人類多能性幹細胞 (human pluripotent stem cells (hPSCs))因具有自我增生能力以及分化能力，在臨床的醫學治療研究上具有極大的淺力。然而，臨床治療需要極大量的人類多性幹細胞，而一般的細胞培養採用的是費時又費力的批次培養。為解決上述問題，本實驗室利用熱敏感材料設計一個連續是細胞培養。將人類胚胎幹細胞培養於熱敏性基材表面，當我們需要收集細胞時，僅需將溫度降低於熱敏性高分子的低臨界溶解溫度 (lower critical solution temperature (LCST)) 以改變基材表面之親疏水性質以達到細胞部分脫附效果。而未脫附細胞將繼續培養於溫度高於低臨界溶解溫度的原熱敏性基材上。 因人類多能幹細胞必須貼附於特定的細胞外基質(Extracellular matrix (ECM))表面上，然而在此研究中，細胞外基質會隨著每一次收集細胞的次數而跟著減少；因此，在此實驗中，為確保細胞維持一定的生長效率，必須再加入一些ECM以確保，細胞能貼附在熱敏感材料，此方法不同於一般的ECM塗佈方法(pre-coating way)，其必須既能同時培養細胞，又能同時加入ECM，我們稱此方法為非塗佈法(un-coating way). 此研究選用重組玻連蛋白(recombinant vitronectin(rVN))及重組層粘連蛋白(recombinant laminin-511(iMatrix-511))兩種細胞外基質。研究在非塗佈且不同比例混合之兩種ECM的環境下，對於細胞生長之影響，並找出最適當的混合比例濃度，再將此非塗佈方法及條件應用於連續式的培養。 本研究成功將人類多能性幹細胞，包括人類胚胎幹細胞(human embryonic stem cells (hESCs))以及人類誘導性多能性幹細胞(human induced pluripotent stem cells (hiPSCs))，培養於非塗佈(un-coating way)的環境及兩種ECM適合的混合比例下長期培養，仍保有原有的多功能性以及分化能力。此非塗佈的方法能避免在塗佈時所使用的溶劑，影響未能自熱敏感材料上脫附的細胞死亡。若此連續培養方式從2D培養方式到3D培養系統，將成為醫學應用領域的一大益處。 ;Stem cells are attractive source for tissue engineering applied on regenerative medicine, translational medicine, and drug discovery. Batch type culture is the typical process for stem cell culture, which is laborious and expansive. Moreover, the digestive enzymes or EDTA solutions to detach stem cells are causing cell damage and increasing production costs. For reducing the costs and laborious processes, I developed the continuous culture system culturing the human pluripotent stem cells (hPSCs) on thermoresponsive polymer surfaces. hPSCs could be detached from the thermoresponsive polymer surfaces by reducing the medium temperature below the lower critical solution temperature (LCST) of the thermoresponsive polymer. After reducing the temperature, the thermoresponsive surface becomes more hydrophilic, creating an unfavorable environment for cell attachment. Subsequently, hPSCs can be partially detached by gentle pipetting. The remained cells could be cultivated and would be confluent again by adding fresh medium. This continuous culture system could be used for 3D-cultivation. However, the barrier of applying this system is that hPSCs became harder to attach on the thermoresponsive surfaces coated with the extracellular matrix (ECM) after multicycles. This should be caused by degradation of ECM after long-term culture. Therefore, I investigated the precoated manner and uncoated manner with different ratios of ECMs; recombinant vitronectin (rVN) and recombinant laminin-511 (iMatrix-511). Comparing with traditional precoated process, the uncoated method where optimal ratio and concentration of ECMs were added into culture medium upon seeding cells, provided cost-effective and time-efficient method and maintained hPSCs pluripotency during long-term culture. This uncoated method that was applied to the continuous culture system would save time cost and accelerate the development of regenerative medicine.
