Research of cell behavior on micro-textured substrate

• Main Authors: Wen-Ta Su, 蘇文達
• Other Authors: I-Ming Chu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/72048201237343935901

博士 === 國立清華大學 === 化學工程學系 === 94 === 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 mm) parallel ridge/groove with 30 mm 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 mm 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.