高通量單細胞篩選與細胞單株化培養之雙微孔微流晶片

High-Throughput Single-Cell Isolation and Clonal Culture Using a Microfl uidic Dual-Well Chip

單細胞的研究在近年來越趨熱門，原因在於一般利用群體細胞進行的研究無法得知細胞間異質性所造成的影響，因而限制了大家對於細胞行為的了解。然而目前仍缺乏一個操作簡單又具有高通量的方法進行單細胞培養實驗。在此研究中，我們設計了一款雙微孔（dual-well(DW)）單細胞篩選微流晶片，能高效率（約77%）地將單細胞個別抓取後置入一個大培養空間中，藉以提供足夠的空間，供細胞貼附及長時間生長。DW晶片的設計是採用一組篩選細胞的小微孔結構對應到另一個培養細胞的大微孔結構。這個設計概念除了能大幅提昇細胞單株化培養的效率外，也讓細胞培養微孔的大小可根據實驗需求做彈性的調整，而不會影響單細胞篩選效率。我們選用小鼠神經幹細胞KT98、人類肺癌細胞株A549及黑色素瘤細胞株和MDA-MB-435進行DW晶片的功能性及效率評估，以及利用A549進行單細胞的群落形成實驗（single-cell colony formation assay），來驗證DW晶片在單細胞長時間培養及細胞單株化應用的可行性。 Single -studies have been gaining growing interests owing to the fact that whole-cell population studies cannot accurately detect cellular heterogeneity presents among the whole cell population, limiting our standing in coordinated cell behaviors. However, traditional methods for studying single cells rely on macro-scale tools that are either labor intensive or difficult to operate. Thus, the development of a high-throughput yet simple method for single cell culture experiments is of great importance. Here, we report a novel microfluidic device with a dualwell(DW) design concept for high-efficiency single-cell capture in large microwells(~77%) , which provides ample space for cells to proliferate and differentiate. The ability of our DW device to simultaneously allow for high-efficiency single cell capture and culture was realized using sets of small microwells for cell capture, paired with large microwells for cell culture. By decoupling the capture and culture functionalities in our device, we were able to flexibly adjust the size of the large culture microwells without compromising single-cell capture efficiency. We also demonstrated single-cell acquiring efficiency of KT98 mouse neural stem cells, A549 and MDA-MB-435 cancer cells, and single-cell colony formation assays using our device, showing the diverse and flexible functionality of the DW device in cell culture applications.