<ol>
	<li>Thery, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+guides+the+orientation+of+the+cell+division+axis.">The extracellular matrix guides the orientation of the cell division axis.</a> Nat Cell Biol. 7, 947-953 (2005).</li><li>Thery, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropy+of+cell+adhesive+microenvironment+governs+cell+internal+organization+and+orientation+of+polarity.">Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity.</a> Proc Natl Acad Sci U S A. 103, 19771-19776 (2006).</li><li>Schauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probabilistic+density+maps+to+study+global+endomembrane+organization.">Probabilistic density maps to study global endomembrane organization.</a> Nat Methods. , (2010).</li><li>Bischofs, I. B., Klein, F., Lehnert, D., Bastmeyer, M., Schwarz, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+network+mechanics+and+active+contractility+determine+cell+and+tissue+shape.">Filamentous network mechanics and active contractility determine cell and tissue shape.</a> Biophys J. 95, 3488-3496 (2008).</li><li>Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cell+life+and+death.">Geometric control of cell life and death.</a> Science. 276, 1425-1428 (1997).</li><li>Dupin, I., Camand, E., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+cadherins+control+nucleus+and+centrosome+position+and+cell+polarity.">Classical cadherins control nucleus and centrosome position and cell polarity.</a> J Cell Biol. 185, 779-786 (2009).</li><li>Gomez, E. W., Chen, Q. K., Gjorevski, N., Nelson, 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=Tissue+geometry+patterns+epithelial-mesenchymal+transition+via+intercellular+mechanotransduction.">Tissue geometry patterns epithelial-mesenchymal transition via intercellular mechanotransduction.</a> J Cell Biochem. 110, 44-51 (2010).</li><li>Kilian, K. A., Bugarija, B., Lahn, B. T., Mrksich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+cues+for+directing+the+differentiation+of+mesenchymal+stem+cells.">Geometric cues for directing the differentiation of mesenchymal stem cells.</a> Proc Natl Acad Sci U S A. 107, 4872-4877 (2010).</li><li>Kwon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+to+suppress+multipolar+divisions+in+cancer+cells+with+extra+centrosomes.">Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes.</a> Genes Dev. 22, 2189-2203 (2008).</li><li>Nelson, 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=Emergent+patterns+of+growth+controlled+by+multicellular+form+and+mechanics.">Emergent patterns of growth controlled by multicellular form and mechanics.</a> Proc Natl Acad Sci U S A. 102, 11594-11599 (2005).</li><li>Pouthas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+migrating+cells,+the+Golgi+complex+and+the+position+of+the+centrosome+depend+on+geometrical+constraints+of+the+substratum.">In migrating cells, the Golgi complex and the position of the centrosome depend on geometrical constraints of the substratum.</a> J Cell Sci. 121, 2406-2414 (2008).</li><li>Thery, M., Jimenez-Dalmaroni, A., Racine, V., Bornens, M., Julicher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+study+of+mitotic+spindle+orientation.">Experimental and theoretical study of mitotic spindle orientation.</a> Nature. 447, 493-496 (2007).</li><li>Thery, M., Pepin, A., Dressaire, E., Chen, Y., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+distribution+of+stress+fibres+in+response+to+the+geometry+of+the+adhesive+environment.">Cell distribution of stress fibres in response to the geometry of the adhesive environment.</a> Cell Motil Cytoskeleton. 63, 341-355 (2006).</li></ol>

Tous les auteurs, sauf Anne Béghin sont des employés de CYTOO SA, qui produit des réactifs et des instruments présentés dans cet article.

Choix de Microdétail Alors que les effets de la 5 blebbistatin uM sont à peine détectables sur des supports de culture standard, la quantification efficace est possible sur micropatterns qui concentrent l&#39;actine dans une fibre de stress majeur unique. Cela améliore la visualisation du cytosquelette d&#39;actine aider quantification précise des effets blebbistatin sur la cellule. Le choix de la forme Microdétail est critique dans la conception de l&#39;essai et est conçu en fonction de l&#39;évolution phénotypique d&#39;être mis en évidence dans l&#39;étude. Conclusions Micropatterns faciliter la visualisation et la quantification des effets des médicaments, en particulier à de faibles concentrations. Le remplacement des substrats homogènes plane avec micropatterns adhésif pour dosages cellulaires améliore la précision, la sensibilité et la qualité des données produites. En conséquence, moins de cellules sont nécessaires à l&#39;analyse afin de parvenir à des résultats robustes statistiques 3. Micropatterns adhésives offrent une réelle opportunité pour l&#39;amélioration des études fonctionnelles et d&#39;explorer les changements phénotypiques à des concentrations inférieures de médicaments afin d&#39;éviter d&#39;induire des effets toxiques des médicaments ou indirecte. Si les plates-formes de dépistage appropriées sont utilisées, micropatterns peut répondre aux exigences des compagnies pharmaceutiques pour améliorer les applications de criblage génétique / drogue. Quelques autres exemples de publications récentes qui ont exploité micropatterns pour analyser et quantifier les phénomènes cellulaires sont cités ci-dessous 3-13.

<table border="1"><tbody><tr><td colspan="2"> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td colspan="5" align="center"> La culture cellulaire </td></tr><tr><td colspan="2"> CYTOOchips 20x20 LM-FN </td><td> CYTOO </td><td> 10-114 </td><td> CYTOOchips sont disponibles pour l&#39;adsorption de la protéine de votre choix </td></tr><tr><td colspan="2"> PBS 1x </td><td> Gibco </td><td> 14040-091 </td><td></td></tr><tr><td colspan="2"> Chambre de comptage (toboggan Kova) </td><td> Prolabo </td><td> 71287.01 </td><td></td></tr><tr><td colspan="2"> DMEM/F-12 + Glutamax-I </td><td> Invitrogen Gibco- </td><td> 25300-054 </td><td></td></tr><tr><td colspan="2"> FBS (f Tal sérum bovin) </td><td> AAP </td><td> 31331-028 </td><td></td></tr><tr><td colspan="2"> La pénicilline et la streptomycine </td><td> AAP </td><td> A15-101 </td><td></td></tr><tr><td colspan="2"> 6 puits à plaques </td><td> Dutscher </td><td> 353046 </td><td></td></tr><tr><td colspan="2"> centrifugeuse </td><td> Fisher Scientific </td><td> M65838 </td><td></td></tr><tr><td colspan="2"> Microscope </td><td> Nikon </td><td></td><td></td></tr><tr><td colspan="5" align="center"> Drogues, la fixation et immunomarquage </td></tr><tr><td colspan="2" > Cytosquelette tampon 1X (CB) </td><td > Sigma </td><td > MES: M8250 KCl: I2636 MgCl: M2393 EGTA: E3889 </td><td > MES 10 mM pH 6,1, KCl 138mm, MgCl 3mm, 2 mM EGTA </td></tr><tr><td colspan="2" > Saccharose </td><td > Sigma </td><td > S2395 </td><td></td></tr><tr><td colspan="2"> PFA 32% </td><td> Sciences Electron Microscopy </td><td> 15714 </td><td></td></tr><tr><td colspan="2"> PFA 5% </td><td></td><td></td><td> Mélanger 10 ml de PFA 32% avec 54 ml de CB 1.185X </td></tr><tr><td colspan="2"> Triton-X100 </td><td> Sigma </td><td> T9284 </td><td></td></tr><tr><td colspan="2"> Comprimés PBS </td><td> Invitrogen Gibco- </td><td> 18912-014 </td><td></td></tr><tr><td colspan="2"> Bovin albumine sérique (BSA) </td><td> Sigma </td><td> A7806 </td><td></td></tr><tr><td colspan="2"> Phallodin-FITC </td><td> Sigma </td><td> P5282 </td><td></td></tr><tr><td colspan="2"> Hoescht </td><td> Invitrogen </td><td> H3570 </td><td></td></tr><tr><td colspan="2"> Blebbistatin </td><td> Euromedex </td><td> BN0640 </td><td></td></tr><tr><td colspan="2"> DMSO </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td colspan="2"> NH 4 Cl 0,1 M </td><td> Sigma </td><td> M11209 </td><td></td></tr><tr><td colspan="5" align="center"> Image Acquisition </td></tr><tr><td colspan="2"> Microscope à épifluorescence </td><td> Nikon</td><td> Eclipse Ti </td><td></td></tr><tr><td colspan="2"> Caméra CCD </td><td> TBC </td><td> TBC </td><td></td></tr><tr><td colspan="2"> Logiciel ImageJ </td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </td><td></td><td></td></tr></tbody></table>

improved visualization and quantitative analysis of drug effects using micropatterned cells

À ce jour, la plupart des HCA (analyse de contenu haute) des études sont réalisées avec des lignes de cellules adhérentes cultivées sur un substrat homogène en culture de tissus traités micro-plaques. Dans ces conditions, les cellules réparties et diviser dans toutes les directions résultant en une variabilité inhérente à la forme des cellules, la morphologie et le comportement. La grande cellule à cellule variance de la population globale font obstacle au succès du HCA, en particulier pour le développement de médicaments. La capacité de micropatterns pour normaliser la forme et la polarité interne de chaque cellule individuelle offre une formidable opportunité pour résoudre ce goulot d&#39;étranglement critiques 1-2. Pour faciliter l&#39;accès et l&#39;utilisation de la technologie micropatterning, CYTOO a développé une gamme de prêt à l&#39;emploi micropatterns, disponible en formats lamelle et micropuits. Dans cet article, vidéo, nous fournissons des protocoles détaillés de toutes les procédures de l&#39;ensemencement de cellules sur micropatterns CYTOOchip, le traitement médicamenteux, la fixation et la coloration à l&#39;acquisition automatisée, traitement d&#39;image automatique et d&#39;analyse des données finales. Avec cet exemple, nous illustrons comment micropatterns peut faciliter dosages cellulaires. Les altérations du cytosquelette de la cellule sont difficiles à quantifier dans les cellules cultivées sur des substrats homogènes, mais la culture de cellules sur les résultats micropatterns dans une organisation reproductible du treillis d&#39;actine due à un positionnement systématique des contacts d&#39;adhérence cellulaire dans chaque cellule. Une telle normalisation de l&#39;architecture intracellulaire permet de quantifier, même de petits effets sur le cytosquelette d&#39;actine comme l&#39;a démontré dans ces ensemble de protocoles utilisant blebbistatin, un inhibiteur de l&#39;interaction actine-myosine.

Watch this Scientific Journal Video about Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells at JoVE.com

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

Micropatterns adhésif qui normalisent l&#39;architecture cellulaire peut être utilisé pour augmenter la sensibilité dans la détection des effets des médicaments, améliorer la reproductibilité et de simplifier l&#39;acquisition automatique d&#39;images et d&#39;analyse. Une telle technologie bénéficieront tests de dépistage de drogue / siARN, réalisée sur conventionnelles soutient la culture cellulaire et par conséquent souffrent d&#39;excès de cellule à cellule variabilité.

Amélioration de la visualisation et l&#39;analyse quantitative des effets des drogues utilisant des cellules Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

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JoVE Journal

Bioengineering

17.2K vues.  Grenoble, France. Micropatterns adhésif qui normalisent l'architecture cellulaire peut être utilisé pour augmenter la sensibilité dans la détection des effets des médicaments, améliorer la reproductibilité et de simplifier l'acquisition automatique d'images et d'analyse. Une telle technologie bénéficieront tests de dépistage de drogue / siARN, réalisée sur conventionnelles soutient la culture cellulaire et par conséquent souffrent d'excès de cellule à ce...

Vidéo: Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Surfaces Micropatterned pour étudier les interactions acide hyaluronique avec les cellules cancéreuses

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Recherche

Bio-ingénierie

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabrication d&#39;hydrogels pour les systèmes de culture Micropatterned neuronaux utilisant la photolithographie dynamique projection de masque

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Les échafaudages auto-déclaration pour les 3-Dimensional Culture cellulaire

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching cellules Micropatterned sur une membrane PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensibilisation à la drogue induit des adénylylcyclase: Rationalisation de dosage et la miniaturisation des petites molécules et siARN préalable Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Transfection génique vers les cellules Spheroid sur microélectrodes plaques de culture destinés à la transplantation de cellules génétiquement modifiées

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Un rapide et quantitative Méthode fluorimétrique pour Small Molecule dépistage du médicament protéique Ciblage

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Utilisation du haut débit MICROBIOREACTEUR système automatisé pour la Production d’IgG1 de modèle dans les cellules CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Purification et analyse d'un anticorps monoclonal à partir de cellules ovaires de hamster chinois à l'aide d'un système automatisé de microbioréacteur

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Clarification primaire du fluide de culture cellulaire récolté par CHO à l’aide d’un séparateur acoustique

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...