跳到主要內容

簡易檢索 / 詳目顯示

回結果列表
研究生： 劉炫志
Hsuan-Chin Liu
論文名稱： 設計與製作圓錐平板型生物反應器以探討剪應力對幹細胞生長與型態之影響
Study of the Effects of Shear Stresses on the Growth and Morphology of Stem Cells with the Cone-Plate Bioreactor
指導教授： 鍾志昂
Chih-Ang Chung
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 機械工程學系
Department of Mechanical Engineering
畢業學年度： 98
語文別： 中文
論文頁數： 90
中文關鍵詞： 組織工程 、剪應力 、生物反應器 、幹細胞 、圓錐平板裝置
外文關鍵詞： Tissue engineering, Stem Cells, Cone-Plate Bioreactor, Bioreactor, Shear Stress
相關次數： 點閱：18  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 近代發展的新領域-組織工程，希望利用體外培養的方式，修護受損的器官與組織，其中生物反應器扮演極為重要的角色，除了提供細胞生存環境外，還可以提供不同的機械刺激調控細胞的基因表現或是分化的路徑。
    本研究主旨為設計圓錐平板型生物反應器，利用圓錐轉動帶動圓錐與平板培養室之間的培養液旋轉，而旋轉的流體會對平板培養室底部產生剪應力，施予平板培養室上的細胞可控制的定量剪應力。並利用荷重元更準確的校正圓錐與平板培養室的間距，減小產生的剪應力誤差。經由測試結果顯示，剪應力值最大可達5.3 dyn/cm2，且經由實驗證明本圓錐平板型生物反應器適合培養細胞。
    利用此生物反應器施予大鼠骨髓幹細胞 (RM1) 0.5、1、2、4dyn/cm2的剪應力，發現細胞的生長速率隨著剪應力的上升而逐漸減緩，當剪應力刺激為2 dyn/cm2時，細胞在受剪應力後的24小時之間有停止增殖的現象。也利用此生物反應器施予人類間葉幹細胞 (M7) 1、2、3、4、5 dyn/cm2的剪應力，發現細胞的生長速率隨著剪應力的上升而逐漸減緩，並在細胞受剪應力後馬上觀察其貼附型態，發現越大的剪應力越容易使細胞貼附不良。
    本研究成功設計出可以穩定培養細胞的生物反應器，可以在一次實驗中同時產生多組不同的定常剪應力刺激，或是一次培養大量的細胞數目，適合細胞進行長時間培養。未來，對於生物反應器裝置方面，可以經由荷重元的應用設計出即時監控剪應力大小的生物反應器，對於細胞培養方面，可以進行生化相關的檢測，並施予細胞不同強度或週期性的剪應力刺激，觀察刺激對細胞的影響。

    Tissue engineering aims to develop biosubstitutes to maintain, restore or replace the injured or damaged tissues and organs. In this emerging biological technology, bioreactors play an important role as to provide not only appropriate environments for cell culture but also can be designed to impose various biomechanical and chemical cues to activate cells’ corresponding gene expressions and pathways. The goal of this work is to develop a shear stress bioreactor. Based on the concept of cone-plate apparatus, we develop the bioreactor that can produce quantified shear stresses for cell cultured on the culture plate. The bioreactor is equipped with load cells that help control the gap between the cone tip and the culture plate such that the produced stress can be precisely controlled. The bioreactor provides shears ranging from zero to 5.3 dyn/cm2, and was demonstrated to be suitable for cell culture by comparison with in the traditional Petri dish.
    We then applied this bioreactor to test the Sprague-Dawley rat bone marrow stem cells (RM1). The growth rate and morphology of the cells are assessed under the action of steady shear stress, which was applied for 4hr with the value of 0.5, 1, 2 and 4 dyn/cm2, respectively. Results show that shear stresses larger than 2 dyn/cm2 apparently retarded the cell proliferation within the 24 hr after imposing the shear stresses. We also tested the biological behavior of human mesenchymal stem cells (M7) using this bioreactor to apply shear stresses of 1, 2, 3, 4 and 5 dyn/cm2. The growth of M7 was demonstrated to gradually reduce with increasing the shear value, and the cell morphology revealed that the larger the shear, the more detached of the cells from the culture plate.
    This work demonstrates a successful design of shear stress bioreactor based on cone-plate apparatus, which can produce three different shear values at the same time, or can carry out the cultivation with a large amount of cells. In the future, the usage of the load cells will be extended to monitor the shear value in real time. Meanwhile, cells stimulated by the shear stress can be continued with biochemical or genomic test to investigate in more detail the influences of shear cues on cell behaviors.

    中文摘要.............................................. I Abstract.............................................. II 誌謝.................................................. IV 目錄.................................................. V 圖目錄................................................ VIII 表目錄................................................ X 第一章 緒論 1.1前言............................................... 1 1.2研究動機........................................... 2 1.3文獻回顧........................................... 2 1.3.1 剪應力生物反應器................................ 2 1.3.2機械刺激對於幹細胞的影響......................... 4 1.4論文架構........................................... 5 第二章 生物反應器設計與製作 2.1 設計前言.......................................... 6 2.2 剪應力產生原理以及剪應力計算...................... 6 2.2.1 剪應力產生原理.................................. 6 2.2.2 定常轉速剪應力計算.............................. 8 2.3 培養室設計........................................ 9 2.3.1培養液性質量測................................... 9 2.3.2圓錐與平板培養室尺寸設計......................... 9 2.3.3系統剪應力表現................................... 11 2.4生物反應器機構設計................................. 13 2.4.1培養環境系統..................................... 13 2.4.2馬達驅動裝置與馬達密封組件....................... 14 2.4.3馬達承載箱體空間................................. 19 2.4.4圓錐與平板培養室間距定位調整..................... 20 2.4生物反應器機構設計................................. 23 2.5.1圓錐錐度對剪應力產生的誤差....................... 23 2.5.2圓錐與平板培養室間距對剪應力產生的誤差........... 24 2.5.3生物反應器對剪應力的操作範圍..................... 24 第三章 實驗方法 3.1 細胞來源.......................................... 25 3.1.1細胞繼代培養 (RM1)............................... 25 3.1.2細胞繼代培養 (M7)................................ 25 3.2 玻片滅菌消毒程序.................................. 26 3.3 細胞種植.......................................... 27 3.3.1細胞種植數之計算................................. 27 3.3.2細胞種植於玻片上的步驟 ........................... 27 3.4實驗前置準備....................................... 28 3.5生物反應器細胞相容性測試........................... 29 3.6實驗步驟........................................... 30 3.6.1細胞種植 (RM1)................................... 30 3.6.2細胞種植 (M7) .................................... 30 3.6.3系統初始種植數量計數與型態觀察................... 31 3.6.4實驗組移入平板培養室............................. 32 3.7系統操作步驟....................................... 32 3.8實驗條件........................................... 33 3.9馬達轉速設定....................................... 33 3.10實驗結果分析方法.................................. 34 3.10.1細胞型態觀察 .................................... 34 3.10.2 細胞計數....................................... 35 第四章 實驗結果 4.1實驗前言........................................... 36 4.2剪應力強度對大鼠骨髓幹細胞生長狀態的影響........... 36 4.2.1剪應力刺激強度：0 dyn/cm2........................ 36 4.2.2剪應力刺激強度：0.5 dyn/cm2...................... 39 4.2.3剪應力刺激強度：1 dyn/cm2........................ 42 4.2.4剪應力刺激強度：2 dyn/cm2........................ 45 4.2.5剪應力刺激強度：4 dyn/cm2........................ 48 4.2.6剪應力對大鼠骨髓幹細胞影響之結論................. 51 4.3剪應力強度對人類間葉幹細胞生長狀態的影響........... 53 4.3.1剪應力刺激強度：0 dyn/cm2........................ 53 4.3.2剪應力刺激強度：0.5 dyn/cm2...................... 56 4.3.3剪應力刺激強度：0.75 dyn/cm2..................... 59 4.3.4剪應力刺激強度：1 dyn/cm2........................ 62 4.3.5剪應力刺激強度：2 dyn/cm2........................ 65 4.3.6剪應力刺激強度：3 dyn/cm2........................ 68 4.3.7剪應力刺激強度：4 dyn/cm2........................ 71 4.3.8剪應力刺激強度：5 dyn/cm2........................ 74 4.3.9剪應力對人類間葉幹細胞影響之結論................. 77 第五章 結論與未來展望 參考文獻.............................................. 81 附錄.................................................. 89

    Angele, P., Yoo, J.U., Smith, C., Mansour, J., Jepsen, K.J., Nerlich, M., and Johnstone, B., 2003. Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. Journal of Orthopaedic Research 21, 451-457.
    Bao, X., Clark, C.B., and Frangos, J.A., 2000. Temporal gradient in shear-induced signaling pathway:involvement of MAP kinase, c-fos, and connexin 43. American Journal of Physiology- Heart and Circulatory Physiology 278, H1598-H1605.
    Blackman, B.R., Barbee, K.A., and Thibault, L.E., 2000. In vitro cell shearing device to investigate the dynamic response of cells in a controlled hydrodynamic environment. Biomedical Engineering Society 28, 363-372.
    Blackman, B.R, García-Cardeña, G., and Gimbrone, M.A., 2002. A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms. Journal of Biomechanical Engineering 124, 397-407.
    Botchwey, E.A., Dupree, M.A., Pollack, S.R., Levine, E.M., and Laurencin, C.T., 2003. Tissue engineered bone:measure of nutrient transport in three-dimensional matrices. Journal of Biomedical Materials Research Part A, 357-367.
    Breen, L.T., McHugh, P.E., McCormack, B.A., Muir, G., Quinlan, N.J., Heraty, K.B., and Murphy, B.P., 2006. Development of a novel bioreactor to apply shear stress and tensile strain simultaneously to cell monolayers. Review of Scientific Instruments 77, 104301-104309.
    Brown, T.D., 2000. Techniques for mechanical stimulation of cells in vitro: A review. Journal of Biomechanics 33, 3-14.
    Bussolari, S.R., Dewey, C.F., and Gimbrone, M.A., 1981. Apparatus for subjecting living cells to fluid shear stress. Review of Scientific Instruments 53, 1851-1854.
    Butcher, J.T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R.M., 2006. Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences：Influence of Shear Stress. Arterioscler Thromb Vasc Biol, 69-77.
    Butler, D.L., 2000. Functional tissue engineering: the role of biomechanics. Journal of Biomechanical Engineering 122, 570-575.
    Chaturani, P. and Narasimman, S., 1990. Flow of power-law fluids in cone-plate viscometer. Acta Mechanica 82, 197-211.
    Chen, K.D., Li, Y.D., Kim, M., Li, S., Yuan, S., Chien, S., and Shyy, J.Y.J., 1999. Mechanotransduction in response to shear stress. The Journal of Biological Chemistry 274, 18393-18400.
    Cheng, D.C.-H., 1968. The effect of secondary flow on the viscosity measurement using a cone-and-plate viscometer. Chemical Engineering Science 23, 895-899.
    Chung, C.A., Tzou, M.R. and Ho, R.W., 2005. Oscillatory flow in a cone-and-plate bioreactor. Journal of Biomechanical Engineering 127, 601-610.
    Chung, C.A., Weng, C.S. and Tu, M.Z., 2006. The periodical Shear environment of a cone-and-plate bioreactor. Journal of Fluids Engineering 128, 388-397.
    Cox, D.B., 1962. Radial flow in the cone-plate viscometer. Nature 193, 670.
    Das, P., Schurman, D.J. and Simth, R.L., 1997. Nitric oxide and G proteins mediate the response of bovine articular chondrocytes to fluid-induced shear. Journal of Orthopaedic Research 15, 87-93.
    Datta, N., Pham Q.P., Sharma, U., Sikavitsas, V.L., Jansen, J.A., and Mikos, A.G., 2006. In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation. Proceeding of the National Academy of Sciences of the United States of America 103, 2488-2493.
    Dewey, C.F., Bussolari, S.R., Gimbrone, M.A. and Davies, P.F., 1981. The dynamic response of vascular endothelial cells to fluid shear stress. Journal of Biomechanical Engineering 103, 177-185.
    Dong, J.D., Gu, Y.Q., Li, C.M., Wang, C.R., Feng, Z.G., Qiq, R.X., Chen, B., Li, J.X., Zhang, S.W., Wang, Z.G., Zhang, J., 2009. Response of mesenchymal stem cells to shear stress in tissue-engineered vascular grafts. Acta Pharmacologica Sinica, 530-536.
    Einav, S., Dewey, C.F., and Hartenbaum, H., 1994. Cone-and-plate apparatus: A compact system for studying well-characterized turbulent flow fields. Experiments in Fluids 16, 196-202.
    Engelmayr Jr, G.C., Sales, V.L., Mayer Jr, J.E., and Sacks, M.S., 2006. Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues. Biomaterials 27, 6083-6095.
    Feugier, P., Black, R.A., Hunt, J.A., How, T.V., 2004. Attachment, morphology and adherence of human endothelial cells to vascular prosthesis materials under the action of shear stress. Biomaterials 26, 1457-1466.
    Garcia, A.M., Lark, M.W., Trippel, S.B. and Grodzinsky, A.J., 1998. Transport of tissue inhibitor of metalloproteinases-1 through cartilage: Contributions of fluid flow and electrical migration. Journal of Orthopaedic Research 16, 734-742.
    Goldestein, A.S, Juarez, T.M, Helmke, C.D, Gustin, M.C., and Mikos, A.G., 2001. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials 22, 1279-1288.
    Griffith, L.G. and Naughton, G., 2002. Tissue engineering-current challenges and expanding opportunities. Science 295, 1009-1016.
    Haseltine, W.A., 2001. The emergence of regenerative medicine: A new field and a new society. Journal of Regenerative Medicine 2, 17-23.
    Hsu, S.H., Chen, C.A., 2003. In vitro evaluation of cell loss: retention and repopulation on substrates upon shear flow by a rheometer. Journal of Medical and Biological Engineering 23, 171-176.
    Huang, C.Y., Hagar, K.L., Frost, L.E. and Sun, Y., 2004. Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem Cells 22, 313-23.
    Isenberg, B.C, Williams, V., and Tranquillo, R.T., 2006. Endothelialization and flow conditioning of fibrin-based media equivalents. Annuals of Biomedical Engineering 34, 971-985.
    Jiang, G.L., White, C.R., Stevens, H.Y. and Frangos, J.A., 2002. Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein. AJP-Endocrinol Metabolism 283, E383-E389.
    Knippenberg, M., Helder, M.N., Doulabi ,B.Z., Semeins, C.M., Wuisman, J.M, and Klrin-Nulend, J., 2005. Adipose tissue-derived mesenchymal stem cell acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation. Tissue Engineering 11, 1780-1788.
    Knothe Tate, M.L., Falls, T.D., Mcbride, S.H., Radhika, A., and Knothe, U.R., 2008. Mechanical modulation of osteochondroprogenitor cell fate. The International Journal of Biochemistry and Cell Biology 40, 2720-2738.
    Kobayashi, N., Yasu, T., Ueba, H., Sata, M., Hashimoto, S., Kuroki, M., Saito, M. and Kawakami, M., 2004. Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells. Experimental Hematology 32, 1238-1245.
    Kreke, M.R., and Goldstein, A.S., 2004. Hydrodynamics shear stimulates osteocalcin expression but not proliferation of bone marrow stromal cells. Tissue Engineering 10, 780-787.
    Langer, R. and Vacanti, J.P., 1993. Tissue Engineering. Science 26, 920-926.
    Lanza, Langer, and Vacanti, 2007. Principles of Tissue Engineering 3rd Edition. Elsevier, N.Y., Chapter 1.
    Lee, A.A., Graham, D.A., Cruz, S.D., Ratcliffe, A., and Karlon, W.J., 2002. Fluid shear stress-induced alignment of cultured vascular smooth muscle cells. Journal of Biomechanical Engineering 124, 37-43.
    Lee, W.C, Maul, T.M, and Vorp, D.A, 2007. Effects of uniaxial cyclic strain on adipose-derived stem cell morphology, proliferation, and differentiation. Biomechanics and Modeling in Mechanobiology 6, 265-273.
    Martin, I., Wendt, D., and Heberer, M., 2004. The role of bioreactor in tissue engineering. TRENDS in Biotechnology 22, 80-86.
    McBeath, R., Pirone, D.M, Nelson, C.M, Bhadriraju, K., and Chen, C.S., 2004. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Developmental Cell 6, 483-495.
    McBride, S.H, Falls, T., and Knothe Tate, M.L., 2008. Modulation of stem cell shape and fate B:Mechanical modulation of stem cell shape and gene expression. Tissue Engineering 14, 1573-1580.
    McKinley, G.H., Oztekin, A., Byars, J.A. and Brown, R.A., 1995. Self-similar instabilities in elastic flows between a cone and a plate. Journal of Fluid Mechanics Digital Archive 285, 123-164.
    Meinel, L., Karageorgiou, V., Fajardo, R., Snyder, B., Shinde-Patil, V., Zichner, L., Kaplan, D., Langer, R. and Vunjak-Novakovic, G., 2004. Bone tissue engineering using human mesenchymal stem cell: Effects of scaffold material and medium flow. Annals of Biomedical Engineering 32, 112-122.
    Miyanishi, K., Trindade, M.C.D., Lindsey, D.P., Beaupre, G.S., Carter, D.R., Goodman, S.B., Schurman, D.J., and Smith, R.L., 2006. Effects of hydrostatic pressure and transforming growth factor-ß3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Engineering 12, 1419-1428.
    Mohtai, M., Gupta, M.K., Doulon, B., Ellison, B., Cooke, J., Gelbbons, G., Schurman, D.J. and Smith, R.L., 1996. Expression of interleukin-6 in osteoarthritic chondrocytes and effects of fluid-induced shear on this expression in normal human chondrocytes in vitro. Journal of Orthopaedic Research 14, 67-73.
    Mooney, M., and Ewart, R.H., 1934. The conicylindrical viscometer. Physis 5, 350-354.
    Nagatomi, J., Arulanandam, B.P., Meunier A., and Bizios, R., 2002. Effect of cyclic pressure on bone marrow cell cultures. Journal of Biomechanical Engineering 124, 319-327.
    O’Cearbhaill, E.D., Punchard, M.A., Murphy, M. Barry, F.P., McHugh, P.E., and Barron, V., 2008. Response of mesenchymal stem cell to the biomechanical environment of the endothelium on a flexible tubular silicone substrate. Biomaterials 29, 1610-1619.
    Park, J.S., Chu, J.S., Cheng, C., Chen, F., Chen, D. and Li, S., 2004. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnology and Bioengineering 88, 359-368.
    Pelech, I. and Shapiro, A.H., 1967. Flexible disk rotating on a gas film next to a wall. Journal of Applied Mechanics-Transactions of the ASME 31, 577–584.
    Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R., 1999. Multilinearge potential of adult human mesenchymal stem cells. Science 284, 143-147.
    Pörtner, R., Nagel-Heyer, S., Goepfert, C., Adamietz, P., and Meenen, N.M., 2005. Bioreactor design for tissue engineering. Journal of Bioscience and Bioengineering 100, 235-245.
    Rabbany, S.Y., Heissig, B., Hattori, K. and Rafii, S., 2003. Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization. TRENDS in Biotechnology 9, 109-117.
    Rüster, B., Göttig, S., Ludwig, R.J., Bistrian, R., Müller, S., Seifried, E., Gille J. and Henschler, R., 2009. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. BLOOD 108, 3938-3944.
    Saxena, A.K., 2005. Tissue engineering: Resent concepts and strategies. Journal of Indian Association of Pediatric Surgeons 10, 14-19.
    Scaglione, S., Wendt, D., Miggino, S., Papadimitropoulos, A., Fato, M., Quarto, R., Martin, I., 2007. Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system. Journal of Biomedical Materials Research A, 411-419
    Sdougos, H.P., Bussolari, S.R., and Dewey, C.F., 1984. Secondary flow and turbulence in a cone-and-plate device. Journal of Fluid Mechanics 138, 379-404.
    Segers, V.F.M., Riet, I.V., Andries, L.J., Lemmens, K., Demolder, M.J., De Becker, A.J.M.L., Kockx M.M. and De Keulenaer, G.W. , 2006. Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. AJP-Heart Circ Physiol 290, H1371-H1377.
    Shieh, S. J., Terada, S. and Vacanti, J.P., 2004. Tissue engineering auricular reconstruction: In vitro and in vivo studies. Biomaterials 25, 1545-57.
    Simmons, C.A., Matlis, S., Thornton, A.J., Chen, S., Wang, C.Y. and Mooney, D.J., 2003. Cyclic strain enhance matrix minerlization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK 1/2) signaling pathway. Journal of Biomechanics 36, 1087-1096.
    Smith, R.L., Carter, D.R., and Schurman, D.J., 2004. Pressure and shear differentially alter human articular chondrocyte metabolism. Clinical Orthopaedics and related Reseach 427S, S89-S95.
    Wang, H., Riha, G.M., Yan, S., Li, M., Chai, H., Yang, H., Yao, Q. and Chen, C., 2005. Shear stress induces endothelial differentiation from a murine embroyonic mesenchymal progenitor cell line. Arterioscler Thrombosis and Vascular Biology 25, 1817-1823.
    Walters, K., Waters, N.D., 1966. Polymer systems, deformation and flow. In Proc. Brit. Soc. Rheol. (ed. R. E. Wetton and R. W. Whorlow). Macmmillan.
    Watt, F.M. and Hogan, B.L., 2000. Out of Eden: Stem cells and their niches. Science 287, 1427-1430.
    王文甫，2008，圓錐平板型生物反應器之設計與製作，中央大學機械工程碩士論文。
    李宜書，2001，「淺談組織工程」，物理雙月刊，廿四卷三期。
    呂明憲，2005，週期式圓錐平板裝置之設計與量測，中央大學機械工程學系碩士論文。
    翁昶生，2004，圓錐平板型生物反應器脈動式二次流場研究，中央大學機械工程碩士論文。

    QR CODE
    :::