雷射直寫微流道晶片製程於肝臟細胞分選之研究

Abstract

本研究利用 CO2雷射於聚甲基丙烯酸甲酯 (PMMA) 基板上進行微加工製造微流道，使用 CO2雷射進行微加工有製造成本低以及加工時間快速等優點，而使用聚甲基丙烯酸甲酯作為微流道的材料則是因為該聚合物具有一定的耐化學性、熱穩定性、生物相容性以及可以媲美玻璃的光學性能，並且與其他材料相比還有重量輕、成本低廉、易於製造優勢，最重要的是聚甲基丙烯酸甲酯對CO2雷射有相當高的吸收率，因此，非常適合使用CO2雷射加工。 實驗過程中探討了各項雷射參數，包含雷射功率、掃描速度以及掃描間距對微流道之影響，發現雷射功率越大，寬度及深度也會隨之增加；掃描速度的增加，寬度及深度則是會減少，這是因為雷射掃描速度愈快，代表施加在聚甲基丙烯酸甲酯基板上的能量就會越少；增加掃描間距則會使寬度及深度減少。根據雷射共軛焦顯微鏡觀察到的結果，CO2雷射所雕刻出的微流道的寬度與深度分別484.55±6.6µm以及139.09±5.6µm，橫截面形狀呈現類似梯形。 本研究所使用的微流道裝置，在2、3以及4mL/min之流量下分離效率都大於80%，而5mL/min的流量下分離效率僅74.6%，使用流量參數為4mL/min，成熟肝細胞分離效率可達87.2%，因此，本研究最佳流量參數為4mL/min。 進行細胞培養時，使用微流道裝置分離所培養之成熟肝細胞，於第3天時可以觀察到生長出肝細胞之型態，並針對所培養之成熟肝細胞進行肝機能測試，在尿素分析的實驗中，發現微流道裝置分離所培養的成熟肝細胞與經過正常離心處理的成熟肝細胞之尿素濃度，具有相同的趨勢，在第3天時尿素濃度最高，到第5天時下降，並且兩者之間並無太大區別，證明了使用本研究的微流道裝置之可行性。

In this study, microfluidic channels were fabricated on poly(methyl methacrylate) (PMMA) substrates using a CO2 laser. The advantages of using CO2 laser for micromachining include low fabrication cost and fast processing time, and the use of PMMA as the material for the microfluidic channels is due to the polymer's chemical resistance, thermal stability, biocompatibility, and optical properties comparable to those of glass, as well as light weight, low cost, and ease of fabrication compared to other materials. Compared with other materials, the polymer is also lightweight, low cost, easy to manufacture, and most importantly, the poly(methyl methacrylate) has a very high absorption rate of CO2 lasers, making it very suitable for CO2 laser processing. The experiment investigated the effects of laser power, scanning speed, and scanning distance on the microfluidic channels. Higher laser power resulted in wider and deeper channels, while increasing scanning speed and distance reduced these dimensions. The average channel width and depth were 484.55±6.6 µm and 139.09±5.6 µm, respectively, with a trapezoidal cross-section. The microfluidic device showed a separation efficiency greater than 80% at flow rates of 2, 3, and 4 mL/min, but only 74.6% at 5 mL/min. The optimal flow rate was 4 mL/min, achieving a separation efficiency of 87.2% for mature hepatocytes. In the Urea assay experiment, the urea concentration of mature liver cells cultured in the microfluidic device followed the same trend as those cultured by normal centrifugation, peaking on day 3 and decreasing by day 5, demonstrating the device's feasibility.