本論文主要目的是利用電子工業微製造技術所製作的微米構形材料來探討細胞生長情形，並用構形反應器來增殖人類造血幹細胞的數目。
我們利用30 □m深，15~100 □m不等寬度微溝槽構形的疏水性PDMS膜在37℃含血清的培養液中，探討老鼠間質細胞的遷移行為。以time-lapse相位差顯微鏡持續25h觀察細胞形態與移動位置的變化。當細胞種入時，大部分的細胞都沉入凹槽內，隨即在材料表面貼附與展延，並往凹槽側面移動，在凹槽/凸脊間不斷地爬上爬下，顯然這30 □m深的落差，並不會造成細胞移動上的障礙，最後大部分的細胞以單層貼附群聚在凸脊上，因接觸抑制而停止增殖。細胞形狀也因溝槽構形而被拉長與溝槽方向平行，形狀指標(FI)是平面上的2.6倍，從移動速率來看，疏水性的表面比親水性表面更能驅動細胞的遷移，移動速率也較快。
當纖維細胞生長在柱狀構形材料上，細胞改變了貼附模式，焦點接觸，細胞骨架重排，細胞形態與移動的方向。所用柱狀構形是直徑1□m，間距3□m或9□m，1□m；5□m；10□m等3種高度的方型排列圓柱，具有相同的表面化學性質。柱狀構形能提供更多細胞貼附的機會，但也是一種限制細胞移動的障礙。當細胞種入時會被一根或數根柱子撐著但沒有刺破細胞，有些細胞則沉入柱子間隙，因而固定了貼附的位置，隨後細胞質膜向外沿著柱子側面貼附往材料底部延伸，形成細長的細胞質膜。Si-1-9基材上的細胞形狀指標是3.7，但 Si-1-3基材上則高達35，是平面上培養細胞的23.6倍。另一個評估細胞形狀變化的指標是細胞高度，構形基材上的細胞在z軸分布的高度是平面上的3.6倍。在細胞移動上，多數的細胞都往同一方向延伸，有些則同時往x與y的方向延伸，形成相當有趣的細胞形態，像是阿拉伯數字7與英文字母Y的字形，這些變化趨勢與柱子高度成正比。因此材料表面的構形會強烈地影響細胞的形態與展延的方向。
我們設計微柱狀反應器模擬體內骨髓微環境，對造血幹細胞作體外增殖培養。首先將老鼠骨髓中培養出的間質細胞( M2-10B4 )種入反應器內當作飼養層細胞，提供一些細胞激素調節造血幹細胞生長。經過21天的培養，在微柱狀反應器中造血幹細胞增殖數目是平面培養的3.69倍，累積的菌落形成能力是平面培養的2.1倍。這些結果顯示微構形基材對造血幹細胞體外增殖培養是有效的，可歸因於柱狀構形基材較能提供適當的細胞與細胞、細胞與胞外基質的互動，因而加速細胞的生長。

The aims of this thesis are to explore the cell behavior on micro-textured substrate by microfabrication techniques and to proliferate hematopoietic stem cells (HSCs) in topographic reactor thereafter.
We used a hydrophobic micro-grooved poly-dimethylsiloxane (PDMS) in medium with 10% FBS at 37℃ to study the motility of mouse stromal fibroblast on variant (15~100 □m) parallel ridge/groove with 30 □m depth. We observed the temporal changes in cell morphology and locomotion by using time-lapse phase-contrast microscopy for 25h. When fibroblasts were seeded onto the micro-grooved substrate, almost all of cells concentrated at the bottom of the grooves. Sequentially, the fibroblasts attached and spread out on the surface, migrated toward the walls of the grooves, climbed up and down the ridges. Apparently, the 30 □m depth of groove was not an obstacle, while cells migrated across the microgrooves. Eventually, they stopped proliferation as a result of contact inhibition and formed a confluent monolayer on the ridges with an orientation parallel to the direction of the ridge/groove. That is to say, cellular motility was directed to the top ridges. Cellular shape of fibroblast was enhanced with the microgrooves, the form index (FI) of nucleus was 2.6-fold greater than that of cells on smooth surfaces. Further, from the velocity of cellular moving，hydrophobic surfaces are more prone to direct cellular motility in comparison with hydrophilic surfaces.
Fibroblasts alter their mode of attachment, focal contact, cytoskeleton arrangement, cellular shape and direction of movement, when placed on square arrays of silicon pillars. All of the pillars that we studied had 1-μm diameters with identical surface chemistry, and were separated by 3 μm and 9 μm, but with different heights (1, 5, or 10 μm). We found that these micro-pillars provided more opportunities for mechanical interlocking of the fibroblasts, rather than specific interactions, and acted as physical barriers that restrain cell migration. When cells were seeded initially on pillar substrate, fibroblasts subsequently were immobilized in situ by one or some pillars that visibly protruded into the cell body, but did not pierce; while some fibroblasts were filled in the intervals between four pillars. Subsequently, cytoplasma migrated outward to the bottom of substrate with long straight lamella along the interval of the pillars and formed several discrete attachment zones at their side walls. The maximal form index (FI) of the cells on the Si-1-9 pillar substrate was 3.7, but form index (FI) was as high as 35 (ca. 23.6-fold greater than that of cells on smooth surfaces) on Si-1-3-10 pillar substrate. Changes were observed in cellular body height, there was 3.6-fold increase in cell height on pillar substrate than that of conventional planar-cultured substrates. Therefore pillar substrate altered the cellular shape entirely. On cellular migration, most of the cells interacted with the pillar substrate by spreading preferentially in a particular direction, but some of them had the ability to undergo coincident two-direction (x and y) migration; right-angle turn orientations led to the growth of dramatic cellular morphologies, for example, the morphology of an Arabic numeral 7 and a letter of Y-shaped structure. Interestingly, this fibroblast’s behavior variation was proportional to the pillar height of substrate. Our results confirm that cellular migration and cellular shape are both strongly affected by the geometry of the growth microenvironment.
We designed micro-pillar reactor to mimic in vivo bone marrow microenvironment and ex vivo expansion of hematopoietic stem cells. Firstly, mouse-derived bone marrow stromal cells (M2-10B4) were seeded on pillar reactor as a feeder layer to provide some cytokines that regulate hematopoiesis. After 21d culturing, the cell expansion was 3.69-fold and accumulative CFUs was 2.1-fold higher on micro-pillar reactor than that of T25 flask culture. These results suggest that micro-textured substrate enhances the expansion of hematopoietic stem cells and pillar substrate may promote proper cell-cell interactions and cell-ECM interactions.

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