利用電濕潤驅動液滴進行動態胚胎培養

Abstract

本研究藉由微流體介電濕潤平台，進行培養液液滴的操控，使之能同時達到胚胎動態培養和保留胚胎本身所分泌之成長因子，進而達到有如輸卵管之動態培養環境。此外，我們以單一胚胎動態培養為目標，以利日後有助於進行胚胎之個別成長狀況之分析。 實驗中，我們利用介電濕潤 (Electrowetting-on-dielectric，EWOD) 之方式進行培養液液滴之驅動，於測試中發現，KSOM + AA (potassium simplex optimized medium)培養液液滴由於含胺基酸等生物分子，易產生生物分子沉積並吸附於晶片表面之現象。因此，我們嘗試在液滴當中加入0.08 % Pluronic F127之界面活性劑，以減少生物分子沉積並吸附於晶片表面之現象，進而幫助液滴驅動藉由介電濕潤力。除了以上培養液液滴之移動試驗外，我們亦嘗試各種不同的方式進行胚胎培養於晶片上。目前所使用之胚胎為ICR鼠胚，於KSOM + AA培養液液滴中進行培養。由實驗結果顯示，胚胎在有旋塗Teflon之晶片表面上(囊胚百分比(blastocyst %)為52.5 %)或者是在含有0.08 % Pluronic F127之界面活性劑的培養液液滴中(blastocyst % = 87.5 %)，皆與petri dish培養之結果無差異性(P > 0.05)，故本實驗所使用的晶片材料與0.08 % Pluronic F127界面活性劑皆具良好的生物相容性。我們也嘗試於晶片系統進行靜態胚胎培養，發現晶片培養系統的blastocyst %值(blastocyst % = 62.5 %)相較於petri dish之培養結果(blastocyst % = 55 %)，有略為上升，故可證實本系統於胚胎培養上之可行性。此外，我們已可長時間移動(72 hr)包覆式培養液液滴，並逐一進行胚胎之動態和培養參數測試，使整個胚胎系統更為完整。 關鍵字：體外培養、介電濕潤、微流體晶片、微液滴、Pluronic F127 界面活性劑

In this study, we manipulate embryo culture medium droplets by electrowetting-on-dielectric (EWOD) to achieve dynamic embryo culture and retain the embryo growth factor, and then mimic the environment of in vivo dynamic culture like the oviduct. In addition, we aim to develop dynamic culture for a single embryo, which will help to analyze the embryo development individually. In the experiment, we actuate the KSOM + AA medium droplet containing amino acids and the other biomolecules by EWOD. The biomolecules could result in deposition and adsorption on the surface. Therefore, we try to add the 0.08 % Pluronic F127 surfactant to the medium droplet reducing the deposition and adsorption on the surface of the chip to help actuation by EWOD force. In addition to the above, we also try different methods to test the embryo culture on a chip. Currently, we use the ICR mouse embryos to cultivate in the KSOM + AA medium droplet. By the experimental results, embryos are on the chip surface that is coated with Teflon (blastocyst% = 52.5 %) or in the medium droplet containing 0.08% Pluronic F127 surfactant (blastocyst% = 87.5 % ), which all have no significant difference with the culture results of petri dish (P > 0.05). Therefore, the chip material and 0.08% Pluronic F127 surfactant all have good biocompatibility. We also try to do the static embryo culture in the chip system, and we find that the blastocyst % value of chip culture system rises slightly (blastocyst% = 62.5 %) comparing to the result of petri dish culture system (blastocyst% = 55 %). Thus we can confirm the feasibility of our system by above results. In addition, we can move the encapsulated medium droplet for a long time (72 hr), and we start to test the parameters of dynamic embryo culture to make the whole embryo system more completion.

Keywords: in vitro culture, electrowetting-on-dielectric (EWOD), microfluidic chip, micro-drop, Pluronic F127 surfactant