| 摘要: | 細胞治療的臨床應用在於保存與運輸可用細胞時面臨關鍵挑戰。雖然冷凍保存為目前細胞長期保存的標準方法，但其高度依賴冷鏈運輸，不僅提高操作成本與風險，更因解凍後細胞極度脆弱、通常僅能存活數小時，難以支撐臨床上必經的檢測流程與時程調度，缺乏必要的緩衝時間。本研究開發並評估一種明膠–海藻酸鈉雙層球型載體，作為短期、非冷凍條件下細胞保存之替代方案。本實驗以高增殖性的 L929 纖維母細胞與人類脂肪來源間質幹細胞為模型。間質幹細胞（mesenchymal stem cells, MSCs）可由骨髓、臍帶或脂肪等組織獲得，其中脂肪來源間質幹細胞（adipose-derived stem cells, HADSCs）因取材容易且具臨床相關性而被廣泛應用。本研究所使用之 MSC 為脂肪來源的 HADSC，以下簡稱為 hMSC。分別於4°C、常溫（指本研究採用之室內自然環境溫度，約20–25 °C，具日常波動）靜態、常溫動態與37°C等不同溫度下，搭配多種儲存液（含或不含血清之DMEM高糖／低糖培養基、HBSS、生理食鹽水），進行為期14天之實驗觀察，評估細胞存活率、粒線體活性與細胞週期變化。結果顯示，L929細胞在含FBS之DMEM-HG中於常溫保存可維持最佳存活率，且靜態與動態條件無顯著差異。相比之下，hMSC亦表現出高保存耐受性，在含FBS之DMEM-LG條件下，於常溫或4°C儲存皆可維持超過90%之存活率與貼附能力長達七天；惟HBSS與生理食鹽水在三天內即無法有效維持任一細胞型態之活性。綜合實驗結果顯示，該雙層載體能有效維持細胞生存率，提供代謝靜止狀態與基本功能穩定性，其效能受溫度與儲存液條件共同調控。本研究證實此雙層球體具備作為短期、非冷凍細胞保存與運輸平台的可行性，不僅降低對冷鏈的依賴與操作風險，更能在臨床流程中提供至少七天的緩衝時間，提升細胞治療與再生醫學中即用型產品的可行性與應用價值。;The clinical application of cell therapy faces critical challenges in the preservation and transportation of viable cells. Although cryopreservation remains the standard approach for long-term storage, it relies heavily on cold-chain logistics, which increases operational cost and risk. More importantly, thawed cells are extremely fragile and typically survive only a few hours, making it difficult to withstand the mandatory testing procedures and scheduling adjustments required prior to clinical use, thereby lacking a necessary buffer period. To address this issue, this study developed and evaluated a gelatin–alginate core–shell carrier as an alternative strategy for short-term, non-cryogenic preservation. Highly proliferative L929 fibroblasts and mesenchymal stem cells (MSCs) were employed as model systems. MSCs can be isolated from various tissues, including bone marrow, umbilical cord, and adipose tissue; among these, adipose-derived stem cells (HADSCs) are particularly accessible and clinically relevant. In this study, HADSCs were used as the MSC source and are hereafter referred to as hMSC. Cells were stored under different temperature conditions, including 4 °C, ambient static, ambient dynamic, and 37 °C, where ambient temperature refers to the natural indoor environment with daily fluctuations of approximately 20–25 °C. Multiple storage media were tested, including DMEM (high- and low-glucose, with or without serum), HBSS, and normal saline, during a 14-day observation period, with assessments of cell viability, mitochondrial activity, and cell cycle distribution. Results showed that L929 cells preserved in DMEM-HG with FBS maintained the highest viability under ambient conditions, with no significant differences between static and dynamic storage. In contrast, hMSC demonstrated strong tolerance, retaining over 90% viability and adhesion capacity for up to seven days in DMEM-LG with FBS under both ambient and refrigerated conditions, whereas HBSS and normal saline failed to sustain either cell type beyond three days. Collectively, the dual-layer carrier effectively maintained cell survival, promoted metabolic quiescence, and preserved fundamental functional stability in a temperature- and medium-dependent manner. This study confirms the feasibility of the dual-layer encapsulation system as a short-term, non-cryogenic preservation and transport platform that not only reduces reliance on cold-chain infrastructure but also provides at least a seven-day buffer period within clinical workflows, thereby enhancing the practicality and translational potential of off-the-shelf therapeutic cells in regenerative medicine. |
