Benutting van microschaal Silicon Cantilevers to Cellular contractiele functie Beoordelen<em> In Vitro</em
Summary
Dit protocol beschrijft het gebruik van microschaal silicium consoles als buigzaam cultuur oppervlakken voor het meten van de contractiliteit van spiercellen in vitro. Cellular samentrekking cantilever buigen, die kan worden gemeten, geregistreerd en omgezet in uitlezingen van kracht, die een non-invasieve en schaalbaar systeem voor het meten contractiele functie in vitro.
Abstract
De ontwikkeling van meer voorspellende en biologisch relevante in vitro testen is gebaseerd op de vooruitgang van de veelzijdige celkweek systemen die de functionele beoordeling van de gezaaide cellen te vergemakkelijken. Daartoe microschaal cantilever technologie biedt een platform waarmee de contractiele functie van verschillende celtypen, waaronder skelet, hart en gladde spiercellen meet, door beoordeling van de contractie geïnduceerd substraat buigen. Toepassing van gemultiplexte cantilever arrays verschaft de middelen matige tot hoge doorvoer protocollen voor het beoordelen van geneesmiddel-effectiviteit en toxiciteit, ziekte fenotype en progressie, alsmede neuromusculaire en andere cel-cel interacties te ontwikkelen. Het manuscript geeft de informatie voor het vervaardigen betrouwbare cantilever arrays hiervoor en de vereiste om succesvol kweek cellen op die oppervlakken methoden. Verdere beschrijving wordt geleverd over de stappen die nodig zijn om functionele anal voerenlyse van contractiele celtypen gehandhaafd op dergelijke arrays met behulp van een nieuwe laser-en foto-detector systeem. De representatieve gegevens wat de nauwkeurigheid en reproduceerbare aard van de analyse van contractiele functie mogelijk met dit systeem, alsmede de diverse studies waaraan dergelijke technologie kunnen worden toegepast. Succesvolle brede invoering van dit systeem zou kunnen bieden onderzoekers de middelen om snelle, goedkope functionele studies uitgevoerd in vitro, wat leidt tot nauwkeurigere voorspellingen weefsel prestaties, ontwikkeling ziekte en de reactie op nieuwe therapeutische behandeling.
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
De kritische stappen in de analyse microschaal cantilevers op tekenen van cellulaire contractie zijn de plaatsing van de cantilever chip binnen de microscoop fase en de daaropvolgende uitlijning van de laser en de fotodetector met de punt van de hoek cantilevers in de array. Als dit niet correct gebeurt, dan zal de software in staat om de posities van de resterende cantilevers extrapoleren in de array, kan leiden tot ophoping van valse negatieven tijdens gegevensverzameling. Exploitanten moeten erop toezien dat de canti…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dit onderzoek werd gefinancierd door het National Institute of Health subsidie nummers R01NS050452 en R01EB009429. Fabricage van de cantilever chips is extern uitgevoerd door medewerkers van de nanofabricage faciliteit gelegen aan de Cornell University. Alle in de cantilever fabricageproces apparatuur werd in deze faciliteit. Speciale dank aan Mandy Esch en Jean-Matthieu Prot voor hun hulp met cantilever micro-fabricage. Video animatie van cantilever functionaliteit werd gegenereerd door Charles Hughes, Alex Zelenin en Eric Imperiale van de Synthetic Reality Lab van 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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