豬胚幹細胞無飼養層培養系統之建立

本試驗之目的在探討無飼養層培養系統對豬胚幹細胞體外增生、群落形成效率、未分化狀態維持、細胞毒性、分化多能性與細胞凋亡之影響，期建立穩定的無飼養層體外培養系統做為胚幹細胞培養之用。試驗之豬胚幹細胞培養於小鼠胎源纖維母細胞為飼養層之培養環境，並以胚幹細胞培養液添加16%胎牛血清為對照組，ESM添加mEF調節培養液或鹼性纖維母細胞生長因子1 ng/mL等不同成分，再以laminin、collagen IV、fibronectin及Matrigel®等飼養層替代性貼附物質，探討無飼養層培養系統對豬胚幹細胞體外培養系統之影響。試驗結果顯示：（1）生長曲線：ESM添加16% FBS之無飼養層培養系統，其生長曲線為對照組之0.17－0.26倍；ESM添加16%FBS及mEF CM之生長曲線為對照組之1.00－1.85倍；ESM 添加16% FBS及bFGF 1ng/mL者則為對照組之1.51－1.95倍。（2）群落形成效率與其維持未分化狀態分析：ESM添加16%FBS為培養液之無飼養層培養系統，於第6天時均無法維持豬胚幹細胞之正常群落形態，但添加16%FBS與mEF CM者可維持5.6－11.1%的群落效率；ESM添加16% FBS及bFGF 1ng/ mL則可維持5.6－23.5%的群落效率。（3）LDH毒性分析：ESM添加16% FBS為培養液之無飼養層培養系統，其細胞毒性為對照組之1.45－1.48倍；ESM添加16%FBS及mEF CM的細胞毒性為對照組之1.38－1.43倍；ESM添加16%FBS及bFGF 1ng/mL則為對照組之1.37－1.43倍。（4）分化多能性分析：以分化多能性標幟之抗體Oct-4、AP、SSEA-3、SSEA-4、TRA-1-60及TRA-1-81進行染色分析，經染色後均具陽性反應，顯示無飼養層之替代性貼附物質可維持豬胚幹細胞之分化多能性。（5）細胞凋亡分析：無飼養層之替代性貼附物質對豬胚幹細胞未發現具有增加細胞凋亡現象。本試驗以laminin、collagen IV、fibronectin及Matrigel®等飼養層替代性貼附物質進行豬胚幹細胞培養，配合以ESM添加16% FBS及Bfgf 1ng/mL之培養液，可獲得較佳之生長曲線、群落形成、未分化狀態與較低的細胞毒性，並能維持分化多能性與避免細胞凋亡等優點，此研究結果可作為未來豬胚幹細胞無飼養層培養系統之參考。

關鍵字

豬胚幹細胞；無飼養層；體外培養

並列摘要

The purposes of this study were to establish a stable feeder-free in vitro culture system and to investigate the effects of laminin (20 mg/mL), collagen IV (10 mg/mL), fibronectin (5 mg/mL) and Matrigel® (1:20) on the cell proliferation, colony formation, undifferentiation status, pluripotency, and apoptosis of porcine embryonic stem cells. The results shown: (1) Growth curve: the growth curves in the feeder-free culture systems which containing 16% FBS, 16% FBS supplemented with mEF conditional medium, and 16% FBS supplemented with bFGF 1 ng/mL were 0.17-0.26, 1.00-1.85 and 1.51-1.95 fold, respectively. (2) Colony efficiency and maintain undifferentiation status: the ESM supplemented with 16% FBS could not maintain the undifferentiated status of pES colonies. The colony efficiency of ESM containing 16% FBS, supplemented with mEF conditional medium or bFGF 1 ng/mL were 5.6-11.1% and 5.6-23.5%, respectively. (3) LDH toxicity: the LDH toxicity shown 1.45-1.48, 1.38-1.43, and 1.37-1.43 fold between the three different medium. (4) Pluripotency: the feeder-free culture system shown positive staining by ICC analysis with Oct-4, AP, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 pluripotent markers, that indicated they could maintain the pluripotency of pES colonies. (5) Apoptosis analysis: there were no apoptosis when used laminin, collagen IV, fibronectin and Matrigel® as feeder-free culture system. Those results could provide as feeder-free culture system for culturing porcine embryonic stem cells. Porcine embryonic stem cells cultured on feeder replacement and ESM containing 16% FBS and bFGF 1 ng/mL in this study performed the better growth curve, colony formation, undifferentiation status, and low cytotoxicity. Furthermore, this strategy could maintain pluripotency and avoid apoptosis. The stable feeder-free culture system can provide as an optimal system for culturing porcine embryonic stem cells.

並列關鍵字

Porcine embryonic stem cell ； Feeder-free ； In vitro culture

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