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標題: | 在聚乙烯醇表面大量製造大小可調控的可注入性真皮乳頭微組織以應用於毛囊再生 Mass production of injectable dermal papilla spheroids of controllable size on poly(vinyl alcohol) surface for hair follicle regeneration |
作者: | Wei-Ting Lin 林韋廷 |
指導教授: | 楊台鴻 |
共同指導教授: | 林頌然 |
關鍵字: | 毛囊再生,三度空間立體培養,真皮乳頭組織,聚乙烯醇,尺寸控制, hair follicle regeneration,three dimension culture,dermal papilla,poly(vinyl alcohol),size control, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 禿髮,一種常見的疾病,其造成的心理創傷遠大於對生理方面的影響,所費不貲的植髮手術僅能重新分佈殘餘毛髮至禿髮部位且無法應用於嚴重落髮患者,因此具有誘導毛囊再生能力的毛囊真皮乳頭細胞成為目前最具潛力的解決方案。
真皮乳頭細胞需在聚集成多細胞球體狀態下,才能表現誘導表皮細胞分化成毛囊結構的能力,已有許多利用組織工程培養立體結構多細胞球的方法,但以特定生醫材料表面使細胞貼附並自我聚集的方法,遭遇如何輕易地收集多細胞球而不造成損壞之困擾。另一方面,懸浮培養方式之球體尺寸往往為一區間分佈而非固定尺寸。 根據細胞於親水性材料表面貼附不佳之特性,我們使用塗佈聚乙烯醇(poly(vinyl alcohol), PVA)的聚合酶鏈鎖反應(polymerase chain reaction, PCR)96孔盤中,由於狹小空間內的高碰撞頻率,我們成功地藉由操縱種植細胞的數量,在一天之內即可迅速獲得尺寸可控制的大鼠與人類真皮乳頭細胞球。 進一步分析顯示,大鼠與人類細胞球內細胞能維持80%以上存活率,均顯著高於懸滴培養方式,組織觀察球體內為緊密的細胞堆疊結構,並能維持真皮乳頭細胞與誘導能力有關的基因表現。我們也驗證真皮乳頭細胞球體再通過注射器之後能維持結構完整與細胞生長能力,於毛囊再生測試中,大鼠與人類真皮乳頭細胞球均能成功誘導毛囊再生,檢驗不同尺寸的真皮乳頭細胞球誘導能力,結果建議細胞球內需要達到一個最少的細胞數量值才能獲得較高的誘導效率。 我們成功地建立一個快速形成尺寸可控制之真皮乳頭細胞球的方法,並具有搭配自動化設備大量生產的潛力,我們的毛源性真皮乳頭細胞球大量生產系統未來也能應用於臨床上毛囊再生之研究。 Hair loss is a common disorder that often causes more intense influence in psychological than physiological aspect. Expensive hair transplantation can only redistribute remaining hair to bald area and can no be applied to patients with extensive hair loss. Regarding this, hair follicle (HF) dermal papilla(DP) cells beome a promising solution due to their ability to induce HF regeneration. DP cells exhibit the potential to guide the transdifferentiation of epidermal cells into follicular structure only when they are kept in multicellular aggregates. A number of tissue engineering approaches have been developed to produce three-dimensional multicellular spheroids. Methods depending on intercellular self-assembly of cells attached to biomaterials encounter difficulty in harmlessly harvesting multicellular spheroids with ease. On the other hand, suspension culture methods can result in variation in spheroid sizes. According to the poor adhesiveness of cells to hydrophilic surface, we culture DP cells in 96-well PCR plate coated with poly(vinyl alcohol)(PVA). Due to high collision frequency in limited space, we can obtain size-controllable human or rat DP spheres rapidly within 24 hours in culture by varying the numbers of cells seeded. Further analysis reveals that more than 80% of cells within the spheres are viable and this viability is higher than that in spheres obtained by hanging drop method. DP spheres have a compact structure in histology and preserve DP signature gene expression profile that is related to the HF induction ability. We also demonstrate that the structure and cellular viability of DP spheres remain intact after they are injected through a cell delivery injection apparatus. Functionally, both human and rat DP spheres can successfully induce the HF regeneration in HF regeneration assay. The HF induction ability is varied when DP spheres of various sizes are tested and our results suggest that a minimum cell number is required within each DP sphere to obtain a higher HF induction efficiency. We successfully set up an easy method for fast generation of size-controllable DP cell spheres and this method is of scalable potential with automatic equipment. Our system of mass preparation of trichogenic DP spheres can also be applied to clinical investigation for HF regeneration in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44655 |
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顯示於系所單位: | 醫學工程學研究所 |
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