Miniaturized single-cell analyses for biomedical applications

Numerous diseases affecting living beings have as a cause a modification of gene expression resulting in anomalous expression of proteins in up- or down-regulated fashion, with altered functions or expression of proteins that are not present in the cell in a normal situation. Among the well-known examples is cancer, where a series of DNA damages turn cells out of control of the rest of the body, from which they do not receive or follow control signals. To have a better understanding of the mechanism of these diseases, in particular the genetic diversity existing in a single affected tissue, it is necessary to be able to perform single-cell analyses that unveil the subpopulations of diseased cells leading to adequate medical treatment. In the course of this thesis, we report on the development of single-cell analysis methods which are of interest for medical applications. The first part focuses on the investigation of the viscoelastic properties of cell membranes by observing the back-relaxation of plasma membrane nanotubes which have been pulled out of a cell by an optical tweezer. Applied to the investigation of the viscoelastic properties of individual tumor cells taken from patients, we could show that this method can distinguish the state of progress of skin melanoma. In the second part, we used beads comprising a chemically modified surface to capture specifically one or several proteins inside single-cells. After extraction out of the cell, the affinity bead is transferred in a microfluidic stream of a fluorescently labeled antibody to detect and quantify the protein(s) of interest. The extraction and detection procedures occur inside a microfluidic platform to allow future automatization of the process. The last chapter focuses on the use of cell-derived extracellular vesicles (EVs) as diagnostic and therapeutic agents. With this goal in mind, we explore the potential of EVs as carriers to transfer genetic material into cells. To demonstrate the feasibility of this approach, we encapsulate EVs inside a giant unilamellar vesicle and release their cargo in a time- and space-controlled manner. This method could have therapeutic applications using a patient's self-EVs for gene therapy.

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