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

NCUIR > Engineering College > Graduate Institute of Mechanical Engineering > Electronic Thesis & Dissertation > Item 987654321/25525

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Title:	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
Authors:	劉炫志;Hsuan-Chin Liu
Contributors:	機械工程研究所
Keywords:	組織工程;剪應力;生物反應器;幹細胞;圓錐平板裝置;Tissue engineering;Stem Cells;Cone-Plate Bioreactor;Bioreactor;Shear Stress
Date:	2009-11-04
Issue Date:	2010-06-10 16:55:07 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	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.
Appears in Collections:	[Graduate Institute of Mechanical Engineering] Electronic Thesis & Dissertation

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