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 × 10⁴ cells/cm²) 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.

解释细胞-细胞和细胞-微环境相互作用对于提高基础生物学知识和促进再生医学的应用至关重要。在以前的研究中已经广泛研究了细胞模式。然而，报道的方法不能同时实现对细胞排列和粘附/扩散的高效率和高通量的精确控制。在这里，提出了一种以微孔阵列作为荫罩的新型固体剥离方法。通过巧妙设计的荫罩同时实现对细胞排列和粘附/扩散的有效和精确控制，其中包含中央区域的大微孔（捕获孔）和周围区域的小微孔（扩散孔），有助于捕获/排列和粘附/分别控制传播。固体剥离的功能如下：（1）通过捕获孔和扩散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，以及（3）通过先前控制细胞粘附/扩散剥离阴影掩模后生成的蛋白质微图案。高通量 (2.4–3.2 × 10以高效率实现了^{4 个}单元格/cm ² ) 单元格对齐（对于单单元格、双单元格和三单元格对齐分别为 86.2 ± 3.2%、56.7 ± 9.4% 和 51.1 ± 4.0%）。使用鼠骨骼肌成肌细胞研究和验证了细胞扩散的精确控制和用于调节细胞骨架和细胞 - 细胞连接的应用。据我们所知，这是第一份通过简单的固体剥离操作同时展示高效和可控的多细胞对齐和粘附/扩散的报告。该研究成功地填补了文献空白，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和可重复应用。