In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.
In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.
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DOI：
10.1016/j.jbiotec.2008.01.015
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发表时间：
2008-04
影响因子：
4.1
通讯作者：
H. Takamatsu;S. Uchida;T. Matsuda
中科院分区：
文献类型：
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作者：
H. Takamatsu;S. Uchida;T. Matsuda
开发了一种在显微镜下从不同细胞类型的多细胞混合物中获取靶细胞的新技术和仪器装置。这项技术的原理是，培养在热敏物质涂层培养皿上的细胞通过特定区域的冷却装置分离，并同时使用微吸管采集，两者都组装在倒置显微镜中。温敏涂层由聚N-异丙基丙烯酰胺(PNIPAAm)和PNIPAAm接枝明胶组成。前者的非细胞粘附性聚合物在低于32.1℃的水中溶解，并在该温度下沉淀(称为低临界溶液温度，LCST)，后者的LCST为34.1°C。这些温敏聚合物的合适混合比例在生理温度下表现出较高的细胞粘附性，在室温下表现出完全的细胞分离。一种只在培养皿底部的一小块区域冷却的装置，在该区域下，显微镜下对准的细胞被组装在显微镜中。研究表明，单个或两个相互粘连的细胞可以在大约30秒内从表面分离并被微吸管采集。
A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1°C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1°C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.