Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes
Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes
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DOI：
10.1039/b405557h
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发表时间：
2005-01-01
期刊：
影响因子：
6.1
通讯作者：
Toner, M
中科院分区：
文献类型：
--
作者：
Revzin, A;Sekine, K;Toner, M
将白细胞组织成高密度阵列使这些细胞易于快速光学表征和随后的分选，从而指向临床和基础科学应用。本论文描述了一种用于创建高密度白细胞阵列的细胞仪平台的开发，并演示了从阵列中检索单细胞。采用聚（乙二醇）（PEG）光刻法来制造由PEG水凝胶壁和大小为20 μ m × 20 μ m和15 μ m × 15 μ m的玻璃附着垫组成的微孔阵列。PEG微图案化的玻璃表面进一步用细胞粘附配体、聚-L-赖氨酸、抗-CD 5和抗-CD 19抗体修饰，以在各个威尔斯孔内设计特异性细胞表面相互作用。通过荧光显微镜观察PEG微孔的玻璃附着垫中荧光标记蛋白的定位。用PEG和细胞粘附配体微图案化的载玻片暴露于T淋巴细胞30分钟。这些锚定非依赖性细胞被选择性地捕获在配体修饰的微孔中，形成高密度细胞阵列。发现微孔中的细胞占有率是抗体依赖性的，对于用T细胞特异性抗CD 5抗体修饰的微孔，达到94.6 +/-2.3%。研究了激光捕获显微切割（LCM）作为从阵列中分选细胞的方法，并证明了单个选定细胞的检索。
Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly( ethylene glycol) (PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 mum x 20 mum and 15 mum x 15 mum in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.