A micropore array-based solid lift-off method for highly efficient and controllable cell alignment and spreading

Tingting Hun,Yechang Guo,Yaoping Liu,Yubo Fan,Wei Wang,Yan Sun

doi:10.1038/s41378-020-00191-5

Abstract

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.

Highlights

Cell patterning is very useful to reveal the mechanisms of cell physiological processes, such as gene expression[1], apoptosis[2], differentiation[3], and migration[4]
In this study, a novel solid lift-off method using a microfabricated micropore array as a shadow mask was successfully developed to obtain high-throughput protein patterning with high precision and cell patterning with high efficiency
The flexibility of Parylene C micropore arrays allowed protein and cell patterning on curved substrates. This solid lift-off method successfully fills a gap in the literature as the first report to demonstrate the precise control of cell alignment and adhesion/spreading simultaneously at a high throughput with high efficiencies

Summary

Introduction

Cell patterning is very useful to reveal the mechanisms of cell physiological processes, such as gene expression[1], apoptosis[2], differentiation[3], and migration[4]. Cell patterning methods can be mainly classified into two types: active and passive approaches. The passive approaches have attracted much attention in biological practices because of their simple operation, which mainly include microwell-assisted cell capture and confinement[17,18,19,20,21] and protein micropattern-controlled cell alignment and spreading[22,23,24,25,26,27,28,29].

Methods

Results

Conclusion