Abstract
Droplet microfluidics has become a powerful tool in many biomedical researches, such as single-cell analysis, drug screening, digital polymerase chain reaction, and clinical diagnostics. Monodisperse droplets as isolated reaction chambers are ideal for profound single-cell analysis, because they provide an independent microenvironment to study the protein secretion, enzyme activity, and proliferation of individual cells. These droplets can also reduce cross-contamination and maintains cell activity at the single-cell level. Compartmentalization of single cell into each individual water-in-oil microdroplets has revolutionized genomics, transcriptomics, and proteomics research. However, droplet generation with single-cell encapsulation is a random process, which also results in a large number of empty and multi-cells droplets. Although the current microfluidics sorting technologies can achieve high-purity enrichment of single-cell droplets, they suffer from drawbacks such as fluorescent labeling, inability to remove multi-cells droplets, or low throughput. This thesis presents both active and passive technologies to achieve single-cell encapsulation, and applies them to mitochondrial transfer for the senescent phenotype reversal of mesenchymal stem cells (MSCs).First, a gray-level guided image-activated droplet sorter (GL-IADS) was developed. The sorter enables label-free, high-accuracy screening of single-cell droplets by rejecting empty and multi-cells droplets. The gray-level based recognition method can accurately classify droplet images (empty, single-cell, multi-cells droplets), especially in differentiating empty and cell-laden droplets (accuracy of 100%). Crucially, this method reduces the image processing time to ~300 microseconds, which makes the GL-IADS possible to reach an ultra-high sorting throughput up to hundreds or even kHz. The GL-IADS integrates the novel recognition method with a detachable acoustofluidic system, achieving sorting purity of 97.9%, 97.4%, and >99% for single-cell, multi-cells, and cell-laden droplets, respectively, with a throughput of 43Hz. The GL-IADS holds promise for numerous biological applications that are previously difficult with fluorescence-based technologies.
Second, an inertial-focusing-assisted droplet microfluidics platform for quantitative high-throughput mitochondrial transfer was developed. A spiral microchannel focuses the cells into a single equilibrium position through an inertial effect. The focused cells reach the droplet-generating junction and are encapsulated into droplets one by one, while the mitochondrial suspension is simultaneously encapsulated in these droplets. The deterministic encapsulation of cells led to a high ratio of single-cell droplets and a low ratio of multi-cells droplets. In this technique, consistent droplet size and a stable volume fraction of mitochondrial solution per droplet keep the number of mitochondria in each droplet within a certain range. The single-cell encapsulation is conducive for quantitative control of mitochondria transferred to each recipient cell. The developed technique provides a cell therapy strategy for mitochondria-related diseases.
Third, the developed inertial-focusing-assisted droplet microfluidics platform was used to transfer mitochondria isolated from young MSCs to senescent MSCs. The in-vitro and in-vivo experiments demonstrated that this mitochondrial transfer with number of 19 can enhance the proliferation capacity and metabolic activities of senescent MSCs, transforming the phenotype of senescent MSCs to young MSC-like phenotype.
In summary, this thesis demonstrates an important significance of droplet microfluidics for single-cell encapsulation and mitochondrial transfer. The proposed GL-IADS enables label-free, high-accuracy screening of single-cell droplets, multi-cells droplets, and both (cell-laden droplets) on demand. The sorter can be integrated into a broad range of droplet microfluidics-based screening applications. The inertial-focusing-assisted droplet microfluidics platform can achieve high-throughput, high-efficiency, and quantitative mitochondrial transfer. Moreover, the platform demonstrates the effectiveness of mitochondrial transfer on ameliorating MSCs aging, which can serve as a novel strategy to improve MSC function in tissue engineering and cell therapy of mitochondria-related diseases.
| Date of Award | 18 Sept 2025 |
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| Original language | English |
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| Supervisor | Gang Gary FENG (Supervisor) & Dong SUN (Co-supervisor) |