| 摘要: | 幹細胞於臨床及組織應用最大的瓶頸是無法在體外快速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，在倫理上剝奪胎牛的生命與不人道的採集常被詬病；在科學上，血清的組成複雜且易變動，常對研究品質造成影響。而在台灣，豬血液的利用效率不佳，僅於食品加工及飼料添加使用，故本研究擬自豬血液開發一合適於幹細胞培養用之胎牛血清替代物，自豬血液以不同離心方式濃縮製成豬血液血小板濃厚血漿 (porcine platelet-rich plasma, P-PRP)，加入不同濃度的膠原蛋白 (collagen)、凝血酶 (thrombin)、氯化鈣 (CaCl2) 配方組合活化血小板並以酵素結合免疫吸附分析 (enzyme-linked immunosorbent assay, ELISA) 測定類胰島素生長因子 (insulin-like growth factor , IGF) 、角質化細胞生長因子 (keratinocyte growth factor, KGF)、血小板衍生生長因子AB (platelet-derived growth AB, PDGF-AB)、轉形生長因子-β (transforming growth factor, TGF-β)之釋出量。經定量分析後以Design-Expert軟體設計最佳之活化劑配方，並檢測重金屬及纖維連結素含量。配製成培養基與胎牛血清及市售無血清培養基，以比較對人類脂肪間葉幹細胞貼附率、增生率及存活率之影響。並以CD73、CD105、CD90、CD34、CD45及HLA-DR鑑定間葉幹細胞經P-PRP培養，是否會因而改變細胞特性。結果顯示P-PRP經活化後所有生長因子分泌量皆顯著提高 (p＜0.05)，TGF-β、PDGF、KGF的分泌量高於FBS，其中經膠原蛋白、氯化鈣活化會增加TGF-β及PDGF的釋放，氯化鈣則增加KGF的分泌，特別是TGF-β達顯著的差異 (p＜0.05)。以生長因子分泌量經Design-Expert軟體藉反應區面法判定Co－T＋Ca－、Co＋T－Ca－及Co＋T＋Ca－為最佳化活化組合。含量分析結果顯示P-PRP不含有害之重金屬，而較高之硒離子濃度可增加細胞抗氧化之能力。P-PRP纖維連結素含量略低於市售胎牛血清，更影響並表現較低細胞貼附率。細胞培養結果顯示三種P-PRP可促進細胞增生率及存活率，並佔有數量上之優勢。此外，間葉幹細胞經P-PRP培養後仍保持原有細胞之特性。未來希望開發出單價合理、成份穩定、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。
關鍵詞：胎牛血清，豬血液血小板濃厚血漿，間葉幹細胞，胎牛血清替代物
Efficient In vitro cell expansion is an obstacle for stem cell research in cell therapy and tissue engineering. Conventionally, stem cell culture medium contains 5-20% fetal bovine serum (FBS). However, due to ethical issues and scientific problems, the FBS concentration of stem cell culture medium must be minimized. On ethical aspect, the preparing procedures of FBS are inhumane. Furthermore, the batches of serum display quantitative and qualitative variations among their compositions and may induce the scientific problems. In Taiwan, the utilizations of porcine blood is mainly used in food manufacturing and animal feed staff. Therefore, the aim of this study is to develop porcine platelet-rich plasma (P-PRP) as the fetal bovine serum substitute in the mesenchymal stem cell cultivation. The effect of platelet-rich plasma will be activated by different formulations (collagen/thrombin/calcium chloride) on release amount of insulin-like growth factor (IGF), keratinocyte growth factor (KGF), platelet-derived growth AB (PDGF-AB), and transforming growth factor (TGF-β) were determined by ELISA (enzyme-linked immunosorbent assay) methodology. The ELISA results were analyzed by Design-Expert software to develop optimal agonist formulations. Moreover, the amount of heavy metal and fibronectin of P-PRP were quantified. P-PRP, fetal bovine serum and serum-free medium were utilized to stem cell cultivation media, and then treated human adipose-derived mesenchymal stem cells (MSC) to observe the efficiency for cell attachment, proliferation and viability. Futhermore, we characterized these MSCs by surface marker expressions, including CD73, CD105, CD90, CD34, CD45 and HLA-DR. Results demonstrated all release amount of growth factors of P-PRP were significantly higher than non-activated P-PRP (p＜0.05). The releasing amount of PDGF-AB and KGF of P-PRP did not reach significant difference from FBS. Furthermore, the levels of TGF-β and PDGF-AB in P-PRP could be increased based on collagen and calcium chloride activation, and calcium chloride could enhance the releasing of KGF in P-PRP. Notably, TGF-β in activated P-PRP was significantly higher than in FBS (p＜0.05). According to the results of growth factor, we used response surface methodology to establish Co－T＋Ca－, Co＋T－Ca－ and Co＋T＋Ca－ as the optimal agonist formulations for P-PRP activation by Design-Expert program. We determined Co＋T＋Ca－, Co＋T－Ca－ and Co－T＋Ca－ as the optimal agonist formulations. These three P-PRP formulations contained no harmful heavy metals but with higher selenium concentration, which would improve the cell ability of anti-oxidation. The fibronectin in P-PRPs were comparatively less than in FBS, which may cause the lower cell attachment on MSCs morphology. P-PRP cell cultivation revealed a higher cell proliferation rate and viability. Furthermore, the results of MSC surface marker expressions presented MSCs do not alter characteristic under P-PRPs cultivation. In summary, we succeed to develop the FBS substitute which would improve in vitro expansion and maintain the differentiation capacity of mesenchymal stem cell with reasonable price, stable composition, and high ethical quality. Further, the P-PRP could be used in human regenerative medicine.
Key words: Fetal bovine serum, Porcine platelet-rich plasma, Mesenchymal stem cell, FBS substitute |
