Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules

Abstract

There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.

Figure 1

Living/Synthetic hybrid cells. (A) Schematic of a biological cell encapsulated inside a vesicle-based artificial cell. (B) The encapsulated cell serves an organelle-like function in the vesicle reactor, processing chemical elements which are then further metabolised downstream by a synthetic enzymatic cascade co-encapsulated in the vesicle.

Figure 2

Schematic and microscopy images of the generation of cells-in-vesicles. (A) A microfluidic chip was used to encapsulate cells in w/o droplets encased in a lipid monolayer. An aqueous phase containing cells was passed through a flow-focusing junction where it met an oil phase containing lipids, leading to droplet generation. Red arrows show the position of a cell before and after encapsulation. Ratio of cell to droplet size varied from 1:4 to 1:10 depending on the cell type, junction geometry, and flow rates. Droplets were then collected in a chamber where their size and encapsulation number was analysed. A mixture of empty droplets and those containing cells (green circles) were observed. Droplets were then expelled from the device to an emulsion phase transfer column. (B) Schematic depicting the transformation of cells-in-droplets to cells-in-vesicles. The droplets descended through the column under gravity. As droplets transferred into the aqueous phase the interfacial monolayer wrapped around them, transforming them into vesicles with cells encapsulated inside.

Figure 3

Cells-in-vesicle hybrids. Brightfield/fluorescence composite images of fluorescent POPC vesicles doped with 1 wt.% Rh-PE with (A) a single BE cell encapsulated (B) two BE cells encapsulated. (C) Image of a suspension cell (Toledo B lymphocytes) and (D) E. coli cells encapsulated in a vesicle. Scale bar = 25 µm.

Figure 4

Cell encapsulation and vesicle size distribution (A) Graph of number of cells encapsulated in the droplet precursors and in the vesicles. These followed a Poisson distribution (dotted lines) where λ = 0.41, 0.13 respectively. (B) Size distribution of the droplet precursors and vesicles. These followed Gaussian distributions (dotted lines).

Figure 5

Encapsulated cell as an organelle-like bioreactor. (A) Reaction scheme (B) Vesicle/cell hybrids were engineered where encapsulated cells performed one step of a multi-step enzymatic pathway (hydrolysis of lactose to glucose). Glucose was then further metabolised in the vesicle interior by an artificial enzymatic cascade, resulting in a fluorescent product (resorufin). (C) (i) Representative brightfield/fluorescence composite images of vesicles/cell hybrids at different time points, showing successful synthesis of reaction products over time compared to control experiments (ii) with no encapsulated cell, which exhibited minimal fluorescence after 180 minutes. Scale bar = 25 µm. (D) Graph demonstrating mean fluorescence of vesicle/cell hybrids with a transfected cell and encapsulated feedstocks 180 minutes after generation, as well as three control scenarios. Error bars represent standard deviations of mean fluorescence after 180 minutes from three independent preparation runs (E) Kinetics trace of bulk fluorescent levels after three hours of the cascade reaction. The presence of transfected cells significantly increased reaction yield, confirming the successful coupling of the cellular pathway to the artificially added elements in the cascade.

Figure 6

Vesicle shielding of cells from toxic surroundings and cellular replication in vesicles. (A) Schematic of assay; Cu²⁺ is permeable to vesicles and is unable to penetrate the membrane and cause cell death. (B) Graph showing the viability of cells over time in a variety of conditions. Error bars represent standard deviations from three independent trials. (C) Fluorescence and brightfield/composite image from a viability assay of cells-in-vesicles with Cu²⁺ in the external solution. Dotted circle represents the location of GUV in the fluorescent channel. Encapsulated cells were viable (arrow) while unencapsulated cells were non-viable (green channel). (D) Composite fluorescence/brightfield image from a metabolic activity assay with Cu²⁺ in the external environment. Cells encapsulated in vesicles remained metabolically active (yellow channel and arrow) despite the presence of the toxic species. (E) E. coli cells encapsulated in POPC vesicles were seen to replicate and increase in number over a period of 24 hours, demonstrated cellular viability over time in the confined environment. Scale bar = 25 µm for all images.