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.

Fig. 1. Droplet laboratory platform.

a-1 Schematic of the acoustic levitator platform setup. Multiple acoustic standing waves (red and blue), generated by the acoustic levitator, can trap microfluidically formed artificial cells with distributed cores (ACDC droplet), at nodal positions. Photographs of full experimental apparatus are available in Fig. S1. a-2 Schematic of patterned ACDC droplets encapsulating multiple reagents within the core networks. b Simulation results of 2 mm diameter droplet trapping in air. The pink circle (droplet levitated) and the black cross (droplet fell) symbols represent the outcome of simulated droplet release positions in the acoustic field (rainbow colour map indicates standing wave pressure profile (symmetric half)). c Droplet trapping dynamics: An ACDC droplet dispensed from the microfluidic device into a standing wave field oscillates vertically in the standing wave node before dampening and assuming a stable position over a period of 4 s. Additional data on droplet release positions are available in Fig. S2. d Top; Simulation and experimental results of the convective flows within levitated water (w) droplet in air (a) (left) and water in oil (o) double emulsion droplets in air (a) (right). Bottom; Water phases containing hydrophilic magnetic microparticles (dia. 1 μm) and minor aggregates (brown), remain suspended and circulate with the fluidic convection, without significant clustering. e Manually adding multiple water droplets to a levitated oil droplet with a micropipette. From top left to bottom right, pink, green, blue and clear droplets. f Deposition of multiple microfluidically formed ACDC droplets at sequential nodes within the acoustic standing wave field. Scale bars = 1 mm.

Fig. 2. Microfluidic control of artificial cells with distributed cores (ACDC droplet) structural organisation.

a Multi-layered 3D printing minimises capillary junction size, improving droplet generation fidelity, in comparison to single-layered devices (left), see Fig. S4 for further details. Uniform, cell-sized, alginate microgels can be formed using 120 µm wide, 3D-printed, multi-layered junctions (right) (flow rates: dispersed phase: 1 ml h⁻¹ and continuous phase: 20 ml h⁻¹. Additional fluidic designs and droplet size data available in Fig. S4). b The addition and combination of parallel fluidic junctions enables formation of multiple different types of water droplets within an oil compartment. Increasing complexity is illustrated left to right with cores of two, three and four different chemical identities. c Programming of fluid inlet flows can be used to generate droplet sequences of defined order and spacing for the creation of customised emulsion droplets. Programmed input profiles regulate droplet formation at each droplet forming junction, on-demand, determining core sequences and therefore providing the chemical encapsulation template for the ACDC construct. Droplet order determines final 3D spatial arrangement of the encapsulated cores. d Examples of patterned, levitated ACDC constructs. The fluidic input flow profile determines the core sequence and packing order in the ACDC construct. From left to right, a specific core (green) is directed to the top, middle and bottom level of ACDC constructs, and the formation of Janus networks comprised of red and green cores. Lipid bilayers segregate cores of the internal droplet network. All scale bars = 500 µm except where otherwise stated.

Fig. 3. Multiple operations on levitated ACDC droplet.

a The combination of controlled chemical compartmentalisation, with subsequent droplet processing and manipulation, as methods to energise the droplet structures, can be used to impart function on the resulting materials. b Pneumatic operations for the spinning of levitated artificial cells with distributed cores (ACDC droplet). (i) A radially tangential air flow to the ACDC droplet rotates the levitated construct. (ii) As the air flow is increased, the droplet rotational speed increases, and the droplet becomes elongated before splitting into two daughter droplets, each with interior core network. (iii) The spinning rate can be precisely controlled by the air flow rate (inset graph: n = 30 for each data point, measured from a 20 s spinning interval). (iv-left column) At high rotational speeds the droplet network disassembles and lipid bilayers are separated between adjoining cores. Spin-stop cycles can be used to reconfigure the internal core network, detaching and re-assembled bilayer connections. (iv-right column) This is depicted for disassembly and reassembly of a core network (green and pink cores) being reconfigured to separate and connect different cores. Scale bars = 1 mm. c Thermal operations for the heating of levitated ACDC droplets. A copper wire coil was used to induce thermal energy within the levitated droplet through thermal convection (i and ii). The droplet can be heated up from room temperature to 50˚C in 1 min with gradual heating (iii and iv). Scale bars = 5 mm. d Magnetic operations for the manipulation of levitated ACDC droplets (i and ii). A levitated ACDC droplet containing a core with encapsulated magnetic particles (iii), can be manipulated by external magnets, providing droplet positional and orientational control (iv). The star symbol (*) indicates the position of the magnetic particles, and their gathering at the core perimeter on application of an external magnetic field. Scale bars = 1 mm.

Fig. 4. Reconstitution of functional alpha-hemolysin pores and bacterial MscL protein channels into the artificial membranes of the core network of levitated ACDC droplets.

a-1 Alpha-hemolysin (aHL) monomers contained in an internal core bind to the membrane separating neighbouring droplets and assemble into heptameric protein pores in the DPhPC bilayer. Ca²⁺ ions diffuse through aHL pores moving from the source cores to the protein-containing core where fluorescence is induced by the presence of the calcium-sensitive fluorophore, Fluo-8. No ion-flux is observed in an identical control droplet without aHL monomer. a-2 The graph indicates the fluorescence intensity over time of both aHL containing and non-aHL containing (control) droplets in the levitated artificial cells with distributed cores (ACDC droplet) over 20 min. (Dotted lines = moving average). b The mechanosensitive channel, MscL, opens in response to membrane tension. Gating of MscL channels in levitated ACDC droplets with membranes of different bilayer leaflet compositions. b-1 In symmetric DPhPC bilayer networks no Ca²⁺ flux is observed, the channel remains closed; b-2 In asymmetric DOPC-DPhPC bilayer networks asymmetric membrane tension is induced, opening MscL channels, and giving rise to Ca²⁺ flux and observed fluorescence. (No fluorescence increase is observed in the absence of MscL (SI)) b-3, Time-course fluorescence in symmetric (DPhPC) and asymmetric (DOPC-DPhPC) membranes with incorporated MscL as depicted in b-1 and b-2. Asymmetric membrane tension-induced activation of MscL channels enables Ca²⁺ flux, eliciting maximal fluorescence response over the course of 250 s (orange trace). No fluorescence increase is observed in the symmetric membrane system (blue trace) indicative of channel inactivation, as well as the asymmetric membrane without MscL reconstitution (Fig. S11). All scale bars = 1 mm.

Fig. 5. Remote control of ion channel gating in levitated ACDC droplets core networks using magnetic manipulation.

a Experimental concept: The incorporation of a core containing magnetic particles enables switching of the levitated artificial cells with distributed cores (ACDC droplet) from a freely rotating to a locked state on the application of an external magnetic field. In the free (spinning) state acoustic forces are dissipated by subtle movement. In the locked state, this is dampened and instead manifests as induction of membrane tension. This may be used to selectively activate MscL channel gating. b Time sequence images of MscL gating in a levitated ACDC droplet. From left to right; t = 5 s in the absence of an applied magnetic field the ACDC droplet is allowed to spin in the acoustic trap. At t = 30 s, the magnetic field is applied, locking the ACDC droplet in position. MscL is activated and Ca²⁺ ion flux induced, observed by the fluorescent reporter, fluo-8, present in the MscL-containing core. At t = 80 s, significant fluorescence increase is observed. With the fluorescence response saturated at t = 170 s. In the absence of MscL no fluorescence increase is observed (Fig. S13, DPhPC symmetric bilayers). c Fluorescence intensity trace of Ca²⁺ flux through MscL channels into a fluo-8 containing core of a locked, levitated, ACDC droplet. (Same experiment labelled (i) in Fig. 6b). (solid line = moving average). d Images of a levitated ACDC droplet with a MscL-containing core (left), a magnetic particle-containing core and a control core containing Fluo-8 only (no MscL) (right). The orientation of the droplet here is controlled by the placement of an external magnet to enable image acquisition from two angles to illustrate the three highlighted cores. Scale bars = 1 mm.

Fig. 6. MscL gating behaviours in levitated ACDC droplets.

a Repeat activation and deactivation of MscL gating in levitated artificial cells with distributed cores (ACDC droplet) can be achieved by repeated lock/spin operations. The orange trace shows an ACDC droplet experiencing one cycle of spin/lock operation, enabling ionic communication (Experiment labelled (ii) in b). The yellow trace shows an ACDC droplet experiencing four sequential cycles of spin/lock operation, enabling repeated activation and shut-down of ionic communication within the ACDC droplet. The relative fluorescence intensity did not increase during the spin intervals (each ~100 s), indicating the MscL channels were mostly deactivated during these periods and activated on application of the magnetic lock. (Experiment labelled (iii) in b). b Relationship of core position and MscL activity: Heatmap depicting the relative rate of ion flux (rate of fluorescence increase) for MscL-containing cores and their positions within different ACDC droplets (x/y offset from ACDC droplet centre). The plot origin is the centroid of each levitated ACDC droplet, and each circular data point represents a separate experiment with the circle’s coordinates corresponding to each MscL-containing core position relative to the centroid of the host ACDC droplet. Marker colour indicates relative activity. Markers i, ii and iii correspond to the individual traces shown in Figs. 5c and b. c Finite element modelling of acoustic streaming induced convective flow patterns in the cores (grey) at different positions within a locked, levitated ACDC droplet. Left: convective flow streamlines in the cores. Middle: flow velocity magnitude (logarithmic scale) within the ACDC droplet. Right: Particles (5 µm) distribution under the convective flow within the cores. These simulations show how acoustic streaming profile varies with core position, corroborating experimental findings in b. d Experimental time sequence images over 4 s show local Ca²⁺ release through MscL channels at the membrane followed by dissipation of the fluorescence with acoustic streaming flow (corresponding to flow simulations in c). Such hot-spots of MscL activation were observed stochastically in regions and periods of high induced local tension. Green spots and arrow dotted lines indicate the flow direction. Scale bars = 1 mm.