L'utilizzo di microscala Silicon cantilever per valutare Cellular contrattile Funzione<em> In Vitro</em
Summary
Questo protocollo descrive l'uso di cantilever silicio microscala come superfici di coltura flessibili per misurare la contrattilità delle cellule muscolari in vitro. Contrazione cellulare provoca cantilever flessione, che può essere misurata, registrata e convertita in letture di forza, fornendo un sistema non invasivo e scalabile per misurare funzione contrattile in vitro.
Abstract
Lo sviluppo di test più predittivi e biologicamente rilevanti in vitro si basa sulla promozione dei sistemi di coltura di cellule versatili che facilitano la valutazione funzionale delle cellule teste di serie. A tal fine, la tecnologia cantilever microscala offre una piattaforma con cui misurare la funzionalità contrattile di una gamma di tipi cellulari, incluse scheletrico, cardiaco e cellule muscolari lisce, attraverso la valutazione della contrazione indotta substrato piegatura. Applicazione di matrici a sbalzo multiplex fornisce i mezzi per sviluppare moderata a protocolli di high-throughput per valutare l'efficacia dei farmaci e la tossicità, fenotipo della malattia e la progressione, così come le interazioni neuromuscolari e altri cellula-cellula. Questo manoscritto fornisce i dettagli per la fabbricazione di matrici a sbalzo affidabili per questo scopo, ed i metodi necessari al successo le cellule di coltura su queste superfici. Ulteriore descrizione è fornita sui passi necessari per eseguire anale funzionaleYsis di tipi di cellule contrattili mantenuta su tali array utilizzando un nuovo sistema laser e foto-rilevatore. I dati rappresentativi forniti evidenzia la precisione e la natura riproducibile dell'analisi della funzione contrattile possibile utilizzare questo sistema, così come la vasta gamma di studi cui tale tecnologia può essere applicata. Adozione diffusa di successo di questo sistema potrebbe fornire gli investigatori i mezzi per eseguire rapidi, studi funzionali a basso costo in vitro, portando a previsioni più accurate delle prestazioni dei tessuti, lo sviluppo della malattia e la risposta al trattamento terapeutico.
Introduction
The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell’s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11.
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney’s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR.
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.
Protocol
Representative Results
Discussion
Le fasi critiche analizzando cantilever microscala per prove di contrazione cellulare sono il posizionamento del chip a sbalzo all'interno della fase di microscopio, e il successivo allineamento del laser e foto-rivelatore con la punta del cantilever angolari nella matrice. Se questo non viene fatto accuratamente, quindi il software sarà in grado di estrapolare le posizioni dei rimanenti cantilever nella matrice, potenzialmente conseguente accumulo di falsi negativi durante la raccolta dei dati. Gli operatori devon…
Declarações
The authors have nothing to disclose.
Acknowledgements
Questa ricerca è stata finanziata dal National Institute of numeri di sovvenzione Salute R01NS050452 e R01EB009429. Fabbricazione dei chip a sbalzo è stata eseguita esternamente da collaboratori presso l'impianto di nanofabbricazione situato presso la Cornell University. Tutte le apparecchiature utilizzate nel processo di fabbricazione a sbalzo era situato in questa struttura. Un ringraziamento speciale a Mandy Esch e Jean-Matthieu Prot per la loro assistenza con cantilever micro-fabbricazione. Animazione Video di funzionalità cantilever è stato generato da Charles Hughes, Alex Zelenin ed Eric Imperiale dal Laboratorio di Realtà sintetica a UCF.
Materials
| Name of material/ equipment | Company | Catalog number | Comments/ Description |
| Primary rat muscle growth medium | |||
| Neurobasal medium | Life Technologies | 21103-049 | N/A |
| B27 (50x) | Life Technologies | 17504044 | 1x |
| Glutamax (100x) | Life Technologies | 35050061 | 1x |
| G5 supplement | Life Technologies | 17503-012 | 1x |
| Glial-Derived Neurotrophic Factor | Cell sciences | CRG400B | 20 ng/ ml |
| Brain-Derived Neurotrophic Factor | Cell sciences | CRB600B | 20 ng/ ml |
| Ciliary Neurotrophic Factor | Cell sciences | CRC400A | 40 ng/ ml |
| Neurotrophin-3 | Cell sciences | CRN500B | 20 ng/ ml |
| Neurotrophin-4 | Cell sciences | CRN501B | 20 ng/ ml |
| Acidic Fibroblast Growth Factor | Life Technologies | 13241-013 | 25 ng/ ml |
| Vascular Endothelial Growth Factor | Life Technologies | P2654 | 20 ng/ ml |
| Cardiotrophin-1 | Cell sciences | CRC700B | 20 ng/ ml |
| Heparin Sulphate | Sigma | D9809 | 100 ng/ ml |
| Leukemia Inhibitory Factor | Sigma | L5158 | 20 ng/ ml |
| Vitronectin | Sigma | V0132 | 100 ng/ ml |
| Primary rat muscle differentiation medium | |||
| NB Activ 4 | Brain Bits LLC | NB4-500 | N/A |
| Equipment | |||
| Class 2 red diode laser | Newport | N/A | |
| Photo-detector | Noah Industries | N/A | |
| Model 2100 Pulse stimulator | A-M systems | N/A | |
| Multiclamp 700B Digitizer | Axon Instruments | N/A | |
| Patch clamp microscope and stage | Olympus | N/A | |
| Delta T4 culture dish controller | Bioptechs | N/A | |
| Axoscope software | Molecular Devices | N/A | |
| LabVIEW software | National Instruments | N/A | |
| 37oC, 5% CO2 incubator | NAPCO | N/A | |
| Class 2 microbiological flow hood | Labconco | N/A | |
| Pipettes and tips | Eppendorf | N/A |
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