 ans, le saumon et ses collaborateurs 1 proposé leur fameux test in vitro de la formation de colonies pour l&#39;évaluation de la chimiosensibilité des cellules tumorales. Malheureusement, ce test a trouvé un succès mitigé en raison du petit nombre de mye multiplesLoma (MM) des échantillons de patients qui étaient capables de former des colonies dans des conditions de contrôle: Il a été estimé que seulement un petit sous-groupe de 0,001% à 0,1% des cellules MM ont été capables de replication, étant désignées comme des cellules souches de myélome multiple 2. Cela a limité de manière significative le taux de succès de l&#39;essai et le nombre de médicaments qui pourraient être testés en même temps, même dans les modèles plus récents 3. Cette question est beaucoup plus important maintenant, quand des agents MM (standard ou expérimentales) sont plus nombreux que il ya quatre décennies. Une limitation majeure de ces premiers essais est leur production dichotomique: soit un patient est «sensible» ou «résistant» à un médicament particulier. Aucune information n&#39;a été fournie concernant le degré de sensibilité et de l&#39;hétérogénéité de la population de la tumeur. Ainsi, les patients avec de petites sous-populations de cellules résistantes à croissance rapide seraient classées comme «sensibles» à la thérapie, mais retombait peu, et donc pas benEFIT du traitement. Compte tenu de l&#39;importance de la durée de la réponse de la survie globale (OS) des patients atteints de cancer de 4,5, il est clair comment ces tests ne sont pas en mesure d&#39;estimer correctement OS cohérente. Afin de contourner ces limitations, et d&#39;être en mesure de générer des modèles de calcul spécifiques à un patient qui permettraient d&#39;estimer la réponse clinique personnalisé à un panel de médicaments, nous avons développé une méthode pour la sensibilité au médicament de contrôle non destructif de myélome multiple (MM) lignées cellulaires et les cellules MM primaires dans une ex vivo reconstruction du microenvironnement de la moelle osseuse, y compris le stroma et la matrice extracellulaire 6. Cet essai, cependant, avait la limitation de compter sur une diapositive microfluidique commerciale particulière, dont les dimensions et le coût limité le nombre d&#39;expériences ou de médicaments qui pourraient être réalisées à la fois dans un équipement donné. Le système décrit ici étend cette analyse originale dans une haute-throughput organotypique plate-forme de dose-réponse, pour le criblage in vitro de médicaments, sur la base d&#39;un algorithme numérique d&#39;analyse d&#39;image de manière non destructive quantifier la viabilité cellulaire. Chaque puits dans 384 ou 1 536 puits des plaques est une reconstruction 3D du microenvironnement de la moelle osseuse, y compris des cellules primaires de MM, une matrice extracellulaire, et le stroma et les facteurs de croissance dérivés de patients. Microscopie directe et une analyse d&#39;image numérique sont utilisés pour détecter des événements de mort cellulaire dans différentes concentrations de médicament, qui sont utilisés pour générer des surfaces de dose-réponse. D&#39;après les données in vitro, un modèle mathématique identifie la taille et la chimiosensibilité des sous-populations à l&#39;intérieur de la charge tumorale du patient, et peut être utilisé pour simuler la manière dont la tumeur répondrait à la drogue (s) dans des conditions physiologiques dans un régime clinique 6 ( Figure 1). Les principales innovations de cette plate-forme sont: (a) petit nombre de cellules cancéreuses nécessaires (1,000-10,000 par médicament concentration); (B) l&#39;évaluation de l&#39;efficacité des médicaments dans des conditions physiologiques (matrice extracellulaire, stroma, des facteurs de croissance provenant de patients); (C) Aucune toxicité de la viabilité des marqueurs 7 puisque seule l&#39;imagerie du champ lumineux est utilisé, donc pas nécessaire de transfecter des cellules avec une fluorescence 8 ou 9 bioluminescence; (D) l&#39;imagerie en continu fournit l&#39;effet du médicament en fonction de la concentration et du temps d&#39;exposition (pharmacodynamie); et (e) l&#39;intégration entre les modèles in vitro et l&#39;évolution de calcul pour estimer le résultat clinique 6 (Silva et al., en préparation). Le facteur clé qui permet à l&#39;algorithme d&#39;analyse d&#39;image numérique de discerner le cancer à partir de cellules stromales est leur affinité différente pour le fond du puits: les cellules MM ne se conforment pas, et restent en suspension dans la matrice, tandis que les cellules stromales adhèrent au fond de la bien et puis étirer. Cette séparation se produit même si MM et le stroma unere ensemencées en même temps, et se produit au cours de la / O N processus d&#39;incubation. L&#39;ensemencement de filature dans la centrifugeuse suivante de la plaque accélère encore ce processus. En conséquence, les cellules MM apparaissent dans l&#39;image que les disques ronds lumineux entouré par un anneau sombre, tandis que le stroma maintient un profil bas et une teinte plus foncée (Figure 2). Ainsi, le dosage dans sa forme actuelle est le mieux adapté pour les cellules cancéreuses non-adhérentes, bien que des ajustements dans l&#39;algorithme pourrait être fait pour le cancer et le stroma séparé reposant sur d&#39;autres caractéristiques morphologiques, comme la taille et la forme. Dans ce protocole, nous décrivons deux types de matrice extracellulaire (collagène et de sous-sol matrice de membrane) ainsi que les deux types de co-cultures, avec (cellules souches mésenchymateuses de moelle extraite d&#39;os) BMSC et HUVEC, qui représente les créneaux interstitiels et périvasculaires de la moelle osseuse respectivement. Utiliser une plaque de 384 puits, 5 concentrations par la drogue et deux répétitions, nous avons été en mesure de test jusqu&#39;à 31 médicaments différents avec l&#39;échantillon d&#39;un seul patient. En 1536 plaques ainsi ce nombre est 127. Figure 3 représente la disposition actuellement utilisée pour l&#39;ensemencement manuel cellulaire, tandis que la figure 1 supplémentaire représente la distribution optimale aide d&#39;une pipette robotisée. Dans la conception de la figure 3, chaque plaque de 384 puits peut transporter 3 échantillons de patients avec 7 différents médicaments, soit un échantillon de patient testés avec 21 médicaments. Chaque médicament est représenté par 5 concentrations, et chaque état est ensemencée en double. 4 puits de contrôle (pas de médicament ajouté) sont ensemencées pour chaque ensemble de 7 médicaments (par exemple, les puits 36-39, 126-129 et 200-203). Pour garantir l&#39;efficacité des médicaments utilisés, une lignée cellulaire de myélome humain (par exemple, H929 ou MM1.S) est également tête de série, et droguée en double à la plus forte concentration de chacun des médicaments utilisés (puits 76-89). Le contrôle de la lignée cellulaire positif assure que les médicaments utilisés ont une puissance suffisante, puisque leurs courbes de réponse de dose sont comparables acexpériences Ross. Deux puits sont ensemencés avec une lignée cellulaire de myélome que le contrôle des conditions environnementales, en d&#39;autres termes, pour détecter d&#39;éventuels problèmes dans la paillasse incubateur lors de l&#39;imagerie (puits 75 et 90). Le dénombrement des puits suit un motif en zigzag afin de réduire la distance parcourue par la platine motorisée du microscope, ce qui réduit le temps d&#39;acquisition et de réduire l&#39;usure de l&#39;équipement.

<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

Dans ce travail, nous décrivons une nouvelle approche qui combine ex vivo tests de sensibilité aux médicaments et l&#39;analyse d&#39;image numérique pour estimer la chimiosensibilité et l&#39;hétérogénéité de myélome multiple (MM) de cellules provenant de patients. Cette approche consiste à ensemencer les cellules MM primaires fraîchement extraites d&#39;aspirats de moelle osseuse dans des chambres microfluidiques mises en œuvre dans des plaques à puits multiples, chacun consistant en une reconstruction du microenvironnement de la moelle osseuse, y compris la matrice extracellulaire (collagène ou de la membrane basale matrice) et le stroma (patient des cellules dérivées de souches mésenchymateuses ou des cellules endotheliales) d&#39;origine humaine (HUVEC). Les chambres sont drogués avec différents agents et concentrations, et sont imagés séquentiellement pendant 96 heures grâce à la microscopie à champ clair, dans un microscope motorisé équipé d&#39;un appareil photo numérique. Logiciel d&#39;analyse d&#39;image numérique détecte cellules vivantes et mortes de présence ou absence de mouvement de la membrane, et génère des courbes d&#39;évolution de la viabilité en fonction of concentration de drogue et le temps d&#39;exposition. On utilise un modèle de calcul pour déterminer les paramètres de la chimiosensibilité de la population ayant une tumeur de chaque médicament, ainsi que le nombre de sous-populations présentes en tant que mesure de l&#39;hétérogénéité de la tumeur. Ces modèles adaptés au patient peuvent alors être utilisées pour simuler des régimes thérapeutiques et estimer la réponse clinique.

Le flux de l&#39;expérience est brièvement décrit à la figure 1 Si toutes les étapes sont terminées avec succès, les images obtenues par le microscope devraient être équivalentes à la figure 2:. Vivent les cellules MM doivent être clairement visibles comme des disques lumineux et BMSCs ou HUVECs devraient être à peine visible et bien réparties dans le fond. Comme on le voit sur ​​la figure 2A, les cellules MM varient en taille d&#39;un patient à l&#39...

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.

Un système organotypiques à haut débit pour la caractérisation de la sensibilité des médicaments cellules du myélome multiple primaire

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

an-organotypic-high-throughput-system-for-characterization-drug

Research

JoVE Journal

Medicine

8.5K vues.  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.

Vidéo: 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.

Système de co-culture avec un cerveau organotypique et 3D sphéroïde de cellules de carcinome

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

Recherche

Médecine

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.

High Throughput Caractérisation des cellules souches adultes d&#39;ingénierie pour la livraison des facteurs thérapeutiques pour les stratégies neuroprotectrices

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.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

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;

Une tumeur au cerveau / organotypiques Slice système de co-culture pour l&#39;étude des tumeurs Microenvironnement et ciblée Thérapies de drogue

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.

Forskoline induite par Gonflement dans Intestinal organites: Un<em&gt; In vitro</em&gt; Essai pour évaluer la réponse des médicaments dans les patients atteints de fibrose kystique

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.

Optimisé méthode LC-MS/MS pour l’analyse de haut débit d’échantillons cliniques de Ivacaftor, ses principaux métabolites et Lumacaftor dans les fluides biologiques des patients atteints de fibrose kystique

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.

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

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.

Isolement de cellules primaires de Decidual humaines des Membranes fœtales des placentas à terme

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.

Un test électrochimiluminescence 7-plex à haut débit dépistant simultanément le diabète de type 1 et de multiples maladies auto-immunes

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.

Caractérisation d’une nouvelle technique de culture rétinienne organotypique humaine

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