Microfluidic construction and operation of artificial cell chassis encapsulating living cells and pharmaceutical compounds towards their controlled interaction

Droplet-based microfluidic devices can generate complex, soft-matter emulsion systems towards drug screening applications and artificial cell membrane studies. This thesis investigates a methodology for the eventual ‘programmed’ release of pharmaceuticals to treat breast cancer cells that are encapsulated and cultured within small diameter (<2 mm), artificial cell chassis hydrogel capsules. A pharmaceutical analogue was compartmentalised within smaller, membrane-bound, inner cores, that are arranged inside the overall hydrogel capsule. The membrane was based upon droplet interface bilayers (DIBs), which are widely employed for the study of artificial cell membrane transport properties. The whole capsule and contents were produced using enclosed 3D-printed multi-material, microfluidic devices. Methods to control the (programmed) release of compounds from the inner cores to the hydrogel shell, were investigated. The application-specific study was used as an exemplar for a more generally applicable model system. Monolithic microfluidic devices were fabricated using 3D printing and filaments of cyclic olefin copolymer (COC) and nylon for the production of single, double and triple emulsions. With these devices, monodispersed single-emulsion microgels suitable for cell encapsulation were produced, whilst dual-junction devices generated double-emulsion capsules with a controlled number of oil cores. Multi-junction devices also produced triple emulsion, encapsulated droplet interface bilayers (eDIBs), which were subsequently monitored and characterised. Additionally, to demonstrate the ability of eDIBs to act as programmed pharmaceutical delivery systems, assays were performed to induce core release, using membrane modulation by lysolipids (LPC). Computational simulations and DIB electrophysiology experiments were performed to investigate the effect of LPC on the system. MCF-7 model breast cancer cells were encapsulated in alginate-collagen emulsion capsules and their viability was assessed. Moreover, multicellular tumour spheroids (MCTSs) in oil core microgels showed no response to tested doxorubicin concentrations, while proliferated at certain LPC concentrations. Encapsulated cells in eDIBs formed tumour spheroids, however, the DIB survival was low. The integration of living cells and artificial cell membranes within a single entity presents a hybrid model for studying their interaction, towards applications in synthetic biology and drug delivery/screening

Label-free volumetric imaging of synthetic cell chassis using optical coherence tomography

Bottom-up, chemically formed synthetic cells are usually imaged by optical microscopy, and the cell sizes and shapes are mostly estimated from acquired 2D images. The three-dimensional (3D) structures of a compartmentalised synthetic cell can be analysed by axially stacking 2D images, typically by using a high-resolution imaging systems, such as laser confocal scanning microscopy and light sheet microscopy. However, these techniques require the synthetic cell to be labelled with fluorescent tags, and have performance limits such as being restricted to volumes less than (approximately) 200 ìm3. Here, we present the label-free, 3D imaging of soft, free-standing, multicompartment synthetic cell using optical coherence tomography (OCT). The volumes of sub-cellular compartments within individual synthetic cells can be obtained via OCT imaging measurement. The spatial arrangements of the compartments and their contact angle information can be illustrated and measured, respectively. This approach provides a new method to evaluate multiphase soft materials spanning the range of micrometres to millimetres, towards the optimisation of synthetic cell construction for novel biomimetic material development

Droplet microfluidics for tumor drug-related studies and programmable artificial cells

Anticancer drug development is a crucial step toward cancer treatment, that requires realistic predictions of malignant tissue development and sophisticated drug delivery. Tumors often acquire drug resistance and drug efficacy, hence cannot be accurately predicted in 2D tumor cell cultures. On the other hand, 3D cultures, including multicellular tumor spheroids (MCTSs), mimic the in vivo cellular arrangement and provide robust platforms for drug testing when grown in hydrogels with characteristics similar to the living body. Microparticles and liposomes are considered smart drug delivery vehicles, are able to target cancerous tissue, and can release entrapped drugs on demand. Microfluidics serve as a high-throughput tool for reproducible, flexible, and automated production of droplet-based microscale constructs, tailored to the desired final application. In this review, it is described how natural hydrogels in combination with droplet microfluidics can generate MCTSs, and the use of microfluidics to produce tumor targeting microparticles and liposomes. One of the highlights of the review documents the use of the bottom-up construction methodologies of synthetic biology for the formation of artificial cellular assemblies, which may additionally incorporate both target cancer cells and prospective drug candidates, as an integrated “droplet incubator” drug assay platform

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Abstract: Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials

Percolation breakdown in binary and ternary monodisperse and polydisperse systems of spherical particles

We perform computer simulations of an agglomeration process for monodisperse and polydisperse systems of spherical particles in a cylindrical container, using a simplified stochastic-hydrodynamic model. We consider a ternary system with three particle types A, B, and C, in which only connections of the type can be forged, while any other connections with particles of the same type or with C-particles are forbidden, and for comparison a binary system with two particle types A and C, in which only connections of the type can be formed. We study the breakdown of the percolation in the agglomeration at the bottom of the cylinder with an increasing fraction of C-particles

Kauffman Model with spatially separated ligation and cleavage reactions

One of the open questions regarding the origin of life is the problem how macromolecules could be created. One possible answer is the existence of autocatalytic sets in which some macromolecules mutually catalyze each other’s formation. This mechanism is theoretically described in the Kauffman model. We introduce and simulate an extension of the Kauffman model, in which ligation and cleavage reactions are spatially separated in different containers connected by diffusion, and provide computational results for instances with and without autocatalytic sets, focusing on the time evolution of the densities of the various molecules. Furthermore, we study the rich behavior of a randomly generated instance containing an autocatalytic metabolism, in which molecules are created by ligation processes and destroyed by cleavage processes and vice versa or generated and destroyed both by ligation processes