從大鼠心臟將細胞溶下做培養的心肌細胞為一個重要且穩定的細胞初代培養來源，用來模擬生物體心肌細胞的功能比一般細胞株更加適合與準確，而在生醫工程上，許多科學家也致力於心肌的研究與器官模型之建立，利用組織工程的概念，將人體器官用微流體整合在一個微小的晶片上甚至在未來可以和其他器官串聯，對於藥物篩選與研發具有非常重要的里程碑。
在心臟的解剖與分析中，可以發現心臟是一條肌肉帶並且具有螺旋型的旋轉與排列，而如何讓在生物體外的心肌具有功能與排列是我們所好奇的，我們在具有生物相容性的PDMS上，利用黃光微影作出各種大小不同的溝槽，來限制細胞的生長行為與約束其排列性，希望透過改變細胞生長的環境來增加心肌的收縮或功能性，並且應用在三維結構上，使其能在三維球狀PDMS上自主收縮，為一種生物體幫浦，並不像其來幫浦一樣需要外接電源或動力來驅動此微型幫浦，可以應用在許多方面，例如微流體的驅動、懸臂樑的致動，作為一種微型致動器。
本實驗利用於器官組織中常見的小鼠纖維母細胞作為初步測試，探討細胞在不同溝槽尺寸下的排列行為，再將此參數應用於大鼠初代培養，並且利用氧電漿接合的方式組成三維PDMS幫浦模型，並且於PDMS膜上培養細胞，模擬微型心臟幫浦的功能，在未來將會做為一個體外心臟模型用於藥物測試系統上。

Neonatal rat heart primary culture cardiomyocytes have been a significant model of chemical or biochemical system. Many scientists are devoted to the study of organ model and integration of different human organ model on one chip. With the concept of tissue engineering, it is an important milestone for drug screening and research.
With the anatomy of human heart, we can discover human heart is the helical ventricular myocardial band structure and cell viability and alignment are still the key issues to facilitate the cell adhesion with substrate to express proper cellular functions. Here we propose a three-dimensional culture system with different size microgrooves on the substrate with photolithography to confine and guide cardiomyocytes for realizing directional 3D artificial heart pump, and the system can be employed as a miniaturized actuators without the supplement of external energy sources.
NIH3T3 is a well-established cell line of fibroblast from mice and commonly used in biological studies so we first incubate this kind of cell to test the cell alignment with different size microgrooves. Depending on those parameters screening on 2D microgrooves, we propose a three-dimensional culture system with pre-designed PDMS microgrooves to confine cardiomyocytes with oxygen plasma treatment and photolithography. The artificial heart pump system will be employed as an in-vitro model for drug testing in the future.

[1] http://chmuseum.klchb.gov.tw/web/Content/Content.aspx?c0=306
[2] Wikswo, J, P,; Curtis, E, L. Scaling and Systems Biology for Integrating Multiple Organs-on-a-chip, 13, 2013.
[3] Dongeun, H.; Geraldine A, H. From 3D Cell Culture to Organs-on-Chips, Trend in Cell Biology, 2011, 21.
[4] LG Griffith, G Naughton - Tissue Engineering—Current Challenges and Expanding Opportunities, Science, 2002.
[5] H.Shin, S.Jo and A.G. Mikos, “Biomimetic materials for tissue engineering.” Biomaterials, 24, 4353-4364, 2003.
[6] Yang, Shoufeng, et al. “The design of scaffolds for use in tissue engineering. Part I. Traditional factors.’’ Tissue engineering 7.6 (2001): 679-689.
[7] Onoe, H.; Okitsu, T. Metre-long Cell-laden microfibers Exhibit Tissue Morphologies and Function. 2013, 12, 584-590.
[8] Higuchi, K. Biomaterials for the Feeder- Free culture of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. 2011, 111, 3021-3035.
[9] Rich,A.; Harris, A. K. “Anomalous Preferences of Cultured Macrophages for Hydrophobic and Roughened Substrata,” Journal of Cell Science. 1981,50,1-7.
[10] X. D. Li, L. Jin, G. Balian et al., “Demineralized bone matrix gelatin as scaffold for osteochondral tissue engineering,” Biomaterials, 27, 2426-2433, Apr, 2006.
[11] 汪俐穎.“溫感型高分子應用於具釋放細胞功能之細胞篩選型晶片’’國立清華大學工程與系統科學所.2010,20-23.
[12] Nagase. K. Temperature-Responsive Intelligent Interfaces for Biomolecular Separation and Cell Sheet Engineering. 2009, 6, S293-S309.
[13] Nichol, J. W.; Koshy, S, T. Cell-laden Microengineered Gelatin Methacrylate Hydrogels. 2010, 5536-5544.
[14] Z. S. Li, H. R. Ramay, K. D. Hauch et al., “Chitosan-alginate hybrid scaffolds for bone tissue engineering,” Biomaterials, vol. 26, no.18,pp. 3919-3928, Jun, 2005.
[15] Rao, C.; Prodromakis, T. The Effect of Microgrooved Culture Substrates on Calcium Cycling of Cardiac Myocytes Derived from Human Induced Pluripotent Stem Cell. Biomaterial. 2013, 34, 2399-2411.
[16] Stefan Neubauer, M.D. The Failing Heart-An Engine Out of Fuel. The New England Journal of Medicine. 2007, 1140-1151
[17] Burridge, P. W. Chemically Defined Generation of Human Cardiomyocytes. Nature Methods. 2014, 11, 855-860.
[18] Lian, X.; Zhang, J. Directed Cardiomyocyte Differentiation from Human Pluripotent Stem Cells by Modulating Wnt/β-catenin Signaling under fully Defined Conditions. Nature Protocols. 2013, 8, 162-175.
[19] Fathi, F. Cardiac Differentiation of P19CL6 cells by oxytocin. International Journal of Cardiology. 2009,75-81.
[20] Kocica, M. J. The Helical Ventricular Myocardial Band: Global,
Three-Dimensional, Functional Architecture of the Ventricular
Myocardium. European Journal of Cardio-Thoracic Surgery 29S. 2006,
s21-s40.
[21] Abbas, N. M.; Morteza, G. Evidence for the Existence of a Functional Helical Myocardial Band. Am J Physiol Heart Circ Physiol. 2009,H127-H131.
[22] Sthphenson, R. S.; Agger, P. The Functional Architecture of Skeletal Compared Musculature. Clinical Anatomy. 2016, 29, 316-322.
[23] Nima, B. A Method to Replicate the Microstructure of Heart Tissure In Vitro Using DTMRI-Based Cell Micropatterning. Annals of Biomedical Engineering. 2009, 37, 2510-2521.
[24] Qiu, Y.; Bayomy, A. F. A Role for Matrix Stiffness in the Regulation of Cardiac Side Population Cell Function. Am J Physiol Heart Circ Physiol. 2015, H990-H997.
[25] Downing, T. L.; Soto, J. Biophysical Regulation of Epigenetic State and Cell Reprogrammimg. Nature Materials. 2013.
[26] Takahashi, H. The Use of Anisotropic Cell Sheets to Control Orientation During the Self-Organization of 3D Muscle Tissue. 2013.
[27] Takahashi, H.; Nakayama, M. Anisotropic Cell Sheets for Constructing Three-Dimensional Tissue with Well-Organized Cell Orientation. 2011.
[28] Hsieh, H. Y. Gradient Static-Strain Stimulation in a Microfluidic Chip for 3D Cellular Alignment. 2014, 14, 482-493.
[29] Grosberg, A. Alford, P. W. Ensemble of engineered cardiac tissue for physiological and pharmacological study: Heart on a chip. 2011, 11, 4165.
[30] Lind, J, U. Instrumented Cardiac Microphysiological Devices via Multimaterial Three-dimentional Printing. 2016.
[31] Tanaka, Y.; Morishima, K. Demonstration of a PDMS-based
Bio-microactuator Using Cultured Cardiomyocytes to Drive Polymer
Micropillars. 2006, 6, 230-255.
[32] Herr, H.; Dennis, R. G. Neuro Engineering Rehabil. 2004.
[33] Park, J.; Ryu, J. Anal. Chem. 2005, 77, 657-6580.
[34] Tanaka, Y.; Sato, K. A Micro-sphere Heart Pump Powered by Cultured Cardiomyocytes. Lab on a Chip. 2007, 7 207-212.
[35] Tanaka, Y.; Morshima, K. An Actuated Pump on-chip Powered by
Cultured Cardiomyocytes. Lab on a Chip. 2006, 6, 362-368.
[36] SU-8 3000 Permanent Epoxy Negative Photoresist.
[37] Louch, W. E.; Sheehan, K. A. “Methods in Cardiomyocyte Isolation, Culture, and Gene Transfer,” J Mol Cell Cardiol, 51(3), 288-289, 2011.
[38] Ehler, E.; Moore-Morris, T. “Isolation and Culture of Neonatal Mouse Cardiomyocytes,” 2013.
[39] Kokkinopoulos, I.; Ishida, H. Cardiomyocyte Differentiation From Mouse Embryonic Stem Cells Using a Simple Defined Protocol, 245,157-165, 2016.
[40] Tohyama, S.; attori, F. Distinct Metabolic Flow Enables Large- scale Purification of Mouse and Human Pluripotent Stem Cell-Derived Cardiomyocytes, 12, 127-137, 2013.