純機械式非酵素型神經球單細胞分離微流晶片

A Microfl uidic Chip for Mechanical Enzyme-Free Dissociation of Neurospheres into Single Cells

到目前為止，還未有一個有效的非酵素型分離方式可以自由神經幹細胞所形成之神經球中分離出大量的活單細胞。因此，本研究利用微流體技術設計一款具有微柱結構的純機械力式非酵素型神經球單細胞分離晶片，我們證明此微流晶片可以將來自老鼠的神經幹細胞KT98及DC115所形成之神經球（培養代數介於3－8代、神經球直徑介於40－250 m m），以90%及95%的高效率分離出大量的單細胞，且經此晶片所分離之單細胞具有80－85%的高存活率。此外，經此晶片所分離出的活單細胞依然保有神經幹細胞之特性，可於神經球培養液中再次培養並形成神經球，並且這些神經幹細胞也保有其幹細胞的分化能力，可在誘導分化的培養條件下成功分化成三種中樞神經系統的主要神經細胞，包含神經元細胞（neurons）、星形膠質細胞（astrocytes）及寡突膠質細胞（oligodendrocytes），故此晶片適合應用於日常性的神經球培養相關實驗上。且因本微流晶片不需酵素參與即可從神經球中分離出大量之活單細胞，故可以降低外源性物質汙染樣品及細胞的風險，很適合使用在需要將神經球分離成單細胞的應用上，並可做為一個廣用的細胞團塊單細胞分離方法。 In this study, we aim to develop new platforms for neurosphere assay and single cell manipulation for neural stem cell research. Neurosphere assay is a common method for identifi cation of neural stem/progenitor cells, but obtaining single cells from dissociated neurospheres is difficult using non-enzymatic methods. We developed a microfl uidic-chip-based approach that utilizes fl ow and microstructures to dissociate neurospheres. Results show that this microfluidic-chip-based neurosphere dissociation method can generate high yields of single cells from dissociated neurospheres of mouse KT98 and DC115 cell models for 90% and 95%, respectively. The microfl uidicchip- dissociated cells had high viabilities(80－85%) and the ability to re-grow into neurospheres, demonstrating the applicability of this device to neurosphere-assay applications. In addition, the dissociated cells retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages(neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions.