Microfluidic Technologies for the Production of Therapeutic Synthetic Cells

Synthetic cell production is at the forefront of bottom-up synthetic biology; whereby non-living building blocks (e.g. amino acids and lipids) are used to assemble cell-mimicking structures. These synthetic cells (also known as artificial cells) offer the potential to be utilised in a range of biological settings. For example, as biosensors, drug delivery vehicles, in evolutionary biology studies, or to form a chassis for embedded organelles.

Unilamellar vesicles (or liposomes), spherical cells containing an aqueous interior enclosed by a single phospholipid bilayer often constitute the structure of synthetic cells. These vesicles can vary in diameter, with certain size ranges being desirable for particular applications. Giant unilamellar vesicles (GUVs) are typically 1micrometer or more in diameter, and are commonly used as a chassis for studying cell behaviour in an environment simpler than that of a living cell.

Small unilamellar vesicles (SUVs) have diameters 100nm or less, and are often used in targeted therapeutics as drug delivery systems. The amphiphilic nature of phospholipids are exploited to encapsulate hydrophilic molecules in the aqueous vesicle interior; or hydrophobic molecules within the lipid bilayer. The composition of the phospholipid bilayer can be engineered in order to make them actively target a given cell, for instance by attaching to complementary surface receptors.

In previous research, engineered vesicles have largely been generated via bulk methods, including ethanol injection, emulsion phase transfer, and lipid film hydration. Though these methods have yielded much success in generating cells for desired applications, such as temperature-controlled content release, they are often limited in both reproducibility and time-efficiency.

The emergence of microfluidic technologies poses solutions to these problems. Microfluidic devices use narrow length scales in order to achieve laminar flow: flow dictated by the geometry of the device, with the absence of turbulence. Small unilamellar vesicles are typically generated by microfluidic hydrodynamic focusing (MHF), a process in which an inner, lipid-containing organic solvent (often ethanol) phase is focused by two aqueous buffer phases.

Due to the laminar nature of these flows and their miscibility, diffusion can occur between them, resulting in the self-assembly of lipid vesicles, as the ethanol molecules diffuse into the aqueous phase. Free lipid molecules, consisting of a hydrophobic head group and hydrophilic tails, will arrange themselves in a bilayer sheet in the presence of water due to the hydrophobic effect.

The exposed edges of the bilayer are both energetically and entropically unfavourable, which drives its global curvature to form a unilamellar, spherical vesicle with an aqueous lumen.

Microfluidic methods carry many advantages, such as increased monodispersity and the ability to incorporate computation. One of the main advantages in the context of generating vesicle populations is the ability to finely tune the diameter of the vesicles by altering the flow rate ratio (FRR) of the two inlet phases. An increased flow rate ratio leads to a smaller vesicle diameter up to a limit determined by the device geometry.