Microstructure-based techniques for single-cell manipulation and analysis
Introduction
Cells are considered the critical functional units integrating living organisms (e.g., human, animals and plant) [[1], [2], [3]]. For this reason, cells have become a critical essential tool for biology and clinical studies [4,5]. The average levels of a group of cells are measured with these conventional single-cell analysis methods, which may be very reasonable in statistics and considered a classical quantitative research method [6]. However, cells are not the bricks adopted to build a tall building because they are heterogeneous and different in numerous characteristics (e.g., mechanical characterization and the protein expression levels) [7]. Thus, such conventional method is likely to miss some vital information for heterogeneity studies [8,9]. In recent years, single-cell based analyzing methods have become frequently adopted to analyze the heterogeneity of cells (e.g., differentiation of stem cells and tumor cell heterogeneity) [10,11]. Mesenchymal stromal cells are heterogeneous in size; it is capable of differentiating fat cells, bone cells, cartilage cells and muscle cells [12]. Cancer stem cells, capable of forming the whole tumor, are a rare population of cancer cells [13].
Conventional single-cell analysis methods consist of dilution method, flow cytometry, laser scanning cytometry, automated microscopy and fluorescence-activated cell sorting [14,15]. These techniques, however, exhibit high processing complexity and time-consuming characteristic, and require expensive and complicated equipment, considerably limiting their applications in cellular heterogeneity study [16]. Accordingly, a novel technique that can remedy the above defects remains a challenge.
Over the past few years, microfluidics-based systems employed to achieve single-cell research have been designed as feasible methods in developing microfluidics techniques [17]. Unlike conventional single-cell analysis method, the advantages of microfluidics-based systems include precise cell capture or manipulation, low reagents consumption, high throughput as well as high integrated functional components [18,19]. Existing microfluidics-based single-cell manipulation techniques could be categorized as active and passive methods. Active methods are capable of isolating single cells based on a range of external forces (e.g., magnetic, optical, acoustic trapping and dielectrophoretic effects) [[20], [21], [22], [23]]. Active methods have been extensively applied for single-cell manipulation and studies. The merits and demerits of active methods have been discussed in a number of recent reviews on single-cell analysis [[24], [25], [26], [27]].
Passive methods refers to an alternative tool to achieve single-cell manipulation and analysis by fluid operation or gravity [28,29], which have been frequently adopted for single-cell manipulation. In comparison with active methods, passive methods are capable of manipulating cell array in a simple and high-density manner without compromising cell viability [30]. Passive methods cover droplet-based methods, flow force, micro-well and microstructure-based method. The merits and demerits of droplet-based methods, flow force and micro-well dedicated to single-cell analysis have also been discussed in several recent reviews [[31], [32], [33], [34]].
In this paper, microstructure-based methods for single-cell manipulation and analysis are mainly highlighted. Microstructure-based methods usually isolate the single cells by the gaps of microtraps. From the general perspective, the microtraps could be categorized as three types (e.g., weir, pillar and pore) [35,36]. Although weir and pore methods have been adopted for single-cell manipulation [[37], [38], [39]], the pillar-based method remains the most frequently used method for single-cell manipulation. Pillar-based method usually comprises of microtrap matrices assembled in a two-dimensional (2D) array. Based on the flow direction, the microstructure-based methods could be classified into two subfields, dead-end mode (DEM) and cross flow mode (CFM) [35,36]. As shown in Fig. 1A, the flow perpendicular to the microtrap and biology micro-particles are driven by the pressure-difference in DEM, the micro-particles larger than the gap of the microtrap are captured by the microtrap, whereas the particles smaller than the microtrap gap could pass through the microtrap. Unlike DEM, the flow of CFM is parallel to the microtraps, and generate two forces (Fig. 1B), one force perpendicular to the microtrap allows biology micro-particles to be captured by microtraps, the other parallel to the microtrap could remove aggregated particles while avoiding microtrap clogging. Furthermore, microvalve, one of the active methods, could operate single-cell and be integrated with subsequent multi-functional detection as microstructure-based methods, and the microvalve for single-cell manipulation is demonstrated in the paper as well (Fig. 1C). Lastly, the challenges required to be overcome in the future are highlighted.
Section snippets
Dead-end mode (DEM)
DEM could capture single cells by the microtrap array, and the geometry and arrange of the microtraps determined the single-cell capture efficiency and throughput. The critical design parameters in microtrap of DEM covers the gap size of microtrap, as well as the fluid flow rate. The fluid flow rate ascertains the force acting on each cell since it is deformed via gap. The cross-sectional opening ascertains the shape and size of the deformed cell that can be taken by the trap. Thus, the
Cross flow mode (CFM)
Though the DEM could be adopted to achieve single cells analysis and manipulation with high throughput, the major demerits of the DEM are potential trap clogging and cell accumulation, causing irregular flow and inefficient separation [6,36]. For this reason, the CFM method for single-cell capture is developed. Unlike the DEM method, the flow of CFM is parallel to the microtraps. This strategy could resolve the problems of trap clogging and cell accumulation during single-cell isolation. In the
Microvalve
Since Quake's group developed the soft lithography to build microvalves [113,114], the microvalve has been applied extensively in biology and medicine. Single-cell manipulation referred to one of the most common areas. By controlling the micro-valve, single cells could be isolated, undergo lysis and receive lysate retrieval, which could be used for complex biological assays by automation and parallelization [115]. The microvalve-based platforms were now being extensively applied in biology and
Conclusions and outlook
In brief, the microstructure-based method for single-cell manipulation and analysis has been significantly developed over the past decade. In this study, defined by the flow direction, the microstructure-based approaches are categorized DEM and CFM methods. Besides, microvalves can operate single-cell and be integrated with subsequent multi-functional detection just like microstructure-based methods, and the microvalves for single-cell manipulation are also demonstrated in this paper.
Acknowledgments
This work is supported by the National Science Foundation of China (81702955 and 81770457), the Natural Science Foundation of Shaanxi Province (2019JQ-885), the Natural Science Foundation of Shaanxi Provincial Department of Education (19JK0771), the National College Students Innovation and Entrepreneurship Training Program, China (201825039); the Project of Shaanxi Key Laboratory of Brain Disorders (18NBZD03) and the Fundamental Research Foundation of Xi’an Medical University (2018PT16).
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