中国激光, 2022, 49 (24): 2407104, 网络出版: 2022-11-09  

基于多光子聚合微笼阵列的单细胞捕获方法 下载: 680次封面文章

Single Cell Capture Method Based on Multiphoton Polymerization Microcage Arrays
杨婷 1,2,3孙丽娜 1,*代国朋 1,2,3吕孝峰 1,2,3王晓朵 2,3,**
作者单位
1 东北大学机械工程与自动化学院,辽宁 沈阳 110819
2 中国科学院沈阳自动化研究所机器人学国家重点实验室,辽宁 沈阳 110016
3 中国科学院机器人与智能制造创新研究院,辽宁 沈阳 110016
摘要
基于飞秒激光单脉冲多光子聚合原理加工出了高纵宽比的微柱阵列,将其与毛细力自组装相结合,有效实现了单细胞阵列的原位限域捕获;通过优化激光加工参数,实现了锥状微柱阵列的高效率加工,并研究了不同激光功率下微柱直径随高度的变化规律;通过优化微柱阵列参数,实现了基于毛细力自组装原理的三维图案化微结构阵列的高通量制备。在此基础上,本团队进行了二氧化硅微球、乳腺癌细胞(MCF-7)单细胞阵列的原位限域捕获实验验证。荧光成像及扫描电子显微镜的表征结果显示,所提方法可以简单、高效地实现单细胞阵列的高通量原位捕获。本研究提供了一种简便、高效的单细胞阵列原位捕获方法,有望应用于生物医学领域单细胞尺寸的相关研究上。
Abstract
Objective

The study of single cell is of great significance in the fields of cell heterogeneity, genetic metabolism, genetic engineering, and toxicity detection. To identify the functional characteristics of individual cells, individual cell capture must first be achieved. However, most trapping methods require a constant force to keep the cell trapped. When the force decreases or disappears, the cells easily revert to a disordered state, which is very harmful to subsequent characterization and analysis. Therefore, a method for capturing individual cells stably without the use of constant external forces is expected to open up a new avenue for single cell research. This paper proposes a method of rapid capture of single cell during the self-assembly process of micropillars based on femtosecond laser multiphoton processing technology and the principle of capillary force self-assembly. It has fast capture, convenient operation, and a broad application range, and has a lot of potential in bioengineering, drug analysis, and other fields.

Methods

High aspect ratio micropillars were prepared by single-pulse femtosecond laser multiphoton polymerization. The height and space of the micropillars can be adjusted by moving the micro/nano translation stages vertically and horizontally. The laser-processed sample was developed upside down by a developer for 6 min to remove the photoresist in the unprocessed area. After the sample was developed, it was washed in isopropanol solution for 10 min to remove any residual developer. To prevent the self-assembly of the sample after developing and cleaning, we put it into a PBS solution immediately. The sample was sealed and degassed for 8 h before being disinfected. Drop the cell solution directly above the micropillar arrays with a density of 1.2×105 cell/mL. After seeding cells, the sample was placed in the cell incubator for 2 h to allow the cells to fully adhere to the Petri dish and fall into the bottom of the micropillar gap. Subsequently, the uncapped cells were washed off with trypsin, and the sample was processed using living dead staining. Using a pipette gun, remove the liquid and allow the residual solution to evaporate naturally. The cells between the micropillars were captured during the self-assembly process of micropillars.

Results and Discussions

The micropillar structure with a high aspect ratio has a large diameter at the bottom and gradually shrinks to the top, showing a conical shape (Fig. 1). The bottom diameter of the micropillar gradually increases as the height and laser power increase. By shortening the distance between micropillars, increasing the distance between self-assembly structures, and reasonably adjusting the height of micropillars, the micropillars close to each other can be self-assembled based on capillary force, to realize the high-efficiency preparation of largescale three-dimensional (3D) complex patterned self-assembly structures (Fig. 2). The experiment of micropillars’ self-assembly driven by capillary force to capture microspheres shows that the micropillars can still be self-assembled into microcage structures when there are particles in the micropillar gap (Fig. 3). Based on the above methods, a single cell array capture experiment was carried out. The results of fluorescence imaging and scanning electron microscope (SEM) images show that this method can realize high-throughput in situ capture of single cell array simply and efficiently (Fig. 5). Additionally, the cell capture experiment of microcage composed of a different number of micropillars provides a relatively simple method for sorting, capturing, and in situ observation of cells of different sizes (Fig. 6). The four micropillar microscage can only capture cell with similar diameter of the microcage, providing a new method for cells sorting. The six micropillar microscage can capture different sizes of cells, and single cell analysis experiment can be carried out by the micropillars’ gap. The eight micropillar microscage can strictly restrict the captured cell well in the microcage and is expected to be used in domain-limited growth characteristics research of single cell.

Conclusions

To meet the application requirements of high throughput single cell capture, a domain-limited passive capture method of single cell arrays based on self-assembly of a micropillar array is proposed. Based on femtosecond laser single pulse multiphoton polymerization technology and the capillary force self-assembly principle, this method realizes the high-throughput in situ capture of the MCF-7 single cell array. Using femtosecond laser single pulse multiphoton polymerization to achieve the high-efficiency preparation of micropillar arrays with a high aspect ratio. By optimizing the spacing and height of micropillars as well as largescale path planning, high throughput capillary force self-assembly of a largescale 3D complex microcage structure was achieved. The in situ capture of a single cell array based on capillary force self-assembly was successfully realized by matching the structural parameters of the microcage with the size of the cells. Different from the traditional single cell capture methods, such as single cell orientation capture method previously proposed by us, this method provides a relatively simple and efficient method for the sorting, capture, and in situ observation of cells with different sizes without the need for continuous additional external force. It has various application prospects in the fields of bioengineering, pharmaceutical analysis, and other domains.

杨婷, 孙丽娜, 代国朋, 吕孝峰, 王晓朵. 基于多光子聚合微笼阵列的单细胞捕获方法[J]. 中国激光, 2022, 49(24): 2407104. Ting Yang, Lina Sun, Guopeng Dai, Xiaofeng Lü, Xiaoduo Wang. Single Cell Capture Method Based on Multiphoton Polymerization Microcage Arrays[J]. Chinese Journal of Lasers, 2022, 49(24): 2407104.

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