使用膜過濾法分離結腸癌幹細胞株

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朱哲維(Zhe-Wei Zhu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

使用膜過濾法分離結腸癌幹細胞株
(Isolation of colon cancer cells using membrane filtration method)

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★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究	★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上

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至系統瀏覽論文 (2025-8-20以後開放)

摘要(中)

結腸癌是全球最致命和常見的腸道癌症之一。癌症起始細胞(CIC)或癌症幹細胞 (CSC)對腫瘤的產生、生長和轉移具有重大意義。從原發性結腸腫瘤組織中純化和分離具有致腫瘤潛能的結腸 CSC(CIC)，將有助於未來開發新的診斷和個性化治療方法。
在本研究中，我們利用耐倫薄膜和聚乳酸-聚乙二醇酸薄膜(PLGA/SK)過濾器的膜過濾法純化了結腸癌細胞株 CoLo205 和 SW480 的 CSC (CIC)並作為原發性結腸癌細胞的模型。純化效率將通過(i)CD44 和 CD133 標記的表達，(ii)集落形成單位試驗，和(iii)癌胚抗原(CEA)的濃度進行檢測。實驗結果顯示，在細胞通過薄膜後，與滲透液和回收液中的細胞相比，CSC(CIC)在遷移的細胞中具有更高的純度。然而，與滲透液和回收液中的細胞相比，遷移的細胞產生了類似的集落形成單位效率。
接著我們將結腸癌細胞株與人類成纖維細胞 CG1639 共培養，且分別用細胞追蹤劑: 紅色與藍色染劑來進行標記。以便在使用膜過濾法將結腸癌細胞與成纖維細胞分離時，可以利用流式細胞儀及螢光顯微鏡進行鑑定。我們的實驗結果表明，回收溶液的 CoLo205 細胞的純度可以從 50%提高到 80%，表明有更高比例的癌細胞殘留。
當結腸癌細胞和成纖維細胞的混合溶液通過膜時，結腸癌細胞保留在回收液中。因此，使用本研究開發的膜過濾法，預計可以從患者的腫瘤組織中分離出高比例的結腸 CSC(CIC)。

摘要(英)

Colon carcinoma is one of the most fatal and common gastrointestinal cancers in the world. Cancer-initiation cells (CICs) or cancer stem cells (CSCs), are responsible for tumor initiation, growth, and metastasis. The purification and isolation of tumorigenic colon CSCs (CICs) from primary colon tumor tissues should be valuable for the development of novel diagnostic and personal therapeutic treatments in the future. In this study, CSCs (CICs) of colon carcinoma cell line CoLo205 and SW480 (model of primary colon carcinoma cells) were purified from human fibroblasts (CG1639). Furthermore, the primary colon carcinoma cells were purified from colon tumor tissues of patients utilizing the membrane filtration method via nylon mesh filters and poly(lactide-co-glycolic acid)/silk screen (PLGA/SK) filters. In the separation of colon carcinoma cells from fibroblasts, both cells were stained with Cell Tracker Red and Blue, respectively for their identification using flow cytometry. CoLo205 purity was enhanced from 50% to 80% in the recovery fraction, which showed the higher cancer cells remaining. The isolation efficiency was characterized using (i) CD44 and CD133 marker expression, (ii) colony-forming unit assay, and (iii) carcinoembryonic antigen (CEA) production. Especially, CSCs (CICs) were purified in the migrated cells compared to the cells in permeation solution and recovery solution, when CoLo205 or SW480 cells were permeated through the membranes. However, the migrated cells generated similar efficiency of colony forming unit compared to the cells in the permeate solution and recovery solution. The colon cancer cells remained in the recovery solution, when the mixed solution of colon cancer cells and fibroblasts were permeated through the membranes. Therefore, a high proportion of colon CSCs (CICs) from the patient’s tumor tissue is expected to be isolated using the membrane filtration method developed in this study.

關鍵字(中)

★ 癌症幹細胞
★ 結腸癌
★ 薄膜過濾法

關鍵字(英)

★ Cancer stem cells
★ Colon cancer
★ Membrane filtration method
★ PLGA/SK membrane

論文目次

Abstract ....................................................................................................................................... I 摘要 ............................................................................................................................................II Index of Contents ..................................................................................................................... III Index of Figure ......................................................................................................................... VI Index of Table.............................................................................................................................. I Chapter 1 Introduction .......................................................................................................... 2
1-1 Stem cells and cancer stem cells .................................................................................. 2 1-1-1 Stem cells.......................................................................................................... 2
1-1-2 Cancer and cancer stem cells............................................................................ 4 1-2 Relationship between cancer cells and microenvironment .......................................... 6 1-2-1 Circulating tumor cells (CTCs) ........................................................................ 6
1-2-2 Cancer associated fibroblasts (CAFs)............................................................... 7
1-2-3 Extracellular Matrix (ECM) ............................................................................. 8 1-3 Isolation process of cancer cells ................................................................................ 10 1-3-1 Fluorescence-activated cell sorting (FACS) ................................................... 10
1-3-2 Membrane filtration method........................................................................... 11 1-4 Identification of cancer stem cells ............................................................................. 13 1-4-1 Surface marker expression of cancer stem cells ............................................. 13
1-4-2 Carcinoembryonic antigen (CEA) detection using ELISA ............................ 14
1-4-3 Colony formation assay (CFA) ....................................................................... 16
1-4-4 Immunofluorescence staining (IF).................................................................. 18
1-5 Goal of this study ....................................................................................................... 19 Chapter 2 Materials and methods............................................................................................. 20 2-1 Experimental materials .............................................................................................. 20
2-1-1 Cell sources..................................................................................................... 20 2-1-2 Membranes ..................................................................................................... 20 2-1-3 Cell cultivation dishes .................................................................................... 21
2-1-4 Digestion and passage process ....................................................................... 21
2-1-5 Ammonium-Chloride-Potassium (ACK) lysing buffer .................................. 21 2-1-6 Phosphate buffered saline solution (PBS) ...................................................... 21 2-1-7 Cell culture medium ....................................................................................... 21 2-1-8 Evaluation of cancer stem cells ...................................................................... 22
2-1-9 CellTracker staining........................................................................................ 22 2-2 Cell culture methods .................................................................................................. 22 2-2-1 Preparation of cell culture medium ................................................................ 22
2-2-2 Cell cultivations .............................................................................................. 23 2-2-3 CellTracker staining and coculture method .................................................... 23 2-2-4 Preparation of phosphate buffered saline solution (PBS)............................... 25 2-2-5 Preparation of Ammonium-Chloride-Potassium (ACK) lysing buffer........... 25 2-2-6 Preparation of collagenase digestion agent .................................................... 26 2-2-7 Passage of cell lines ........................................................................................ 26 2-2-8 Cell density measurement............................................................................... 27 2-2-9 Extraction of primary cancer cells.................................................................. 28
2-2-10 Preparation of PLGA-silk screen (PLGA/SK) membranes .......................... 29 2-3 Cell sorting method.................................................................................................... 30 2-3-1 Membrane filtration method ........................................................................... 30 2-4 Identification of cancer stem cells ............................................................................. 32 2-4-1 Surface marker analysis (flow cytometry) of cancer stem cells ..................... 32
2-4-2 Soft agar colony formation assay ................................................................... 32
2-4-3 Evaluation of carcinoembryonic antigen concentration (CEA) in cell culture
medium..................................................................................................................... 35 2-5 Characterization of membranes ................................................................................. 37 2-5-1 Primos-CR measurements .............................................................................. 37
2-5-2 Zeta potential measurements .......................................................................... 37 Chapter 3 Results and discussion ............................................................................................. 39
3-1 Characterization of membranes ................................................................................. 39 3-1-1 Primos-CR measurements .............................................................................. 39
3-1-2 Zeta potential measurements .......................................................................... 41 3-2 Characterization of colon cancer cell lines ................................................................ 43
3-2-1 Isolation of cancer stem cells from CoLo205 cell line by membrane filtration method...................................................................................................................... 43 3-2-2 Isolation of cancer stem cells from SW480 cell line by membrane filtration method...................................................................................................................... 45 3-2-3 Flow cytometry analysis of cancer stem cells surface marker ....................... 47 3-2-4 Soft agar colony formation assay of cancer cell lines .................................... 60
3-2-5 Carcinoembryonic antigen (CEA) concentration of cancer cell lines ............ 67 3-3 Characterization of primary colon cancer cells ......................................................... 69 3-3-1 Extraction of cancer cells from primary colon cancer tissue.......................... 69
3-3-2 Isolation of primary colon cancer cells by membrane filtration method........ 71
3-3-3 Flow cytometry analysis of primary colon cancer cells ................................. 72
3-3-4 Soft agar colony formation assay of primary colon cancer cells.................... 76 3-4 Coculture cancer cells and fibroblasts cells and Celltracker staining........................ 78 3-4-1 Flow cytometry analysis of cancer cells and fibroblasts ................................ 78
3-4-2 Isolation cancer cells from different ratio mixing solution by membrane
filtration method ....................................................................................................... 81
Chapter 4 Conclusion ............................................................................................................... 85 Reference.................................................................................................................................. 87

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2023-8-14

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