開發人工利基應用於常溫細胞儲存和運輸

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姓名

李冠霖(Kuan-Lin Li) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

開發人工利基應用於常溫細胞儲存和運輸
(To Establish an Artificial Niche for Facile Cell Storage and Transportation under Ambient Temperature)

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摘要(中)

醫學領域快速發展促使人類平均壽命延長，進而大幅提升老年人口比例，當世界全面進入超高齡化社會，人類將面臨一系列與醫療相關的挑戰。傳統的手術治療及藥物療法已不再能滿足需求，再生醫學透過替代已受損細胞或組織的方式，成為一項重要策略。在多個再生醫學領域中，細胞治療作為其中規模最大的發展項目之一，具有顯著的前景；而細胞治療的成功關鍵，在於確保細胞保存及運輸過程中，維持細胞的活性及功能性。冷凍保存技術（cryopreservation）為現行的細胞保存技術主要方法，然而冷凍過程會導致細胞內水分形成冰晶，引發細胞結構性破壞，需要藉由冷凍保護劑（cryoprotective agents，CPA）來防止細胞受損，臨床實際應用卻發現，部分患者在治療後出現低血壓、心律失常等嚴重反應，研究顯示與冷凍保護劑無法完全去除有關。有鑑於此，本研究開發高度生物相容性之人工利基（artificial niche）「Traveling Pearl」。Traveling Pearl具有殼/核結構，外層使用海藻酸（alginate），能與二價陽離子迅速交聯，細胞運輸過程具有保護功能；而內層則選用具有黏滯性的明膠溶液（gelatin solution），這有助於在細胞保存時提供緩衝作用，確保細胞穩定。本研究設計不同溫度條件參數，包括常溫（ambient temperature，AT，泛指15-30℃）、37℃、4℃以及液態氮冷凍（liquid nitrogen，LN2）組，也模擬保存及運輸時差異，分為靜態（static）及動態（dynamic）。實驗結果證實，在AT組別，細胞在經過3天和7天保存後仍然保持良好的細胞活性及貼附能力，且保存效果穩定。綜上所述，本研究開發Traveling Pearl，旨在解決冷凍保存衍生的問題，並提高生物相容性來強化細胞治療的成功率，期許這項研究成果能為再生醫療提供新工具。

摘要(英)

Rapid advancements in the field of medicine have led to an extended average human lifespan, resulting in a substantial increase in the proportion of elderly individuals within the population. As global society transitions into a super-aged demographic, humanity is poised to confront an array of medical challenges. Conventional surgical interventions and pharmaceutical therapies are no longer adequate to meet these demands; thus, regenerative medicine has emerged as a pivotal strategy for cell and tissue replacement of damaged constituents. Among the myriad domains of regenerative medicine, cell therapy stands out as one of the most expansive avenues, exhibiting considerable promise. The success of cell therapy depends critically on the preservation of cellular viability and functionality during storage and transport. Cryopreservation, currently the predominant method for cell preservation, entails freezing procedures that, unfortunately, engender intracellular ice crystal formation, thereby inducing structural detriment. Cryoprotective agents (CPAs) are employed to mitigate cellular damage caused by freezing; however, clinical application has revealed instances of severe post-treatment patient reactions, such as hypotension and arrhythmia, indicating residual issues independent of CPA usage. In light of these challenges, this study introduces the development of a highly biocompatible artificial niche termed "Traveling Pearl." The Traveling Pearl is characterized by a shell/core architecture, where the outer layer employs alginate derived from brown algae, exhibiting rapid crosslinking with divalent cations to confer protective attributes during cellular transport. The inner layer comprises a viscous gelatin solution that serves as a buffering agent during cell preservation, thereby ensuring stability. Various temperature conditions were investigated in this study, including ambient temperature (AT, encompassing 15-30°C), 37°C, 4°C, and liquid nitrogen freezing (LN2), in addition to static and dynamic simulations, to mimic disparities during storage and transportation. Experimental findings revealed that within the AT group, cells exhibited sustained viability and adhesive capabilities following 3-day and 7-day preservation periods, thereby underscoring the stability of the preservation effects. In summary, this study presents the development of the Traveling Pearl as a strategic intervention aimed at addressing complications arising from cryopreservation techniques, while enhancing biocompatibility to bolster the success rate of cell therapy. The results of this study may provide a novel tool for augmenting the domain of regenerative medicine.

關鍵字(中)

★ 細胞保存
★ 細胞運輸
★ Traveling Pearl載體
★ 常溫

關鍵字(英)

★ cell preservation
★ cell transportation
★ Traveling Pearl
★ ambient temperature

論文目次

摘要 i
ABSTRACT ii
符號縮寫說明 x
第一章研究背景暨文獻回顧 1
1-1 再生醫學 1
1-1-1細胞治療流程 2
1-1-2細胞治療市場 3
1-2 細胞運輸保存 4
1-2-1冷凍保存 4
1-2-2細胞冰損傷 5
1-2-3冷凍保護劑 6
1-2-4 冷凍保護劑不良反應 7
1-3 競品比較 7
1-4 材料選用 9
1-4-1海藻酸 9
1-4-2海藻酸的交聯 10
1-4-3海藻酸細胞包覆 11
1-4-4明膠 12
第二章研究動機與實驗目的 13
2-1研究動機 13
2-2研究目的 13
第三章材料與方法 16
3-1 使用儀器與耗材 16
3-1-1藥品清單 16
3-1-2耗材清單 17
3-1-3設備器材清單 17
3-2 實驗架構 18
3-3 實驗選用細胞 20
3-3-1小鼠纖維母細胞 20
3-4 Traveling Pearl製作 21
3-4-1海藻酸鈉以及明膠製備 21
3-4-2注射幫浦參數設定 21
3-4-3熱板攪拌器參數設定 22
3-4-4細胞包覆效率分析 22
3-4-5生物相容性測試 23
3-5 Traveling Pearl內細胞分析 24
3-5-1 Traveling Pearl細胞死活染色分析 24
3-5-2 Traveling Pearl細胞團分析 24
3-6 Traveling Pearl材料去除及後續實驗 24
3-6-1材料去除 24
3-6-2粒線體活性檢測 25
3-6-3細胞貼附性測試 25
3-7 細胞運輸碰撞測試 26
3-8 細胞週期測試 26
3-9 Traveling Pearl細胞團包覆 27
第四章結果與討論 28
4-1 Traveling Pearl製作 28
4-1-1 Traveling Pearl分層結構 28
4-1-2置於不同環境下之細胞觀察 29
4-1-3細胞包覆效率分析 30
4-1-4生物相容性分析 31
4-2 Traveling Pearl細胞存活率測試 32
4-2-1 Traveling Pearl包裹分析 32
4-2-2 Traveling Pearl細胞保存率 33
4-2-3細胞團觀察 35
4-3 Traveling Pearl包裹後細胞觀察 35
4-4 Traveling Pearl包裹後粒線體活性檢測 38
4-5 Traveling Pearl包裹後細胞貼附分析 40
4-6 Traveling Pearl運輸碰撞測試 40
4-7 細胞週期分析 42
4-8 Traveling Pearl細胞團包裹 46
4-8-1 Traveling Pearl細胞團包裹分析 46
4-8-2 Traveling Pearl細胞團保存率 47
4-8-3 Traveling Pearl包裹後細胞團貼附情況 48
第五章結論 49
第六章參考文獻 50

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指導教授

陳靖昀柯承志(Ching-Yun Chen Cherng-Jyh Ke)

審核日期

2023-8-21

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