設計製作靜水壓生物反應器以應用於幹細胞分化為成骨細胞之實驗研究

Design of a Hydrostatic Pressure Bioreactor for Discussing the Differentiation of Placenta-Derived Multipotent Cells into Osteogenic Cells

郭哲易(Che-I Kuo)

指導教授 ： 鍾志昂

國立中央大學/工學院/機械工程學系/碩士(2011年)

摘要

組織工程利用體外培養的方式，使細胞分化及生長成所需要的組織及器官，進而修護受損的器官與組織，由於是體外培養因此生物反應器扮演極為重要的角色，生物反應器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。
本文探討胎盤導出幹細胞遭受壓力刺激後是否會加速朝向成骨細胞分化。有鑑於前人的論文研究指出胚胎導出幹細胞 (PDMCs) 無法承受太大的壓力刺激，因此本研究主旨為設計一套低壓力且壓力精度高之生物反應器，為了得到穩定且正確的壓力值，採用回授控制系統，使得生物反應器可施加定壓或循環壓力。測試結果顯示，壓力值最大可達到300 kPa，最小值為10 kPa，頻率可達1 Hz，精度約2 kPa左右。
我們使用此生物反應器給予胚胎導出幹細胞 (PDMCs) 定常壓力 (0 kPa、10 kPa、30 kPa、50 kPa)，及添加Osteogeneic(Ost) 一群可誘導幹細胞朝向成骨細胞分化的藥物，再用Alizarin Red S stain (ASR) 檢測等方式觀察壓力對細胞分化表現的影響。而結果顯示，添加Ost後再輔以壓力刺激的確明顯在相同細胞密度下讓細胞加速朝向成骨細胞分化。未來將可使用這套系統實驗何種壓力形式或週期更能達到成骨分化的目標。

關鍵字

幹細胞 ； 生物反應器 ； 壓力 ； 組織工程

並列摘要

Tissue engineering aims to repair damaged tissue and organs by developing artificial tissue substitutes. To this end, cells are seeded onto three-dimensional scaffolds and cultured in vitro. When cells grow to a certain amount, the cellular scaffold is implanted into patients to repair the impaired tissue. To this end, bioreactor plays an important role. Except providing suitable biochemical environments, a variety of bioreactors have been designed to apply mechanical stimulation to facilitate cell growth, differentiation and the formation of extracellular matrix.
This thesis used a bioreactor to test the hypothesis that pressure may promote human placenta-derived multipotent cells (PDMCs) to differentiate toward osteoblasts. A self-designed bioreactor that could impose precisely hydrostatic pressure was applied. The bioreactor was able to produce both constant and sinusoidal pressure forms, with precision to 2kPa, the maximum and minimum value at 300 kPa and 10 kPa respectively, and maximum operable frequency at 1 Hz. Experiments were then conducted to test PDMCs under 0 kPa, 10 kPa, 30kPa and 50 kPa with the biochemical Osteogeneic agents added to guide the cells differentiate toward osteoblasts. The levels of cell differentiation were assessed qualitatively by using Alizarin Red S stain (ARS). Results from the experiments showed that applying constant pressure one hour a day was able to promote the differentiation of PDMCs toward osteoblasts. Bigger the pressure value with more intense ARS appeared until the enhancing effect approached a saturated condition at about 30 kPa. In the future, the bioreactor can be used to test the influence of other pressure forms on the cell differentiation as well as growth, and the results may serve as a reference for the development of bone tissue engineering.

並列關鍵字

Placenta-derived multipotent cells ； Hydro-static pressure ； Bioreactor ； Tissue engineering

參考文獻

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