The Evaluation of Effect of Platelet-Rich Plasma on Culturing Corneal Endothelial Cells

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Project title

Code/計畫編號

NSC101-2324-B075-001

Translated Name/計畫中文名

豬血液血小板濃厚血漿取代幹細胞培養基中胎牛血清及在再生醫療之應用-〈子計畫三〉血小板濃厚血漿取代角膜內皮細胞培養基中胎牛血清之可行性評估

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

陳克華(計畫主持人)

Department/Unit

Department of Ophthalmology, Taipei Veterans General Hospital

Website

https://www.grb.gov.tw/search/planDetail?id=2588101

Year

2012

Start date/計畫起

01-08-2012

Expected Completion/計畫迄

31-07-2013

Co-Investigator(s)

Yung-Kai Lin

Bugetid/研究經費

540千元

ResearchField/研究領域

臨床醫學
生物技術(醫)

血小板濃厚血漿取代角膜內皮細胞培養基中胎牛血清之可行性評估人類角膜內皮細胞 (human corneal endothelial cells, HCECs) 對於視覺的維持極為關鍵，其可控制體液的進出並維持角膜組織的水合作用。由於角膜內皮細胞於體內不可逆的退化現象、創傷及白內障手術引起的傷害，因此體外培養角膜內皮細胞成為角膜移植(keratoplasty) 的替代方案。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。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HCECs 於體外增生且可取代培養基中FBS。本計畫擬自開發猪血液中產製幹細胞培養用胎牛血清之替代物，自猪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後與市售FBS 比較生長因子如IGF、TGF-α、PDGF 與及纖維連結素(fibronectin)之濃度。進一步以Design Expert 軟體輔助計算出不同比例之FBS 與PRP 之培養液配方組合，並評估其對間葉幹細胞之型態、顯微結構、增生曲線、存活率、細胞貼附率、細胞外基質生合成及BrdU incorporation。最後以反應曲面法推算最佳之培養基配方。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HCEC 體外增生且維持功能特性之胎牛血清替代物並應用於再生醫療領域。 Feasibility study of porcine platelet-rich plasma as fetal bovine serum substitute in human corneal endothelial cell cultivation The main role of the corneal corneal endothelium is maintaining corneal transpancy by regulating stromal hydration. Since the human corneal endothelial cell (HCECs) density naturally decreases with age or trauma, many patient loss their vision because of corneal edema when their HCECs decreased to a level of 500/cm2. The tissue engineering corneal replacement are regarded as the alternative for keratoplasty. However, the in vitro proliferative capacity of HCECs were retained. Ex vivo expansion of HCECs is conventionally achieved in the medium containing fetal bovine serum (FBS). However, in the future the FBS concentration of HCECs culture medium should be minimized since ethical and scientific problems. On ethical respect, fetuses are exposed to pain and discomfort and therefore current practice of fetal blood is inhumane. Moreover, the serum batches display quantitative and qualitative variations in their composition and may induces the 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 on HCECs 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. Due to the P-PRP containing higher concentration of TGF-β and PDGF than commercial FBS. Present project will be conducted to observing the effects of different proportion of FBS and P-PRP and exploring the optimal formulation by Design Expert. The morphological properties, proliferation, viability, cell attachment, biosynthesis of extracellular matrix and BrdU incorporation of HCECs in various culture media will be evaluated. The purposer wish to developing a reasonable price, stable composition, high ethical quality FBS substitute which would be improving ex vivo expansion and maintain the functionality of HCECs and apply in regenerative medicine.

Keyword(s)

人類角膜內皮細胞
胎牛血清
血小板濃厚血漿
Human corneal endothelial cells
fetal bovine serum
porcine platelet-rich plasma

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The Evaluation of Effect of Platelet-Rich Plasma on Culturing Corneal Endothelial Cells

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