大腸直腸癌是世界上常見的惡性腫瘤，癌症幹細胞在其發展中有著至關重要的作用。儘管結腸癌患者得到晚期治療，但由於癌症幹細胞對化療具有抗藥性，導致癌症復發和轉移，使他們的治療面臨挑戰。本研究旨在從HT-29結腸癌細胞系以及我們實驗室所建立的原代結腸癌細胞系中分離癌症幹細胞並開發了兩種改質PLGA膜：一種是與帶負電荷的PVA-IA進行混合，另一種是與帶正電荷的PLL混合。我們也製備了未改質PLGA膜作為對照組，以驗證改質膜是否改善了癌症幹細胞的分離。本研究的目的是建立一種使用膜過濾法來富集結腸癌細胞中癌症幹細胞的有效方法。 本研究使用三種方法評估了滲透溶液、回收溶液和遷移的細胞以確定癌症幹細胞的純化：使用菌落形成試驗評估細胞特徵、流式細胞儀檢測CD44和CD133(癌症幹細胞標誌物)的表達、以及酵素結合免疫吸附分析法(ELISA) 試劑盒測量癌胚抗原的產生。為了進一步顯示細胞的分子特徵，進行了免疫螢光染色實驗，並呈現可視化癌症幹細胞標誌物的表達影像。 除了研究膜特性對結腸癌細胞純化的影響外，還對混合細胞進行膜過濾：一種溶液含有HT-29結腸癌細胞和CG1639纖維母細胞，另一種溶液含有原代結腸癌細胞和CG1639纖維母細胞，並使用細胞追蹤染色進行識別。分離實驗的目的是了解純化過程中癌細胞和周圍非癌細胞之間的相互作用，以便更深入地了解使用膜過濾法從原代結腸組織細胞中純化結腸癌細胞的潛在機制。;Colorectal cancer (CRC) is a prevalent malignant tumor worldwide, and cancer stem cells (CSCs) are critical in tumor progression. Despite advanced treatment of patients with CRC, their therapy faces challenges due to CSCs resistance to chemotherapy, leading to recurrence and metastasis of the cancer. This study mainly isolated cancer stem cells from HT-29 colon cancer cells and primary colon cancer cells established in our laboratory. We developed two types of modified PLGA membranes: one membrane blended with a negatively charged poly (vinyl alcohol-co-itaconic acid) (PVA-IA) and another membrane blended with a positively charged poly(L-lysine) (PLL). We also developed unmodified PLGA membranes as a control group to validate whether the modified membranes improved the isolation of CSCs. The goal of this study is to establish a reliable and effective method for enriching CSCs of colon cancer cells using a membrane filtration method. The cells in the permeation solution, recovery solution, and migration were evaluated using three approaches to decide the purity of CSCs: a colony formation assay to assess cellular characteristics, flow cytometry to evaluate CD44 and CD133 (CSCs markers) expression, and an enzyme-linked immunosorbent assay (ELISA) kit to measure carcinoembryonic antigen (CEA) production. To further show the molecular characteristics of the cells, immunofluorescence staining (IF) was performed to visualize CSC marker expression. In addition to studying the impact of membrane characteristics on the purification of colon cancer cells, membrane filtration of mixed cell solutions: one solution holding HT-29 colon cancer cells and CG1639 human fibroblasts, and another solution holding primary colon cancer cells and CG1639 human fibroblasts, which were identified using cell tracker staining. The aim of separation experiments was to understand the interactions between cancer cells and surrounding non-cancer cells (Fibroblasts) during the purification process via membranes in order to gain deeper insights into the underlying mechanisms for purifying colon cancer cells from primary colon tissue cells using a membrane filtration method.
