Mikro Silikon konsolun Kullanımı Hücresel kasılma fonksiyonunu değerlendirmek için<em> İn Vitro</em
Summary
Bu protokol, in vitro kas hücrelerinin kasılma ölçmek için esnek kültür yüzeyleri olarak mikro silikon konsolun kullanımını tarif etmektedir. Hücresel kasılması, ölçülen kaydedilen ve in vitro kontraktil fonksiyon ölçmek için non-invazif ve ölçeklenebilir sisteminin temin edilmesiyle, kuvvet çıktılan dönüştürülebilir dirsekli bükme neden olur.
Abstract
Daha fazla ön ve biyolojik olarak uygun in vitro tahlillerin gelişimi tohumlanmış hücrelerin fonksiyonel değerlendirilmesini sağlamak çok yönlü hücre kültür sistemlerinin ilerlemesi dayanmaktadır. Bu amaçla, mikro konsol bükme teknolojisi daralma kaynaklı alt tabakanın değerlendirilmesi yoluyla, iskelet, kalp ve düz kas hücreleri dahil hücre tipleri, bir dizi kasılma işlevselliğini ölçmek için hangi ile bir platform sunuyor. Birden fazla mesaj göndermiş konsol dizilerin uygulanması ilaç yeterlilik ve toksisitesini, hastalığın ilerlemesini fenotip ve yanı sıra, kas ve diğer hücre-hücre etkileşimleri değerlendirmek için yüksek verimli protokollere orta geliştirmek için bir araç sağlar. Bu yazıda, bu amaç için güvenilir bir konsol dizileri imal edilmesi için ayrıntıları ve bu yüzeyler üzerinde başarıyla kültürü hücreleri için gerekli olan bir yöntem sağlar. Ayrıca açıklama fonksiyonel anal gerçekleştirmek için gerekli adımlar sağlanırkontraktil hücre tipleri Ysis yeni lazer ve foto-dedektör sistemi kullanarak bu tür diziler üzerinde muhafaza. Vurgular hassasiyet ve bu sistem ile mümkün kontraktil fonksiyonun tekrar analiz tekrarlanabilir niteliği verilen temsili veriler, hem de çalışma geniş olan bu tür önlemlerin hale gelebilir. Bu sistemin başarılı yaygınlaşmasının araçlarla araştırmacılar doku performansı, hastalık gelişimi ve yeni terapötik tedaviye yanıtın daha doğru tahminlere yol açan, in vitro hızlı, düşük maliyetli fonksiyonel çalışmalar yapmak sağlayabilir.
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
Hücresel daralma kanıt için mikro konsol analiz kritik adımlar mikroskop sahne içindeki konsol çip yerleştirme ve dizideki köşe konsolun ucuyla lazer ve foto-dedektör sonraki hizalama vardır. Bu doğru yapılmazsa, o zaman yazılım potansiyel veri toplama sırasında yanlış negatif birikimine neden, dizideki kalan konsolun pozisyonlarını hesaplamak için mümkün olacaktır. Operatörler konsol çip lazer pozisyonları kalibre önce kültür çanak alt ile aynı hizada yalan emin olmak için dikkatli olm…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Bu araştırma Sağlık hibe numaraları R01NS050452 ve R01EB009429 Ulusal Enstitüsü tarafından finanse edildi. Konsol cips imalatı Cornell Üniversitesi'nde bulunan Nanofabrikasyona Tesisinde işbirlikçileri tarafından dışarıdan gerçekleştirildi. Konsol üretim sürecinde kullanılan tüm ekipmanlar bu tesiste yer oldu. Konsol mikro-üretim ile yardım için Mandy Esch ve Jean-Matthieu Prot Special thanks. Konsol işlevsellik video animasyon UCF de Sentetik Gerçeklik Lab Charles Hughes, Alex Zelenin ve Eric Imperiale tarafından oluşturulmuştur.
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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