現雖有不同幹細胞分離技術，其中薄膜分離技術是由本實驗室提出且為最簡便、易保持無菌、及維持細胞活性的方法，為臨床醫師認為最具潛力的細胞分離術。臍帶血之血液幹細胞經由膜分離技術，可得到7-20%的回收率。此研究也發展臍帶血幹細胞之體外直接膜培養，可得到6.6倍的血液幹細胞增殖倍數。為了最終應用於臨床醫療之目的，此研究更進ㄧ步設計閉鎖式循環膜培養系統，封閉的培養環境可降低污染的可能性，而不斷循環的培養基則可提供充足的養分給予附著在膜上的血液幹細胞，利用此ㄧ系統可得到14.6倍的血液幹細胞增殖率。除了創新的膜分離培養技術外，此研究也比較了目前普遍使用的免疫磁珠法分離血液幹細胞，被分離的血液幹細胞進而培養在不同的二維及三維培養材料上，由此研究結果發現，血液幹細胞培養在三維材料上，其增殖率小於二維材料，這可能由於血液幹細胞表面表現黏著的分子(CD164)，容易附著於3D膜的表面之緣故。此研究也進行了血液幹細胞的分化能力鑑定。閉鎖式循環膜培養系統與免疫磁珠法分離的血液幹細胞培養可得到接近的血液幹細胞增殖倍率，但閉鎖式循環膜培養系統從分離到進行培養只需30分中的操作時間，免疫磁珠法卻需耗費5-8個小時，閉鎖式循環膜培養系統為一具有潛力之血液幹細胞體外增殖的方法。 Stem cell separation through membrane filtration method is simple and inexpensive, and sterility is easy to maintain during the process. The recovery ratio of HSCs from umbilical cord blood (UCB) was found to be 7-20% depending on the recovery solution. A high recovery ratio of HSCs was obtained when 20 wt% of dextran was used as the recovery solution. Direct ex vivo expansion of hematopoietic stem cells (HSCs) from UCB by membrane filtration method has been developed in this study. This study also reports the expansion ratio and ability of colony-forming units of HSCs from UCB purified by conventional Ficoll-Paque and MACS medthod, batch type method of direct ex vivo expansion through membranes and bioreactor of perfusion method of direct ex vivo expansion through membranes, and compared these results. Ex vivo expansion of HSCs compared to initial number of HSCs in UCB was found to be 6.6 folds when was used as a rinsing solution in batch type of direct ex vivo expansion of HSCs through the PU-COOH membranes. Ex vivo expansion of HSCs in 3D culture was found to be worse than that in 2D culture using HSCs purified by Ficoll-Paque and MACS method. This is because pluripotent HSCs adhered on PU membranes tightly, and HSCs in PU membranes of 3D culture could not be detached even after pipetting with washing solution. However, ex vivo expansion of HSCs in bioreactor of perfusion method was found to be similar ex vivo expansion fold compared to that in 2D culture using HSCs purified by conventional Ficoll-Paque and MACS method, which needs longer working time (e.g., 5 hours). The bioassay of HSCs by colony-forming units where HSCs were cultured after purification of HSCs from UCB by conventional Ficoll-Paque and MACS method and by our direct ex vivo expansion method was also investigated. Perfusion type of direct ex vivo expansion method of HSCs from UCB through membranes can provide high ex vivo expansion fold with simple procedure and short working time for HSC purification before HSC culture.
