| 研究生: |
藍雋凱 Jyun-kai Lan |
|---|---|
| 論文名稱: |
利用生物反應器培養細胞以探討壓力對骨髓幹細胞型態之影響 Investigating the Influence of Pressure on Cell Morphology by Hydrostatic Pressure Bioreactor. |
| 指導教授: |
曾清秀
Ching-Shiow Teseng |
| 口試委員: | |
| 學位類別: |
碩士 Master |
| 系所名稱: |
生醫理工學院 - 生物醫學工程研究所 Graduate Institute of Biomedical Engineering |
| 畢業學年度: | 98 |
| 語文別: | 中文 |
| 論文頁數: | 85 |
| 中文關鍵詞: | 生物反應器 、靜水壓 、細胞遷移 |
| 外文關鍵詞: | Bioreactor, Hydrostatic pressure, Cell migration |
| 相關次數: | 點閱:16 下載:0 |
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許多研究利用不同的訊號因子促進細胞生長與分化,其中機械刺激對於細胞型態與功能為一重要議題。多數的研究利用生物反應器提供循環壓力來促進幹細胞的分化,但是壓力對於細胞的即時形態影響卻很少提及。因此本研究主要為設計一套可於顯微鏡下進行細胞培養的靜水壓力式生物反應器,藉由顯微鏡拍攝之連續影像探討定常壓力對於細胞型態、生長與遷移之影響。
本研究設計之生物反應器可與顯微鏡的活細胞培養設備結合,並連結壓力驅動器,以提供細胞培養時所需之生長環境與靜水壓刺激。生物反應器結構具良好的封閉性,內部為渠道式設計,以確保細胞培養液能連續流動。腔內孔洞位置與尺寸也經過精確計算,使顯微鏡能拍攝到清晰的細胞影像。
實驗使用大鼠骨髓幹細胞並種植於玻璃玻片上,放入淨水壓力式生物反應器中培養。實驗分為控制組(0MPa)與加定常壓組(0.4MPa、0.8MPa、1MPa)。利用影像處理方式擷取細胞影像邊界,進而得到細胞輪廓面積變化,並以細胞輪廓的形心位置變化量計算細胞遷移量及速率。分析實驗結果發現越大的壓力造成細胞生長週期的延遲,且細胞面積於加壓期間隨壓力越大面積越小,而越大的壓力值使細胞回復正常細胞型態時間越長。細胞遷移在加壓狀態下之細胞遷移速率有些微高於未加壓狀態之細胞。
Many studies applied different signals to improve cell proliferation and differentiation. Mechanical forces have been shown to be important stimuli for the cellular phenotype and function. Many studies applied cyclic pressure by bioreactors to improve cell differentiation, but few studies had been discussed with the influence of pressure on real-time cell morphology. Therefore, we designed a hydrostatic pressure bioreactor system, and analyzed the continuous images took by an Inversion Microscope to estimate that the effect of hydrostatic pressure on cell morphology, proliferation and migration.
The bioreactor integrated with peripheral equipment of the live cell culture system of the microscope and a pressure actuator to provide suitable environment and hydrostatic pressure stimulation required for cell proliferation. The bioreactor has a air-tight structure and a media channel for media circulation. The location and dimension of holes in the chamber were carefully designed to enable the microscope to take clear cell images.
Rat bone marrow stem cells were seeded onto the cover glasses and cultured the bioreactor with hydrostatic pressure. The research can be divided into control group (0MPa) and static pressure group (0.4MPa、0.8MPa、1MPa). Using image processing method to find cell boundary, reprocessed the boundary images to find out the cell area variation. Furthermore, calculate the cell migration distance and speed from cell center of form. Our findings showed that the cell cycle will increase when the pressure was increase. The cell area proportion to pressure when exposed to pressure phase. Nevertheless, cell morphology will change while the pressure stimulated, and it took a longer time to recover from higher pressure. The cell migration speed is higher under pressurized state than atmospheric state.
[1] R. Langer and J. Vacanti, “Tissue Engineering”, Science, Vol. 260, pp. 920-926,1993.
[2] E. Lavik and R. Langer, “Tissue engineering: current state and perspectives”, Biotechnol, Vol. 65, pp. 1-8, 2004.
[3] R.P. Lanza, R. Langer, J. Vacanti and W.L. Chick, “Principles of Tissue Engineering”, Annals of Biomedical Engineering , Vol. 27, pp. 580, 1999.
[4] A.K. Saxena, “Tissue Engineering: Resent Concepts an Strategies”, Journal of Indian Association of Pediatric Surgeons, Vol. 10, pp. 14-19, 2005.
[5] S.J. Shieh, S. Terada and J.P. Vacanti, “Tissue Engineering Auricular Reconstruction: in vitro and in vivo studies”, Biomaterials, Vol. 25, pp. 1545-1557, 2004.
[6] E. Lavik and R. Langer, “Tissue engineering: current state and perspectives”, Biotechnol, Vol. 65, pp. 1-8, 2004.
[7] V. Karageorgiou and D. Kaplan, “Porosity of 3D biomaterial scaffolds and osteogenesis”, Biomaterials, Vol. 26, pp. 5474-5491, 2005.
[8] Y. Ikada and H. Tsuji, “Biodegradable polyesters for medical and ecological applications”, Macromolecular Rapid Communications, Vol. 21, pp. 117-132, 2000.
[9] V. Karageorgiou and D. Kaplan, “Porosity of 3D biomaterial scaffolds and osteogenesis”, Biomaterials, Vol. 26, pp. 5474-5491, 2005.
[10] 黃仁波, “以冷凍式快速原型法製作組織工程支架”, 國立中央大學機械工程研究所碩士論文, 民94.
[11] S. R. Frenkel, R. M. Clancy, J. L. Ricci, P. E. DiCesare, J. J. Rediske and S. B. Abransom, “Effects of Nitric Oxide on Chondrocyte Migration, Adhesion and Cytoskeletal Assembly”, Arthritis Rheum, Vol. 39, pp. 1905-1912, 1996.
[12] S. Maniwa, M. Ochi, T. Motomura, T. Nishikori, J. Chen and H. Naora, “Effects of Hyaluronic Acid and Basic Fibroblast Growth Factor on Motility of Chondrocytes and Synovial Cells in Culture”, Acta Orthop, Vol. 72, pp. 299-303, Scand 2001.
[13] C. Hidaka, C. Cheng, D. Alexandre, M. Bhargava and P.A. Torzilli, “Maturational Differences in Superficial and Deep Zone Articular Chondrocytes”, Cell Tissue, Vol. 323, pp. 127-135, Res 2006.
[14] C. Chang, D.A. Lauffenburger and T.I. Morales, “Motile Chondrocytes from Newborn Calf: Migration Properties and Synthesis of Collagen II”, Osteoarthritis Cartilage, Vol. 11, pp. 603-612, 2003.
[15] I. Martin, D. Wendt and M. Heberer, “The role of bioreactors in tissue engineering”, Trends in Biotechnology, Vol. 22, pp. 80-86, 2004.
[16] U. Hansen, M. Schunke, C. Domm, N. Ioannidis, J. Hassenpflug and T. Gehrke, “Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering”, J Biomech, Vol. 34, pp. 941–949, 2001.
[17] L.R. Smith, S.F. Rusk, B.E. Ellison, P. Wessells, K. Tsuchiya, D.R. Carter, W.E. Caler, L.J. Sandell and D.J. Schurman, “In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure”, Journal of Orthopaedic Research, Vol. 14, pp. 53-60, 1996.
[18] G. Rh. Owen, J. Jackson, B. Chehroudi, H. Burt and D. M. Brunette, “A PLGA Membrane Controlling Cell Behaviour for Promoting Tissue Regeneration”, Biomaterials, Vol. 26 , pp. 7447-7456, 2005.
[19] J.F. Cornhill and R.M. Nerem, “The role of fluid mechanics in atherogenesis”, ASME J. Biomech. Eng, Vol. 102, pp. 181-189, 1980.
[20] 王偉儒, “以細胞染色影像分析支架上的細胞分佈”, 國立中央大學機械工程研究所碩士論文, 民94.
[21] 張恆鐘, “組織工程用支架表面細胞遷移行為研究”, 國立中央大學機械工程研究所論文,民96.
[22] 劉于綸, “細胞顯微影像分割與運動分析”, 國立中央大學機械工程研究所碩士論文, 民93.
[23] S. Mizuno, T. Ushida, T. Tateishi and T. Glowacki, “Effects of physical stimulation on chondrogenesis in vitro”, Materials Science and Engineering, Vol. 6, pp. 301-306, 1998.
[24] T.M. Maul, D.W. Hamilton, A. Nieponice, L. Soletti and D.A. Vorp, “A new experimental system for the extended application of cyclic hydrostatic pressure to cell culture”, Jornal of Biomechanical Engineering, Vol. 129, pp. 110-116, 2007.
[25] S. Mizuno, T. Tateishi, T. Ushida and J. Glowacki, “Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture”, Journal of Cellular Physiology, Vol. 193, pp. 319-327, 2002.
[26] J. Nagatomi, B.P. Arulanadam, D.W. Metzger, A. Meunier and R. Bizios, “Cyclic pressure affects osteoblast functions pertinent to osteogensis”, Annals of Biomedical Engineering, Vol. 31, pp. 917-923, 2003.
[27] S.E. Carver and C.A. Heath, “Semi-continuous perfusion system for delivering intermittent physiological pressure to regenerating cartilage”, Tissue Engineering, Vol. 5, pp. 1-11, 1999.
[28] S.Mizuno, T. Tateishi, T. Ushida and J. Glowacki, “Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture”, Journal of Cellular Physiology, Vol. 193, pp. 319-327, 2002.
[29] Z.J. Luo and B.B. Seedhom, “Light and low-frequency pulsatile hydrostatic pressure enhances extracellular matrix formation by bone marrow mesenchymal cells: an in-vitro study with special reference to cartilage repair”, Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, Vol. 221, pp. 499-507, 2007.
[30] M. Fujiwara, S. Koizumi and M. Takagi, “Effect of Static Pressure on Intracellular pH of Adhesive Chinese Hamster Ovary Cells”, Journal of Bioscience and Bioengineering, Vol. 104, pp. 510-512, 2007.
[31] J. Pugin, I. Dunn, P. Jolliet, D. Tassau, J.L. Magnenat, L.P. Nicod and J.C. Chevrolet. “Activation of Human Macrophages by Mechanical Ventilation in Vitro”, Am J Physiol Lung Cell Mol Physiol, Vol. 275, pp. 1040–1050, 1998.
[32] N. Kataoka, S. Ujita and M. Sato, “Effect of Flow Direction on the Morphological Responses of Cultured Bovine Aortic Endothelial Cells”, Medical and Biological Engineering and Computing, Vol. 36, pp. 122–128, 1998.
[33] H. Takamatsu and B. Rubinsky, “Viability of Deformed Cells”, Cryobiology, Vol. 39, pp. 243-251, 1999.
[34] S.R. Frenkel, R.M. Clancy, J.L. Ricci, P.E. DiCesare, J.J. Rediske and S.B. Abransom, “Effects of Nitric Oxide on Chondrocyte Migration, Adhesion and Cytoskeletal Assembly”, Arthritis Rheum, Vol. 39, pp. 1905-1912, 1996.
[35] C. Hidaka, C. Cheng, D. Alexandre, M. Bhargava and P.A. Torzilli, “Maturational Differences in Superficial and Deep Zone Articular Chondrocytes”, Cell Tissue, Vol. 323, pp. 127-135, 2006.
[36] S. Maniwa, M. Ochi, T. Motomura, T. Nishikori, J. Chen and H. Naora, “Effects of Hyaluronic Acid and Basic Fibroblast Growth Factor on Motility of Chondrocytes and Synovial Cells in Culture”, Acta Orthop, Vol. 72, pp. 299-303, 2001.
[37] C. Chang, D.A. Lauffenburger and T.I. Morales, “Motile Chondrocytes from Newborn Calf: Migration Properties and Synthesis of Collagen II”, Osteoarthritis Cartilage, Vol. 11, pp. 603-612, 2003.
[38] E. D. Miller, G. W. Fisher, L. E. Weiss, L. M. Walker and P.G. Campbell, “Dose Dependent Cell Growth in Response to Concentration Modulated Patterns of FGF-2 Printed on Fibrin”, Biomaterials, Vol. 27, pp. 2213-21, 2005.
[39] IWAKI EHN-R Instruction Manual, IWAKI PUMPS, 2006.
[40] Axiovert 200 / Axiovert 200M Operating Manual, Carl ZEISS Light Microscopy, 2001.
[41] AxioVision Manual Release 3.1, Carl ZEISS, 2002.
[42] AxioVision 3.1 Reference, Carl ZEISS, 2000.
[43] R. C. Gonzalez, R. E. Woods and S. L. Eddins, “Digital Image Processing Using MATLAB”, 359-405頁, 謬紹綱譯, 初版, “數位影像處理運用MATLAB”, 東華書局, 台灣, 2005.
[44] B. Albert, D. Bray, K. Hopkin, A. Johnson, J.Lewis, M. Raff, K. Roberts and P. Walter, “Essential Cell Biology”, Garland Science, pp. 611-658, 2003.
[45] B. Albert, A. Johnson, J.Lewis, M. Raff, K. Roberts and P. Walter, “The Cell ”, Garland Science, pp. 983-1026, 2001.
[46] K. Wu, D. Gauthier and M. D. Leivine, “Live Cell Image Segmentation”, IEEE Trans. On Biomedical Engineering, Vol. 42, No. 1, pp. 1-12, January 1995. Proceeding IEEE Southwest Symposium on Image Analysis and Interpretation, pp. 117-122, 1996.