Cell proliferation and migration inside single cell arrays

Cell proliferation and migration are fundamental processes in determining cell and tissue behaviour. In this study we show the design and fabrication of a new single cell microfluidic structure, called a “vertically integrated array” or “VIA” trap to explore quantitative functional assays including single cell attachment, proliferation and migration studies. The chip can be used in a continuous (flow-through) manner, with a continuous supply of new media, as well as in a quiescent mode. We show the fabrication of the device, together with the flow characteristics inside the network of channels and the single cell traps. The flow patterns inside the device not only facilitate cell trapping, but also protect the cells from mechanical flow-induced stress. MDA-MB-231 human breast cancer cells were used to study attachment and detachment during the cell cycle as well as explore the influences of the chemokine SDF-1 (enabling the quantification of the role of chemokine gradients both on pseudopod formation and directional cell migration)

Expression of membrane-associated proteins within single emulsion cell facsimiles

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB–RFP). We follow the aggregation and localisation of the fusion protein MreB–RFP in this artificial cell-like environment. The expression of MreB–RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface

Spatially selecting single cell for lysis using light induced electric fields

An optoelectronic tweezing (OET) device, within an integrated microfluidic channel, is used to precisely select single cells for lysis among dense populations. Cells to be lysed are exposed to higher electrical fields than their neighbours by illuminating a photoconductive film underneath them. Using beam spot sizes as low as 2.5 μm, 100% lysis efficiency is reached in <1 min allowing the targeted lysis of cells

Mixing-Performance Evaluation of a Multiple Dilution Microfluidic Chip for a Human Serum Dilution Process

This paper is aimed to propose a numerically designed multiple dilution microfluidic chip that can simultaneously deliver several serum dilutions in parallel. The passive mixing scheme is selected for dilution and achieved by the serpentine mixing channel in which Dean vortices are induced to increase the contact area and time for better diffusion. The mixing performance at the exit of this dilution chip is numerically evaluated using five commonly-used mixing indices with the goal that the homogeneity of the mixture over the exit cross-sectional area of the mixing channel must be greater than 93.319% to fulfill the six-sigma quality control

Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from 50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells

Single cell devices for migration and division studies

Microfluidic technologies and devices now provide powerful tools for many biological studies to gain knowledge and insight into cell behaviour because of their potential to control the local in vitro environment. This thesis aims to develop microfluidic devices for the single cell proliferation and migration studies that are fundamental in determining cell and tissue behaviour. There are two designs of microfluidic devices that have been used in this project. The first one is hydrodynamic single cell trap device having a bagatelle- like structure. The bagatelle-like devices were used to trap modified MCF7 cells expressing both mcherry-tubulin and GFP-actin and also to study the influences of the oestrogen hormone on MCF7 cells. It was found that the MCF7 cell proliferation could not be seen in the bagatelle-like devices either in the presence or absence of oestrogen. It was hypothesised that this might be due to cell stresses arising from being in a constrained area (trap) and subjected to strong fluid flow forces. The second, novel, device consists of three segregated layers and is termed a microhole device. It was specifically designed, fabricated, characterised and utilised in cancer cell proliferation and migration studies in this thesis. The microhole devices were designed to address the limitations of the bagatelle-like device. In each microhole device, the lower layer comprises of a network of submerged channels linked to an upper layer through cavity-like holes. The networks of submerged channels provide a route through which cells can migrate. The middle layer consists of an array of circular holes used to organise single cells into the cavities beneath. The top layer is a PDMS chamber for cell loading and culture medium perfusion. It was found that the recirculatory flow patterns inside the devices facilitate cell trapping, while also serving to separate high velocity flow in the top chamber from the middle and the bottom layer thereby protecting the cells from shear stress. MDA-MB-231 cells were used in this study. It was found that they can undergo cell cycling normally in the microhole devices, and migrate along an SDF-1α solution gradient produced inside the device, towards high SDF-1α concentration. To explore whether the cells were sensitive to SDF-1α on the surface to which they adhered (as opposed to solution gradients), the microhole devices were modified to have SDF-1α immobilised on selected interior surfaces. Despite each stage of the immobilisation process being verified using the appropriate fluorescence assays, relatively low levels of SDF-1α were detected in the completed devices. This may be due to fabrication processes that might deteriorate the immobilised SDF-1α functionality. It was found that unlike the situation when SDF-1α is in solution form, the MDA-MB-231 cells showed no migratory preference toward the immobilised SDF-1α. Taken together, the microhole devices developed in this thesis provide suitable environments for study cell migration toward stimuli under perfusion conditions. The geometry and the flow characteristics inside the array facilitate cell trapping and serve to protect cells from shear stress caused by high fluid flow. Further applications of the multilayer microhole devices can be found through modifying the different layers to accommodate different geometries for different cell types as well as more complex stimulation conditions, or in other application areas associated with droplet microfluidics and synthetic biology

Extracellular Vesicle-Mediated IL-1 Signaling in Response to Doxorubicin Activates PD-L1 Expression in Osteosarcoma Models

The expression of programmed cell death ligand 1 (PD-L1) in tumors is associated with tumor cell escape from T-cell cytotoxicity, and is considered a crucial effector in chemoresistance and tumor relapse. Although PD-L1 induction has been observed in patients after chemotherapy treatment, the mechanism by which the drug activates PD-L1 expression remains elusive. Here, we identified the extracellular vesicles (EVs) as a molecular mediator that determines the effect of doxorubicin on PD-L1 expression in osteosarcoma models. Mechanistically, doxorubicin dependently stimulates the release of extracellular vesicles, which mediate autocrine/paracrine signals in osteosarcoma cells. The recipient cells were stimulated by these EVs and acquired the ability to promote the expression of inflammatory cytokines interleukin (IL)-1β and IL-6. In response to doxorubicin, IL-1β, but not IL-6, allowed- osteosarcoma cells to promote the expression of PD-L1, and the elimination of IL-1β/IL-1 receptor signaling with IL-1 receptor antagonist reduced PD-L1 expression. Together, these findings provided insights into the role of EV release in response to chemotherapy that mediates PD-L1 expression via the IL-1 signaling pathway, and suggested that the combination of a drug targeting IL-1 or PD-L1 with chemotherapy could be an effective treatment option for osteosarcoma patients

Cell proliferation and migration inside single cell arrays

Cell proliferation and migration are fundamental processes in determining cell and tissue behaviour. In this study we show the design and fabrication of a new single cell microfluidic structure, called a “vertically integrated array” or “VIA” trap to explore quantitative functional assays including single cell attachment, proliferation and migration studies. The chip can be used in a continuous (flow-through) manner, with a continuous supply of new media, as well as in a quiescent mode. We show the fabrication of the device, together with the flow characteristics inside the network of channels and the single cell traps. The flow patterns inside the device not only facilitate cell trapping, but also protect the cells from mechanical flow-induced stress. MDA-MB-231 human breast cancer cells were used to study attachment and detachment during the cell cycle as well as explore the influences of the chemokine SDF-1 (enabling the quantification of the role of chemokine gradients both on pseudopod formation and directional cell migration)