Microfluidic systems for quantitative analysis of secreted proteins at the single-cell level
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Author
Date
2024Type
- Doctoral Thesis
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Abstract
Cancer is responsible for million of deaths in every population across all continents. Extensive research is conducted to fundamentally understand the disease; specifically, the formation and progression of tumours and their surrounding environment. The highly heterogeneous cell population in the tumour microenvironment consists of a vast array of different cells. Particularly, the body’s own immune cells are a significant portion of the tumour microenvironment and interact with their surrounding by secretion of various stimuli. They play a crucial role in the development of the tumour and have the potential to alter the course of the disease. A key classification parameter of anti- or pro-tumoural cells is their secretion profile. However, the detection and quantification of secreted proteins from single cells is still challenging. Specifically, thousands of single cells need to be analysed to obtain significant findings which necessitates high throughput.
Microfluidic devices are well suited to overcome these challenges. Their small geometries are on the same length scale as mammalian cells and are perfectly suited to control and handle cell suspensions. The laminar flow in micro-scaled channels can be utilized to capture and isolate single cells. The secreted proteins are concentrated in the small volumes increasing the sensitivity of commonly applied detection and quantification assays. This thesis exploits microfluidic systems to tackle the challenges for single-cell protein secretion analysis and presents two microfluidic platforms.
The first device developed in this thesis is capable of measuring the secreted proteins of up to 1084 single cells. The cells are captured with hydrodynamic traps and isolated with pneumatically activated donut-shaped valves. The cells are co-captured with magnetic beads embedded with a fluorescent barcode enabling multiplexed secretion measurements. As a proof of concept, the versatile platform was used to characterize different macrophage polarization states based on their secretion profile. The profile of pro- and anti-inflammatory macrophages were compared to macrophages stimulated with cancerous cell supernatant. The findings help to understand the impact of the tumour microenvironment on the immune cells in it.
The second device increased the throughput of analysed cells from 10³ to 10⁵ by two orders of magnitude while reducing the complexity of the system drastically. Instead of the actively controlled valves to isolate single cells, a two-phase system in combination with a microwell array was developed. The open system is operated by only using standard laboratory equipment resulting in an easy-to-use system. With the developed device, the impact of different stimuli on the secretion profile of native macrophages was investigated. Additionally, the impact of anti-cancer drugs on the macrophages was examined and compared to pro- and antiinflammatory stimuli.
Recently, it became apparent that the cellular heterogeneity is a crucial factor in characterization of populations, disease development, and fundamental research. Specifically for the realization of personalized medicine, it is crucial to develop easy-to-use devices which can be applied in standard laboratories. The final platform presented in this thesis is a step towards adaptable and easily operatable microfluidic devices. Show more
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https://doi.org/10.3929/ethz-b-000662093Publication status
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Publisher
ETH ZurichOrganisational unit
03807 - Dittrich, Petra / Dittrich, Petra
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