마이크로 실리콘 캔틸레버의 이용은 세포 수축 기능을 평가하기 위해<em> 체외</em
Summary
이 프로토콜은 시험 관내에서 근육 세포의 수축력을 측정하기위한 유연한 배양 표면과 같은 실리콘 마이크로 캔틸레버의 사용을 설명한다. 셀룰러 수축 측정 기록하고, 시험 관내에서 수축력을 측정하는 비 침습적이고 확장 가능한 시스템을 제공하고, 힘의 판독 값으로 변환 할 수 외팔보 굽힘을 야기.
Abstract
더 많은 예측과 관련된 생물학적 시험 관내 분석법의 개발은 시드 세포의 기능 평가를 용이 다목적 세포 배양 시스템의 발전에 입각한다. 이를 위해 마이크로 캔틸레버 벤딩 기술은 기판의 수축에 의한 평가를 통해, 뼈, 심장, 및 평활근 세포를 포함한 세포 유형의 범위의 수축 기능을 측정 할 수있는 플랫폼을 제공한다. 다중화 된 캔틸레버 어레이의 적용은 약물의 효능과 독성 질환 표현형 및 진행뿐만 아니라, 신경 근육 및 기타 세포 – 세포 상호 작용을 평가하기위한 높은 처리량 프로토콜 중등도 개발 수단을 제공한다. 이 논문은이 목적을 위해 신뢰할 수있는 캔틸레버 어레이를 제조하기위한 상세하고, 이들 표면에 성공적으로 세포 배양에 필요한 방법을 제공한다. 또한 설명은 기능적 항문을 수행하는 데 필요한 단계를 제공한다수축성 세포 유형의 ysis는 신규 한 레이저 및 광 검출기 시스템을 사용하여 이러한 배열에서 유지. 하이라이트 정밀도 및이 시스템을 사용하여 가능 수축력의 분석 재현성을 제공하는 자연 대표 데이터뿐만 아니라, 연구의 넓은 범위는 이러한 기술이 적용될 수있다. 이 시스템의 성공은 널리 채택 수단 조사관 티슈 성능 질환 개발 및 신규 한 치료 적 처치에 대한 응답의 더 정확한 예측 선도, 시험관 내에서 신속, 저비용으로 기능 연구를 수행하기 위해 제공 할 수있다.
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
셀룰러 수축 증거 마이크로 캔틸레버를 분석하는데 중요한 단계는 현미경 스테이지 내의 캔틸레버 칩의 배치 및 배열 코너 캔틸레버 팁과 레이저와 광 검출기의 후속 정렬이다. 이 정확하게 수행하지 않은 경우, 소프트웨어는 잠재적으로 데이터를 수집하는 동안 위음성의 축적으로 이어지는 배열 잔존 캔틸레버의 위치를 추정 할 수 없습니다. 운영자는 캔틸레버 칩이 레이저의 위치를 ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
이 연구는 건강 허가 번호 R01NS050452 및 R01EB009429의 국립 연구소에 의해 투자되었다. 캔틸레버 칩의 제조는 코넬 대학교 (Cornell University)에있는 나노 제조 시설에서 공동 작업자에 의해 외부에서 수행 하였다. 캔틸레버의 제조 공정에서 사용되는 모든 설비는이 설비에 위치했다. 캔틸레버 마이크로 제조 그들의 도움 맨디 에슈와 Jean-마티유 보호 해주는 특별 감사. 캔틸레버 기능의 비디오 애니메이션 UCF의 합성 현실 연구실에서 찰스 휴즈, 알렉스 Zelenin 에릭 임페리얼에 의해 생성되었습니다.
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 |
References
- Bischoff, R. Enzymatic liberation of myogenic cells from adult rat muscle. Anat. Rec. 180, 645-661 (1974).
- Yasin, R., et al. A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells. J. Neurol. Sci. 32, 347-360 (1977).
- Dennis, R. G., Kosnik, P. E., 2nd, Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro. In Vitro Cell Dev. Biol. Anim. 36, 327-335 (2000).
- Khodabukus, A., Baar, K. Defined electrical stimulation emphasizing excitability for the development and testing of engineered skeletal muscle. Tissue Eng Part C Methods. 18, 349-357 (2012).
- Langelaan, M. L. P., et al. Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells. Journal of tissue engineering and regenerative medicine. 5, 529-539 (2011).
- Stephenson, G. M., O’Callaghan, A., Stephenson, D. G. Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats. Diabetes. 43, 622-628 (1994).
- Harber, M., Trappe, S. Single muscle fiber contractile properties of young competitive distance runners. Journal of applied physiology (Bethesda, Md: 1985). 105, 629-636 (2008).
- Hvid, L. G., et al. Four days of muscle disuse impairs single fiber contractile function in young and old healthy men. Experimental gerontology. 48, 154-161 (2013).
- Edman, K. A. Contractile performance of striated muscle. Advances in experimental medicine and biology. 682, 7-40 (2010).
- Krivickas, L. S., Walsh, R., Amato, A. A. Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029. Muscle Nerve. 39, 3-9 (2009).
- Sung, J. H., et al. Microfabricated mammalian organ systems and their integration into models of whole animals and humans. Lab on a chip. 13, 1201-1212 (2013).
- Wilson, K., Molnar, P., Hickman, J. J. Integration of functional myotubes with a Bio-MEMS device for non-invasive interrogation. Lab on a chip. 7, 920-922 (2007).
- Wilson, K., Das, M., Wahl, K. J., Colton, R. J., Hickman, J. Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement. PLoS ONE. 5, (2010).
- Binnig, G., Quate, C. F., Gerber, C. Atomic Force Microscope. Physical Review Letters. 56, 930-933 (1986).
- Pirozzi, K. L., Long, C. J., McAleer, C. W., Smith, A. S., Hickman, J. J. Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device. Applied physics letters. 103, 83108 (2013).
- Smith, A., Long, C., Pirozzi, K., Hickman, J. A functional system for high-content screening of neuromuscular junctions in vitro. Technology. 1, 37-48 (2013).
- Stenger, D. A., et al. Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth. Journal of the American Chemical Society. 114, 8435-8442 (1992).
- Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. A defined long-term in vitro tissue engineered model of neuromuscular junctions. Biomaterials. 31, 4880-4888 (2010).
- Rumsey, J. W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers. Biomaterials. 31, 8218-8227 (2010).
- Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression. Biomaterials. 30, 5392-5402 (2009).
- Das, M., et al. Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation. In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).
- Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. Surface determinants of neuronal survival and growth on self-assembled monolayers in culture. Brain research. 630, 136-147 (1993).
- Hickman, J. J., et al. Rational pattern design for in vitro cellular networks using surface photochemistry. Journal of Vacuum Scienc., & Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).
- Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system. Biotechnology progress. 19, 1756-1761 (2003).
- Rumsey, J. W., et al. Node of Ranvier formation on motor neurons in vitro. Biomaterials. 30, 3567-3572 (2009).
- Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior. International journal of biomedical engineering and technology. 2, 104-134 (2009).
- Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons. Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).
- Varghese, K., et al. A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology. PLoS ONE. 5, e8643 (2010).
- Natarajan, A., et al. Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform. Biomaterials. 32, 4267-4274 (2011).
- Davis, H., et al. Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model. Journal of biomaterials and tissue. 2, 206-214 (2012).
- Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. Engineered In Vitro Feed-Forward Networks. J Biotechnol Biomater. 3, 2 (2013).
