Использование микромасштабных кремниевые зонды для оценки Сотовый сократительной функции<em> In Vitro</em
Summary
Этот протокол описывает использование микромасштабных кремниевых кантилеверов, гибких культуры поверхностей для измерения сократимость мышечных клеток в пробирке. Сотовый сокращение вызывает изгиба кантилевера, которое может быть измерено, записанного, и превращается в считываниями силы, обеспечивая неинвазивный и масштабируемую систему для измерения сократительной функции в пробирке.
Abstract
Развитие более интеллектуального и биологически соответствующих в пробирке анализов основывается на продвижении универсальных системах клеточных культур, которые облегчают функциональную оценку посеянных клеток. С этой целью, микромасштабная технологии консольные предлагает платформу, с которой для измерения функциональность сократительную диапазоне типов клеток, в том числе скелетных, сердечных, и гладкомышечных клеток, путем оценки сокращения, вызванного субстратом изгиба. Применение мультиплексированных массивов консольных предоставляет средства для разработки протоколов умеренной до высокой пропускной оценки эффективности лекарственного средства и токсичности, фенотип болезни и прогрессирования, а также нервно-мышечных и других межклеточных взаимодействий. Эта рукопись предоставляет детали для изготовления надежных массивов консольные для этой цели, и методы, необходимые для успешного культивирования клетки на этих поверхностях. Далее краткое описание приведено на ряд шагов, необходимых для выполнения приллиз типов сократительная клеток поддерживается на таких массивах, используя лазер нового и системы фото-детектора. Представительные данные, предоставленные моменты точность и воспроизводимость характер анализа функции возможного сократительной используя эту систему, а также широкий спектр исследований, в которой такая технология может быть применена. Успешное широкое внедрение этой системы может предоставить следователи с помощью выполнять быстрые, низкая стоимость функциональные исследования в пробирке, что приводит к более точными предсказаниями производительности тканей, развития заболевания и ответ на нового терапевтического лечения.
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
Критические шаги в анализе микромасштабной консолей для доказательства сотовой сокращения являются размещение чипа консольной в столике микроскопа, и последующее выравнивание лазера и фотодетектора с наконечником из угловых консолей в массиве. Если это не будет сделано точно, то пр?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Это исследование было профинансировано Национальным институтом номера грантов Здоровье R01NS050452 и R01EB009429. Изготовление консольных чипов была выполнена внешне сотрудниками в нанофабрикацию фонда, расположенного в Корнельском университете. Все оборудование, используемое в процессе изготовления кантилевера был расположен в этом учреждении. Отдельное спасибо Мэнди Эш и Жан-Матье Prot за помощь с консольной микро-производства. Видео анимация функциональности консольной был создан Карлом Хьюз, Алекс Зеленин и Эрик Imperiale из синтетической реальности Lab на УКУ.
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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