Hun Tingting, Liu Yaoping, Guo Yechang, Sun Yan, Fan Yubo, Wang Wei
Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, 100191 Beijing, China.
Institute of Microelectronics, Peking University, 100871 Beijing, China.
Microsyst Nanoeng. 2020 Sep 7;6:86. doi: 10.1038/s41378-020-00191-5. eCollection 2020.
Interpretation of cell-cell and cell-microenvironment interactions is critical for both advancing knowledge of basic biology and promoting applications of regenerative medicine. Cell patterning has been widely investigated in previous studies. However, the reported methods cannot simultaneously realize precise control of cell alignment and adhesion/spreading with a high efficiency at a high throughput. Here, a novel solid lift-off method with a micropore array as a shadow mask was proposed. Efficient and precise control of cell alignment and adhesion/spreading are simultaneously achieved via an ingeniously designed shadow mask, which contains large micropores (capture pores) in central areas and small micropores (spreading pores) in surrounding areas contributing to capture/alignment and adhesion/spreading control, respectively. The solid lift-off functions as follows: (1) protein micropattern generates through both the capture and spreading pores, (2) cell capture/alignment control is realized through the capture pores, and (3) cell adhesion/spreading is controlled through previously generated protein micropatterns after lift-off of the shadow mask. High-throughput (2.4-3.2 × 10 cells/cm) cell alignments were achieved with high efficiencies (86.2 ± 3.2%, 56.7 ± 9.4% and 51.1 ± 4.0% for single-cell, double-cell, and triple-cell alignments, respectively). Precise control of cell spreading and applications for regulating cell skeletons and cell-cell junctions were investigated and verified using murine skeletal muscle myoblasts. To the best of our knowledge, this is the first report to demonstrate highly efficient and controllable multicell alignment and adhesion/spreading simultaneously via a simple solid lift-off operation. This study successfully fills a gap in literatures and promotes the effective and reproducible application of cell patterning in the fields of both basic mechanism studies and applied medicine.
细胞间以及细胞与微环境相互作用的阐释对于推进基础生物学知识和促进再生医学应用都至关重要。细胞图案化在先前的研究中已得到广泛探究。然而，所报道的方法无法同时在高通量条件下高效地实现对细胞排列以及黏附/铺展的精确控制。在此，提出了一种以微孔阵列作为荫罩的新型固体剥离方法。通过一个巧妙设计的荫罩可同时高效且精确地控制细胞排列以及黏附/铺展，该荫罩在中心区域包含大微孔（捕获孔），在周边区域包含小微孔（铺展孔），分别有助于捕获/排列以及黏附/铺展控制。固体剥离的作用如下：(1) 通过捕获孔和铺展孔生成蛋白质微图案；(2) 通过捕获孔实现细胞捕获/排列控制；(3) 在荫罩剥离后，通过先前生成的蛋白质微图案控制细胞黏附/铺展。实现了高通量（2.4 - 3.2×10个细胞/平方厘米）的细胞排列，且效率较高（单细胞、双细胞和三细胞排列的效率分别为86.2±3.2%、56.7±9.4%和51.1±4.0%）。使用小鼠骨骼肌成肌细胞对细胞铺展的精确控制以及调节细胞骨架和细胞间连接的应用进行了研究和验证。据我们所知，这是第一份通过简单的固体剥离操作同时证明高效且可控的多细胞排列以及黏附/铺展的报告。本研究成功填补了文献空白，并促进了细胞图案化在基础机制研究和应用医学领域的有效且可重复的应用。
