Abstract
Mitochondria, which are basic organelles in cells, play important functions in cell life activities, including energy production, cell proliferation, aging, apoptosis, innate immunity, calcium homeostasis, and even stem cell differentiation potential. The mutations of mtDNA impair the functions of cells and tissues. The spontaneous transfer of mitochondria between healthy and damaged cells can occur in nature via different mechanisms, and this process protects damaged cells and restores cellular function. Mitochondrial transfer is a technique to alter mtDNA in cells and has already been used in cell therapy for mtDNA-related diseases. Transferring exogenous mitochondria into recipient cells can restore or improve cell and tissue functions. Although previous studies have shown that exogenous isolated mitochondria can be delivered to cells via simply coculture or microinjection techniques, quantity control and throughput remain challenging. This thesis presents an advanced mitochondrial transfer method based on droplet microfluidics. This method can control the number of mitochondria transferred to recipient cells at the single-cell level in a high throughput manner. The study mainly consists of the following three aspects.First, a droplet microfluidics-based mitochondrial transfer system was developed. This system can perform mitochondrial transfer in a high efficiency, quantity control and high throughput manner at the single-cell level. The co-flowing of cell suspension that received mitochondria and isolated mitochondria suspension was dispersed into droplets with diameters of approximately 40 µm in a droplet generation chip. Single-cell encapsulation efficiency, which is an important factor for the quantitative control of mitochondrial transfer, was improved by using a wave-like structure to align randomly distributed cells from the inlet into a line. The closed microenvironment of droplets limited the travelling distance of isolated mitochondria and increased the probability of contact between isolated mitochondria and recipient cells, thereby facilitating the uptake of mitochondria by the cell and lead to a high mitochondrial transfer efficiency. The number of isolated mitochondria encapsulated together with cells can be controlled by adjusting the concentration of isolated mitochondria suspension used, thus allows the quantitative control of the number of mitochondria transferred into recipient cells. The high-throughput property of droplet microfluidics enabled the proposed system to yield 2 × 106 recipient cells in droplets for mitochondrial transfer in 30 min, thereby making the system a potential tool for manipulating cell therapy products in the clinical application.
Second, the developed droplet microfluidics system was used to transfer mitochondria at varying amounts into C2C12 myoblasts to improve cell functions. In vitro experiments demonstrated that C2C12 myoblasts with 31 transferred mitochondria showed significant improvements in cellular functions, including proliferation rate, energy production, and differentiation ability into myotubes, compared with those with 0, 8, and 14 transferred mitochondria. Hence, C2C12 myoblasts with 31 transferred mitochondria were used in studying the functional outcome and dose effect of cell therapy treatment for injured muscle in in vivo experiments. The selected C2C12 myoblasts showed better therapeutic effects than C2C12 myoblasts without transferred mitochondria.
Third, a low-cost and low-complexity image-activated droplet sorting system was developed to further improve the purity of single-cell encapsulated droplets and quantity control of mitochondrial transfer. The well-known YOLO algorithm was trained and used to distinguish among empty droplets, single-cell encapsulated droplets, and multiple-cell encapsulated droplets in the images acquired through a high-speed camera. A homemade high-voltage generator, which generated pulse voltage of up to 1.2 kV from a low voltage input of 12 V, was connect to 3D electrodes, which were fabricated by perfusion of liquid metal into channels preformed in the droplet sorting chip. Single-cell encapsulated droplets were sorted from the generated or reinjected droplets via the pulse dielectrophoretic forces generated. The high-speed camera was fixed on the microscope eyepiece during the system’s operation, and the high-voltage generator was consisted of an Arduino MCU, a DC voltage supply, and DC-DC voltage amplifier. This design constituted the core part of the sorting system and was simple. All the electric components used were readily available and had low costs. Thus, unlike high-cost fluorescence-activated droplet sorting systems, the developed system is affordable for all interested laboratory, which can considerably contribute to droplet microfluidics-related research, such as single-cell qPCR and clonal expansion.
In summary, the works of this thesis demonstrated an important significance in the field of mitochondrial transfer and droplet microfluidics. The droplet microfluidics-based mitochondrial transfer system exhibited abilities of high efficiency, quantity control, and high throughput on performing mitochondrial transfer. Moreover, and demonstrated that enhancing the effectiveness of cell therapy with mitochondrial transfer before administration is feasible. The proposed image-activated droplet sorting system, which has a low cost and complexity, can further purify the droplets encapsulating single cells. These features can not only improve the quantity control ability of our proposed mitochondrial transfer system but also contribute to other single-cell studies based on droplet microfluidics. The novel methods introduced in this thesis delivered novel concepts of mitochondrial transfer and droplet microfluidics research.
| Date of Award | 7 Nov 2022 |
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| Original language | English |
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| Supervisor | Dong SUN (Supervisor) |