豬血液血小板濃厚血漿取代間葉幹細胞培養基中胎牛血清之可行性評估 幹細胞於臨床及組織應用最大的瓶頸是無法在體外快速的培養至治療所需之數量， 目前許多研究使用胎牛血清(fetal bovine serum, FBS)做為培養基補充劑，但胎牛血清的 生產與使用始終伴隨著倫理及科學之爭議。由於血小板中的 α 顆粒會在適當之活化劑存 在 下 被 激 活 並 釋 放 多 種 生 長 因 子 可 並 促 進 組 織 重 建 ， 經 濃 縮 之 血 小 板 濃 厚 血 漿 (platelet-rich plasma, PRP)應可促進幹細胞於體外增生與分化並扮演與胎牛血清類似之 角色。本計畫擬自開發豬血液中產製幹細胞培養用胎牛血清之替代物，自豬血液以不同 離心方式濃縮並找出血小板濃度最高之方法，加入不同活化劑活化與市售 FBS 比較 IGF、TGF-β、PDGF 與 KGF 濃度、物化特性與生化組成之差異並評估其對間葉幹細胞 之型態、增生曲線、存活率、細胞貼附率、細胞外基質生合成、生長因子分泌量與群落 形成能力之影響，並藉分析間葉幹細胞表面細胞標記如 CD 45, CD 73 與 CD 105 之表 現。申請者希望藉執行本研究計畫開發單價合理、成分穩定、符合動物福祉且能促進幹 細胞體外增生且維持原始特性之胎牛血清替代品並應用於再生醫療領域。 Feasibility study of porcine platelet-rich plasma as fetal bovine serum substitute in mesenchymal stem cell cultivation The major concern of stem cell for cell-therapy or tissue engineering approach is the restrained ex vivo expansion ability of stem cell. Ex vivo expansion of stem cell is conventionally achieved in the medium contain fetal bovine serum (FBS). However, the FBS concentration of stem cell culture medium must be minimized since ethical and scientific problems. Due to the α-granule of platelet can be active by suitable agonist to release numerous growth factors and considered as a FBS substitute. Present project will be conduct to developing the porcine platelet-rich plasma (P-PRP) as fetal bovine serum substitute in mesenchymal stem cell cultivation. The preparations of maximum platelet concentration factor will active by various agonists to explore the optimal condition of α-granule activation and highest release amount of IGF, TGF-β, PDGF and KGF. Furthermore, the biochemical composition and physical-chemical properties of FBS and P-PRP also compared. The morphological properties, proliferation, viability, cell attachment, biosynthesis of extracellular matrix, growth factors secretion, and colony forming unit of adipose-mesenchymal stem cell in various culture media will be evaluated. Moreover, the expression of MSC surface markers of CD 45, CD 73 and CD 105 will be determined. Proposer will be optimized suitable media formulation by response surface method and wish to develop a reasonable price, stable composition, high ethical quality FBS substitute which would be improving ex vivo expansion and maintain the primitive properties of mesenchymal stem cell and apply in regenerative medcine.
