臍帶血是造血幹細胞移殖可行的幹細胞來源。然而從單一臍帶血中能取得的造血幹細胞數量極低，限制了對病患的臍帶血移殖療。 在本研究中，我們使用兩種不同的培養方式，各別是二維平面與三維立體培養法。對2維平面培養，我們使用具有細胞外間質(ECM)與寡糖胜肽蛋白質(如Fibronectin與CS-1)或者是化學官能基(如氨基)固定化接枝的的表面改殖材料進行培養。除此之外，在三維立體培養法上，我們也發展了使用膜過濾法的造血幹細胞直接體外培養法；我們研究如何使用奈米接枝對材料或膜表面改殖，並模仿骨髓幹細胞龕環境，以適用於體外造血幹細胞培養。 對直接體外培養法而言，使用具有COOH基接枝的PU膜具有較高的幹細胞增殖倍率；對膜過濾直接體外培養法而言，潤洗液必需含有超過50%的低血小板血清。使用膜過濾直接體外培養法可藉由簡單的臍帶血分離操作提供三維培養環境與高幹細胞增殖倍率。在二維培養方面，高表面接枝密度氨基不適合培養造血幹細胞，我們發現對造血幹細胞培養而言，氨基具有最適化表面接枝密度。除此之外，在最適化接枝密度下且具有良好表面接枝度控制的細胞外間質表面改殖，有較高的幹細胞增殖倍率。總結而言，PS-FN、PS-CS1-H與PS-Ozone-AMA-0.3mM是出色的造血幹細胞培養生醫材料。 Umbilical cord blood (UCB) is a promising source of stem cells for hematopoietic stem cell (HSC) transplantation. However, the low number of HSCs that can be obtained from a single UCB donor limits the transplantation of UCB for patients. We investigated two culture methods of HSCs, 2D and 3D culture. The 2D-culture used the surface-modified materials immobilized extracellular matrix (ECM) and oligopeptides such as fibronectin and CS-1, or nano-segments having amino group. We also developed a direct ex vivo HSC expansion method followed by 3D-culture of HSCs on the membranes. We investigated how to create and mimic the bone marrow niche for the expansion of HSCs by using nano-segments and ECMs on the surface of the membranes and culture materials. In the direct ex vivo expansion of HSCs, PU membranes having nano-segments of carboxylic acid showed higher expansion fold of HSCs among surface-modified PU membranes investigated in this study. In this case, the rinsing solution should contain platelet-poor plasma (PPP) more than 50%. The direct ex vivo expansion of HSCs from UCB filtered through membranes can provide a simple 3-D culture method of HSCs and a high fold ex vivo expansion of HSCs with a simple operation. In 2D-culture of HSCs, high surface density of amino group on the culture dishes was not favor, and the optimal surface density of amino group was found for HSC expansion. There found optimal amino groups with well controlled surface density and ECMs (CS1-H and Fibronectin) with optimal surface density for high expansion folds of HSCs in 2-D culture. In summary, PS-FN, PS-CS1-H and PS-Ozone-AMA-0.3mM dishes are excellent cultivation materials of HSCs.
