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.
