The Feasibility of Human Platelet-Rich Plasma as a Substitute for Fetal Bovine Serum in the Cultivation and Differentiation of Adipose-Derived Stem Cells: A Preliminary Study

以血小板濃厚液配方組合取代胎牛血清作為脂肪幹細胞之培養液之初步研究

邱彥豪(Yen-Hao Chiu) ； 林詠凱(Yung-Kai Lin) ； 蔡錡函(Chi-Han Tsai) ； 廖文傑(Wen-Chieh Liao) ； Fendi Sugiri ； 馬旭(Hsu Ma)

《臺灣整形外科醫學會雜誌》 25卷2期 (2016/06) Pp. 87-100

摘要

背景：胎牛血清是細胞培養液的重要成分。然而要將其應用於臨床則具有爭議性。包含血清取得衍生的動保議題、可能潛在傳播Prion疾病，以及過敏免疫反應之風險、以及批間偏差。因此我們試著尋找足以取代胎牛血清的細胞培養液，以及建構不含胎牛血清的細胞培養系統。目的及目標：活化後的人類血小板濃厚血漿目前已廣泛應用於美容醫學及重建手術。我們試著比較不同濃度的活化後人類血小板濃厚血漿製劑與胎牛血清，用以培養脂肪幹細胞的生長與分化能力。材料及方法：從醫院血庫申請新鮮的人類血小板濃厚血漿，將其加入凝血酶、鈣離子或置於數次冷凍-解凍循環的環境，活化後以酵素免疫分析法測得生長因子PDGF-AB 與TGF-β1之濃度。濃度最高的組別則用以培養脂肪幹細胞至第五代，並以流式細胞技術測量細胞表面標記與細胞凋亡。同時也觀測其分化為脂肪或硬骨細胞的能力。結果：含有10 U/mL凝血酶，1%氯化鈣，以及合併有二者以的活化人類血小板濃厚血漿，能釋放出最高濃度的生長因子。與胎牛血清比較，以含有1%氯化鈣活化的20%人類血小板濃厚血漿培養脂肪幹細胞，在第5，7天能得到最佳的生長曲線。含有1%氯化鈣活化的10%與20%人類血小板濃厚血漿培養脂肪幹細胞於第十代有較低的細胞老化程度。所有以人類血小板濃厚血漿培養的脂肪幹細胞都具有分化成脂肪或硬骨細胞的潛力。結論：我們的初步研究結果確認人類血小板濃厚血漿取代胎牛血清成為脂肪幹細胞培養液的方向，亦為未來細胞治療的展望。

並列摘要

Background: Despite its importance and ubiquity in cell culture, the use of fetal bovine serum (FBS) remains controversial. Concerns over animal welfare, safety risks due to the risk of prion disease transmission and immune reactions, and significant batch variations. There is great interest in finding a viable replacement for FBS and motivated the development of a serum-free cell culture system. Aim and Objectives: Activated human platelet-rich plasma (PRP) has been used in aesthetic and reconstructive surgery and touted as a potential FBS replacement. We compared the effectiveness of various formulationsof PRP in adipose-derived stem cells (ASCs) cultures and differentiation. Materials and Methods: PRP samples obtained from a blood bank were pooled and activated with freeze-thaw cycles and thrombin and calcium solutions combinations, resulting in eight distinct formulations. The concentrations of platelet-derived growth factor-AB (PDGF-AB) and transforming growth factor-β1 (TGF-β1) were measured for each formulation using enzyme-linked immunosorbent assays (ELISA). The PRP groups with the highest growth factor levels were tested using fifth-passage ASCs culture. Immunophenotypic and apoptosis analyses were also performed using flow cytometry, adipogenesis and osteogenesis assays. Results: Activated human PRP with 10 U/mL thrombin, 1% CaCl2, and the combination of 10 U/mL thrombin and 1% CaCl2 provided optimum growth factor levels. Cell culture assays showed that 20% human PRP with 1% CaCl2 exhibited significant higher cell count on days 5 and 7 (p＜ 0.001). Significant lower overall apoptotic rate was noted in group of 10% human PRP with 1% CaCl2 (p＜ 0.001) and 20% human PRP with 1% CaCl2 (p＜ 0.05) in passage ten. All groups of ASCs cultured in serum-free PRP in early-passage showed differentiation potential in qualitative analysis. Conclusion: Our results confirmed the viability of human calcium-activated PRP as an FBS replacement in ASCs culture and would be promising for cell therapy. (J Taiwan Soc of Plast Surg 2016;25:87～100)

並列關鍵字

platelet-rich plasma (PRP) ； adipose-derived stem cells (ASCs)