Droplet assembly process

Abstract

The invention relates to a process for reducing the size of droplets in a synthetic droplet assembly, wherein the synthetic droplet assembly comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets, wherein the process comprises removing water from the aqueous medium of droplets in the droplet assembly. The invention also relates to a process for increasing the size of droplets in such a synthetic droplet assembly, which process comprises adding water to the aqueous medium of droplets in the droplet assembly. The invention also provides a synthetic droplet assemblies, including synthetic droplet assemblies obtainable by the processes of the invention.

Description

[0001] DROPLET ASSEMBLY PROCESS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a process for reducing the size of droplets in a synthetic droplet assembly, a process for increasing the size of droplets in a synthetic droplet assembly, and to synthetic droplet assemblies obtainable by these processes.

[0004] BACKGROUND TO THE INVENTION

[0005] 3D droplet printing technology may be used to produce synthetic droplet assemblies in which multiple droplets are connected to one another via a layer (e.g. a droplet interface bilayer, DIB) of amphipathic molecules at each droplet interface. Such 3D printing of droplet assemblies is described, for instance, in Gabriel Villar, Alexander D. Graham, and Hagan Bayley “A Tissue-Like Printed Material”, Science 340.6128 (Apr. 2013), pp. 48-52, and in WO 2014 / 087175 Al, WO 2021 / 234394 Al and I. Cazimoglu et al, ACS Nano 2021, 15, 20214. 3D droplet printing methods such as these are advantageously able to produce very complex and diverse, three-dimensional droplet assemblies, which may contain any number of droplets, including hundreds of thousands, millions or even billions of droplets. Very large, self-supporting, droplet assemblies, on the millimetre or centimetre scale, may be produced. Such droplet assemblies have an exciting range of uses in fields such as medicine and synthetic biology, including the production of mimic cells and tissues, their use as cell and tissue scaffolds, and their use in drug delivery, to name but a few. The droplet assemblies can easily be functionalised, for instance by the incorporation of membrane protein pores into layers of amphipathic molecules between specific contacting droplets, or by the inclusion of other materials, including small molecules, enzymes, cell-free expression systems, and living cells, within specific droplets. Thus, functional mimics of biological tissue can be produced by the inclusion of such materials in droplets within an assembly. Indeed, such droplet assemblies are often referred to as synthetic tissues. Alternatively, living cells may, be allowed to grow within the droplets of a droplet assembly scaffold, and to break down the layers of amphipathic molecules between droplets sometime after printing.

[0006] However, such 3D droplet printing technology has only a limited ability to minimize the droplet size while maintaining high spatial precision. This is because smaller droplets are more difficult to generate consistently. Also, smaller droplets sink slowly through the viscous lipid-in-oil solution, making them more susceptible to drag forces caused by nozzle movements and subsequent displacement of droplets from their intended location. This significantly extends printing times. Moreover, the smaller the droplet size, the higher the likelihood for inaccuracies to evolve during printing, which leads to defects in droplet packing. Furthermore, variations in droplet volume during the printing of small droplets are more pronounced, exacerbating such inaccuracies.Accordingly, useful assemblies of droplets below a certain size have not thus far been obtainable by such 3D droplet printing technology.

[0007] There is therefore an ongoing need for a process that is able to produce useful synthetic droplet assemblies of the kind discussed above, but with a smaller droplet size. There is also an ongoing need for processes that allow control of the droplet size in synthetic droplet assemblies obtainable by 3D printing, without the abovementioned disadvantages of loss of high spatial precision, significantly extended printing times, inaccuracies during printing, and droplet packing defects.

[0008] SUMMARY OF THE INVENTION

[0009] It is a finding of the invention that the sizes of droplets in a synthetic droplet assembly may be adjusted in a separate process, independently of any 3D printing process that may have been used to produce the droplet assembly. In particular, the invention provides processes for tuning the droplet size in a synthetic droplet assembly, and the processes may be carried out post-printing, either to reduce or increase the sizes of the droplets. The overall volume of the droplet assembly may also thereby be reduced or increased, independently of any printing process used to produce the assembly.

[0010] Advantageously, the general morphology of the synthetic droplet assembly is unaffected by the processes of the invention. The overall shape and structure of the droplet assembly is retained, as are the droplet interface layers (e.g. DIBs) within the synthetic tissues. The processes of the invention also usefully provide access to synthetic droplet assemblies with much smaller droplet sizes than are currently obtainable through known 3D printing processes. This allows for increased droplet resolution - a higher number of droplets per unit length in each dimension - in the products produced, whether they be synthetic tissues or delivery vehicles for patterned delivery of an effector molecule. It is demonstrated herein, for instance, that such droplet assemblies can achieve an even-higher spatial resolution of patterned gene expression than was possible using an equivalent assembly with larger droplets obtained by 3D printing alone.

[0011] Accordingly, the invention provides a process for reducing the size of droplets in a synthetic droplet assembly,

[0012] wherein the synthetic droplet assembly comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0013] and wherein the process comprises removing water from the aqueous medium of droplets in the droplet assembly.The invention further provides a synthetic droplet assembly obtainable by the process of the invention for reducing the size of droplets in a synthetic droplet assembly.

[0014] The invention also provides a process for increasing the size of droplets in a synthetic droplet assembly, wherein the synthetic droplet assembly comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,

[0015] and wherein the process comprises adding water to the aqueous medium of droplets in the droplet assembly.

[0016] The invention also provides a synthetic droplet assembly obtainable by the process of the invention for increasing the size of droplets in a synthetic droplet assembly.

[0017] The invention further provides a synthetic droplet assembly which comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets, wherein:

[0018] the mean diameter of the droplets in the droplet assembly is less than 10 µm; and / or the mean volume of the droplets in the droplet assembly is less than 0.5 pL.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 shows localized gene expression, and in particular: (a) a schematic of the droplet printing process, where droplet interface bilayers (DIBs) are formed between the droplets (left image). Through 3D printing, networks of droplets are formed (center image), producing droplet networks, which can be transferred with a pipette (right image), (b) A schematic of a 3D-printed droplet network interfacing a bacterium -laden hydrogel 18 h after transfer to the lipid-in-oil solution on top of the hydrogel. Magenta and yellow represent bacteria in the hydrogel expressing mCherry at high and low levels, respectively, (c) Zoomed-in schematic of (b) at the droplet network-hydrogel interface. A droplet hydrogel bilayer (DHB) forms between the droplet network and the hydrogel, -hemolysin (aHL) is incorporated into the bilayer, allowing arabinose to be released from the droplet network onto the immobilized bacteria, activating the expression of mCherry. (d) and (e) Epi-fluorescence microscopy images of a droplet network containing 33 mM arabinose, 50 μg mL⁻¹ αHL and 250 μM cascade blue dextran on top of a bacterium -laden hydrogel (composite bright-field (d) and fluorescence images (e) at t = 0 h (when the droplet network is placed on top of the hydrogel) and t = 18 h ( 18 hours after the droplet network is placed on top of the hydrogel)), (f) A z-stacked 3D confocal microscopy image of a droplet network on top of a bacterium -laden hydrogel at 18 h. In (d)-(f), cyan is cascade blue dextran fluorescence,yellow represents sfGFP fluorescence and magenta represents mCherry fluorescence, (g) A graph of the total mCherry expression, IT, overtime in bacterium-laden hydrogels with droplet networks on top containing 33 mM arabinose and 50 μg mL⁻¹ αHL (orange line), only 33 mM arabinose (blue line), or no arabinose (green line). Solid lines and shaded regions are the mean and standard deviation values of n = 3 technical repeats.

[0021] Figure 2 shows patterned gene expression by controlled chemical signal release, and in particular: (a) a schematic of the image analysis workflow for calculating the pattern fidelity index (PF). First, raw epi-fluorescent microscopy images of both mCherry expression and the droplet network containing cascade blue dextran are binarized, before comparing the binarized pixel values of the two epi-fluorescent channels. Based on the area of intended gene expression (Ai) the area of unintended expression (Au) and the area of no gene expression (AN) within Ai were computed. Then, PF as a measure of controlled release of the chemical signal was calculated by subtracting Au from Ai before normalizing to Ai (see Equation (1) in text and Methods). Total gene expression, IT, and mean gene expression, IM, were determined based on raw pixel values, (b) and (c) Graphs of PF and total mCherry expression, IT (see Methods), against aHL concentration (b) and arabinose concentration (c) within the droplets of the droplet networks. Solid lines and shaded regions, respectively, are the mean and standard deviation values of n = 4 technical repeats for droplet networks on top of bacterium -laden hydrogels after 18 h. (d) Schematic of cross-sectional patterns incorporated into all layers of the 3D-printed droplet networks. White, droplets with no arabinose, no aHL, and no cascade blue dextran, and cyan, droplets containing 33 mM arabinose, 25 μg mL⁻¹ αHL and 250 μM cascade blue dextran, (e) Corresponding epi-fluorescent images of droplet networks constructed according to the patterns in (d). Cyan fluorescence is cascade blue dextran, (f), Binarized fluorescence images of the corresponding expression of mCherry (magenta) in bacteria underneath the droplet networks as shown in (e). (e) and (f) Images 18 h after placement of the droplet networks on top of bacteriumladen hydrogels, (g), PF, unintended area of gene expression, Au (normalized to the intended area of gene expression, Ai), area of no gene expression, AN (normalized to the intended area of gene expression, Ai), and mean mCherry expression, I, (see Methods) corresponding to the patterns in (e)-(f).

[0022] Figure 3 shows chemical signal storage and release in droplet networks, in particular: (a) a schematic of droplet networks of 4, 8 and 16 droplet layers on top of a bacterium -laden hydrogel, (b) Graph of the relationship between PF, total mCherry expression, IT, and normalized areas of no expression, AN, within Ai (normalized to Ai) with increasing droplet layer numbers. Solid lines and shaded regions, respectively, are the mean and standard deviation values of n = 3 technical repeats for droplet networks on top of bacterium -laden hydrogels after 18 h. (c) Epi-fluorescence images of mCherry at 18 h after transfer of droplet network networks composed of 4, 8 and 16 layers, (d) and (e) Schematics depicting a droplet network composed of a mask (cross-like pattern) with areservoir on top (in cyan) from the side or bottom, respectively. Magnetic beads (grey spheres) are attached to the comers of the droplet network, (f) Schematic of a droplet network composed of a mask and reservoir on top of a bacterium -laden hydrogel after 18 h. A magnet (grey oval) directs controlled landing during the transfer of the droplet network to the bacterium -laden hydrogel, (g) Epi -fluorescence image of mCherry at 18 h after droplet network transfer. The red dashed line indicates an area where the bottom droplets did not form the intended contact with the hydrogel. In the schematics (a) and (d)-(f) and the corresponding experiments in (b) and (g) cyan droplets contain 33 mM arabinose, 50 μg mL⁻¹ αHL and 250 μM cascade blue dextran (see Methods), whereas white droplets in the schematics do not contain these three components. Magenta and yellow are representations of bacteria in the hydrogel expressing mCherry at high and low levels, respectively.

[0023] Figure 4 shows increased resolution of patterned gene expression. In particular (a)-(c) are schematic, composite and epi-fluorescence images, respectively, of a droplet network containing an aHL-mediated diffusion pathway comprising a single linear chain of droplets after 18 h on top of a bacterium -laden hydrogel, (d) Schematic of a droplet network during the heat-induced shrinking process, where a droplet network is printed in lipid-in-oil (left image), before placement on top of a heat plate (center image), which initiates the shrinking process through droplet annealing and volume decrease (right image), (e) Bright-field microscopy images over time of a droplet network in lipid-in-oil solution on top of a heat plate set to 43 °C. (f) Time-dependence of the percentage changes in network side length (normalized to initial side length) during heating at 36 °C and 43 °C (see Methods), (g) Diameters overtime of single droplets, droplets forming a single droplet interface bilayer with another droplet, and central and peripheral droplets of one-layered droplet networks and eight-layered droplet networks heated at 36 °C. In (f) and (g) solid lines and shaded regions, respectively, are the mean values and standard deviations of n = 3 technical repeats, (h)-(j) Composite (bright-field and fluorescence) and epi-fluorescence images of an unheated droplet network (triangular pattern, left) and a shrunken network (rectangular pattern, right) at 18 h after transfer on top of a bacterial -laden hydrogel. The droplet network in (i) was shrunken in lipid-in-oil solution for 170 min at 43 °C before transfer on top of the bacterium-laden hydrogel. In (a)-(d) and (h)-(j) cyan droplets contain 33 mM arabinose, 50 μg mL⁻¹ αHL and cascade blue dextran, while white droplets do not contain these components. Magenta and yellow represent bacteria in the hydrogel expressing mCherry at high and low levels, respectively. In (j) the dashed lines represent the widths of the frame-like gene expression pattern.

[0024] Figure 5 shows change of θDIBduring temperature-mediated shrinkage of synthetic tissues, (a) Bright-field microscopy images of synthetic tissues heated at 36 °C for 80 minutes. Time-resolved images depict the annealing of droplets within 24 minutes (left to second left image) and decrease in volume throughout the evaporation process (second to right and right image), (b) Bright-field images of droplet pairs (that each form a DIB) composed of a range of fractions of thetarget printing solution (0 - 1) by diluting the target printing solution with water. Red circles indicate the circumference of the droplets forming DIBs, which was used by the imaging program (Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105) to determine θDIB. (c) Graph depicting θDIBof droplet pairs as a function of the fraction of the target printing solution. The solid line and shaded area represent the mean and standard deviation of droplet pair contact angles of n = 3 technical replicates.

[0025] Figure 6 shows bilayer retention during heat-mediated shrinking of synthetic tissues. (a,b) Composite (bright-field and epi-fluorescent) microscopy images of synthetic tissues (single -droplet (a) or stripe-like pattern (b) before (t = 0 min) and after (t = 95 min) heat-mediated shrinkage. In (b) the image on the right shows the shrunken synthetic tissue upon heat removal and placement on top of a bacterium-laden hydrogel. Cyan fluorescence is cascade blue dextran, (c) Epi-fluorescent images of synthetic tissues containing Atto 550 maleimide (red fluorescence), of which the fluorescence intensity of Atto 550 maleimide is increased when partitioned within a bilayer (Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105). The white outlines show droplets (1, 2 and 3) that are retained throughout the heat-induced evaporation process.

[0026] Figure 7 shows osmosis-driven growth and shrinkage of synthetic tissues at an aqueous-lipid-in-oil interface, (a) Schematics depicting a synthetic tissue at the interface between an aqueous solution (Wout) and the lipid-in-oil solution. By introducing an aqueous solution of increased osmolarity into Wout, a net water efflux from the synthetic tissue into Woutcauses the synthetic tissue to shrink. In contrast, by introducing an aqueous solution of decreased osmolarity into Wout, a net water influx from Woutinto the synthetic tissue causes the synthetic tissue to grow in volume. (b,d) Bright-field microscopy images depicting a synthetic tissue during osmosis-driven shrinkage or growth, respectively. (c,e) Graphs depicting the fold-change in volume (AV ) during osmosis-driven shrinkage or growth, respectively.

[0027] Figure 8 shows optimized resolution of patterned gene expression, (a-f) Composite (bright-field and fluorescence) and epi-fluorescence images of an unheated synthetic tissue (triangular pattern, (a and d) and a shrunken tissue (rectangular pattern, b, c, e and f) at 18 h after transfer on top of a bacterial -laden hydrogel. The synthetic tissue in b and e was shrunken in lipid-in-oil solution for 170 min at 43 °C before the transfer on top of the bacterium -laden hydrogel. In a, b and e cyan droplets contain 33 mM arabinose, 50 μg mL⁻¹ αHL monomer and cascade blue dextran, while white droplets do not contain these components. In c, d and f, magenta represents bacteria in the hydrogel expressing mCherry at high levels.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Droplet assembly features applicable to all aspects of the invention

[0030] The invention provides a process for reducing the size of droplets in a synthetic droplet assembly. The invention also provides a process for increasing the size of droplets in a syntheticdroplet assembly. In both of these processes of the invention, and in the synthetic droplet assembly of the invention, the synthetic droplet assembly comprises a plurality of droplets, and each of the droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium. Furthermore, each of the droplets contacts another of the droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets.

[0031] In each aspect of the invention - the processes of the invention and the synthetic droplet assembly of the invention - the synthetic droplet assembly comprises a plurality of said droplets. Thus, the number of droplets in the synthetic droplet assembly, which may be denoted “n”, is equal to or greater than 2. Thus, n is an integer equal to or greater than 2.

[0032] Often, however, the synthetic droplet assembly comprises more than 2 droplets. For instance, typically, the number of droplets, n, in the synthetic droplet assembly is equal to or greater than 3, or equal to or greater than 5. Usually, the number of droplets, n, in the synthetic droplet assembly is equal to or greater than 10, or, for instance, equal to or greater than 20, or equal to or greater than 30. Often, the number of droplets, n, is equal to or greater than 50, or, for instance, equal to or greater than 100. For instance, n may be equal to or greater than 200, equal to or greater than 500, or equal to or greater than 1,000. Indeed, the number of droplets, n, may be equal to or greater than 20,000, equal to or greater than 50,000, or, for instance, equal to or greater than 100,000.

[0033] There is in principle no upper limit to the number of droplets, n, in the synthetic droplet assembly because very large droplet assemblies of this kind may be produced. Indeed, the number of droplets, n, in the synthetic droplet assembly of the system of the invention may be very large, for instance, at least 1,000,000 or even larger. The number of droplets, n, may for instance be at least 10,000,000, or, for instance, at least 1,000,000,000. In some embodiments, the number of droplets, n, is at least 10,000,000,000, for instance at least 50,000,000,000. If, for instance the droplet assembly is a cube comprising at least 4000 layers of droplets, the number of droplets, n, in the droplet assembly may be at least 64 billion droplets.

[0034] Thus, for instance, the number of droplets, n, in the synthetic droplet assembly may be from 2 to 100,000,000,000, for instance from 2 to 50,000,000,000, from 2 to 10,000,000,000, from 2 to 1,000,000,000, from 2 to 100,000,000, from 2 to 10,000,000, or from 2 to 1,000,000. The number of droplets, n, may for instance be from 2 to 100,000, for instance from 2 to 10,000, or, for example, from 2 to 5,000, or from 2 to 2,000, for instance from 2 to 1,000. The number of droplets, n, in the synthetic droplet assembly may alternatively for instance be from 10 to 100,000,000,000, for instance from 10 to 50,000,000,000, from 10 to 10,000,000,000, from 10 to 1,000,000,000, from 10 to 100,000,000, from 10 to 10,000,000, or from 10 to 1,000,000. The number of droplets, n, may for instance be from 10 to 100,000, for instance from 10 to 10,000, or, for example, from 10 to 5,000, or from 10 to 2,000, for instance from 10 to 1,000. Alternatively, for instance, the numberof droplets, n, in the synthetic droplet assembly may be from 100 to 100,000,000,000, for instance from 100 to 50,000,000,000, from 100 to 10,000,000,000, from 100 to 1,000,000,000, from 100 to 100,000,000, from 100 to 10,000,000, or from 100 to 1,000,000. The number of droplets, n, may for instance be from 100 to 100,000, for instance from 100 to 10,000, or, for example, from 100 to 5,000, or from 100 to 2,000, for instance from 100 to 1,000. The number of droplets, n, in the synthetic droplet assembly may alternatively for instance be from 200 to 100,000,000,000, for instance from 200 to 50,000,000,000, from 200 to 10,000,000,000, from 200 to 1,000,000,000, from 200 to 100,000,000, from 200 to 10,000,000, or from 200 to 1,000,000. The number of droplets, n, may for instance be from 200 to 100,000, for instance from 200 to 10,000, or, for example, from 200 to 5,000, or from 200 to 2,000, for instance from 200 to 1,000. The number of droplets, n, in the synthetic droplet assembly may alternatively for instance be from 500 to 100,000,000,000, for instance from 500 to 50,000,000,000, from 500 to 10,000,000,000, from 500 to 1,000,000,000, from 500 to 100,000,000, from 500 to 10,000,000, or from 500 to 1,000,000. The number of droplets, n, may for instance be from 500 to 100,000, for instance from 500 to 10,000, or, for example, from 500 to 5,000, or from 500 to 2,000, for instance from 500 to 1,000.

[0035] Alternatively, the number of droplets, n, in the synthetic droplet assembly may be from 1,000 to 100,000,000,000, for instance from 1,000 to 50,000,000,000, from 1,000 to 10,000,000,000, from 1,000 to 1,000,000,000, from 1,000 to 100,000,000, from 1,000 to 10,000,000, or from 1,000 to 1,000,000.

[0036] At least some, if not all, of the n droplets in the synthetic droplet assembly may be arranged in a close-packed structure, for instance in a hexagonal close-packed structure.

[0037] Droplet assemblies of this kind may be produced by a process for producing droplet assemblies by 3D-printing of droplets, for instance as described in any ofWO 2014 / 087175 Al, WO 2021 / 234394 Al, G. Villar et al. Science 2013, 340, 48, and I. Cazimoglu et al, ACS Nano 2021, 15, 20214.

[0038] As mentioned above, each of the n droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium. The aqueous medium may be any suitable aqueous medium. For instance, the aqueous medium may be pure water, or an aqueous buffer solution, or an aqueous solution of one or more salts. The aqueous medium may for instance be an aqueous solution comprising Dulbecco's phosphate-buffered saline (DPBS). The aqueous solution may for instance comprise DPBS and sucrose. Alternatively, the aqueous medium may comprise a hydrogel.

[0039] Often, the aqueous medium is an aqueous solution.

[0040] The aqueous medium may for instance comprise a growth medium or a culture medium, for instance a cell growth medium or a cell culture medium. The aqueous medium may for instance comprise culture medium for eukaryotic cells or tissue, or a growth medium for bacteria, such as for example M9 minimal medium. Often, the aqueous medium comprises a growth medium orculture medium which is suitable for supporting the growth of the biological cells or tissue present in the target region of the system of the invention.

[0041] Thus, for instance, the aqueous medium may be an aqueous solution which comprises M9 minimal salts. The aqueous solution which comprises M9 minimal salts may further comprise one or more amino acids. It may additionally further comprise glucose or glycerol. Often, the aqueous solution which comprises M9 minimal salts further comprises at least one of MgSO4, CaCl2, one or more amino acids, glucose or glycerol. Typically, the aqueous solution which comprises M9 minimal salts further comprises MgSO4, CaCl2, casamino acids, and glucose or glycerol.

[0042] When the aqueous medium comprises a hydrogel, the aqueous medium may, for instance, comprise agarose and water. The concentration of the agarose in water is typically less than or equal to 10% w / v agarose. For instance, the concentration of the agarose in said water may be from 0.25 to 5% w / v agarose. Hydrogels other than agarose may also be used. For instance the aqueous medium may comprise methylcellulose, polyethylene glycol diacrylate, polyacrylamide, matrigel, hyaluronan, polyethylene oxide, poly AMPS (poly(2-acrylamido-2-methyl-l -propanesulfonic acid)), polyvinylpyrrolidone, polyvinyl alcohol, sodium polyacrylate, acrylate polymers or poly(N-isopropylacrylamide). Alternatively, the aqueous medium body may comprise a silicone hydrogel or LB (Luria broth) agar.

[0043] The pH of the aqueous medium can be varied over a wide range. In some embodiments, for instance, the pH of the aqueous medium within the aqueous droplet or droplets may be in the range of from 5 to 9 (or for instance in the range of from 6 to 8) although higher and lower pH values are also possible. The aqueous medium may therefore be an aqueous buffer solution. Any suitable buffer can be employed, depending on the desired pH. The buffer solution may for instance comprise Tris-HCl and / or KC1. In some embodiments the pH of the aqueous buffer solution is from 5 to 9, or for instance from 6 to 8. The nature and concentration of the solutes can be varied to vary the properties of the solution.

[0044] Usually, the aqueous medium is an aqueous solution. Thus, often, each of the n droplets comprises (i) an aqueous medium which is an aqueous solution, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium.

[0045] The aqueous medium of each droplet in the droplet assembly may be the same or different. For instance, one or more of the droplets in the synthetic droplet assembly may further comprise an effector molecule (for instance a drug or other molecule suitable for modulating a biochemical process in biological cells or tissue, or any other molecule which may be released to cause an effect), whereas other droplets in the synthetic droplet assembly may not comprise an effector molecule. Similarly, one or more of the droplets in the synthetic droplet assembly may further comprise a protein pore, for allowing passage of a molecule (e.g. an effector molecule) into and / or out of the droplet in question, whereas other droplets in the synthetic droplet assembly may notcomprise any such pore. In these cases, the aqueous medium will not be exactly the same in every droplet in the droplet assembly because it could comprise different components.

[0046] Each droplet of the aqueous medium is usually formed initially by dispensing it into a hydrophobic medium in the presence of amphipathic molecules. The amphipathic molecules may, for instance, be disposed in the aqueous medium or in the hydrophobic medium. Typically, the amphipathic molecules are disposed in the hydrophobic medium. The concentration of the amphipathic molecules in the hydrophobic medium may, for instance, be from 0.1 mM to 10 mM, or, for instance, from 0.2 mM to 5 mM, such as from 1 mM to 3 mM, e.g. about 2 mM. When the aqueous medium is dispensed into the hydrophobic medium in the presence of the amphipathic molecules, an aqueous droplet forms, which droplet comprises (i) an aqueous medium and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium.

[0047] As discussed, the droplet assembly comprises n (at least two, but often much more than two) droplets in contact with each other. The boundary that is shared between contacting droplets, at the point of contact between the droplets, is referred to herein as an interface. An interface is formed when part of the outer layer of one droplet contacts part of the outer layer of another droplet. For instance, when the droplet is brought into contact with the other droplet, a layer of amphipathic molecules typically forms at the interface between the two objects. Depending on the amphipathic molecules, the layer of amphipathic molecules that forms at the interface between two droplets may be a bilayer. Typically, each layer of said amphipathic molecules which is an interface between contacting droplets is a bilayer of said amphipathic molecules. This is typically the case when the amphipathic molecules are lipids, for instance phospholipids or fatty acids. Such a bilayer comprises amphipathic molecules from the outer layer of amphipathic molecules around the surface of the aqueous medium of each droplet at the interface. The bilayer (known in the art as a droplet interface bilayer, or “DIB”) forms as it is an energetically more favourable configuration for the amphipathic molecules to adopt. As the skilled person will appreciate, the contacting droplets will acquire the geometry with the lowest free surface energy.

[0048] The amphipathic molecules may not necessarily comprise lipids but may comprise molecules of a block copolymer instead, for instance a triblock copolymer. In such cases the layer of the amphipathic molecules which is an interface between contacting droplets is not necessarily be a bilayer. It may, for instance, be a monolayer of triblock copolymer molecules, in which two, outer, polar blocks of each triblock molecule contact the respective aqueous media of two adjacent droplets, and a central, apolar block of each triblock molecule resides at the centre of the monolayer (in between the two polar blocks of each molecule). Suitable block copolymers are described in WO 2014 / 064444, and include the triblock copolymer amphipathic molecule poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA). The or each layer of amphipathic molecules which is an interface between contactingdroplets may for instance be a layer of a block copolymer, for example a layer of a triblock copolymer such as PMOXA-PDMS-PMOXA.

[0049] Thus, often, the or each layer of said amphipathic molecules which is an interface between contacting droplets is (i) a bilayer of said amphipathic molecules or (ii) a layer of said amphipathic molecules wherein the amphipathic molecules comprise a block copolymer (for instance a triblock copolymer). In case (ii), the layer may, for instance, comprise a monolayer of said amphipathic molecules, for instance a monolayer of the block (e.g. triblock) copolymer molecules.

[0050] Alternatively, in case (ii) the layer may comprise a bilayer of the amphipathic molecules, for instance a bilayer of the block (e.g. triblock) copolymer molecules.

[0051] Usually, each layer of the amphipathic molecules which is an interface between contacting droplets is a bilayer of the amphipathic molecules.

[0052] Thus, often, each layer of amphipathic molecules which is an interface between contacting droplets is a bilayer of the amphipathic molecules. In other words, in the synthetic droplet assembly employed in any of the processes of the invention, or in the synthetic droplet assembly of the invention, preferably each of said droplets contacts another of said droplets to form a bilayer of said amphipathic molecules as an interface between the contacting droplets.

[0053] The amphipathic molecules of a droplet need not be all of the same type. Rather, the amphipathic molecules may in some embodiments be a mixture of two or more different kinds of amphipathic molecule. Another important example is that the amphipathic molecules in the respective outer layers of different droplets in a droplet assembly may be of different types so that any bilayer(s) formed between the different droplets may be asymmetric.

[0054] The amphipathic molecules may be any suitable amphipathic molecule. Often, the amphipathic molecules will be ones which are capable, when present in a high enough concentration, of forming a bilayer at any one of said interfaces. The type of amphipathic molecule that is capable of forming a bilayer may, for instance, depend on additional components of the contacting droplets. For example, if the droplets are disposed in a hydrophobic medium, the amphipathic molecules may be any suitable amphipathic molecules capable of forming a bilayer within a hydrophobic medium. The type of amphipathic molecules capable of forming a bilayer within the hydrophobic medium would typically depend on the nature of the hydrophobic medium and the aqueous medium of the droplets, but a wide range of amphipathic molecules are possible.

[0055] Amphipathic molecules are molecules which have both hydrophobic and hydrophilic groups. The outer layer of amphipathic molecules usually comprises a monolayer of amphipathic molecules on the surface of the droplet. The monolayer is typically formed and maintained naturally by the interaction of the hydrophilic and hydrophobic groups with the aqueous medium and the bulk medium so that the molecules align on the surface of the droplet with the hydrophilic groups facing inwards towards the aqueous medium and the hydrophobic groups facing outwards, for instance towards a hydrophobic medium.The amphipathic molecules may, for instance, be non-polymeric amphipathic molecules. Alternatively, the amphipathic molecules may be polymeric amphipathic molecules.

[0056] An important class of amphipathic molecules which can be used in the droplet assembly is lipid molecules. The lipid molecules may be any of the major classes of lipid, including phospholipids, fatty acids, fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides. Some important examples include phospholipids and fatty acids, for instance phospholipids. The lipid molecules may be naturally occurring or synthetic. Whilst the formation of a bilayer from lipid molecules has been demonstrated the method is expected to be appropriate for any amphipathic molecules.

[0057] A common class of hydrophobic group that may be present in an amphipathic molecule is a hydrocarbon group, as for instance in most lipids. However, another suitable kind of hydrophobic group that may be employed is a fluorocarbon group. Thus, a further important class of amphipathic molecule is an amphipathic molecule that comprises at least one fluorocarbon group. An example of such a molecule would be a lipid-like molecule which comprises a hydrophobic fluorocarbon tail and a hydrophilic head group.

[0058] The amphipathic molecules of the droplet need not be all of the same type. Rather, the amphipathic molecules may in some embodiments be a mixture of two or more different kinds of amphipathic molecule. Another important example is that the amphipathic molecules in the respective outer layers of different droplets in a droplet assembly may be of different types so that, if bilayers are formed, the bilayer(s) formed between the different droplets may be asymmetric. In some embodiments, the lipid leaflets of two contacting droplets are different.

[0059] Typically, therefore, the amphipathic molecules comprise lipid molecules. The lipid molecules need not be all of the same type. Thus, the amphipathic molecules may comprise a single type of lipid or a mixture of two or more different types of lipid molecules. Likewise, when the droplet is in contact with another droplet, the lipid compositions of the outer layers of the individual droplets may be the same as or different from one another. Lipid molecules are particularly advantageous because lipid bilayers, or more generally bilayers of amphipathic molecules, are models of cell membranes and the droplet assembly may therefore serve as an excellent platform for a range of experimental studies, including for instance as novel platforms for the fundamental study of membrane proteins, or as multi-compartment protocellular chassis for “bottom-up” synthetic biology.

[0060] The lipid may, for instance, be sensitive to its environments (i.e. be a smart lipid). The lipid may, for instance, be sensitive to changes is pH, light or temperature. Thus the lipid may be a pH-sensitive lipid, a temperature -sensitive lipid or a light-sensitive lipid.

[0061] The lipid may allow a membrane protein (such as a natural, engineered or synthetic membrane protein) to act as a functional component of the minimal tissue.Phospholipids are particularly preferred for reasons outlined above and also because they are a major component of all cell membranes, making droplets comprising phospholipids particularly suitable for synthetic biology applications, as well as for drug delivery.

[0062] Accordingly, the amphipathic molecules that form an outer layer on at least part of the surface of the aqueous medium typically comprise phospholipid molecules. The phospholipid molecules may be the same or different, i.e. the amphipathic molecules comprise a single kind of phospholipid, or a mixture of two or more different phospholipids. Phospholipids are well known to the skilled person and many are commercially available, from suppliers such as Avanti Polar Lipids. The phospholipid molecules may be glycerophospholipids or phosphosphingolipids or a mixture of the two. The phospholipid molecules may comprise anionic phospholipids, phospholipids comprising primary amines, choline -containing phospholipids and / or glycosphingoplipids. Usually, the amphipathic molecules comprise one or more glycerophospholipids. As the skilled person will appreciate, glycerophospholipids include, but are not limited to glycerophospholipids having a structure as defined in the following formula (I):

[0063] O

[0064] 111

[0065] R— C— CL

[0066] R— C— 0—

[0067] O

[0068] o113

[0069] O— P— OR

[0070] O’

[0071]

[0072] (I)

[0073] wherein:

[0074] R1and R2, which are the same or different, are selected from C10-C25 alkyl groups and C₁₀-C₂₅ alkylene groups;

[0075] either R3is absent such that OR3is O', or R3is present and is H, CH2CH2N(R4)3+, a sugar group, or an amino acid group; and

[0076] each R4, which is the same or different, is independently selected from H and unsubstituted C1-C4 alkyl.

[0077] Typically, when R3is CH2CH2N(R4)3+, each R4, which is the same or different, is selected from H and methyl. As the skilled person will appreciate, when each and every R4is methyl, the R3group is a choline group, and when each and every R4is H, the R3group is an ethanolamine group.

[0078] When R3is an amino acid group it may for instance be a serine group, i.e.

[0079] -CH2CH(NH2)(COOH). When R3is a sugar group, it may for instance be glycerol, i.e.

[0080] -CH2CHOHCH2OH, or for instance inositol, i.e. -CH(CHOH)5.

[0081] Typical examples of R1and R2groups are C10-C25 alkyl groups, including, but not limited to linear C10-C25 alkyl groups such as, for instance, CH₃(CH₂)₁₀-, CH₃(CH₂)₁₂-, CH₃(CH₂)₁₄-, CH₃(CH₂)₁₆-, CH₃(CH₂)₁₈-, CH₃(CH₂)₂₂- and branched C10-C25 alkyl groups such as for instance -CH2-CH(CH3)-(CH2)3-CH(CH3)-(CH2)3-CH(CH3)-(CH2)3-CH(CH3)2.Further typical examples of R1and R2groups are unsubstituted C10-C25 alkylene groups, including, but not limited to, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4(CH=CHCH2)3CH=CH(CH2)3-, and CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-.

[0082] As the skilled person will appreciate, the O' group in the phosphate group adjacent to the OR3group may in some embodiments be protonated, or associated with a suitable cation, for instance a metal cation such as Na+.

[0083] Thus, the amphipathic molecules may comprise one or more glycerophospholipids having the structure of formula (I) as defined above.

[0084] Thus, the amphipathic molecules may comprise lipid molecules. The lipid molecules may comprise phospholipids. Often, amphipathic molecules comprise one or more glycerophospholipids. The amphipathic molecules may for instance comprise any one or more of the following glycerophospholipids

[0085] For instance, the amphipathic molecules may comprise any one or more of the following glycerophospholipids: l,2-diphytanoyl-5«-glycero-3-phosphocholine (DPhPC), 1 -palmitoyl -2-oleoyl-glycero-3 -phosphocholine (POPC), l,2-distearoyl-5«-glycero-3 -phosphocholine (DSPC), l,2-dipalmitoyl-5«-glycero-3 -phosphocholine (DPPC), l.2-dipalmito l-s77-glyccro-3-|phospho-rac-(1 -glycerol)] (DPPG), 1 -palmitoyl -2 -oleoyl -s77-glyccro-3 -phosphoglycerol (POPG), or glycerophospholipid l,2-dioleoyl-5«-glycero-3 -phosphoethanolamine (DOPE). Any one or more of the aforementioned glycerophospholipids can be employed as the amphiphilic molecules herein, including as the amphiphilic molecules in the n droplets of the droplet assembly, or a mixture of two or more thereof may be employed. Often, the amphipathic molecules comprise 1,2-diphytanoyl-s77-glyccro-phosphatidylcholinc (DPhPC). The amphipathic molecules may for instance comprise l,2-diphytanoyl-5«-glycero-phosphatidylcholine (DPhPC) and 1 -palmitoyl -2-oleoyl-glycero-3 -phosphocholine (POPC). The amphipathic molecules may for instance comprise DPhPC and POPC in a molar ratio of from 3: 1 to 1:1. Often, for instance, the amphipathic molecules comprise DPhPC and POPC in a 2: 1 molar ratio. Alternatively, the amphipathic molecules may all be DPhPC.

[0086] Additionally or alternatively, the amphipathic molecules may comprise a steroid, which steroid comprises an alkyl side-chain. The amphipathic molecules may, for instance, comprise cholesterol, P-sitosterol and lanosterol.

[0087] In some embodiments, the amphipathic molecules comprise glycerophospholipids which are a phosphatidylcholine, such as POPC (1 -palmitoyl -2 -oleoyl -s77-glyccro-3 -phosphocholine) or DPPC ( l.2-dipahnitoyl-s77-glyccro-3-phosphocholinc). or which are a phosphatidylglycerol, such as POPG (1 -palmitoyl -2 -oleoyl -s77-glyccro-3 -phosphoglycerol).

[0088] Preferably, the amphipathic molecules comprise DPhPC.The amphipathic molecules may, for instance, comprise one or more fatty acids, e.g. oleic acid. Fatty acids are of course well known to the skilled person and a wide range of these are commercially available.

[0089] The amphipathic molecules may for instance comprise a mixture comprising: (a) one or more phospholipids, and (b) one or more fatty acids.

[0090] In addition to the amphipathic molecules, the outer layer of amphipathic molecules of one or more of, or each of, the n droplets of the synthetic droplet assembly, may further comprise a PEGylated lipid. The term “PEGylated lipid”, as used herein, refers to a lipid which has been derivatised with polyethylene glycol). The inclusion of one or more PEGylated lipids may stabilise the droplet assembly in vivo, and in particular prolong the life of the droplet assembly. This means that the inclusion of one or more PEGylated lipids may also have the useful effect of prolonging the plasma half-life of the effector molecule within the droplet assembly. Such effects have been observed previously when PEGylated lipids are used in liposomal drug formulations. PEGylated lipids are known in the art and are commercially available from suppliers such as NOF Corporation, Japan (see http: / / www.phospholipid.jp / phospholipid_2-3.html). Any suitable PEGylated lipid may be employed, including, but not limited to, PEG-phospholipids, diacylglycerol-PEG, cholesterol-PEG derivatives, and mixtures thereof.

[0091] The polyethylene glycol) (PEG) component of the PEGylated lipid may have any one of several different geometries. Thus, it could be substantially linear PEG or branched PEG. The branched PEG may for instance have from three to ten PEG chains emanating from a central core group. Alternatively, the branched PEG could be a star PEG, having from 10 to 100 PEG chains emanating from a central core group. Alternatively, the PEG may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0092] The one or more PEGylated lipids employed may for instance comprise a PEG-phospholipid of the following formula (II)

[0093] O

[0094] 1

[0095] R— C11

[0096] — Ch

[0097] 7

[0098] R— C— O—

[0099] II o

[0100] o L IIS

[0101] ^O— P— O-R

[0102]

[0103] °’ (II)

[0104] wherein R1and R2are as defined above for the glycerophospholipids of formula (I), and R5is a group which comprises polyethylene glycol).

[0105] The group which comprises polyethylene glycol) may for instance have the formula -CH2CH2NHC(O)-X, or for instance -CH2CH2NHC(O)(CH2)3C(O)-X wherein X comprises said poly(ethylene glycol). The group X may for instance comprise substantially linear PEG, or for instance a branched PEG, having, for instance, from three to ten PEG chains emanating from acentral core group. Alternatively, it can be a star PEG, having, for instance, from 10 to 100 PEG chains emanating from a central core group. Or for instance it may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0106] Thus, R5may for instance be -CH₂CH₂NHC(O)-(OCH₂CH₂)qOCH₃, -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOCH3, -CH2CH2NHC(O)-(OCH2CH2)qOH, or -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOH, wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0107] Alternatively, R5may be -(CEECEEO^CEE or -(CEECEEO^EI, wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0108] Additionally or alternatively, the one or more PEGylated lipids may comprise a diacylglycerol-PEG of formula (III)

[0109] O

[0110] 1

[0111] R— C11

[0112] — (k

[0113] 7

[0114] R— C— O—

[0115] II

[0116] ° ^O

[0117]

[0118] URK6(III)

[0119] wherein R1and R2are as defined above for the glycerophospholipids of formula (I), and R6is a group which comprises polyethylene glycol).

[0120] The polyethylene glycol) may for instance comprise substantially linear PEG, or for instance a branched PEG, having, for instance, from three to ten PEG chains emanating from a central core group. Alternatively, it can be a star PEG, having, for instance, from 10 to 100 PEG chains emanating from a central core group. Or for instance it may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0121] R⁶ may for instance be -(CH₂CH₂O)qCH₃, -(CH₂CH₂O)qH,

[0122] -CH2CH2NHC(O)-(OCH2CH2)qOCH3, -CH2CH2NHC(O)-(OCH2CH2)qOH, -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOCH3 or

[0123] -CH2CH2NHC(O)(CH2)3C(O)-(OCH2CH2)qOH wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0124] Additionally or alternatively, the one or more PEGylated lipids may comprise a cholesterol-PEG derivative of formula (IV)

[0125]

[0126] wherein R7is a group which comprises polyethylene glycol).

[0127] Again, the poly(ethylene glycol) may comprise substantially linear PEG, or for instance a branched PEG, having, for instance, from three to ten PEG chains emanating from a central core group. Alternatively, it can be a star PEG, having, for instance, from 10 to 100 PEG chains emanating from a central core group. Or for instance it may be a comb PEG, having multiple PEG chains grafted to a polymer backbone.

[0128] R7may for instance be -(OCH₂CH₂)qOH or -(OCH₂CH₂)qOCH₃ wherein q is a positive integer. The integer q may for instance be from 5 to 10,000, or for instance from 10 to 1,000.

[0129] Polyglycerine may be used instead of poly(ethylene glycol).

[0130] The amphipathic molecules may alternatively be polymeric amphipathic molecules. For instance, the amphipathic molecules may comprise a block copolymer, for instance a triblock copolymer. Suitable block copolymers and triblock copolymers are described in WO 2014 / 064444, and include the triblock copolymer PMOXA-PDMS-PMOXA.

[0131] Droplets can exchange chemical species with each other, and with the external environment, through protein pores incorporated in the layer of amphipathic molecules between droplets, or between a droplet and the external environment. Thus, one or more of the droplets in the synthetic droplet assembly may further comprise a protein pore. The protein pore is a membrane protein which is able to form a pore, channel or pump in a layer of amphipathic molecules. Thus, in the synthetic droplet assembly described herein, the protein pore may form a pore, channel or pump in any of the layers (e.g. bilayers) of amphipathic molecules which is an interface between contacting droplets.

[0132] Thus, often, in the synthetic droplet assembly, at least one layer of said amphipathic molecules which is an interface between contacting droplets further comprises a protein pore. In some embodiments, each layer of said amphipathic molecules which is an interface between contacting droplets further comprises a protein pore.

[0133] Often said interfaces between droplets are bilayers of the amphipathic molecules, i.e. each layer of said amphipathic molecules which is an interface between contacting droplets is a bilayer of said amphipathic molecules.

[0134] Often, therefore, the protein pore is a membrane protein which is able to form a pore, channel or pump in a bilayer of amphipathic molecules.

[0135] Often, each layer of said amphipathic molecules which is an interface between contacting droplets is a bilayer of said amphipathic molecules, and at least one bilayer of said amphipathic molecules which is an interface between contacting droplets further comprises a protein pore. For instance, each bilayer of said amphipathic molecules which is an interface between contacting droplets may further comprise a protein pore.The use of integral membrane proteins in droplet interface layers of amphipathic molecules (e.g. in DIBs) has been demonstrated, but it is equally expected that peripheral membrane proteins could be used.

[0136] Membrane pumps, channels and pores can allow for precise control over the exchange of the effector molecule and / or other materials, between (i) individual droplets within the synthetic assembly and (ii) the synthetic droplet assembly and an external region, for instance a target region comprising biological cells or tissue. The protein pore could for instance be an a-hemolysin (aHL) pore, such as a staphylococcal a-hemolysin pore. However, any suitable membrane protein can be used including one from the two major classes, that is, P-barrels or a-helical bundles, to form the protein pore. An example of another protein pore which may be employed, which has a larger internal diameter than the aHL pore is perfringolysin O. The protein pore may also, for instance be a channel, which may be a voltage-gated ion channel, a light-sensitive channel such as bacteriorhodopsin, a ligand-gated channel or a mechano-sensitive channel.

[0137] Suitable protein pores which allow for exchange of materials are known and readily available to the skilled person; many such proteins are either commercially available or can be prepared by known methods. For instance, wild type (WT) aHL monomers can be prepared by in vitro transcription-translation (IVTT), and heptamerised by incubation with rabbit red blood cell membranes. The heptamers are typically purified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Maglia, G. et al., Method. Enzymol. 475, 591-623, (2010). Also, Bayley, H. et al., Droplet interface bilayers. Mol. BioSyst. 4, 1191-1208 (2008) lists several proteins that were tested for insertion into droplet interface bilayers made in bulk oil. Other suitable membrane proteins include, but are not limited to, bacterial peptides and ionophores.

[0138] The protein pore may, alternatively, be an engineered protein pore or a synthetic protein pore. The engineered protein pore may, for instance, be a genetically engineered protein pore, or a covalent or non-covalent chemically engineering protein pore. The synthetic protein pore may, for instance, be a peptide or an organic molecule. The protein pore need not comprise only protein or peptide molecules but may comprise other molecules in addition to the protein component. For instance, the protein pore may be a hybrid nucleic acid-protein pore of the kind described in WO 2020 / 025909. Droplets can exchange chemical species with each other through protein pores incorporated in the layer of amphipathic molecules at the interface between the droplets.

[0139] Often, the protein pore is a-hemolysin (aHL).

[0140] Usually, when present, the concentration of protein pore is equal to or greater than 0.1 ng mL1, for instance, equal to or greater than 1 ng mL'1. For instance, the concentration of the protein pore may be equal to or greater than 2 ng mL1, for instance, equal to or greater than 10 ng mL1. Often, the concentration of protein pore is equal to or greater than 100 ng mL1, for instance, equal to or greater than 1 pg mL'1. For instance, the concentration of the protein pore maybe equal to or greater than 2 μg mL⁻¹, for instance, equal to or greater than 5 μg mL⁻¹, or equal to or greater than 10 μg mL⁻¹.

[0141] The concentration of the protein pore may, for instance, be from 0.1 ng mL'1to 100 mg mL1. Typically, the concentration of the protein pore is from 1 ng mL1to 1 mg mL1, for instance from 100 ng mL'1to 100 pg mL1. More typically, the concentration of the protein pore is from 1 pg mL'1to 100 pg mL1, for instance, from 2 pg mL'1to 80 pg mL1. In some embodiments, the concentration of the protein pore is from about 1 pg mL'1to about 60 pg mL1, or from about 5 pg mL'1to about 50 pg mL1, or about 10 pg mL'1to about 50 pg mL1. It may for instance be from 10 pg mL'1to 40 pg mL1, or from 20 pg mL'1to 40 pg mL1, for instance from 25 pg mL'1to 35 pg mL1. In other embodiments, the concentration of the protein pore is from about 10 pg mL'1to about 80 pg mL1, or from about 20 pg mL'1to about 70 pg mL1, or about 20 pg mL1to about 60 pg mL1. It may for instance be from 30 pg mL1to 60 pg mL1, or from 40 pg mL1to 60 pg mL1, for instance about 50 pg mL1.

[0142] Such concentrations are particularly suitable for the aHL pore, especially when employed in combination with the effector molecule arabinose, at any of the concentrations for the effector molecule specified herein.

[0143] Typically, the concentration of the protein pore is the concentration of the protein pore (i.e. the pore-forming protein) in the aqueous medium of the droplet, when the droplet is formed. When a droplet comprising a protein pore (pore-forming protein) is contacted with another droplet (which may or may not comprise a protein pore), a layer of amphipathic molecules (often a bilayer of amphipathic molecules) is formed at the interface. That interface layer of amphipathic molecules typically then comprises the protein pore. Therefore a protein pore initially in the aqueous medium may move to the layer of amphipathic molecules (often a bilayer of amphipathic molecules) at the interface of the droplet with another droplet and / or the target region.

[0144] Suitable concentrations of the protein pore may depend on a number of factors. The rate of insertion of the membrane protein into the bilayer may, for instance, decrease with time. Typically, this will put a lower limit on the concentrations of the protein pore that may be used.

[0145] The concentration of protein pore in one droplet of the droplet assembly and another droplet may be the same or different. Thus, the concentration of protein pores in each droplet of the droplet assembly may be the same or different.

[0146] Typically, at least one layer (e.g. bilayer) of amphipathic molecules in the synthetic droplet assembly comprises a protein pore. A layer (e.g. bilayer) of amphipathic molecules at an interface between contacting droplets, or at an interface between a target interface droplet and the external environment (e.g. a target region comprising biological cells or tissue), may comprise more than one protein pore. For instance, a particular layer (e.g. bilayer) of amphipathic molecules may contain multiple copies of the same protein pore, or two or more different classes of protein pores.Where more than one class is present, the layer (e.g. bilayer) of amphipathic molecules may contain multiple copies of each different class.

[0147] The protein pore may be as further defined herein, for instance it may be a-hemolysin (aHL) or a different pore, for instance a protein pore with a larger internal diameter such as perfringolysin O.

[0148] It is not necessary for the synthetic droplet assembly (or any of the droplets within it, or any of the layers of amphipathic molecules within it) to comprise a protein pore in order for flux of water in or out of the droplet assembly in accordance with any of the processes of the invention to be possible. Indeed, flux of water may be achieved by osmosis, or evaporation, for instance, in the absence of any protein pores.

[0149] The synthetic droplet assembly employed in either of the processes of the invention, or the synthetic droplet assembly of the invention, may be in contact with a hydrophobic medium. The synthetic droplet assembly may additionally be in contact with a region other than the hydrophobic medium. For instance, the synthetic droplet assembly may also be in contact with an aqueous region. The aqueous region may be referred to as an external aqueous medium, to distinguish that aqueous medium from the aqueous medium of the droplets of the droplet assembly. The aqueous region (or external aqueous medium) may for instance be an aqueous solution or a hydrogel. The aqueous region may for instance be a “target region” comprising biological cells or tissue, which may optionally further comprise an aqueous solution or a hydrogel. Alternatively, the synthetic droplet assembly may be in contact only with the hydrophobic medium. Often, the synthetic droplet assembly employed in either of the processes of the invention, or the synthetic droplet assembly of the invention, is in (i.e. it is disposed in) a hydrophobic medium.

[0150] The presence of the hydrophobic medium in such cases may have a stabilising effect on the synthetic droplet assembly. For instance, the outer layer of amphipathic molecules of each of the droplets of the droplet assembly that is at an external surface of the synthetic droplet assembly may form a stable monolayer at the interface between the droplet and the hydrophobic medium. In such a monolayer, polar head groups of the amphipathic molecules may be in contact with the aqueous medium of the droplet and non-polar tail groups of the amphipathic molecules may be in contact with the hydrophobic medium.

[0151] The hydrophobic medium may be selected from a wide range of materials. It may be the same hydrophobic medium as a hydrophobic medium in which the droplet assembly was first produced by 3D-droplet printing (e.g. as described in any ofWO 2014 / 087175 Al, WO 2021 / 234394 Al, G. Villar et al. Science 2013, 340, 48, and I. Cazimoglu et al, ACS Nano 2021, 15, 20214). The hydrophobic medium can, for instance, be selected to affect the buoyancy of the droplets and the speed of formation of the layer of amphipathic molecules around the droplets after the droplets are first introduced into the hydrophobic medium during the droplet printing process.The hydrophobic medium may comprise a single hydrophobic compound. Alternatively, it may comprise a mixture of two or more different hydrophobic compounds. The hydrophobic medium employed may, for instance, have a density close to that of water, for instance a density of less than or equal to about 1 g cm-3.

[0152] The hydrophobic medium is typically an oil. The oil may be a single, pure, compound, or the oil may comprise a mixture of two or more compounds. It is usually desirable that the oil does not significantly destabilize any layers of amphipathic molecules (e.g. bilayers of amphipathic molecules) formed.

[0153] The oil may for instance comprise silicone oil, for instance poly(methylphenylsiloxane). Silicone oil is advantageous on account of its density being close to that of water, which ensures that the droplet is approximately neutrally buoyant in water. The silicone oil may for instance be poly(methylphenylsiloxane), which has a density of about 1 g cm-3. The oil may consist of a single silicone oil, for instance poly(methylphenylsiloxane), such as AR20. Alternatively, the oil may comprise a mixture of two or more different silicone oils. Any suitable silicone oil may be used. For instance, the oil may comprise silicon oil DC200 (a polymer comprising monomer units of -O-Si(CH3)2-), poly(dimethylsiloxane) (PDMS), hydroxy terminated, or PDMS 200. Often, however, the silicone oil is a poly(methylphenylsiloxane), such as AR20.

[0154] Additionally, or alternatively, the oil may comprise a hydrocarbon. The hydrocarbon typically has from 5 to 40 carbon atoms (a C5-C40 hydrocarbon), more typically from 10 to 30 carbon atoms (a C10-C30 hydrocarbon). Typically, it is an alkane or an alkene. Thus, the hydrocarbon may be a C5-C30 alkane, or a C10-C20 alkane. In another embodiment, the hydrocarbon may be a C5-C20 alkene, or a C10-C20 alkene. The hydrocarbon is typically unsubstituted. In one embodiment it is undecane. In a preferred embodiment, the hydrocarbon is an unsubstituted C5-C20 alkane, preferably an unsubstituted C10-C20 alkane. The hydrocarbon may for instance be squalene, hexadecane, undecane or decane. However, in some embodiments the hydrocarbon may be substituted with a halogen atom. For example the oil may be a fluorocarbon, or a bromo-substituted C10-C30 alkane, or for instance a bromo-substituted C10-C20 alkane, e.g. bromododecane. When the oil comprises a hydrocarbon, it may comprise a single hydrocarbon compound, or a mixture of two or more hydrocarbons.

[0155] In some embodiments, the oil is a mixture comprising: (a) one or more hydrocarbons, and (b) one or more silicone oils. The one or more silicone oils may be as defined above, for instance they may comprise poly(methylphenylsiloxane) (for instance AR20). The one or more hydrocarbons may, for instance, comprise any suitable liquid hydrocarbon. Whether a particular hydrocarbon is liquid will depend upon the temperature of the hydrophobic medium. Thus, the term liquid hydrocarbon refers to a hydrocarbon that is a liquid at the temperature that the hydrophobic medium is at. Typically, the hydrophobic medium will be at room temperature. However, in some embodiments, the hydrophobic medium may be above or below room temperature. In someembodiments, the oil may comprise a solid. A solid hydrocarbon may, for instance, be used in combination with a silicone oil. The oil may, for instance, be a mixture of solids that dissolve to form a liquid. The hydrocarbon may be branched or unbranched, for example a hydrocarbon having from 5 to 40 carbon atoms, or from 5 to 30 carbon atoms (although hydrocarbons of lower molecular weight would require control of evaporation). Preferably, the hydrocarbon is a liquid at the operating temperature of the synthetic droplet assembly employed in the invention. Suitable examples include alkanes or alkenes, such as hexadecane, undecane, decane, pentane or squalene. Often, it is a liquid alkane hydrocarbon, for instance a C10-C20 alkane such as hexadecane, undecane, decane. Undecane may be preferred. Usually, the oil comprises a hydrocarbon and the hydrocarbon comprises undecane. Often the hydrocarbon is undecane. Thus, the oil may comprise undecane.

[0156] In one embodiment, the hydrophobic medium comprises a silicone oil and an unsubstituted C10-C20 alkane, and the amphipathic molecules employed comprise one or more glycerophospholipids. For instance, the hydrophobic medium may comprise poly(methylphenylsiloxane) (for instance AR20) and undecane, and the amphipathic molecules may comprise DPhPC and / or POPC. For instance, the amphipathic molecules may be DPhPC, or the amphipathic molecules may comprise both DPhPC and POPC.

[0157] In some embodiments, the hydrophobic medium comprises a silicone oil and a hydrocarbon. Such mixtures have been found to provide advantageously short incubation times required for stable bilayers to be formed during the printing process for producing synthetic droplet assemblies. The silicone oil and hydrocarbon in the mixture may be as further defined above. Typically, the hydrocarbon is an unsubstituted C10-C20 alkane, preferably undecane. The silicone oil usually has a density close to, but less than, that of water, to control the sinking rate of droplets during printing. The silicon oil may be as further defined above, and is often poly(methylphenylsiloxane) (for instance AR20). Thus, often, the hydrophobic medium is an oil, and the oil comprises a hydrocarbon and a silicone oil, wherein the hydrocarbon is undecane and the silicone oil is a poly(methylphenylsiloxane) (for instance AR20). Usually, the volume:volume ratio of the hydrocarbon to the silicone oil is from 1: 10 to 10:1, for instance from 1:5 to 5: 1 or from 1:4 to 4: 1. The volume ratio of the hydrocarbon to the silicone oil may for instance be about 20: 80. It is often from 1:3 to 3: 1. It is often, for instance from 1:2 to 1:1. The volume ratio of the hydrocarbon to the silicone oil may for instance be about 35:65.

[0158] Usually, amphipathic molecules are disposed in the hydrophobic medium. The amphipathic molecules disposed in the hydrophobic medium are typically the same as those employed in the synthetic droplet assembly itself, and may therefore be as further defined anywhere herein. The concentration of the amphipathic molecules in the hydrophobic medium may be any suitable concentration. For instance, often, the concentration of the amphipathic molecules in the hydrophobic medium is less than or equal to 15 mg mL-1. For instance, the concentration ofamphipathic molecules may be from 0 to 10 mg mL-1. Usually, the concentration of amphipathic molecules is from 0.05 mg mL-1to 10 mg mL-1, for instance, from 0.05 mg mL-1to 5 mg mL-1. More typically, the concentration of amphipathic molecules is from 0.1 mg mL-1to 2.5 mg mL-1, for instance, from 0.2 mg mL-1to 0.5 mg mL-1. In some embodiments, the concentration of the amphipathic molecules in the hydrophobic medium is, for instance, from 0.1 mM to 10 mM, or, for instance, from 0.2 mM to 5 mM, such as from 0.5 mM to 3 mM, e.g. about 1 mM, or about 2 mM. Process for reducing the size of droplets

[0159] The invention provides a process of for reducing the size of droplets in a synthetic droplet assembly of the kind defined above, and this process allows access to droplet assemblies in which droplet sizes are smaller than previously obtainable by 3D droplet printing. This advantageously allows for patterned release of an effector molecule at very high resolution, amongst other new applications.

[0160] In the process of the invention for reducing the size of droplets in a synthetic droplet assembly, the synthetic droplet assembly is as defined above, i.e. it comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets. The process comprises removing water from the aqueous medium of droplets in the droplet assembly.

[0161] The size of droplets in the synthetic droplet assembly may be reduced by any method of achieving flux of water out of the droplets. Indeed, any technique suitable for achieving flux of water out of the droplets, and thereby removing water from the aqueous medium of droplets in the droplet assembly, may be employed. Two important options for this that are discussed further herein are the removal of water by evaporation or by osmosis. However, any other suitable technique for the removal of water could in principle be employed.

[0162] Reducing the size of the droplets in the synthetic droplet assembly, in this way, by the process of the invention, may comprise reducing the diameter of the droplets to a diameter of less than 60 pm. Often, however, reducing the size of the droplets in the synthetic droplet assembly, comprises reducing the diameter of the droplets to a diameter of less than 10 pm. For instance, the diameter of each of the n droplets in the synthetic droplet assembly may be reduced to a diameter of less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm, or less than or equal to 3 pm, for example less than or equal to 2 pm, by the process of the invention for reducing the size of droplets. Thus, each of the n droplets in the synthetic droplet assembly may be reduced to a diameter of from 50 nm to less than 10 pm, for instance from 50 nm to 9 pm, or from 50 nm to 8 pm. Often, each of the n droplets in the synthetic droplet assembly is reduced to a diameter of from 50 nm to 6 pm, for instance from 70 nm to 5 pm, or for example from 100 nm to 3 pm. For instance, each of the n droplets in the synthetic droplet assembly may bereduced to a diameter of from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm.

[0163] Similarly, reducing the size of the droplets in the synthetic droplet assembly may comprise reducing the volume of the droplets to a volume of less than 113 pL. Often, reducing the size of the droplets in the synthetic droplet assembly comprises reducing the volume of the droplets to a volume of less than 0.5 pL. For instance, each of the n droplets in the synthetic droplet assembly may be reduced to a volume of less than or equal to 382 fL (femtolitres), less than or equal to 268 fL, or for instance less than or equal to 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL. Thus, each of the n droplets in the synthetic droplet assembly may be reduced to a volume of from 65 zL to less than 0.5 pL, for instance from 65 zL to 382 fL, or from 65 zL to 268 fL. Often, each of the n droplets in the synthetic droplet assembly is reduced to a volume of from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL. For instance, each of the n droplets in the synthetic droplet assembly may be reduced to a volume of from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0164] Droplet diameters and volumes can be determined by microscopy.

[0165] Removal of water by evaporation

[0166] The Example hereinbelow and Figure 4(d) describe a heat-induced post-printing shrinking process, in which droplet size was reduced post-printing, by release of water from the droplets through evaporation.

[0167] Thus, often, in the process of the invention, removing water from the aqueous medium of droplets in the synthetic droplet assembly comprises evaporating said water.

[0168] The water may be evaporated by heating the droplet assembly or by subjecting the droplet assembly to reduced pressure. The water may be evaporated by heating the droplet assembly and by subjecting the droplet assembly to reduced pressure.

[0169] Usually, evaporating the water comprises heating the droplet assembly. For instance, evaporating the water may comprise heating the droplet assembly without subjecting it to reduced pressure. In other embodiments, however, evaporating the water may comprise subjecting the droplet assembly to reduced pressure, for instance by subjecting the droplet assembly to reduced pressure without heating the droplet assembly.

[0170] Preferably, however, evaporating the water comprises heating the droplet assembly.

[0171] Typically, heating the droplet assembly comprises exposing the droplet assembly to a temperature of at least 30 °C, for instance at least 35 °C. Heating the droplet assembly may for instance comprise exposing the droplet assembly to a temperature of at least 40 °C, for instance at least 45 °C, or at least 50 °C.

[0172] Often, heating the droplet assembly comprises exposing the droplet assembly to a temperature of from 30 °C to 90 °C, for instance from 30 °C to 80 °C, or from 30 °C to 70 °C, or for instance from 35 °C to 70 °C. Heating the droplet assembly may for instance comprise exposingthe droplet assembly to a temperature of from 40 °C to 70 °C. Usually, however, heating the droplet assembly comprises exposing the droplet assembly to a temperature of from 30 °C to 50 °C, for instance to a temperature of from 35 °C to 45 °C.

[0173] Often, heating the droplet assembly comprises exposing the droplet assembly to said temperature for a duration of at least 5 minutes, optionally for a duration of at least 20 minutes, or for a duration of at least 1 hour, for instance for a duration of from 1 to 4 hours. The time needed for water to evaporate and droplets to shrink depends on the temperature. The evaporation time can be minimized by increasing temperature.

[0174] Typically, in the process of the invention for reducing the size of droplets, the droplet assembly is in a hydrophobic medium. This is particularly often the case when removing water from the aqueous medium of droplets in the droplet assembly comprises evaporating said water and evaporating said water comprises heating the droplet assembly. In this case, heating the droplet assembly typically comprises heating the hydrophobic medium. Indeed, typically, the hydrophobic medium is heated to a temperature as defined above, for a duration as defined above. Thus, often, evaporating said water comprises heating the droplet assembly by heating the hydrophobic medium, optionally wherein the hydrophobic medium is heated to a temperature as defined above and optionally for a duration as defined above. Often, the droplet assembly is in a hydrophobic medium, and the hydrophobic medium is in a container, and heating the hydrophobic medium comprises heating the container. Any suitable container may be employed, for instance a glass container such as a beaker, petri dish or cuvette, and the container may be disposed on a heating plate, for heating the container.

[0175] The hydrophobic medium may be as further defined anywhere herein. For instance, as discussed above, the hydrophobic medium may be an oil. It may for instance comprise silicone oil and a hydrocarbon, for instance an unsubstituted C10-C20 alkane. The hydrophobic medium may for instance comprise poly(methylphenylsiloxane) (e.g. AR20) and undecane. It typically further comprises said amphipathic molecules. The volume ratio of the hydrocarbon to the silicone oil may be as defined above.

[0176] Removal of water by osmosis

[0177] The size of droplets in the synthetic droplet assembly may be reduced by any method of achieving flux of water out of the droplets. One method of achieving this is to use osmosis.

[0178] Thus, typically, in the process of the invention for reducing the size of droplets in the synthetic droplet assembly, removing water from the aqueous medium of the droplets comprises removing said water by osmosis.

[0179] Often, removing said water by osmosis comprises contacting the droplet assembly with an external aqueous medium, wherein the external aqueous medium has an osmolarity which is greater than the osmolarity of the aqueous medium of the droplets. Typically, said contacting comprises (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting theosmolarity of the external aqueous medium (while keeping the droplet assembly in contact with the external aqueous medium) so that the external aqueous medium has an osmolarity which is greater than the osmolarity of the aqueous medium of the droplets, and (iii) maintaining contact between the droplet assembly and the external aqueous medium. Adjusting the osmolarity of the external aqueous medium typically comprises increasing the osmolarity of the external aqueous medium, and optionally comprises increasing the osmolarity of the external aqueous medium in a stepwise manner. For instance, said contacting may comprise (i) bringing the droplet assembly into contact with an external aqueous medium whose osmolarity is (about) the same as the osmolarity of the aqueous medium of the droplets, and then (ii) adjusting the osmolarity of the external aqueous medium so that the osmolarity of the external aqueous medium is greater than the osmolarity of the aqueous medium of the droplets, and then (iii) maintaining contact between the droplet assembly and the external aqueous medium.

[0180] Adjusting the osmolarity of the external aqueous medium typically comprises adding to the external aqueous medium an aqueous solution whose osmolarity is greater than the osmolarity of the aqueous medium of the droplets. The aqueous solution may for instance be pipetted into the external aqueous medium. Said aqueous solution may be added (e.g. pipetted) until the external aqueous medium has the desired osmolarity that is greater than the osmolarity of the aqueous medium of the droplets. The aqueous solution may be added in a stepwise manner (i.e. in a plurality of steps, each comprising adding the aqueous solution to the external aqueous medium) until the desired osmolarity is achieved. The stepwise addition may be performed over a period of time, for instance over a period of from 0.5 to 20 hours, for instance from 2 to 15 hours, or from 5 to 11 hours.

[0181] When the osmolarity of the external aqueous medium is greater than the osmolarity of the aqueous medium of the droplets, and contact between the droplet assembly and the external aqueous medium is maintained, water flows out of the droplets and into the external aqueous medium by osmosis to reduce the size of the droplets. Indeed, step (iii) above may be performed until enough water has flowed for the osmolarities of the droplet assembly and the external aqueous medium to be equal.

[0182] The droplet assembly is typically in contact with a hydrophobic medium. For instance, the droplet assembly may be disposed in a hydrophobic medium. The hydrophobic medium may be as further defined anywhere herein. For instance, as discussed above, the hydrophobic medium may be an oil. It may for instance comprise silicone oil and a hydrocarbon, for instance an unsubstituted C10-C20 alkane. The hydrophobic medium may for instance comprise poly(methylphenylsiloxane) (e.g. AR20) and undecane. It typically further comprises said amphipathic molecules. The volume ratio of the hydrocarbon to the silicone oil may be as defined above.

[0183] When the droplet assembly is in contact with a hydrophobic medium, for instance disposed in a hydrophobic medium, the hydrophobic medium may also be in contact with the externalaqueous medium. For instance, the hydrophobic medium may be layered on top of the external aqueous medium, e.g. within a container such as a beaker, petri dish, cuvette (e.g. a special optical glass (SOG) cuvette), vat or any other vessel.

[0184] Thus, often, contacting the droplet assembly with the external aqueous medium comprises moving the synthetic droplet assembly within the hydrophobic medium in order to bring the synthetic droplet assembly into contact with the external aqueous medium. For instance, contacting the droplet assembly with the external aqueous medium may comprise introducing the droplet assembly into the hydrophobic medium and moving the synthetic droplet assembly within the hydrophobic medium in order to bring the synthetic droplet assembly into contact with the external aqueous medium. Moving the synthetic droplet assembly within the hydrophobic medium in order to bring it into contact with the external aqueous medium may comprise allowing the droplet assembly to sink through the hydrophobic medium until the droplet assembly contacts the external aqueous medium.

[0185] As with the aqueous medium of the droplets, the external aqueous medium may be any suitable aqueous medium. For instance, the external aqueous medium may be an aqueous solution. It may for instance be an aqueous solution of one or more salts, or an aqueous buffer solution. The aqueous medium may for instance be an aqueous solution comprising Dulbecco's phosphate-buffered saline (DPBS). The aqueous solution may for instance comprise DPBS and sucrose. The concentration of sucrose may be selected to achieve a desired osmolarity, although any other suitable solute could be employed, instead of sucrose, for the purpose of adjusting osmolarity. The external aqueous medium may for instance comprise a growth medium or a culture medium, for instance a cell growth medium or a cell culture medium. The external aqueous medium may for instance comprise culture medium for eukaryotic cells or tissue, or a growth medium for bacteria, such as for example M9 minimal medium. Thus, for instance, the external aqueous medium may be an aqueous solution which comprises M9 minimal salts. The aqueous solution which comprises M9 minimal salts may further comprise one or more amino acids. It may additionally further comprise glucose or glycerol. Often, the aqueous solution which comprises M9 minimal salts further comprises at least one of MgSO4, CaCl2, one or more amino acids, glucose or glycerol. Typically, the aqueous solution which comprises M9 minimal salts further comprises MgSO4, CaCl2, casamino acids, and glucose or glycerol. Alternatively, the external aqueous medium may comprise a hydrogel. When the external aqueous medium comprises a hydrogel, the aqueous medium may be as further defined above for the aqueous medium of the droplets.

[0186] Contacting the droplet assembly with the external aqueous medium typically forms a layer of the amphipathic molecules as an interface between droplets of the synthetic droplet assembly and the external aqueous medium. Often, said layer of the amphipathic molecules is a bilayer of the amphipathic molecules. Water may flow through the layer or bilayer by osmosis.Typically, the aqueous medium in the droplets of the droplet assembly has a first osmolarity, and removing said water by osmosis comprises: contacting the droplet assembly with said external aqueous medium which has a second osmolarity, wherein the second osmolarity is greater than the first osmolarity.

[0187] Often, the aqueous medium in the droplets of the droplet assembly has a first osmolarity, and said contacting comprises: (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium (while keeping the droplet assembly in contact with the external aqueous medium) so that the external aqueous medium has a second osmolarity wherein the second osmolarity is greater than the first osmolarity, and (iii) maintaining contact between the droplet assembly and the external aqueous medium. Adjusting the osmolarity of the external aqueous medium typically comprises increasing the osmolarity of the external aqueous medium, and optionally comprises increasing the osmolarity of the external aqueous medium in a stepwise manner. For instance, the aqueous medium in the droplets of the droplet assembly may have a first osmolarity, and said contacting may comprise: (i) bringing the droplet assembly into contact with an external aqueous medium whose osmolarity is (about) the same as the first osmolarity, and then (ii) adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has a second osmolarity wherein the second osmolarity is greater than the first osmolarity, and then (iii) maintaining contact between the droplet assembly and the external aqueous medium.

[0188] Adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has said second osmolarity typically comprises adding an aqueous solution to the external aqueous medium, wherein the osmolarity of said aqueous solution is greater than the first osmolarity. The aqueous solution may for instance be pipetted into the external aqueous medium. Said aqueous solution may be added (e.g. pipetted) until the external aqueous medium has said second osmolarity as desired. The aqueous solution may be added in a stepwise manner (i.e. in a plurality of steps, each comprising adding the aqueous solution to the external aqueous medium) until the desired second osmolarity is achieved. The stepwise addition may be performed over a period of time, for instance over a period of from 0.5 to 20 hours, for instance from 2 to 15 hours, or from 4 to 11 hours.

[0189] When the external aqueous medium has said second osmolarity - which is greater than the first osmolarity of the aqueous medium of the droplets - and contact between the droplet assembly and the external aqueous medium is maintained, water flows out of the droplets and into the external aqueous medium by osmosis to reduce the size of the droplets. Indeed, step (iii) may be performed until enough water has flowed for the osmolarities of the droplet assembly and the external aqueous medium to be equal. Alternatively, step (iii) may be performed until a desired reduction in the size of the droplets, or droplet assembly, is achieved. Often, step (iii) is performed for a duration of at least 0.5 hours, and typically up to 7 hours.The process may further comprise separating the droplet assembly and the external aqueous medium. Often, the separating is performed when a desired reduction in the size of the droplets, or droplet assembly, is achieved.

[0190] Often, the first osmolarity is from 0 osm to 7 osm, for instance from 0 osm to 3 osm, or from 0 osm to 2 osm. The first osmolarity may for instance be from 0 osm to 1 osm, for instance from 0.001 osm to 0.8 osm, or from 0.01 osm to 0.6 osm, for instance from 0.1 osm to 0.5 osm. The first osmolarity may for instance be about 0.3 osm (300 mosm) or for instance about 0.4 osm (400 mosm).

[0191] The second osmolarity is often from 0.001 osm to 7.001 osm. The second osmolarity may for instance be from 0.01 osm to 7.001 osm, for instance from 0.1 osm to 7 osm, or from 0.2 osm to 7 osm. It may for instance be from 0.3 osm to 7 osm, or for instance from 0.4 osm to 7 osm. The second osmolarity may for instance be about 0.3 osm (300 mosm) or about 6.5 osm, or it may be anywhere in between these values. Often, the second osmolarity is towards the higher end of this range. For instance, the second osmolarity may be from 0.5 osm to 7 osm, for instance from 1 osm to 7 osm, for instance from 3 osm to 7 osm, or from 5 osm to 7 osm. The second osmolarity may for instance be about 6.5 osm.

[0192] Often, the first osmolarity is from 0 osm to 7 osm and the second osmolarity is from 0.001 osm to 7.001 osm.

[0193] Thus, the first osmolarity may be from 0 osm to 3 osm and the second osmolarity may be from 0.01 osm to 7 osm. For instance, the first osmolarity may be from 0 osm to 1 osm and the second osmolarity may be from 0.1 osm to 7 osm. The first osmolarity may for instance be from 0 osm to 1 osm and the second osmolarity may be from 0.2 osm to 7 osm. The first osmolarity may for instance be from 0.001 osm to 0.8 osm and the second osmolarity may be from 0.3 osm to 7 osm. The first osmolarity may for instance be from 0.01 osm to 0.6 osm and the second osmolarity may be from 0.3 osm to 7 osm. The first osmolarity may for instance be from 0.1 osm to 0.5 osm and the second osmolarity may be from 0.3 osm to 0.3 osm. The first osmolarity may for instance be from 0.2 osm to 0.5 osm and the second osmolarity may be from 0.4 osm to 7 osm. The first osmolarity may for instance be from 0.01 osm to 0.6 osm and the second osmolarity may be from 0.5 osm to 7 osm. The first osmolarity may for instance be from 0.1 osm to 0.5 osm and the second osmolarity may be from 1 osm to 7 osm. The first osmolarity may for instance be from 0.2 osm to 0.5 osm and the second osmolarity may be from 3 osm to 7 osm, or for instance from 5 osm to 7 osm.

[0194] Often, removing said water by osmosis comprises contacting the droplet assembly with said external aqueous medium (i.e. with said external aqueous medium which has an osmolarity which is greater than the osmolarity of the aqueous medium of the droplets, or with said external aqueous medium which has said second osmolarity) for a duration of at least 0.2 hours, for instance at least 0.5 hours. For instance, removing said water by osmosis may comprise contacting thedroplet assembly with said external aqueous medium for from 0.2 hours to 7 hours, for instance from 0.5 hours to 7 hours. The duration required to achieve the desired size reduction depends on various factors including the difference in osmolarity, and the size of the synthetic droplet assembly. The duration can be minimized or maximized depending on the application.

[0195] Thus, step (iii) above, of maintaining contact between the droplet assembly and the external aqueous medium typically comprises maintaining contact between the droplet assembly and the external aqueous medium for at least 0.2 hours, for instance for at least 0.5 hours, for instance from 0.2 hours to 7 hours, or from 0.5 hours to 7 hours.

[0196] Often, removing said water by osmosis comprises contacting the droplet assembly with said external aqueous medium (i.e. with said external aqueous medium which has an osmolarity which is greater than the osmolarity of the aqueous medium of the droplets, or with said external aqueous medium which has said second osmolarity) until the osmolarities of the aqueous medium of the droplets and the external aqueous medium are equal. Alternatively, contacting the droplet assembly with said external aqueous medium may be performed until a desired reduction in the size of the droplets, or droplet assembly, is achieved.

[0197] Typically, in the process of the invention for reducing the size of droplets in a synthetic droplet assembly (whether said removing water is by evaporation, by osmosis or by other means) removing water from the aqueous medium of droplets in the droplet assembly reduces the mean diameter of the droplets in the droplet assembly from a first droplet diameter to a second droplet diameter. Droplet diameters can be determined by microscopy.

[0198] Often, the first droplet diameter is at least 10 pm, for instance at least 60 pm.

[0199] Typically, the second droplet diameter is less than 60 pm, for instance less than 10 pm, or less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm. The second droplet diameter may for instance be less than or equal to 3 pm, or say, less than or equal to 2 pm.

[0200] Thus, typically, the first droplet diameter is at least 10 pm and the second droplet diameter is less than 10 pm, or less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm. The first droplet diameter may be at least 10 pm and the second droplet diameter may be less than or equal to 3 pm, such as less than or equal to 2 pm. The first droplet diameter may for instance be at least 60 pm, and the second droplet diameter may be less than 60 pm, for instance less than 10 pm, or less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm. The first droplet diameter may be at least 60 pm and the second droplet diameter may be less than or equal to 3 pm, for instance less than or equal to 2 pm.

[0201] Often, the first droplet diameter is from 10 pm to 500 pm, for instance from 60 pm to 400 pm.

[0202] Typically, the second droplet diameter is from 50 nm to 60 pm, or for instance from 50 nm to less than 10 pm, for instance from 50 nm to 9 pm, or from 50 nm to 8 pm, for instance from 50nm to 6 pm, or from 70 nm to 5 pm. The second droplet diameter may for instance be from 100 nm to 10 pm, or from 100 nm to less than 10 pm, for example from 100 nm to 3 pm. The second droplet diameter may for instance be from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm, for instance from 500 nm to 5 pm.

[0203] Thus, typically, the first droplet diameter is from 10 pm to 500 pm and the second droplet diameter is from 50 nm to 10 pm (typically to less than 10 pm), or from 100 nm to 10 pm (typically to less than 10 pm). For instance, the first droplet diameter may be from 10 pm to 500 pm and the second droplet diameter may be from 50 nm to 9 pm, or from 50 nm to 8 pm, for instance from 50 nm to 6 pm, or from 70 nm to 5 pm, for example from 100 nm to 3 pm. The first droplet diameter may be from 10 pm to 500 pm and the second droplet diameter may for instance be from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm, for instance from 500 nm to 5 pm.

[0204] The first droplet diameter may for instance be from 60 pm to 400 pm, and the second droplet diameter may be from 50 nm to 60 pm (typically to less than 60 pm), for instance the second droplet diameter may be from 100 nm to 10 pm, or from 500 nm to 5 pm. The first droplet diameter may be from 60 pm to 400 pm, and the second droplet diameter may be from 50 nm to 9 pm, or from 50 nm to 8 pm, for instance from 50 nm to 6 pm, or from 70 nm to 5 pm, for example from 100 nm to 3 pm. The first droplet diameter may be from 60 pm to 400 pm and the second droplet diameter may for instance be from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm, for instance from 500 nm to 5 pm.

[0205] Often, in the process of the invention for reducing the size of droplets in a synthetic droplet assembly (whether said removing water is by evaporation, by osmosis or by other means) removing water from the aqueous medium of droplets in the droplet assembly reduces the mean volume of the droplets in the droplet assembly from a first droplet volume to a second droplet volume.

[0206] Droplet volumes can be determined by microscopy.

[0207] Often, the first droplet volume is at least 0.5 picolitres (pL). For instance, the first droplet volume may be at least 113 pL.

[0208] The second droplet volume is typically less than 113 pL, optionally less than 0.5 pL, for instance less than 65 femtolitres (fL).

[0209] For instance, the first droplet volume may be at least 0.5 pL and the second droplet volume may be less than 0.5 pL, for instance less than or equal to 382 fL, less than or equal to 268 fL, or for instance less than or equal to 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL.

[0210] The first droplet volume may for instance be at least 113 pL and the second droplet volume may be less than 113 pL, for instance less than 0.5 pL, less than or equal to 382 fL, less than or equal to 268 fL, or for instance less than or equal to 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL.Often, the first droplet volume is from 0.5 pL to 65 nL. For instance, the first droplet volume may be from 113 pL to 34 nL.

[0211] The second droplet volume is often from 65 zeptolitres (zL) to 113 pL, or from 65 zL to 0.5 pL (typically to less than 0.5 pL), for instance from 65 zL to 382 fL, or from 65 zL to 268 fL. The second droplet volume may for instance be from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL. The second droplet volume may for instance be from 524 zL to 0.5 pL (typically to less than 0.5 pL), from 4 aL to 65 fL, or from 65 attolitres (aL) to 65 fL. The second droplet volume may for instance be from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0212] The first droplet volume may for instance be from 0.5 pL to 65 nL, and the second droplet volume may be from 524 zL to 0.5 pL (typically to less than 0.5 pL), for instance from 4 aL to 65 fL, or from 65 attolitres (aL) to 65 fL. The first droplet volume may for instance be from 0.5 pL to 65 nL, and the second droplet volume may be from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0213] The first droplet volume may be from 113 pL to 34 nL, and the second droplet volume may be from 65 zeptolitres (zL) to 113 pL (typically to less than 113 pL), for instance from 524 zL to 0.5 pL, or from 4 aL to 65 fL, or from 65 attolitres (aL) to 65 fL. The first droplet volume may for instance be from 113 pL to 34 nL, and the second droplet volume may be from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0214] Typically, removing water from the aqueous medium of droplets in the droplet assembly reduces the volume of the droplet assembly from a first droplet assembly volume to a second droplet assembly volume. The second droplet assembly volume is typically less than 99 % of the first droplet assembly volume, and more typically less than 70 % of the first droplet assembly volume. The second droplet assembly volume may for instance be less than 50 % of the first droplet assembly volume, for instance less than 40 % of the first droplet assembly volume, less than 20 % of the first droplet assembly volume, or less than 10 % of the first droplet assembly volume. It may for instance be less than 1 % of the first droplet assembly volume. Often, the second droplet assembly volume is from 0.5 %to 99% of the first droplet assembly volume, optionally from 1 % to 70 % of the first droplet assembly volume, for instance from 2 % to 50 % of the first droplet assembly volume. The second droplet assembly volume may for instance be from 5 % to 40 % of the first droplet assembly volume, for instance from 10 % to 40 % of the first droplet assembly volume. Alternatively the second droplet assembly volume may be from 0.1 % to 10 % of the first droplet assembly volume, for instance from 0.5 % to 5 % of the first droplet assembly volume.

[0215] Often, in the process of the invention, removing water from the aqueous medium of droplets in the droplet assembly reduces the contact angle between droplets (θDIB) from a firstcontact angle to a second contact angle, wherein the second contact angle is less than the first contact angle.

[0216] Typically, the first contact angle is at least 50°, more typically at least 55°, for instance at least 60°. The first contact angle may for instance be from 50° to 75°, for instance from 55° to 70°, from 60° to 68°, or from 61° to 67°. The second contact angle is less than the first contact angle. The second contact angle is usually less than 60°, and typically is less than 55°, for instance less than 50°. The second contact angle is often for instance less than 45°, and may for instance be less than 40°. The second contact angle may for instance be from 25° to 59°, for instance from 30° to 54°, or from 30° to 50°. The second contact angle may for instance be from 33° to 50°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0217] For instance, the first contact angle may be at least 50°, and the second contact angle may be less than 50°, for instance less than 45°, or less than 40°. The first contact angle may be at least 55°, and the second contact angle may be less than 55°, for instance less than 50°, for instance less than 45°, or less than 40°. The first contact angle may be at least 60°, and the second contact angle may be less than 60°, for instance less than 55°, such as less than 50°, for instance less than 45°, or less than 40°.

[0218] The first contact angle may for instance be from 50° to 75°, and the second contact angle may be from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0219] The first contact angle may for instance be from 55° to 70°, and the second contact angle may be from 30° to 54°, for instance from 30° to 50°, or from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0220] The first contact angle may for instance be from 60° to 68°, and the second contact angle may be from 25° to 59°, for instance from 30° to 54°, from 30° to 50°, or from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0221] The first contact angle may for instance be from 61° to 67°, and the second contact angle may be from 25° to 60°, from 25° to 59°, for instance from 30° to 54°, or from 30° to 50°; the second contact angle may for instance be from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0222] The contact angle between droplets (θDIB) is measured using image detection. In particular, it may be measured using the image detection algorithm disclosed in Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105.

[0223] The process of the invention for reducing the size of droplets in a synthetic droplet assembly may further comprise, prior to removing water from the aqueous medium of droplets in the droplet assembly: producing the droplet assembly. Producing the droplet assembly may be carried out using a 3D droplet printing process, for instance as described in WO 2014 / 087175.Thus, often, producing the droplet assembly comprises producing the droplet assembly using an apparatus for producing the droplet assembly,

[0224] wherein the droplet assembly is as defined herein and comprises: a plurality of droplets, wherein each of said droplets comprises: (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, wherein each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets; and

[0225] the apparatus comprises: at least one droplet generator; a container which is moveable relative to the at least one droplet generator; and a control unit, which control unit is adapted to control the dispensing of droplets from the at least one droplet generator and the movement of the container relative to the at least one droplet generator; and

[0226] said container of the apparatus contains a bulk medium which is a hydrophobic medium; which process comprises:

[0227] (a) a plurality of dispensing steps, wherein each dispensing step comprises dispensing a droplet of the aqueous medium from a said droplet generator into the bulk medium, in the presence of amphipathic molecules, and thereby forming in the bulk medium a droplet which comprises (i) said aqueous medium and (ii) an outer layer of amphipathic molecules around the surface of the medium; and

[0228] (b) moving the container relative to the at least one droplet generator, to control the relative positioning of the droplets in the bulk medium.

[0229] This process of producing the droplet assembly using an apparatus for producing the droplet assembly may be as further defined anywhere in WO 2014 / 087175.

[0230] Process for increasing the size of droplets

[0231] The invention also provides a process for increasing the size of droplets in a synthetic droplet assembly of the kind defined above. Thus, the synthetic droplet assembly comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets. The synthetic droplet assembly may be as further defined anywhere herein. The process for increasing the size of droplets in the synthetic droplet assembly comprises adding water to the aqueous medium of droplets in the droplet assembly.

[0232] The size of droplets in the synthetic droplet assembly may be increased by any method of achieving flux of water into the droplets. Thus, any technique suitable for achieving flux of water into the droplets, and thereby adding water to the aqueous medium of droplets in the droplet assembly, may be employed. An important option for this that is discussed further herein is the addition of water by osmosis. However, any other suitable technique for adding of water to the aqueous medium of the droplets could in principle be employed.Thus, typically, in the process of the invention for increasing the size of droplets in the synthetic droplet assembly, adding water to the aqueous medium of droplets in the droplet assembly comprises adding said water by osmosis. Example 1 hereinbelow describes the addition of water by osmosis.

[0233] Typically, adding said water by osmosis comprises contacting the droplet assembly with an external aqueous medium, wherein the external aqueous medium has an osmolarity which is less than the osmolarity of the aqueous medium in the droplets. Typically, said contacting comprises (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium (while keeping the droplet assembly in contact with the external aqueous medium) so that the external aqueous medium has an osmolarity which is less than the osmolarity of the aqueous medium of the droplets, and (iii) maintaining contact between the droplet assembly and the external aqueous medium. Adjusting the osmolarity of the external aqueous medium typically comprises decreasing the osmolarity of the external aqueous medium, and optionally comprises decreasing the osmolarity of the external aqueous medium in a stepwise manner. For instance, said contacting may comprise (i) bringing the droplet assembly into contact with an external aqueous medium whose osmolarity is (about) the same as the osmolarity of the aqueous medium of the droplets, and then (ii) adjusting the osmolarity of the external aqueous medium so that the osmolarity of the external aqueous medium is less than the osmolarity of the aqueous medium of the droplets, and then (iii) maintaining contact between the droplet assembly and the external aqueous medium.

[0234] Adjusting the osmolarity of the external aqueous medium typically comprises adding to the external aqueous medium water (for instance, pure water) or an aqueous solution whose osmolarity is less than the osmolarity of the aqueous medium of the droplets. The water or aqueous solution may for instance be pipetted into the external aqueous medium. Said water or aqueous solution may be added (e.g. pipetted) until the external aqueous medium has the desired osmolarity that is less than the osmolarity of the aqueous medium of the droplets. The water (for instance, pure water) or aqueous solution may be added in a stepwise manner (i.e. in a plurality of steps, each comprising adding the water or aqueous solution to the external aqueous medium) until the desired osmolarity is achieved. The stepwise addition may be performed over a period of time, for instance over a period of from 0.5 to 20 hours, for instance from 2 to 15 hours, or from 5 to 11 hours.

[0235] When the osmolarity of the external aqueous medium is less than the osmolarity of the aqueous medium of the droplets, and contact between the droplet assembly and the external aqueous medium is maintained, water flows out of the external aqueous medium and into the droplets by osmosis to increase the size of the droplets. Indeed, step (iii) above may be performed until enough water has flowed for the osmolarities of the droplet assembly and the external aqueous medium to be equal. Alternatively, step (iii) may be performed until a desired reduction in the sizeof the droplets, or droplet assembly, is achieved. Often, step (iii) is performed for a duration of at least 0.5 hours, and typically up to 7 hours.

[0236] The droplet assembly is typically in contact with a hydrophobic medium. For instance, the droplet assembly may be disposed in a hydrophobic medium. The hydrophobic medium may be as further defined anywhere herein. For instance, as discussed above, the hydrophobic medium may be an oil. It may for instance comprise silicone oil and a hydrocarbon, for instance an unsubstituted C10-C20 alkane. The hydrophobic medium may for instance comprise poly(methylphenylsiloxane) (e.g. AR20) and undecane. It typically further comprises said amphipathic molecules. The volume ratio of the hydrocarbon to the silicone oil may be as defined above.

[0237] When the droplet assembly is in contact with a hydrophobic medium, for instance disposed in a hydrophobic medium, the hydrophobic medium may also be in contact with the external aqueous medium. For instance, the hydrophobic medium may be layered on top of the external aqueous medium, e.g. within a container such as a beaker, petri dish, cuvette (e.g. a special optical glass (SOG) cuvette), vat or any other vessel.

[0238] Thus, often, contacting the droplet assembly with the external aqueous medium comprises moving the synthetic droplet assembly within the hydrophobic medium in order to bring the synthetic droplet assembly into contact with the external aqueous medium. For instance, contacting the droplet assembly with the external aqueous medium may comprise introducing the droplet assembly into the hydrophobic medium and moving the synthetic droplet assembly within the hydrophobic medium in order to bring the synthetic droplet assembly into contact with the external aqueous medium. Moving the synthetic droplet assembly within the hydrophobic medium in order to bring it into contact with the external aqueous medium may comprise allowing the droplet assembly to sink through the hydrophobic medium until the droplet assembly contacts the external aqueous medium.

[0239] As with the aqueous medium of the droplets, the external aqueous medium may be any suitable aqueous medium. For instance, the external aqueous medium may be water (for instance pure water) or an aqueous solution. It may for instance be an aqueous solution of one or more salts, or an aqueous buffer solution. The aqueous medium may for instance be an aqueous solution comprising Dulbecco's phosphate-buffered saline (DPBS). The aqueous solution may for instance comprise DPBS and sucrose. The concentration of sucrose may be selected to achieve a desired osmolarity, although any other suitable solute could be employed, instead of sucrose, for the purpose of adjusting osmolarity. The external aqueous medium may for instance comprise a growth medium or a culture medium, for instance a cell growth medium or a cell culture medium. The external aqueous medium may for instance comprise culture medium for eukaryotic cells or tissue, or a growth medium for bacteria, such as for example M9 minimal medium. Thus, for instance, the external aqueous medium may be an aqueous solution which comprises M9 minimal salts. The aqueous solution which comprises M9 minimal salts may further comprise one or more aminoacids. It may additionally further comprise glucose or glycerol. Often, the aqueous solution which comprises M9 minimal salts further comprises at least one of MgSO4, CaCl2, one or more amino acids, glucose or glycerol. Typically, the aqueous solution which comprises M9 minimal salts further comprises MgSO4, CaCl2, casamino acids, and glucose or glycerol. Alternatively, the external aqueous medium may comprise a hydrogel. When the external aqueous medium comprises a hydrogel, the aqueous medium may be as further defined above for the aqueous medium of the droplets.

[0240] Contacting the droplet assembly with the external aqueous medium typically forms a layer of the amphipathic molecules as an interface between droplets of the synthetic droplet assembly and the external aqueous medium. Often, said layer of the amphipathic molecules is a bilayer of the amphipathic molecules. Water may flow through the layer or bilayer by osmosis.

[0241] Typically, the aqueous medium in the droplets of the droplet assembly has a first osmolarity, and adding said water by osmosis comprises: contacting the droplet assembly with said external aqueous medium which has a second osmolarity, wherein the second osmolarity is less than the first osmolarity.

[0242] Often, the aqueous medium in the droplets of the droplet assembly has a first osmolarity, and said contacting comprises: (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium (while keeping the droplet assembly in contact with the external aqueous medium) so that the external aqueous medium has a second osmolarity wherein the second osmolarity is less than the first osmolarity, and (iii) maintaining contact between the droplet assembly and the external aqueous medium. Adjusting the osmolarity of the external aqueous medium typically comprises increasing the osmolarity of the external aqueous medium, and optionally comprises increasing the osmolarity of the external aqueous medium in a stepwise manner. For instance, the aqueous medium in the droplets of the droplet assembly may have a first osmolarity, and said contacting may comprise: (i) bringing the droplet assembly into contact with an external aqueous medium whose osmolarity is (about) the same as the first osmolarity, and then (ii) adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has a second osmolarity wherein the second osmolarity is less than the first osmolarity, and then (iii) maintaining contact between the droplet assembly and the external aqueous medium.

[0243] Adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has said second osmolarity typically comprises adding to the external aqueous medium water (for instance, pure water) or an aqueous solution, wherein the osmolarity of said water or aqueous solution is less than the first osmolarity. The water or aqueous solution may for instance be pipetted into the external aqueous medium. Said water or aqueous solution may be added (e.g. pipetted) until the external aqueous medium has said second osmolarity as desired. The water or aqueous solution may be added in a stepwise manner (i.e. in a plurality of steps, each comprisingadding the water or aqueous solution to the external aqueous medium) until the desired second osmolarity is achieved. The stepwise addition may be performed over a period of time, for instance over a period of from 0.5 to 20 hours, for instance from 2 to 15 hours, or from 4 to 11 hours.

[0244] When the external aqueous medium has said second osmolarity - which is less than the first osmolarity of the aqueous medium of the droplets - and contact between the droplet assembly and the external aqueous medium is maintained, water flows out of the external aqueous medium and into the droplets by osmosis to increase the size of the droplets. Indeed, step (iii) may be performed until enough water has flowed for the osmolarities of the droplet assembly and the external aqueous medium to be equal. Alternatively, step (iii) may be performed until a desired reduction in the size of the droplets, or droplet assembly, is achieved. Often, step (iii) is performed for a duration of at least 0.5 hours, and typically up to 7 hours.

[0245] The process may further comprise separating the droplet assembly and the external aqueous medium. Often, the separating is performed when a desired reduction in the size of the droplets, or droplet assembly, is achieved.

[0246] The first osmolarity is often from 0.001 osm to 7.001 osm. The first osmolarity may for instance be from 0.01 osm to 7.001 osm, for instance from 0.05 osm to 7 osm, or from 0.1 osm to 7 osm. Typically, the first osmolarity is from 0.01 osm to 3 osm, for instance from 0.05 osm to 1 osm, such as from 0.1 osm to 0.8 osm, or from 0.1 osm to 0.5 osm, for instance from 0.1 osm to 0.4 osm. The first osmolarity may for instance be about 0.24 osm (240 mosm) or for instance about 0.3 osm (300 mosm).

[0247] Often, the second osmolarity is from 0 osm to 7 osm, for instance from 0 osm to 3 osm, or from 0 osm to 1 osm. The second osmolarity may for instance be from 0 osm to 0.5 osm, for instance from 0.001 osm to 0.3 osm, or from 0.002 osm to 0.25 osm, for instance from 0.003 osm to 0.3 osm. The second osmolarity may for instance be from 0.004 osm (4 mosm) to 0.25 osm (250 mosm). The second osmolarity may for instance be about 0.0048 osm (4.8 mosm) or for instance about 0.24 osm (240 mosm). The second osmolarity may for instance be from 0.005 osm to 0.24 osm, or from 0.006 osm to 0.23 osm, or from 0.01 osm to 0.15 osm.

[0248] Typically, the first osmolarity is from 0.001 osm to 7.001 osm and the second osmolarity is from 0 osm to 7 osm. Thus, the first osmolarity may be from 0.01 osm to 7.001 osm and the second osmolarity may be from 0 osm to 3 osm. For instance, the first osmolarity may be from 0.05 osm to 7 osm and the second osmolarity may be from 0 osm to 1 osm. The first osmolarity may for instance be from 0.1 osm to 7 osm and the second osmolarity may be from 0 osm to 0.5 osm. The first osmolarity may for instance be from 0.01 osm to 3 osm and the second osmolarity may be from 0.001 osm to 0.3 osm. The first osmolarity may for instance be from 0.05 osm to 1 osm osm and the second osmolarity may be from 0.002 osm to 0.25 osm. The first osmolarity may for instance be from 0.1 osm to 0.8 osm and the second osmolarity may be from 0.003 osm to 0.3 osm. The first osmolarity may for instance be from 0.1 osm to 0.5 osm and the second osmolarity maybe from 0.003 osm to 0.3 osm. The first osmolarity may for instance be from 0.1 osm to 0.4 osm and the second osmolarity may be from 0.003 osm to 0.3 osm. The first osmolarity may for instance be from 0.1 osm to 0.8 osm and the second osmolarity may be from 0.005 osm to 0.24 osm. The first osmolarity may for instance be from 0.1 osm to 0.5 osm and the second osmolarity may be from 0.006 osm to 0.23 osm. The first osmolarity may for instance be from 0.1 osm to 0.4 osm and the second osmolarity may be from 0.01 osm to 0.15 osm.

[0249] Often, adding said water by osmosis comprises contacting the droplet assembly with said external aqueous medium (i.e. with said external aqueous medium which has an osmolarity which is less than the osmolarity of the aqueous medium of the droplets, or with said external aqueous medium which has said second osmolarity) for a duration of at least 0.2 hours, for instance at least 0.5 hours. For instance, adding said water by osmosis may comprise contacting the droplet assembly with said external aqueous medium for from 0.2 hours to 7 hours, for instance from 0.5 hours to 7 hours. The duration required to achieve the desired size increase depends on various factors including the difference in osmolarity, and the size of the synthetic droplet assembly. The duration can be minimized or maximized depending on the application.

[0250] Thus, step (iii) above, of maintaining contact between the droplet assembly and the external aqueous medium typically comprises maintaining contact between the droplet assembly and the external aqueous medium for at least 0.2 hours, for instance for at least 0.5 hours, for instance from 0.2 hours to 7 hours, or from 0.5 hours to 7 hours.

[0251] Often, adding said water by osmosis comprises contacting the droplet assembly with said external aqueous medium (i.e. with said external aqueous medium which has an osmolarity which is less than the osmolarity of the aqueous medium of the droplets, or with said external aqueous medium which has said second osmolarity) until the osmolarities of the aqueous medium of the droplets and the external aqueous medium are equal. Alternatively, contacting the droplet assembly with said external aqueous medium may be performed until a desired increase in the size of the droplets, or the synthetic droplet assembly, is achieved.

[0252] Typically, in the process of the invention for increasing the size of droplets in a synthetic droplet assembly, adding water to the aqueous medium of droplets in the droplet assembly increases the mean diameter of the droplets in the droplet assembly from a first droplet diameter to a second droplet diameter. Droplet diameters can be determined by microscopy.

[0253] Often, the first droplet diameter is less than 60 pm, for instance less than 10 pm. The first diameter may for instance be less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal 5 pm. The first droplet diameter may for instance be less than or equal to 3 pm, or say, less than or equal to 2 pm.

[0254] The second droplet diameter is typically at least 10 pm. For instance, the second droplet diameter may be at least 60 pm, or for instance at least 100 pm.Thus, typically, the first droplet diameter is less than 60 pm, for instance less than 10 pm, or less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm, and the second droplet diameter is at least 60 pm. For instance, the first droplet diameter may be less than or equal to 3 pm, for instance less than or equal to 2 pm, and the second droplet diameter may be at least 60 pm. The second droplet diameter may in these embodiments be at least 100 pm.

[0255] Often, the first droplet diameter is less than 10 pm, for instance less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm, and the second droplet diameter is at least 10 pm. For instance, the first droplet diameter may be less than or equal to 3 pm, for instance less than or equal to 2 pm, and the second droplet diameter may be at least 10 pm.

[0256] Often, the first droplet diameter is from 50 nm to 60 pm, for instance from 100 nm to 10 pm, or from 500 nm to 5 pm.

[0257] The second droplet diameter is typically from 10 pm to 500 pm, for instance from 60 pm to 400 pm.

[0258] Thus, typically, the first droplet diameter is from 50 nm to 60 pm (typically to less than 60 pm), for instance from 50 nm to 10 pm, or from 50 nm to 9 pm, or from 50 nm to 8 pm, for instance from 50 nm to 6 pm, or from 70 nm to 5 pm, and the second droplet diameter is from 60 pm to 500 pm. The first droplet diameter may for instance be from 100 nm to 10 pm, or from 100 nm to 3 pm, or from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm, for instance from 500 nm to 5 pm, and the second droplet diameter may be from 60 pm to 500 pm. The second droplet diameter may in any of these embodiments be from 60 pm to 400 pm. The first droplet diameter may for instance be from 50 nm to 10 pm (typically to less than 10 pm), or from 50 nm to 9 pm, or from 50 nm to 8 pm, for instance from 50 nm to 6 pm, or from 70 nm to 5 pm, and the second droplet diameter is from 10 pm to 500 pm. The first droplet diameter may for instance be from 100 nm to 10 pm (typically to less than 10 pm), or from 100 nm to 3 pm, or from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm, for instance from 500 nm to 5 pm, and the second droplet diameter may be from 10 pm to 500 pm.

[0259] Often, in the process of the invention for increasing the size of droplets in a synthetic droplet assembly, adding water to the aqueous medium of droplets in the droplet assembly increases the mean volume of the droplets in the droplet assembly from a first droplet volume to a second droplet volume. Droplet volumes can be determined by microscopy.

[0260] Typically, the first droplet volume is less than 113 pL, for instance less than 0.5 pL. The first droplet volume may for instance be less than 65 femtolitres (fL).

[0261] Often, the second droplet volume is at least 0.5 picolitres (pL). For instance, the second droplet volume may be at least 113 pL.

[0262] Thus, typically, the first droplet volume is less than 113 pL, for instance less than 0.5 pL, or less than or equal to 382 fL, for instance less than or equal to 268 fL, and the second dropletvolume is at least 113 pL. Often, the first droplet volume is less than less than 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL, and the second droplet volume is at least 113 pL. The first droplet volume may be less than 0.5 pL, for instance less than 382 fL, or less than or equal to 268 fL, and the second droplet volume may be at least 0.5 pL. Often, the first droplet volume is less than 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL, and the second droplet volume is at least 0.5 pL.

[0263] Typically, the first droplet volume is from 65 zeptolitres (zL) to 113 pL, for instance from 524 zL to 0.5 pL, or from 65 attolitres (aL) to 65 fL.

[0264] Often, the second droplet volume is from 0.5 pL to 65 nL, for instance from 113 pL to 65 nL or from 113 pL to 34 nL.

[0265] Thus, typically, the first droplet volume is from 65 zeptolitres (zL) to 113 pL, for instance from 65 zL to 0.5 pL, or from 65 zL to 382 fL, or from 65 zL to 268 fL and the second droplet volume is from 113 pL to 65 nL. For instance, the first droplet volume may be from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL, and the second droplet volume may be from 113 pL to 65 nL. The first droplet volume may be from 524 zL to 0.5 pL, from 4 aL to 65 fL, or from 65 aL to 65 fL, for instance from 4 aL to 4 fL, or from 14 aL to 4 fL, for instance from 65 aL to 4 fL, and the second droplet volume may be from 113 pL to 65 nL. The second droplet volume in these embodiments may for instance be from 113 pL to 34 nL.

[0266] For instance, the first droplet volume may be from 65 zeptolitres (zL) to 0.5 pL (typically to less than 0.5 pL), for instance from 65 zL to 382 fL, or from 65 zL to 268 fL and the second droplet volume may be from 0.5 pL to 65 nL. For instance, the first droplet volume may be from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL, and the second droplet volume may be from 0.5 pL to 65 nL. The first droplet volume may be from 524 zL to 0.5 pL, from 4 aL to 65 fL, or from 65 aL to 65 fL, for instance from 4 aL to 4 fL, or from 14 aL to 4 fL, or for instance from 65 aL to 4 fL, and the second droplet volume may be from 0.5 pL to 65 nL.

[0267] Typically, adding water to the aqueous medium of droplets in the droplet assembly increases the volume of the droplet assembly from a first droplet assembly volume to a second droplet assembly volume wherein the second droplet assembly volume is greater than the first droplet assembly volume. The second droplet assembly volume is often at least double the first droplet assembly volume. For instance, it may be at least three times, at least four times, at least five times, or at least ten times, the first droplet assembly volume. Typically, the second droplet assembly volume is from 2 to 50 times the first droplet assembly volume, for instance from 3 to 20 times the first droplet assembly volume, or from 5 to 10 times the first droplet assembly volume. It may for instance be from 5 to 50 times the first droplet assembly volume, for instance from 10 to 50 times, or from 20 to 50 times, the first droplet assembly volume.Often, in the process of the invention, adding water to the aqueous medium of droplets in the droplet assembly increases the contact angle between droplets (θDIB) from a second contact angle to a first contact angle, wherein the first contact angle is greater than the second contact angle.

[0268] Typically, the first contact angle is at least 50°, more typically at least 55°, for instance at least 60°. The first contact angle may for instance be from 50° to 75°, for instance from 55° to 70°, from 60° to 68°, or from 61° to 67°. The second contact angle is less than the first contact angle. The second contact angle is usually less than 60°, and typically is less than 55°, for instance less than 50°. The second contact angle is often for instance less than 45°, and may for instance be less than 40°. The second contact angle may for instance be from 25° to 59°, for instance from 30° to 54°, or from 30° to 50°. The second contact angle may for instance be from 33° to 50°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0269] For instance, the first contact angle may be at least 50°, and the second contact angle may be less than 50°, for instance less than 45°, or less than 40°. The first contact angle may be at least 55°, and the second contact angle may be less than 55°, for instance less than 50°, for instance less than 45°, or less than 40°. The first contact angle may be at least 60°, and the second contact angle may be less than 60°, for instance less than 55°, such as less than 50°, for instance less than 45°, or less than 40°.

[0270] The first contact angle may for instance be from 50° to 75°, and the second contact angle may be from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0271] The first contact angle may for instance be from 55° to 70°, and the second contact angle may be from 30° to 54°, for instance from 30° to 50°, or from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0272] The first contact angle may for instance be from 60° to 68°, and the second contact angle may be from 25° to 59°, for instance from 30° to 54°, from 30° to 50°, or from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0273] The first contact angle may for instance be from 61° to 67°, and the second contact angle may be from 25° to 60°, from 25° to 59°, for instance from 30° to 54°, or from 30° to 50°; the second contact angle may for instance be from 33° to 49°, for instance from 33° to 45°, from 33° to 43°, or from 35° to 41°, for instance from 37° to 40°.

[0274] The contact angle between droplets (θDIB) is measured using image detection. In particular, it may be measured using the image detection algorithm disclosed in Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105.

[0275] The process of the invention for increasing the size of droplets in a synthetic droplet assembly may further comprise, prior to adding water to the aqueous medium of droplets in the droplet assembly: producing the droplet assembly. Producing the droplet assembly may be carriedout using a 3D droplet printing process, for instance as described in WO 2014 / 087175. Thus, often, producing the droplet assembly comprises producing the droplet assembly using an apparatus for producing the droplet assembly,

[0276] wherein the droplet assembly is as defined herein and comprises: a plurality of droplets, wherein each of said droplets comprises: (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, wherein each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets; and

[0277] the apparatus comprises: at least one droplet generator; a container which is moveable relative to the at least one droplet generator; and a control unit, which control unit is adapted to control the dispensing of droplets from the at least one droplet generator and the movement of the container relative to the at least one droplet generator; and

[0278] said container of the apparatus contains a bulk medium which is a hydrophobic medium; which process comprises:

[0279] (a) a plurality of dispensing steps, wherein each dispensing step comprises dispensing a droplet of the aqueous medium from a said droplet generator into the bulk medium, in the presence of amphipathic molecules, and thereby forming in the bulk medium a droplet which comprises (i) said aqueous medium and (ii) an outer layer of amphipathic molecules around the surface of the medium; and

[0280] (b) moving the container relative to the at least one droplet generator, to control the relative positioning of the droplets in the bulk medium.

[0281] The process of producing the droplet assembly using an apparatus for producing the droplet assembly may be as further defined anywhere in WO 2014 / 087175.

[0282] As will be understood by the skilled person, the process of the invention for increasing the size of droplets in a synthetic droplet assembly may be carried out after the process of the invention for reducing the size of droplets in a synthetic droplet assembly, on the same synthetic droplet assembly. Similarly, the process of the invention for reducing the size of droplets in a synthetic droplet assembly may be carried out after the process of the invention for increasing the size of droplets in a synthetic droplet assembly, on the same synthetic droplet assembly.

[0283] Novel synthetic droplet assemblies

[0284] The above-described processes of the invention usefully provide access to synthetic droplet assemblies with smaller droplet sizes than are currently obtainable through known 3D droplet printing processes. This allows for increased droplet resolution - a higher number of droplets per unit length in each dimension - in the products produced, whether they be synthetic tissues or delivery vehicles for patterned delivery of an effector molecule. It is demonstrated in Example 2 herein, for instance, that such droplet assemblies can achieve an even-higher spatial resolution ofpaterned gene expression than was possible using an equivalent assembly with larger droplets obtained by 3D printing alone.

[0285] Accordingly, the invention provides a synthetic droplet assembly which comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets. Typically, in the synthetic droplet assembly of the invention: the mean diameter of the droplets in the droplet assembly is less than 10 µm; and / or the mean volume of the droplets in the droplet assembly is less than 0.5 pL.

[0286] The synthetic droplet assembly of the invention may be as further defined anywhere herein, for instance it may be as further defined hereinbefore under the heading " Droplet assembly features applicable to all aspects of the invention".

[0287] In the synthetic droplet assembly of the invention, the mean diameter of the droplets in the synthetic droplet assembly of the invention is usually less than 10 pm. The diameters of droplets in the synthetic droplet assembly of the invention can readily be measured by microscopy, thus the mean droplet diameter may be determined.

[0288] Often, the mean diameter of the droplets in the synthetic droplet assembly of the invention is less than or equal to 9 pm, for instance less than or equal to 8 pm, or for instance less than or equal to 5 pm. It may for instance be less than or equal to 3 pm, for example less than or equal to 2 pm. The mean diameter of the droplets in the synthetic droplet assembly of the invention may for instance be from 50 nm to less than 10 pm, for instance from 50 nm to 9 pm, or from 50 nm to 8 pm. Often, the mean diameter is from 50 nm to 6 pm, for instance from 70 nm to 5 pm, or for example from 100 nm to 3 pm. For instance, the mean diameter may be from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm. For instance, the mean diameter may be from 100 nm to 1000 nm, for instance from 200 nm to 900 nm, or for instance from 300 nm to 800 nm.

[0289] Usually, the mean volume of the droplets in the synthetic droplet assembly of the invention is less than or equal to 382 fL (femtolitres), less than or equal to 268 fL, or for instance less than or equal to 65 fL. It may for instance be less than or equal to 14 fL, for example less than or equal to 4 fL. The mean volume of the droplets in the synthetic droplet assembly of the invention may for instance be from 65 zL to less than 0.5 pL, for instance from 65 zL to 382 fL, or from 65 zL to 268 fL. Often, the mean volume is from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL. For instance, the mean volume may be from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL. For instance, the mean volume may be from 524 zL to 4 fL, for instance from 4 aL to 4 fL, or for instance from 14 aL to 4 fL.

[0290] Thus, often, the mean diameter of the droplets in the synthetic droplet assembly of the invention is as defined above, for instance it may be less than 5 pm; and the mean volume of thedroplets in the droplet assembly is as defined above, for instance it may be less than 65 femtolitres (fL).

[0291] The number of droplets, n, in the synthetic droplet assembly of the invention is at least two, but typically larger than that, and it may be as defined anywhere hereinbefore. The number of droplets, n, may for instance be at least 64, for instance at least 500, at least 10,000, or at least 1,000,000, but n may be as further defined above under the heading " Droplet assembly features applicable to all aspects of the invention".

[0292] Often, in the synthetic droplet assembly of the invention, each of the n droplets in the synthetic droplet assembly has a diameter of less than 10 pm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of less than or equal to 9 pm, less than or equal to 8 pm, or for instance less than or equal to 5 pm, or less than or equal to 3 pm, for example less than or equal to 2 pm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of from 50 nm to less than 10 pm, for instance from 50 nm to 9 pm, or from 50 nm to 8 pm. Often, each of the n droplets in the synthetic droplet assembly has a diameter of from 50 nm to 6 pm, for instance from 70 nm to 5 pm, or for example from 100 nm to 3 pm. For instance, each of the n droplets in the synthetic droplet assembly may have a diameter of from 200 nm to 2 pm, for instance from 300 nm to 2 pm, or for instance from 500 nm to 2 pm.

[0293] Similarly, in the synthetic droplet assembly of the invention, each of the n droplets in the synthetic droplet assembly may have a volume of less than 0.5 pL. For instance, each of the n droplets in the synthetic droplet assembly may have a volume of less than or equal to 382 fL (femtolitres), less than or equal to 268 fL, or for instance less than or equal to 65 fL, or less than or equal to 14 fL, for example less than or equal to 4 fL. Thus, each of the n droplets in the synthetic droplet assembly may have a volume of from 65 zL to less than 0.5 pL, for instance from 65 zL to 382 fL, or from 65 zL to 268 fL. Often, each of the n droplets in the synthetic droplet assembly has a volume of from 65 zL to 113 fL, for instance from 180 zL to 65 fL, or for example from 524 zL to 14 fL. For instance, each of the n droplets in the synthetic droplet assembly may have a volume of from 4 aL to 4 fL, for instance from 14 aL to 4 fL, or for instance from 65 aL to 4 fL.

[0294] In some embodiments of the synthetic droplet assembly of the invention, the number of droplets, n, is at least 64 (for instance n may be at least 500, at least 10,000, or at least 1,000,000 or n may be as further defined anywhere herein) and the volume of the droplet assembly is less than or equal to 0.5 pL multiplied by said number of droplets in the droplet assembly, n. For instance, the volume of the droplet assembly may be: less than or equal to 382 fL multiplied by n, or less than or equal to 268 fL multiplied by n, or for instance less than or equal to 65 fL multiplied by n. For instance, the number of droplets, n, may be at least 64 (for instance n may be at least 500, at least 10,000, or at least 1,000,000 or n may be as further defined anywhere herein) and the volume of the droplet assembly may be less than or equal to 14 fL multiplied by n, for example less than or equal to 4 fL multiplied by n.The synthetic droplet assembly of the invention may comprise one or more effector droplets, each of which, in addition to comprising (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, further comprises (iii) an effector molecule or means for producing the effector molecule. The number of said effector droplets in the synthetic droplet assembly, p, is from 1 to n. Thus, at least one of the n droplets of the synthetic droplet assembly is an effector droplet.

[0295] A purpose of each effector droplet may be to house, or to be able to produce, an effector molecule therein. Then, in use, the effector molecule may pass from the effector droplet in question to an external target region, such as biological cells or tissue, which is outside of the synthetic droplet assembly. The effector molecule and / or the means for producing the effector molecule are typically disposed in the aqueous medium of the effector droplet. The effector molecule and / or means for producing the effector molecule are often, for instance, suspended in, or dissolved in, the aqueous medium of the effector droplet. Often, the effector molecule is dissolved in the aqueous medium of the effector droplet.

[0296] An effector droplet may comprise more than one type of effector molecule (i.e. more than one compound which acts an effector), and / or the means for producing more than one type of effector molecule.

[0297] Usually, each effector droplet comprises an effector molecule. Usually, when present, the concentration of the effector molecule (for instance its concentration in the aqueous medium of the effector droplet) is equal to or greater than 0.001 mM, for instance, equal to or greater than 0.01 mM. For instance, the concentration of the effector molecule may be equal to or greater than 0.1 mM, for instance, equal to or greater than 0.5 mM. Often, the concentration of effector molecule is equal to or greater than 1 mM, for instance, equal to or greater than 2 mM. For instance, the concentration of the effector molecule may be equal to or greater than 5 mM, for instance, equal to or greater than 10 mM, or equal to or greater than 15 mM. The concentration of the effector molecule may be equal to or greater than 20 mM, for instance, equal to or greater than 25 mM, or equal to or greater than 30 mM.

[0298] The concentration of the effector molecule may, for instance, be from 0.001 mM to 1,000 mM. Typically, the concentration of the effector molecule is from 0.01 mM to 800 mM, for instance from 0.1 mM to 600 mM. More typically, the concentration of the effector molecule is from 0.5 mM to 400 mM, for instance, from 1 mM to 300 mM. In some embodiments, the concentration of the effector molecule is from about 2 mM to about 200 mM, or from about 5 mM to about 100 mM, or about 10 mM to about 80 mM. It may for instance be from 15 mM to 60 mM, or from 20 mM to 50 mM, for instance from 25 mM to 40 mM, or from 30 mM to 35 mM, for instance about 33 mM.

[0299] Such concentrations are particularly suitable for the effector molecule arabinose, especially when employed in combination with the αHL pore in the effector droplet, at any of the concentrations for the protein pore specified herein.The effector molecule may be natural or synthetic. The effector molecule may be of any size. For instance, it may be a small molecule, an oligomer, or a macromolecule, such as for instance a natural or synthetic polymer. It may for instance be a small molecule drug, a peptide or protein, or a polynucleotide such as DNA or RNA.

[0300] The effector molecule may be any molecule that is desired to be delivered to an external target region, for instance to biological cells or tissue, or for instance any molecule that has any kind of effect on the biological cells or tissue, or any other desired effect. The effector molecule may for instance have a beneficial effect on the cells or tissue, for instance it may be a nutrient or other molecule that promotes cell or tissue growth, or it may be a pharmaceutical that treats a disease in the cells or tissue. Alternatively, the effector molecule may have a detrimental effect on the target cells or tissue, for example it may cause cell death or tissue necrosis, or it may prevent or retard cell proliferation. This may be useful, for example, in cases where the target cells or tissue are diseased cells or tissue, for instance cancer cells or tumour tissue, and destruction of those cells may be an objective. Often the effector molecule is a molecule that is suitable for modulating a biochemical process in said biological cells or tissue. Said biochemical process may for instance be the expression of a gene. Thus, the effector molecule may be a molecule capable of inducing protein expression in said biological cells or tissue. The protein expression may be protein expression in eukaryotic cells or tissue, or bacterial protein expression. For instance, the target region may comprise biological cells which are bacteria, and the effector molecule may be a molecule capable of inducing protein expression in said bacteria. Once possibility, as described in the Example herein, is that the effector molecule may be L-(+)-arabinose, which is capable of inducing expression of the mCherry gene in the described bacteria. Thus, the effector molecule may for instance be L-(+)-arabinose. Other possibilities are that the effector molecule may be a pharmaceutical, or, for instance, a signalling molecule.

[0301] As discussed above, the effector molecule may be of any size. In some embodiments, however, the effector molecule has a molecular weight of equal to or less than 2,000 Da. An advantage of an effector molecule having a molecular weight of equal to or less than 2,000 Da is that it may pass through an a -hemolysin (aHL) protein pore. Such a membrane protein (protein pore) may be present in a layer of the amphipathic molecules which is an interface between contacting droplets in the droplet assembly, or in a layer of the amphipathic molecules which is an interface between a target interface droplet and an external region, outsider of the droplet assembly. Such a protein pore may thereby allow passage of the effector molecule from one droplet to another (adjacent) droplet in the synthetic droplet assembly, or from the droplet assembly to the external region. Other protein pores may be employed instead of aHL if the effector molecule is larger, for instance if it has a molecular weight greater than 2,000 Da. Indeed, whereas the narrowest inner diameter of aHL is 1.4 nm, other membrane protein pores such as perfringolysin O have largerinternal diameters (between 25 nm and 30 nm in the case of perfringolysin O) that facilitate passage of larger molecules.

[0302] As discussed above, each effector droplet may comprise means for producing the effector molecule. Such means may be a molecule which is a precursor to the effector molecule, that can easily be converted into the effector molecule by chemical or biochemical reaction. Alternatively, the means may be two or more molecules that can be reacted together, for instance coupled together, to produce the effector molecule. Thus, the means for producing the effector molecule may comprise one or more precursors to the effector molecule. The one or more precursors to the effector molecule comprise one or more reactants capable of conversion into the effector molecule.

[0303] Alternatively, the means for producing the effector molecule may comprise one or more molecules which enable production of the effector molecule. The one or more molecules that enable production of the effector molecule may for example be molecular machinery that enables production of the effector molecule by cell-free protein expression, for instance an in vitro transcription-translation system. Alternatively, they may be a catalyst that catalyses the production of the effector molecule. The catalyst may for instance be a small molecule or an enzyme.

[0304] The means for producing the effector molecule may comprise any combination of the above; for instance, the means may comprise one or more precursors to the effector molecule and a catalyst for catalysing a reaction which converts said one or more precursors into the effector molecule. A purpose of the effector droplets is to house the effector molecule therein, and / or to be able to produce the effector molecule therein, so that the effector molecule can be delivered, for instance to an external region outside of the droplet assembly.

[0305] As will be apparent from the above discussion and the Examples herein, the synthetic droplet assembly typically comprises a plurality of effector droplets, often multiple effector droplets. Thus, the number of effector droplets, p, is often from two to n.

[0306] Thus, the number of effector droplets, p, is often at least two, more typically at least three, and even more typically at least four.

[0307] For instance, the number of effector droplets, p, may be at least 10, for instance at least 16, at least 25, or for instance at least 64. Often, the number of effector droplets is at least 100, for instance at least 150. For instance, the number of effector droplets, p, may be at least 500, or, for instance, at least 1,000, or at least 10,000. Often, in these cases, some or all of the effector droplets together form one or more rows of effector droplets, a layer of effector droplets, or a plurality of layers of effector droplets, as discussed above.

[0308] Each effector droplet typically further comprises a protein pore, for allowing passage of the effector molecule from the droplet to an adjacent droplet or to an external region outside of the synthetic droplet assembly.

[0309] The synthetic droplet assembly of the invention may further comprise magnetic particles attached to the synthetic droplet assembly, suitable for moving the synthetic droplet assemblyrelative to its external environment using a magnet. Typically, the magnetic particles are particles which comprise nickel, iron or cobalt, or an alloy of one or more of nickel, iron and cobalt.

[0310] Magnetic particles which comprise nickel (Ni) may for instance be employed, for instance MagneHis™ Ni-particles. Often, the magnetic particles are disposed in a plurality of hydrogel droplets, wherein each hydrogel droplet is attached to the synthetic droplet assembly, usually via a droplet interface layer (e.g. bilayer) of amphipathic molecules at an interface between the hydrogel droplet and one or more droplets of the synthetic droplet assembly. The hydrogel of the droplets containing the magnetic particles may be as defined above for the aqueous medium; the hydrogel may for instance be agarose (for example ultra-low gelling agarose), optionally at a concentration as defined above for agarose (for instance 1.5% w / v).

[0311] The synthetic droplet assembly of the invention may be as further defined anywhere herein, including in hereinbefore under the heading " Droplet assembly features applicable to all aspects of the invention”.

[0312] The invention will be described further in the following Examples.

[0313] EXAMPLES

[0314] Example 1 - Temperature- and osmosis- driven size-adjustment of synthetic tissues

[0315] This Example presents two means of tuning the droplet size of synthetic tissues post-printing: a temperature-mediated shrinking process and an osmosis-mediated, reversible process, whereby the volume of the synthetic tissues can be tuned based on controlled water flux. By shrinking synthetic tissues, the droplet size can be minimized. As a result, improved spatial controllability over patterned delivery of a chemical signal towards bacterial cells could be achieved.

[0316] 3D droplet printing technology (e.g. G. Villar et al., Science 340.6128 (Apr. 2013), pp. 48-52) is limited in terms of being able to minimize droplet size while maintaining high spatial precision. This is because smaller droplets are more difficult to generate consistently and smaller droplets sink slowly through the viscous lipid-in-oil solution making them more susceptible to drag forces caused by nozzle movements and subsequent displacement of droplets from their intended location. Therefore, to overcome printing constraints and further increase the printing resolution, two postprinting shrinking methods have been developed - one being driven through temperature (Section 1.2 of Example 1) and one being driven through osmosis (Section 1.3 of Example 1).

[0317] 1.2 Temperature-driven shrinkage of synthetic tissues

[0318] In order to shrink 3D-printed synthetic tissues post printing (Fig. 4d, left image), water molecules have to be removed from the synthetic tissue through the lipid-in-oil solution into the atmosphere.One way of achieving this is by exposing synthetic tissues in lipid-in-oil solutions to elevated temperatures (in this Example, 36 ± 0.68 °C to 43 ± 0.68 °C), inducing an evaporative process (Fig.

[0319] 4d, middle image). When developing this method, increasing contact angles were observed between droplets at the start of this heat-induced evaporation process, forming more tightly packed synthetic tissues (droplet annealing, Fig. 4d, right image). This was followed by a continuous decrease in volume of the compartments, presumably as water molecules partitioned into the lipid-in-oil solution (Fig. 4a), from which water molecules eventually got released to the unsaturated atmosphere in an evaporative process. Crucially, the general morphology of the synthetic tissues was unaffected and in this case the cube-shaped droplet assembly retained its cubic structure (Fig.

[0320] 4e).

[0321] 1.2.1 Droplet volumes

[0322] It was found that synthetic tissues shrink more quickly with increasing temperatures, as indicated by the rate of decrease in side lengths of synthetic tissues (see Fig. 4f for

[0323] definition) when comparing synthetic tissues heated at 36 °C and 43 °C (Fig. 4f). We thought that this was due to the exponential increase in water vapour pressure (Arnold Wexler, “Vapor Pressure Formulation for Water in Range 0 to 100 °C. A Revision”, Journal of Research of the National Bureau of Standards. Section A, Physics and Chemistry 80A.5-6 (1976), pp. 775-785) and an increased solubility of water molecules in the oil phase with increasing temperature, causing water flow from synthetic tissues through the oil into the atmosphere. In addition, it was observed that the volume change of a given single compartment is dependent on their immediate number of neighbouring droplets (Fig. 4g). Single droplets shrink the quickest, followed by droplet pairs forming DIBs, peripheral and then central droplets within one-layered synthetic tissues, and peripheral and then central droplets within eight-layered synthetic tissues (Fig. 4g). It was assumed this was as with an increasing number of neighbouring droplets, the surface area exposed to the lipid-in-oil solution was decreased, decelerating the rate at which water molecules partition into the lipid-in-oil solution. However, due to imaging limitations (Alessandro Alcinesio et al. “Controlled Packing and Single -Droplet Resolution of 3D-printed Functional Synthetic Tissues”, Nature Communications 11.1, Apr. 2020, p. 2105) we could only quantify volume changes of the bottom layer of an 8 -layered network.

[0324] 1.2.2 Contact angles

[0325] During the heat-induced evaporation method, it was found that the contact angle between droplets (θDIB) decreased after significant volume reduction (Fig. 5a, right image). We reasoned that this was because of increasing solute concentrations caused by the decrease in droplet volume, which is known to both affect bilayer and monolayer surface tensions, which affect θDIB (Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105). To investigate this effect, ODIBwas measured (using the image detection algorithm disclosed in Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105) in droplet pairs that contained varying fractions of the target printing solution (i.e. the final concentration of solutes once the synthetic tissue was shrunk, Fig. 5b). For example, droplets comprised of 9: 1 (v / v) water: target printing solution was 0.1 fraction of the target printing solution, whereas droplets labelled 0, or 1, contained either water only, or the target printing solution only, respectively. These experiments were performed to simulate the change in solute concentration within the droplets during the shrinkage process of synthetic tissues. Interestingly, it was found that θDIB was constant (63.9 ± 2.9°) at all target printing solution fractions ranging from 0 to 0.9, but dropped significantly when DIBs were composed of the target printing solution only (38.2 ± 1.1, Fig. 5c).

[0326] These results confirmed that a decrease in θDIB occurred when synthetic tissues were shrunk because of changes in droplet solute concentration (through loss of water), rather than effects caused by elevated temperatures.

[0327] 1.2.3 Bilayer retention

[0328] Critically, during synthetic tissue shrinking, DIBs within the synthetic tissues were retained, as confirmed by pattern retention within synthetic tissues (Fig. 6a, b). The change in droplet packing can cause synthetic tissues to initially form spherical structures, while patterns were still retained (Fig. 6a, b). However, once the glass cuvette was removed from the heat plate, causing the oil temperature to decrease back to room temperature, synthetic tissues returned to their initial cubic morphology (Fig. 6b), implying a sort of shape-memory mechanism. Bilayer stability was further investigated during heat-induced evaporation by introducing a dye that sequesters into the bilayers of our synthetic tissues (Atto 550 maleimide (245 nM)) (Alessandro Alcinesio et al., Nature Communications 11.1, Apr.2020, p. 2105). By comparing the bilayers of droplets within 8-layered synthetic tissues before and after heat-induced evaporation at 36 °C (t = 0 min and t = 53 min) it was found that the droplet packing changed, as visualized by the change in morphology of droplets, such as droplets 1, 2 and 3, while the bilayers between droplets were retained (Fig. 6c). No buckling or fission of DIBs was encountered throughout the process of evaporation.

[0329] 1.3 Osmosis-driven growth and shrinkage of synthetic tissues

[0330] The presented method of applying elevated temperatures to shrink synthetic tissues post printing through an evaporative process is limited, as, once removed from the synthetic tissues through the lipid-in-oil solution into the atmosphere, water molecules cannot be reintroduced easily for the purpose of rehydrating the synthetic tissues. However, a method that would allow both the shrinkage and growth (rehydration) of synthetic tissues would be of interest, for example, so to reversibly change the volume and, hence, solute concentrations within the synthetic tissues. Forthis, an osmosis-mediated mechanism is provided herein to reversibly control water influx or outflux from synthetic tissues, allowing for water to be introduced (growth) or removed (shrinkage) from the synthetic tissues. This method comprises the formation of a lipid bilayer between the synthetic tissue and an aqueous solution (Wout) at a liquid-liquid interface between Woutand a lipid-in-oil solution. First, the synthetic tissues were printed in lipid-in-oil solution (DPhPC (1 mM) in 20: 80=undecane: silicone AR20) before transferring them to a lipid-in-oil solution of the same composition on top of Wout, which initially was composed of the same solution as the synthetic tissues (i.e. M9 medium, Wm). Next, the osmolarity of the outer aqueous solution (aqueous solution forming the liquid-liquid interface, Wout) was altered by manually adding an aqueous solution of different osmolarity through pipetting (Fig. 7a, schematic in the middle). By introducing aqueous solution of increased osmolarity as compared to Wm, the resulting imbalance in osmolarity between Wmand Woutcaused a net water flux outward of the synthetic tissue, causing the synthetic tissue to shrink (Fig. 7a, left schematic). In contrast, when introducing aqueous solution of decreased osmolarity, the osmotic imbalance caused a net water flux into the synthetic tissue, causing the synthetic tissue to grow (Fig. 7a, schematic on the right). To test, whether the introduction of an aqueous solution of higher osmolarity could be used to shrink synthetic tissues at the liquid-liquid interface, a synthetic tissue composed of 1 x DPBS was printed and placed at the interface between Wout(lxDulbecco’s phosphate-buffered saline (DPBS), 300 mosm) and the lipid-in-oil solution. Next, the osmolarity of Wout(OOut) was increased step-wise within a time frame of 400 min to reach 6.5 osm (Section 1.6.2.2). It was found that an increase in

[0331] indeed caused a volume decrease of the synthetic tissue (Fig. 7b), confirming the hypothesis that a difference in osmolarity between Wmand Woutcan control water flux.

[0332] To quantify the effect of osmotic imbalance, the side lengths of the synthetic tissue were measured based on bright-field microscopy images of the synthetic tissue at the interface (Fig. 7b), which allowed the approximation of its volume for Ooutranging between 0.3 osm and 6.5 osm (Fig. 7c, Section 1.6.2.3). Additionally, we sought to compare the measured volume to the expected volume as a consequence of water outflux from the synthetic tissue. For this, it was assumed that the difference in osmolarity between Wmand Woutcaused a net water outflux from the synthetic tissue until the osmolarity of Wmreached the osmolarity of Wout. Furthermore, it was assumed that the outflux of water from the synthetic tissue into Wouthad negligible influence on the osmolarity of

[0333]

[0334] because the volume of (400 pL) was significantly larger than the volume of the synthetic tissue 0.5 pL). Based on this, the expected volume fold-change, AV, of the synthetic tissue as a consequence of water outflux from the synthetic tissue to reach Ooutwas calculated as follows:

[0335] C) ■

[0336]

[0337] (1.1)where Omand Ooutare the osmotic concentrations of Wmand Wout, respectively. It was found that the actual volume fold-change did not follow the expected volume fold-change. This may be due to inaccuracies when approximating the volume of the synthetic tissue or time-related aspects. For example, the time it takes for Omand Ooutto equilibrate may increase with increasing osmolarities, as the osmotic difference between Omand Oout relative to their absolute values decreases. To test whether the volume of synthetic tissues could be increased by introducing an aqueous solution of lower osmolarity as compared to Omand Oout, pure water was manually added into Wout(M9 medium). In doing so, it was found that the volume of the synthetic tissue could be increased, resulting in the synthetic tissue to grow at the aqueous-lipid-in-oil interface (Fig. 7d). Ooutranged between 240 mosm and 4.8 mosm, which lead to the synthetic tissue to increase in volume by a factor of 7.4. Despite the significant growth, the measured value was significantly lower than the expected volume fold change (Fig. 7e). At Oout < 50 mosm we encountered multiple coalescence events between droplets of the synthetic tissue, suggesting that DIBs become less stable with decreasing solute content or that water influx has to be precisely monitored so to prevent rapid water influx, leading to destabilizing effects on DIBs. Taken together, it has been shown that by using both a temperature-driven and osmosis-driven mechanism synthetic tissues can be minimized in size post printing. Crucially, osmosis-driven influx and outflux of water presents a reversible means to adjust the volume of synthetic tissues.

[0338] 1.4 Improved resolution of gene induction in bacterial cells

[0339] We aimed to leverage the temperature-driven shrinkage of synthetic tissues to maximize the resolution in delivering chemical signals to living cells (Fig. 8). Specifically, we investigated whether shrunken synthetic tissues could release arabinose through aHL incorporated into DHBs between the synthetic tissues and a bacterium-laden hydrogel in a patterned manner. By using shrunken synthetic tissues, we significantly increased spatial resolution compared to single -droplet pathways

[0340] 100 pm) and previously presented patterns, such as a triangular pattern (Fig. 8a-d). Shrunken tissues activated gene expression within a frame-like pattern, the width of which was <50 pm (Fig. 8b, c and 8e,f). Further details of the experiments on improved resolution of gene induction in bacterial cells are presented in Example 2 hereinbelow.

[0341] 1.5 Example 1 conclusions

[0342] The currently-known 3D-droplet printing technology is limited when it comes to minimizing the droplet size. This is because smaller droplets sink slowly through the viscous lipid-in-oil solution, significantly extending printing times. Moreover, the smaller the droplet size the higher the likelihood for inaccuracies to evolve during printing, which leads to defects in droplet packing. Moreover, variations in droplet volume during the printing of small droplets is more pronounced,exacerbating such inaccuracies. Therefore, in order to minimize droplet volume while maintaining or even improving printing accuracy it is desirable to print larger droplets at first. Based on this, we have presented two different means to shrink synthetic tissues post printing through temperature-mediated evaporation of water molecules from synthetic tissues and through osmosis-mediated efflux of water molecules. Crucially, internal bilayers and patterned arrangements of droplets were retained throughout the process of temperature-mediated shrinkage. Although this has yet to be confirmed for the osmosis driven, reversible volume change, the bright-field microscopy images look promising. Interestingly, we observed a change in contact angle after significant volume reduction, presumably due to changes in solute concentration, rather than as a consequence of the applied temperature. In some cases, synthetic tissues adapted a spherical shape, whereby the synthetic tissues returned to their initial cubic shape upon removal of the heat. Shrunken synthetic tissues achieved patterned delivery of a chemical signal, arabinose, for the purpose of patterned activation of gene expression in E. coli at a spatial resolution of ~50 pm. Furthermore, the osmosis-mediated volume change at an aqueous-lipid-in-oil interface allows for reversible volume change (shrinkage and growth).

[0343] 1.6 Methods - Reversible volume change of synthetic tissues

[0344] 1.6.1 Temperature-driven dehydration of synthetic tissues

[0345] Synthetic tissues were printed in SOG cuvettes, which contained 500 pL of lipid-in-oil solution. The cuvettes were then placed in the centre of a transparent heating plate (Leica Thermo Plate), which was set to 36 °C or 43 °C. The temperature in the lipid-in-oil solution was allowed to reach equilibrium as determined with a sensor (Thorlabs, TSP01). The printing solution was diluted with Milli-Q® water so that the concentrations of the components in the synthetic tissues would be as desired after shrinkage (36 °C or 43 °C). Once the desired decrease in volume was attained as determined by microscopy, a shrunken tissue was brought to room temperature for 30 min before transfer on top of the bacterium -laden hydrogel (as described further in Example 2).

[0346] 1.6.2 Osmosis-driven dehydration and growth of synthetic tissues

[0347] Synthetic tissues were printed in SOG cuvettes, which contained 500 pL of lipid-in-oil solution. The synthetic tissues were then transferred to SOG cuvettes containing an aqueous solution at the bottom of the cuvette covered by a lipid-in-oil solution (same composition as the lipid-in-oil solution for 3D-printing). Synthetic tissues sank to the liquid-liquid interface and remained afloat. Following this, aqueous solution from the bottom was removed step-wise by pipetting without disturbing the synthetic tissue at the liquid-liquid interface. Equal amounts of aqueous solution (either lower or higher in osmolarity) were then added directly into the aqueous solution at thebottom of the liquid-liquid interface to reach the initial volume.

[0348] 1.6.2.1 Measuring the osmotic concentration

[0349] Osmotic concentrations were measured using the ‘i osmometer M’ by Loser Messtechnik. The device was calibrated using the standards provided (300 mosm and 900 mosm) prior to every use. The sample volumes were kept constant (25 pL) for both calibration and sample measurements. The measurement range of the device was 0 - 2500 mosm, however, all measurements were taken within the range of calibration samples (0 - 900 mosm). So, if the measurement was outside of this range, the sample was diluted until the measurement fell into the calibration range. The osmotic concentration was then calculated based on the osmotic concentration measured and the dilution factor.

[0350] 1.6.2.2 Osmosis-driven dehydration

[0351] Here, the synthetic tissue comprised of 0.2 x DPBS supplemented with 10% (w / v) of sucrose, which had an osmolarity of 406 mosm. It was printed in a lipid-in-oil solution comprising DPhPC (1 mM) in 20:80=undecane:silicone oil. The step-wise addition of aqueous solution comprised the removal of 200 pL from the initial 400 pL first, before adding the 200 pL of a solution of increasing osmolarity. The first aqueous solution that was added had an osmotic concentration of 500 mosm, which was added to the initial solution of 409 mosm. Based on this, the resulting osmotic concentration was 453 mosm. This procedure was repeated 26 times in total, whereby the osmotic concentration of the added aqueous solution increased by 100 mosm each step, giving a final osmotic concentration of 3000 mosm, which is the osmotic concentration of 10 x DPBS. From there, the step-size was increased to 300 mosm: 11 x DPBS, 12 x DPBS,..., 18 x DPBS. The last two aqueous solutions that were added showed osmolarities of 6 osm (20 x DPBS) and 7.5 osm (25 x DPBS), leading to a final osmotic concentration of 6.525 osm in the aqueous solution at the bottom of the cuvette, which is a 21.75-fold increase as compared to the initial solution. All steps were carried out within a time frame of 395 min.

[0352] 1.6.2.3 Osmosis-driven rehydration

[0353] In contrast, in order to induce tissue growth, 100 pL ofMilli-Q® water was added to 400 pL of the previous dilution. That way, the osmotic concentration in the aqueous solution at the bottom of the cuvette was reduced and water molecules could be introduced into the synthetic tissue. The synthetic tissue was composed of M9 medium initially, which showed an osmolarity of 240 mosm. Once the synthetic tissue was placed at the interface, Milli-Q® water was added step-wise to give dilutions of 0.8 x M9, 0.64 x M9, 0.51 x M9, 0.41 x M9, 0.33 x M9, 0.26 x M9, 0.16 x M9, 0.08 x M9 and 0.02 x M9. All dilutions were conducted within a time frame of 293 min.Example 2 - detailed Example of patterned release of chemical signals, both from “as-produced” and “shrunken” droplet networks

[0354] 2.1. Introduction

[0355] The present inventors have appreciated that functional droplet networks (M. A. Holden et al., J. Am. Chem. Soc. 2007, 129, 8650; M. J. Booth et al., Molecular BioSystems 2017, 13, 1658; G. Villar et al., Nat Nanotechnol 2011, 6, 803; C. E. G. Hoskin et al., Nat. Chem. 2022, 1) in other contexts also referred to as synthetic tissues (H. Bayley et al., Emerg Top Life Sci 2019, 3, 615; G. Villar et al., Science 2013, 340, 48; A. J. Lin et al., ACS Synth. Biol. 2023, 12, 1889; A. Alcinesio et al., Nat Commun 2020, 11, 2105; A. Alcinesio et al., ChemSystemsChem 2022, 4, e202100036; I. Cazimoglu et al., ACS Nano 2021, 15, 20214), exhibit potential for releasing chemical signals with high spatial and temporal resolution, as they have the ability to feature the patterning of compartments and signalling between compartments both within the droplet network and with the immediate external environment at micrometre resolution.

[0356] In this Example 2, 3D-printed picolitre droplet networks are provided as a universal platform that can direct cellular activity by the patterned release of chemical signals. Specifically, the controlled release of chemical signals onto populations of homogeneously-distributed Escherichia coli microcolonies is demonstrated, eliciting patterned changes in gene expression by the precise tuning of cargo release dynamics. In addition, a method to reliably shrink our droplet networks to achieve chemical signalling with micrometre resolution (~50 pm) has been developed. The system of the invention shows improved cargo storage capacity compared to synthetic cell-based systems, while retaining the ability of releasing chemical signals from a single compartment. Further, it is shown herein that the droplet assemblies of the invention can be orientated in space through magnetism and that cargo release can be activated at defined times by the connection of reservoir networks. The versatility of the inventive system is illustrated by the spatiotemporal control of the expression of colicin E7, a proteinaceous toxin produced by specific E. coli strains (E. Cascales et al., Microbiology and Molecular Biology Reviews 2007, 71, 158), hence directing the outcome of toxin-driven bacterial competition. Taken together, the results described herein pave the way towards applications of functional droplet networks in directing cellular patterning for applications in fundamental biology and medicine that require the local control of gene expression within a target group of cells.

[0357] 2.2. Results and Discussion

[0358] 2.2.1. Interfacing Bacterial Cells with Droplet Networks

[0359] Patterned picoliter droplet networks - comprising 500 to 1000 droplets (-150 pL per droplet) connected through droplet interface bilayers (DIB) - were constructed by using a 3D dropletprinter (Figure 1(a)) (G. Villar et al., Science 2013, 340, 48; WO 2014 / 087175). In brief, aqueous droplets containing L-(+)-arabinose (the chemical signal used to induce bacterial protein expression) and monomers of the pore-forming membrane protein a -hemolysin (aHL) were ejected into a lipid-in-oil solution: 1,2-diphytanoyl-sn-glycero-phosphatidylcholine (DPhPC) and 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC, 2:1 molar ratio) in 35:65 (v:v) undecane: silicone oil AR20). Monomers of aHL assemble to form heptameric transmembrane pores in the DIBs, which allow the diffusion of small molecules (less than 2 kDa; L. Song et al., Science 1996, 274, 1859) between the droplets. Other protein pore types may be selected for allowing passage of larger molecules. We have previously optimized the packing of our droplets within our 3D-printed droplet networks to maximize hexagonal close-packing (A. Alcinesio et al., Nat Commun 2020, 11, 2105).

[0360] To interface these droplet networks with bacteria, droplet networks were transferred from the printing chamber (Figure 1(a), right graphic) to a lipid-in-oil solution on top of a bacterium -laden hydrogel (Escherichia coli in 30 pL of 1.5% (w / v) ultra-low gelling temperature agarose (ULGA), forming a cylindrically-shaped hydrogel with a circular area of -0.32 cm2and a thickness of -1 mm). E. coli cells were dispersed within the hydrogel at starting densities ranging from

[0361] 1.6 x 107to 4.0 x 1010cells mL-1. Once a droplet network (8 x 8 x 8 droplets) came into contact with the hydrogel, bilayers formed between the external droplets of the tissue and the lipid monolayer at the hydrogel surface that were stable for weeks, with a quadratically-shaped area of -0.3 mm2to -0.4 mm2(Figure 1(b)) (J. R. Thompson et al., Nano Lett. 2007, 7, 3875).

[0362] To observe gene expression in the E. coli population (BZB1011 Pmax:sfgfp:: Tn7 pJS1-PBAD:-mCherry-AMP – mCherry-inducible) by the release of arabinose from droplet networks, the bacteria contained the plasmid pJSl-P BAD: -mCherry-AMP encoding a fluorescent protein, m Cherry, downstream of the promoter (PBAD), which is regulated by arabinose. Once droplet networks (initially containing 33 mM arabinose and 25 pg mb1aHL monomer) were placed on top of the bacterium-laden hydrogel (t = 0 h), aHL inserted into the DHB, establishing a flux of arabinose through the pores into the bacterium-laden hydrogel (Figure 1(c)). Over 18 h, we observed that E. coli cells were actively growing and dividing from single-cell dispersions (t = 0 h, Figure 1(d) and Figure 1(e)) to form 3D micro-colonies (R. Krishna Kumar et al., Nat Commun 2021, 12, 857), and cells directly below the droplet networks (within -50 pm to 100 pm underneath the contact area) and in close proximity to the droplet networks (within -100 pm from the edge) expressed high amounts of mCherry, quantified as the mean gene expression, IM (see Methods), of activated cells (634 a.u., Figure 1(e) and Figure 1(f)). Cells further away from the droplet network expressed only baseline levels of mCherry (179 a.u., Figure 1(e) and Figure 1(f)). An increase in fluorescence was detected at approximately t = 10 h (Figure 1(g)). This was followed by a rapid increase in mCherry expression, consistent with the all-or-nothing nature of the pBADsystem (Supplementary Note 1). After ~18 h, no further increase in mCherry expression was detected. Droplet networks without aHL barely induced gene expression (Figure 1(g)), confirming the limited permeability of the lipid bilayers to arabinose (M. G. Sacerdote et al., Proceedings of the National Academy of Sciences 2005, 102, 6004) compared to other commonly used chemical signals, such as IPTG (A. Dupin et al., Nature Chem 2019, 11, 32). Therefore, localized arabinose flux from droplet networks can be controlled by the permeabilization of bilayers with aHL pores, allowing localized gene expression within a homogenous population of bacterial cells.

[0363] 2.2.2 Patterned Gene Expression Through Optimized Arabinose Flux

[0364] To understand and quantify the factors that control spatiotemporal release of arabinose and thus local gene expression of the bacterial population, we introduced the pattern fidelity (PF) index:

[0365] PF = (AI-AU) / AI

[0366] A ’ ’

[0367] where Ai is the intended area of expression (the x, v-planc cross-sectional area of a droplet network where aHL inserts into the DHBs), and where Au is the area of unintended expression (see Methods). We sought a PF approaching a value of 1, reflecting minimal gene expression in unintended areas, Au (Figure 2(a)), while maintaining a high level of gene expression, by exploring the aHL and arabinose concentrations in the droplet networks. To evaluate control over gene expression within intended areas of gene expression, Ai, we further introduced the normalized measure, AN, which indicated areas where no gene expression was induced within Ai (Figure 2(a)). At a fixed concentration of arabinose (33 mM), we found that increased concentrations of aHL monomer (0 - 50 pg mL1) led to decreased PF due to increasing A u, presumably caused by a greater flux of arabinose (Figure 2(b)). Moreover, we observed an increase in total gene expression, IT (sum of all pixel values in activated pixels, see Methods), with increased aHL

[0368] concentration (Figure 2(b)), which we reasoned was due to both increased gene expression in unintended areas of gene expression, Au, and increased gene expression within Ai, which was confirmed by decreased areas of AN. At a fixed concentration of aHL monomer (50 pg mL1), arabinose concentrations higher than 33 mM also led to decreased values of PF, associated with increasing Au and IT and decreasing areas of no gene expression within intended areas of gene expression, AN (Figure 2(c)).

[0369] We found that at low aHL monomer (<10 pg mL1) and low arabinose (<10 mM) concentrations we could attain PFs close to 1, however, ITwas low (<1.2 x 108a.u., Figure 2(b) and Figure 2(c)). Therefore, we settled on concentrations of aHL monomer (25 pg mL1) and arabinose (33 mM) where PF was 0.77, but ITwas high (≥1.4 × 108a.u., Figure 2(b) and Figure 2(c)) and AN was low (0.12±0.07, Figure 2(b) and Figure 2(c)). At these concentrations, we printed a range of patterned droplet networks designed to produce different release patterns of chemical signals, byusing droplets that did or did not contain arabinose and aHL. These printed networks produced gene expression patterns within a homogenous population of bacteria at PFs of >0.89 (Figure 2(d)-(g), Supplementary Note 2). To further evaluate the control over induced gene expression as a consequence of arabinose release from 3D-printed synthetic tissues, we introduced AN as a means to quantify areas of no gene expression AN) within areas of intended gene expression (d / ). We noticed AN values between 0.48 and 0.85. We next investigated whether we could store larger amounts of arabinose within printed tissues while maintaining spatial control over gene expression. Therefore, we sought to vary the number of layers (4, 8, 16 in total), while keeping Ai unchanged (Figure 3(a)). The volume-to-surface-area ratio (RVSA) was defined as:

[0370] RVSA=’ (2)

[0371] where V is the total volume of the droplet networks containing arabinose and aHL. According to this definition, droplet networks with an increasing number of network layers (4, 8, 16) are characterized by RVSA values of 0.25 mm, 0.49 mm and 1.07 mm, respectively. Increased RVSA indeed led to enhanced total gene expression, IT, and decreased areas of AN (with mean AN / AI values of 0.81±0.17, 0.57±0.18 and 0.38±0.15 for RVSA values of 0.25 mm, 0.49 mm and 1.07 mm, respectively, Figure 3(b,c)). Importantly, unintended expression, Au, barely increased (from Au / Aj values of 0.02 with 4-layered networks to 0.08 with 16-layered networks, corresponding to high PF values of 0.98 and 0.92, respectively, Figure 3(b)). To reduce printing time and further increase storage capacity (Supplementary Note 3), we printed droplet networks composed of 4 patterned layers at the bottom (mask) and 4 uniform layers at the top entirely composed of arabinose and aHL-containing compartments (reservoir, Figure 3(d) and Figure 3(e)). By equipping our droplet networks with magnetic handles (agarose droplets containing MagneHis™ Ni-particles) and placing a magnet placed underneath the bacterium-laden hydrogel, droplet networks were precisely guided to interface with the bacterium-laden hydrogel through the mask layers rather than the reservoir layers (Figure 3(f), Supplementary Note 3), retaining patterned gene

[0372] induction (Figure 3(g)).

[0373] 2.2.3 Improved Resolution of Gene Expression with " Shrunken" Tissues

[0374] We next sought to achieve even higher spatial resolution of patterned gene expression with droplet networks. We found that chemical signal release can be achieved through a linear, single -droplet pathway (with droplet diameters of 65 pm) containing arabinose and aHL (Figure 4(a) and Figure 4(b)). In this case, gene expression was induced within a circular area of -100 pm diameter (Figure 4(c)). To further increase the resolution, we developed a heat-induced postprinting shrinking process (Figure 4(d)). At the start of this process, we observed increasing contact angles between droplets, forming more tightly packed droplet networks (droplet annealing). Then, the volume of the compartments continuously decreased, presumably as water moleculespartitioned into the oil, from which water molecules eventually got released to the unsaturated atmosphere in an evaporative process (Figure 4(d)). Importantly, the general morphology of the droplet networks (Figure 4(e)) and the patterned arrangements of the chemical signal -containing droplets were barely affected (Figure 4(i)). We found that the rate at which the droplets shrank increased with temperature (Figure 4(f)), which we reasoned was due to the exponential increase in water vapor pressure (A. Wexler, J Res Natl Bur Stand A Phys Chem 1976, 80A, 775) and an increased solubility of water molecules in the oil phase with increasing temperature, causing water flow from droplet networks through the oil into the atmosphere. Additionally, we discovered that for a given droplet, the shrinking rate decreased with an increasing number of neighboring droplets. For example, we found that droplets at the periphery of droplet networks shrank more quickly than droplets in the center (Figure 4(g)), likely because water molecules from peripheral droplets partition into the oil phase first. Solute concentrations were diluted prior to printing, such that target concentrations were reached within the droplets after the evaporation process. By using shrunken droplet networks, we significantly increased spatial control compared to single-droplet pathways (Figure 4(a)) and previous patterns, such as a triangular pattern (Figure 4(h)). Shrunken networks activated gene expression within a frame-like pattern, the width of which was

[0375] <50 pm (Figure 4(i) and (j)).

[0376] 2.3. Remarks in relation to Example 2

[0377] In this Example, chemical signal-based communication between 3D-printed droplet networks and bacterial cells is presented. The flux of arabinose, the chemical signal, can be precisely controlled, by adjusting signal and pore concentrations within the droplet networks, to produce patterned gene expression in bacterial populations. We can magnetically guide our droplet networks to land precisely onto bacterial populations, controlling where chemical signal molecules can be released. In addition, we can store chemical signals in printed droplet reservoirs, while maintaining high spatial control over gene expression. We further increased the resolution of patterned gene expression significantly to 50 pm by using evaporative shrinkage of the networks, which represents a significant improvement when compared with the millimeter resolution of previous work (T. Danino et al., Nature 2010, 463, 326; A. Tamsir et al., Nature 2011, 469, 212; T. Sohka et al., Proceedings of the National Academy of Sciences 2009, 106, 10135; J. J. Tabor et al., Cell 2009, 137, 1272; S. Basu et al, Nature 2005, 434, 1130; J. Zhang et al., J Mol Biol 2020, 432, 3137). Further, by using our key-lock mechanism the release of chemical signals can be activated at a controlled point in time.

[0378] Critically, we show the utility of our system by inducing the expression of bacteriocins in E. coli. which drives patterned DNA damage in co-cultured susceptible cells and local competition between toxin-producing strains with different potencies. Therefore, the present work demonstrateschemical signal release from 3D-printed droplet networks as a tool for the study of spatiotemporal dynamics of complex cellular behaviors within microbial communities.

[0379] Additionally, we envision that our droplet networks might encapsulate cell-free protein expression systems (M. J. Booth et al., Science Advances 2016, 2, el600056) which could mediate the release of chemical signals in response to the environment. For example, n-acyl homoserine lactones (O. D. Toparlak et al., Science Advances 2020, 6, eabb4920) released from bacterial cells could drive the expression of aHL inside droplet networks, which would then allow chemical signals, such as arabinose, to be released back onto the bacterial cells, inducing the expression of a gene of interest, thereby, establishing bidirectional communication between droplet networks and bacterial cells. To further control chemical signal release, membrane proteins that gate in response to stimuli, such as Zn2+(A. Alcinesio et al., Advanced Functional Materials 2022, 32, 2107773; O. Braha et al., Chemistry & Biology 1997, 4, 497; M. J. Booth et al., Commun Chem 2019, 2, 1) or light (C. Chang et al., Chemistry & Biology 1995, 2, 391) could be used to permeabilize membranes to achieve light-sensitive chemical signal release (C. Chang et al., Chemistry & Biology 1995, 2, 391). We envisage that our system can encapsulate different types of chemical signals (such as drugs, peptide / proteins, or DNA / RNA), as well as having the ability to integrate with other effector technologies (for example lipid nanoparticles) for interactions with various cell types. Membrane pores with larger internal diameters could be used to allow larger signaling molecules to be released from droplet networks (O. D. Toparlak et al., Science Advances 2020, 6, eabb4920). For example, perfringolysin O has a diameter between 25 nm and 30 nm (T. X. Dang, Journal of Structural Biology 2005, 150, 100) as compared with the aHL pore, whose narrowest inner diameter is 1.4 nm (L. Song et al., Science 1996, 274, 1859). Moreover, chemical signals could be produced in droplet networks by cell-free protein expression or multiple types of droplet network could be connected to restore chemical signals once depleted (e.g. by exchanging storage modules) and to release multiple types of chemical signals (e.g. by connecting storage modules containing different chemical signals to one release module). Further, our droplet networks could function in bulk aqueous environments by adapting previous work of forming external lipid bilayers surrounding the droplet networks to interface with cells in 3D (A. Alcinesio et al., ChemSystemsChem 2022, 4, e202100036). Lastly, 3D-printed hybrid tissues that contain living cells might be constructed (R. Krishna Kumar et al., Nat Commun 2021, 12, 857; A. D. Graham et al., Sci Rep 2017, 7, 7004; L. Zhou et al., Advanced Materials 2020, 32, 2002183) where gradients of growth factors control the differentiation of cells at defined locations, thereby allowing the formation of complex tissues (Z. Liu et al, Progress in Materials Science 2017, 88, 467; R. Subbiah et al, Advanced Healthcare Materials 2019, 8, 1801000; L. Sardelli et al, J Appl Biomater Funct Mater 2019, 17, 2280800019829023). In summary, the technology of the invention demonstrates patterned spatiotemporal communication between droplet networks and living cells based onchemical signals. The platform might be modified to investigate the patterning of various groups of cells through the release of various natural and synthetic signals, such as quorum sensing molecules, antibiotics and growth factors. Such an approach will prove useful in fundamental research, including the modulation of species diversity in bacterial communities or the spatiotemporal dynamics of tissue development and morphogenesis. Furthermore, the use of droplet networks in medical applications (G. Chen et al., Proceedings of the National Academy of Sciences 2022, 119, e2207525119; Z. Li et al., Science Advances 2020, 6, eaay0589; R.

[0380] Chandrawati et al., Advanced Materials 2017, 29, 1604932; S. Kartha et al, Advanced Healthcare Materials 2017, 6, 1700500) is envisioned, such as in the treatment of chronic wounds, cancer, neurodegenerative diseases and spinal cord injuries.

[0381] 2.4. Methods

[0382] Preparing Aqueous Phases. Lysogeny broth Miller (LB) medium (Invitrogen) was prepared by adding LB powder (25 g) to Milli-Q® water (I L) and autoclaving. 5 x M9 minimal salts solution was prepared by adding M9 minimal salts (2.82 g, Sigma- Aldrich) to Milli-Q® water (100 mL) and autoclaving. Casamino acid solution was prepared by adding Calbiochem® OmniPur® Casamino acid (5 g, Merck) to Milli-Q® water (50 mL) and autoclaving. The final M9 minimal medium consisted of 1 x M9 minimal salts, MgSO4 (2 mM), CaCl₂ (0.1 mM, filter-sterilized using a 0.22-pm polyethersulfone membrane (Millex-GP Syringe Filter Unit)), casamino acids (0.2% (w / v), Sigma) and D-glucose (24 mM) or glycerol (24 mM). Bacterium -laden hydrogels were prepared by adding ultra-low gelling agarose (Sigma- Aldrich) to the M9 minimal medium (at a final concentration of 1.5% (w / v)) and autoclaving. The hydrogel solution was kept molten in a water bath at 37°C and prepared freshly before every experiment. Antibiotics were dissolved in Milli-Q® water, filter-sterilised (0.22-pm polyethersulfone membrane) and frozen (-20°C) as stock solutions (ampicillin: 100 mg mL-1, kanamycin: 50 mg mL⁻¹, Sigma-Aldrich). The final concentrations of antibiotics in media were 100 μg mL-1and 50 μg mL-1for ampicillin and kanamycin, respectively.

[0383] Construction of Recombinant DNA'. The E. coli strain BZB1011 was used for all Golden Gate cloning experiments in this study. E. coli cells were cultured in LB medium at 37°C with shaking at 250 rpm and plated on LB agar, incubated at 37°C. The LB medium and agar were supplemented with the appropriate antibiotics, either ampicillin (100 μg mL⁻¹) or kanamycin (50 μg mL⁻¹). For blue-white screening, agar plates were additionally supplemented with IPTG (0.1 mM, Sigma Aldrich) and Xgal (40 μg mL-1, Thermo Fisher). Constructs (pKC1-PBAD:-ColE7-AMP and pYY1-PBAD:-ColE7-mCherry-AMP) were made using the MoClo kit and cloning method89. The following components were added to a 0.2 mL PCRtube: DNA components (10 fmol each), Bsal (10 U) or BbsI (10 U) restriction enzyme (NEB), T4 Ligase (20 U, NEB), and 1× T4 DNA ligase buffer (NEB). Milli-Q® water was then added to bring the total volume to 10 pL. Reaction mixtures were incubated in a thermocycler for 40 cycles of digestion and ligation (37°C fordigestion for 2 min, 16°C for ligation for 5 min), followed by 5 min at 50°C and a heat kill step at 80°C for 10 min. The mixtures were then held at 4°C, and 4 pL was used to transform cells.

[0384] Transformants were selected using lacZa blue-white screening.

[0385] Competent Cell Preparation and Cell Transformation'. An overnight culture of E. coli BZB1011 cells was set up by inoculating the cells into LB medium, followed by shaking (225 rpm) at 37°C for no longer than 12 h. The overnight culture was inoculated into LB (50 mL) at an OD600of 0.05 and grown at 37°C with shaking (225 rpm) until the OD600reached 0.4-0.6. The culture was centrifuged at 4481 x g at 4°C for 10 min. The supernatant was removed and the cell pellet was resuspended in a solution (25 mL) containing CaCl₂ (100 mM) and glycerol (15% (v / v)) at 4°C. After 45 min, the cells were pelleted again by centrifugation at 4481 x g at 4°C for 10 min. The supernatant was removed and the cell pellet was re-suspended in a solution (5 mL) containing CaCl₂ (100 mM) and glycerol (15% (v / v)) at 4°C before aliquoting and storing at -80°C.

[0386] Chemically competent E. coli cells (BZB1011, 750 pL) were thawed on ice and of the plasmid of interest (100 ng) was added. After 30 min on ice, the cells were heat-shocked for 45 sec at 42°C. After 2 min on ice, the cells were added to pre-warmed super optimal broth (SOC, 1 mL) at 37°C and incubated for 1 h. The cells were then inoculated onto LB-agar plates supplemented with antibiotics (100 μg mL-1ampicillin, 50 μg mL-1kanamycin). After overnight culture at 37°C, a single colony was picked and inoculated into LB (4 mL), supplemented with antibiotics (100 μg mL-1ampicillin, 50 μg mL-1kanamycin), and shaken for no longer than 12 h at 37°C (225 rpm). The culture was then mixed with an equal volume of glycerol (50% (v / v)) solution. Glycerol stocks were stored at -80°C.

[0387] Preparation of Bacterium-laden Hydrogels'. E. coli cells (BZB1011) were pipetted from glycerol stocks into a round bottom tube containing LB (4 mL) supplemented with antibiotics (100 μg mL-1ampicillin, 50 μg mL-1kanamycin). Cells were grown for no longer than 12 h at 37°C with shaking (225 rpm). 40 pL of overnight culture were then transferred to a round bottom tube containing LB (4 mL) supplemented with antibiotics (100 μg mL-1ampicillin, 50 μg mL-1kanamycin) and grown for 3 h at 37°C with shaking (225 rpm). Based on the ODeoo, which was measured using a spectrophotometer (BioRad SmartSpec™ Plus, with a conversion factor of 1.0 = 108cells mL-1), appropriate amounts of culture were added to a 1.5 mL tube and centrifuged for 8 min at 8000 x g. The supernatant was removed and the cells were re-suspended in appropriate amounts of pre-heated (37°C) M9 medium containing ultra-low gelling agarose (final concentration: 1.5% (w / v)) to form a molten bacterium-laden hydrogel solution with the desired cell density (i.e. ODeoo 0.02, 2 or 4.5). In case of experiments involving two bacterial strains (i.e. bacterial interference competition) the amount of cells were adjusted according to the starting ratio to reach the desired total cell concentration. The resulting cell suspension (30 pL) was added to anumber of wells (<30) of a 96-well plate according to the number of experimental conditions and solidified at 4°C for 35 min.

[0388] Expression and Purification of aHL '. The aHL monomers were prepared by transforming E. coli BL21(DE3) pLysS cells (Agilent) with the pT7-αHL-D₈H₆ plasmid90and inoculated onto LB-agar plates containing antibiotics (carbenicillin, 50 μg mL-1, chloramphenicol, 34 μg mL-1). A single colony from the plate was picked and inoculated in LB (10 mL) for the pre-culture. An LB culture (400 mL) containing the same antibiotics was inoculated with overnight pre-culture (4 mL). This expression culture was shaken at 37°C at 250 rpm for approximately 3 h until the ODeoo reached 0.6, when it was cooled to 18°C before the addition of IPTG (2 mL, 0.1 M, Fluorochem) to induce protein expression. The culture was further shaken at 18°C at 200 rpm overnight. The cells were then harvested by centrifugation in a Beckman J25 centrifuge at 5000 rpm for 20 min at 4°C and resuspended in lysis buffer (10 mL, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, 5% glycerol, 2 mM TCEP with an EDTA-free protease-inhibitor tablet (ThermoFisher)). Lysis was then performed by the addition of lysozyme (250 μL, 40 mg mL-1, ThermoFisher), universal nuclease (2 μL, 250 U μL-1, ThermoFisher) and MgCl₂-containing solution (25 μL, 2 M), and incubation on ice for 1 h. The lysate was sonicated at 40% amplitude for 3 min in a 30 s-ON-30 s-OFF pulse train on ice (VCX 500, Sonics). The supernatant was cleared by centrifugation at 29000 x g for 45 min at 4°C and transferred to a gravity column containing Ni-NTA resin (1 mL, bed volume, ThermoFisher). The lysate supernatant and resin mixture were mixed at 4°C on a platform rotator for 1 h. The column was washed with washing buffer (2 x 15 mL, 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 2 mM TCEP, 0.1% Triton X-100 and 5% glycerol) and eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole, 2 mM TCEP, 0.1% Triton X-100 and 5% glycerol). The fractions (~10 mL) containing aHL were combined and loaded onto a HiLoad 26 / 600 Superdex 200pg (Cytiva) SEC column equilibrated with SEC buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM TCEP and 5% glycerol) at 4°C. Fractions containing monomers of aHL were concentrated (1 mg mL-1) and stored at -80°C as aliquots. The mass of the monomer was verified by LC-MS. On average, 9 mg pure aHL monomer after SEC purification was obtained from culture (400 mL).

[0389] Preparation of Lipid-in-oil Solutions'. Lipids (l,2-diphytanoyl-sn-glycero-3 -phosphocholine (DPhPC, 4ME 16:0-18:1 PC), 1 -palmitoyl -2 -oleoyl -glycero-3 -phosphocholine (POPC, 16:0-18:1 PC); Avanti Polar Lipids) were dissolved in anhydrous chloroform (2.5 mL, 10 mg mL-1, Sigma-Aldrich). The final lipid composition DPhPC: POPC (2: 1 molar ratio) was prepared in chloroformcleaned, Teflon capped glass vials (Supelco®). The chloroform was evaporated under nitrogen and the remaining solvent removed by placing the vials under vacuum for 24 h. The vials were stored at -80°C under argon. Before use, the vials were brought to room temperature for 15 minutes and a pre-mixed oil solution consisting of undecane (Sigma-Aldrich) and silicone oil (AR20, Wacker) ina ratio of 35:65 (v / v) was added. The lipid-in-oil solution was vortexed and then sonicated (Branson 2800 ultrasonic bath 230 V) for 1 h at 25 to 35°C and vortexed again before use. The total concentration of lipids was 2 mM.

[0390] 3D-Printing of Droplet Networks'. The droplet networks used in this work were formed by using a 3D-printing device as described elsewhere (I. Cazimoglu, M. J. Booth, H. Bayley, ACS Nano 2021, 15, 20214). Briefly, an aqueous solution (M9 minimal medium supplemented with various concentrations of L-(+)-arabinose (0 - 333 mM, Sigma-Aldrich), aHL monomer (0 - 50 pg ml1) and cascade blue dextran (250 μM, Invitrogen, Cat. D1976) was ejected from a glass nozzle into the lipid-in-oil solution in a printing cuvette. The printing cuvettes (composed of special optical glass (SOG), Starma Scientific) were mounted on a micromanipulator stage (Patch Star 7000, Scientifica), which moved in xyz-direction so to position the static glass nozzle according to a printing map. During droplet ejection, monolayers of lipid assemble spontaneously at the interface between the aqueous droplets and the lipid-in-oil solution. Lipid bilayers form between neighboring droplets when lipid monolayers contact one another. The placement of individual droplets with various contents can be controlled by using a multiple nozzle setup, where patterned droplet networks are formed by initializing the printing software according to the relative position of the glass nozzles. The droplet size (60 - 120 pm in diameter) was controlled by adjusting pulse voltage and / or pulse duration of the piezo driver.

[0391] Placement of Droplet Networks on Bacterium-laden Hydrogels'. Lipid-in-oil solution (50 pL) was pipetted on top of bacterium -laden hydrogels in a 96-well plate and incubated at room temperature for 15 min. Then, droplet networks were transferred to the lipid-in-oil solution using a pipette, when they sank forming robust DHBs at the interface with the hydrogel. After network placement the 96-well plates were incubated at 37°C for 18 h until further analysis (see pattern fidelity quantification by image analysis).

[0392] Pattern Fidelity Quantification by Image Analysis'. Bacterium -laden hydrogels and droplet networks were imaged with an epi-fluorescence microscope (Leica DMI8, camera: DFC7000T) and a laser scanning confocal microscope (Zeiss LSM780). Epi-fluorescence images were recorded after placement of the droplet networks (at t = 0 h and t = 18 h, unless stated otherwise). The same settings were used throughout this work with 5x magnification (objective: N PLAN 5x / 0.12 DRY): cascade blue dextran: λex: 327 – 383 nm, λem: 435 – 485 nm, exposure time: 600 ms, gain: 1; sfGFP: λex: 450 – 490 nm, λem: 500 – 550 nm, exposure time: 200 ms (for constitutive expression of sfGFP) or 1 s (for bacterial interference competition), gain: 1; mCherry: λex: 540 – 552 nm, λem: 567 – 643 nm, exposure time: 2 s, gain: 1. Images were composed of 1920 pixels in x-direction (xmax) and 1440 pixels in y-direction (ymax), spanning a field of view of 2.4887 mm (x-direction) by 1.8662 mm (y-direction) and were analyzed by using MATLAB R2021b. The pixel value range spanned 0 to 4095. Baseline gene expression of mCherry, lB. arising from bacterium-laden hydrogels without arabinose, was computed based on the mean of all pixel intensity values Ix,yin fluorescence images, where x is the xthpixel in x-direction and y is the ythpixel in y-direction of an image):

[0393] IB= (Σx_maxx=1Σy_maxy=1Ix,y) / (xmaxymax) = 179 a.u. (3)

[0394]

[0395] xmaxymax Accordingly, pixel intensity values >179 a.u. were considered to reflect induced gene expression of mCherry (termed activated pixels). The mean gene expression, IM, of all activated pixels in mCherry fluorescence images was computed as follows:

[0396] IM= (Σx_maxx=1Σy_maxy=1Ix,y× H(Ix,y− IB)) / Na

[0397] (4)

[0398]

[0399] where, H(z) is the Heaviside step function, defined as:

[0400] (0 if z < 0

[0401]

[0402] (5)

[0403] and where Nais the total number of activated pixels.

[0404] The total gene expression, IT, was calculated as the sum of pixel values of all activated pixels in mCherry fluorescence images:

[0405]

[0406] IT= Σx_maxx=1Σy_maxy=1Ix,y× H(Ix,y− IB), (6) using Equation (5).

[0407] The area of intended gene expression, Ai, was determined by multiplying the total number of activated pixels in cascade blue dextran (CBD) fluorescence images with the area of a single pixel, Apix(1.68 x 10-6mm2):

[0408] A

[0409]

[0410] AI= Σx_maxx=1Σy_maxy=1H(Ix,y− ICBD) × Apix, (7) where Ix,ycBDare the pixel values of cascade blue dextran fluorescence images and ICBD = 500 a.u. was set as an appropriate threshold to capture the outline of the area comprising of droplets containing cascade blue dextran and, hence, arabinose and aHL. The same step function was used (see Equation (5)).

[0411] The area of unintended gene expression, Au, was calculated by multiplying the total number of pixels outside of Ai with Apix

[0412] AU= Σx_maxx=1Σy_maxy=1[H(Ix,y− IB) × H(ICBD− Ix,y)] × Apix(8)

[0413]

[0414] The area of no gene expression, AN, was calculated by multiplying the total number of pixels within Ai with Apix

[0415]

[0416] AN= Σx_maxx=1Σy_maxy=1[H(Ix,y− IB) × H(Ix,y− ICBD)] × Apix, (9) was computed by using Equation (1).

[0417] For bacterial interference competition, PF was determined as described above, with sfGFP as the read-out for induced gene expression, as opposed to mCherry. Here, a threshold value of >200 was used, which was determined from the baseline expression of sfGFP as a consequence of colicin E7 baseline expression levels in bacteria without the presence of arabinose.

[0418] Volume -to-surface -area Ratio (RVSA)'. First, the area of intended gene expression, Ai, of a droplet network was determined by multiplying the number of cascade blue dextran pixels reaching the threshold of 500 with Apix. Based on Ai, the pixel size (mm2) and the number of droplets per layer, the cross-sectional area of a single droplet was determined (mm2). This information was used to determine the volume of a single droplet (mm3) and the volume of a droplet network based on the total number of droplets (mm3). The volume-to-surface-area ratio RVSA) was then determined as shown in Equation (2), yielding a value (mm) describing the relation between volume and area of intended gene expression, Ai.

[0419] Magnetic Handles for Guided Landing of Droplet Networks'. Magnetic handles were prepared by adding a pre-heated (60°C) solution (composition not stated by manufacturer) containing Ni- particles (MagneHis™ Ni-Particles, Promega) to pre-heated (60°C) M9 medium containing 1.5% (w / v) of ULGA at a volume ratio of 2:3. Droplets (50 - 250 pm diameter) were then ejected (FemtoJet 4x, Eppendorf) into lipid-in-oil (same composition used for printing of droplet networks) and cooled at 4°C for 35 min. The gelled droplets were transferred next to the comers of a droplet network by pipetting and pushed onto the droplet network to which they adhered through DIB formation. The droplet network was then placed on top of a bacterium-laden hydrogel, under which a magnet had been placed to direct the orientation of the droplet network.

[0420] Heat-induced Dehydration of Droplet Networks'. Droplet networks were printed in SOG cuvettes, which contained lipid-in-oil solution (500 p. L). The cuvettes were then placed in the center of a transparent heating plate (Leica Thermo Plate), which was set to 36 °C or 43 °C. The temperature in the lipid-in-oil solution was allowed to reach equilibrium as determined with a sensor (Thorlabs, TSP01). The printing solution was diluted with Milli-Q® water so that the concentrations of the components in the droplet networks would be as desired after shrinkage (36 °C or 43 °C). Once the desired decrease in volume was attained as determined by microscopy, a shrunken network was brought to room temperature for 30 min before transfer on top of the bacterium -laden hydrogel. Definitions and Calculations'.

[0421] Mean Gene Expression IM'. Mean gene expression (mCherry or sfGFP) refers to the sum of all pixel values in activated pixels divided by the total number of activated pixels, where pixels areconsidered activated when baseline expression levels are reached (see Pattern fidelity quantification by image analysis).

[0422] Total Gene Expression IT. Total gene expression (mCherry or sfGFP) refers to the sum of all pixel values in activated pixels, where pixels are considered activated when baseline expression levels are reached (see pattern fidelity quantification by image analysis).

[0423] Starting Ratio: Starting ratio refers to the ratio between cells of one over the other genotype in a bacterial community at the start of an experiment when the two genotypes were mixed homogeneously in an ULGA hydrogel solution (t = 0 h).

[0424] Bacterial competition: Bacterial competition refers to the process by which one individual decreases the survival or reproduction of others.

[0085]

[0425] Statistical Analysis'.

[0426] Epi-fluorescent microscopy images were processed according to “Pattern Fidelity Quantification by Image Analysis”, which was visualized in Figure 2(a). The data is presented as mean ± standard deviation (SD) and the number of replicates is stated in each figure caption. For analysis of statistical differences between groups it was first tested whether the data was normally distributed using the Shapiro-Wilk test (p < 0.05). Given groups were normally distributed, significance between groups was tested performing a two-sample t-test. If data of at least one group was not normally distributed, significance between groups was tested by performing a Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0427] Supplementary Note 1: Arabinose-induced Gene Expression: pBAD Rationale

[0428] We chose pBAD as an inducer system for multiple reasons: 1) The permeability of arabinose (chemical signal) was minimal (Figure 1(h)) through lipid bilayers (comprising of a lipid composition 2: 1 (molar ratio) DPhPC: POPC) formed between compartments within droplet networks (droplet interface bilayers = DIBs) and between droplet networks and the

[0429] hydrogel (droplet hydrogel bilayers = DHBs). This allowed us to control the arabinose flux from the droplet networks into the bacterium-laden hydrogels by changes in both arabinose and aHL concentration, and prevented diffusion of arabinose between compartments within the droplet networks unless aHL was present in the DIBs. 2) pBAD systems have been reported to behave as an all-or-nothing system (D. A. Siegele et al., Proc Natl Acad Sci U S A 1997, 94, 8168), which we reasoned would be ideal to achieve patterned gene expression based on arabinose flux from droplet networks. We hypothesized that below or above a critical arabinose concentration, the gene expression in cells would be low and high, respectively, rather than a gradient going from low to high. Hence, a tight population gene expression pattern could be achieved depending on the arabinose gradient released over time. This all-or-nothing system works once a criticalconcentration of arabinose is reached, resulting in high expression levels of genes downstream of the PBAD promoter, such as mCherry or cxE7. At the same time, increased activity of the transcriptional regulator AraC induces the expression of araE. which encodes for the arabinose transporter AraE. This increases the uptake of arabinose, leading to a positive feedback loop that rapidly increases protein expression in the cells until a maximum is reached.

[0430] Supplementary Note 2: Pattern Fidelity as a Measure of Gene Expression Controllability

[0431] As described, our F measure describes the controllability of gene expression in E. coli populations by comparing the area of unintended gene expression, Au, to the area of intended gene expression, Ai. Another measure we considered was factoring AN, which refers to the area within Ai, where no gene expression was induced. However, this measure was not chosen to optimize arabinose release from 3D-printed droplet networks, as we did not encounter areas within Ai where gene expression was not induced (unless droplets did not form DHBs with the hydrogel (see Figure 3(g))). Therefore, our PF measure was chosen to quantify gene expression in areas outside of the intended gene expression area as a measure over controlled release of arabinose. However, in addition we reported values of AN (Figure 2(g)), which takes into account areas within Ai where insufficient DHB formation as a consequence of printing defects and irregularities of the surface of bacterium -laden hydrogels prevented arabinose release.

[0432] By using cascade blue dextran to reveal where arabinose was released from droplet networks, we could determine the area of unintended gene expression, Au and AN (Figure 2(a)). However, in some cases, the definitive location of arabinose release was imperfect due to imaging limitations (A. Alcinesio et al, Nat Commun 2020, 11, 2105). This is because the layers of droplets in our droplet networks are stacked by shifting every other layer in both the x- and v-dircctions such that hexagonally-packed structures can form. So, droplets of the same position in x and y within the droplet networks were offset depending on the layer. Therefore, accurate determination of the droplets from which arabinose was being released into the bacterium-laden hydrogel was difficult in some cases. In particular, this may cause inaccuracies in locating fine patterns, such as singledroplet diffusive pathways (Figure 4(b)) or patterns in mask layers, where the fluorescence signal from cascade blue dextran in droplets comprising reservoir layers may interfere with the fluorescence signal from cascade blue dextran droplets in the mask layers.

[0433] Supplementary Note 3: Magnetic Beads for Guiding Tissue Placement

[0434] Our 3D droplet printing technology can be time-consuming when printing intricate patterns. This is because, 1) droplets are positioned one after another, 2) when printing droplets of different compositions (e.g. with or without arabinose and aHL), two printing nozzles are used of which theprinting stage has to be moved between the nozzle positions. For example, printing droplet networks composed of 10 x 10 x 8 droplets (in xi’z-dircction) takes 67 minutes for a cross-like pattern and 87 minutes for an arrow-like pattern. To overcome this, we reasoned that only the bottom layers (masks) in the droplet networks are necessary to be patterned, as long as this ‘mask’ layer was connected to a reservoir of arabinose and αHL-containing droplets to supply arabinose to the mask layers. Therefore, we printed cubic, arabinose-containing ‘reservoirs’ (4-8 layers) on top of the mask layers (4 layers) connecting reservoirs to the mask via aHL-mediated droplet diffusive pathways. Using this method, we reduced our printing time significantly, for example, the printing time for droplet networks composed of twelve cross-like patterned layers (9 x 9 x 12 droplets) was 56 minutes, while the printing time for droplet networks composed of 4 mask layers (9 x 9 x 4 droplets) of the same pattern and 8 reservoir layers (7 x 7 x 8) on top was 41 minutes. Moreover, this method stored arabinose more efficiently within the droplet network in terms of occupied volume, as 8 reservoir layers were composed of a total of 392 aHL and cascade blue dextran-containing droplets as opposed to 136 in 8 patterned layers.

[0435] To avoid reservoir droplets from accidentally rolling to the patterned bottom layer of droplet networks during printing and, hence, disrupting intended patterns, reservoirs were designed to be slightly smaller than bottom layers (e.g. 6 x 6 droplets (reservoir) instead of 8 x 8 droplets (mask)). As a result, we found that droplet networks predominantly flipped before they landed on top of the bacterium -laden hydrogel (i.e. the reservoir interfaced with the hydrogel instead of the mask layer). Out of 45 droplet networks composed of patterned masks and a reservoir, 88.9% flipped during the transfer and landed with the reservoir facing the hydrogel. In this case, arabinose would diffuse directly from the reservoir to the cells, resulting in gene expression patterns reflecting the shape of the reservoir, rather than the intended pattern). This occurred likely due to hydrodynamic forces in the viscous lipid-in-oil solution, aligning the smaller reservoir towards the hydrogel. To overcome the flipping, we developed a mechanism to control the landing of droplet networks on top of the hydrogel. This comprised of attaching droplets containing magnetic beads (1.5% w / v ultra-low gelling agarose and nickel magnetic beads) to the comers of the mask layers before the transfer and then placing a magnet underneath the bacterium-laden hydrogel to guide the mask layer towards the hydrogel. Using this method, the correct landing was achieved in 76.5% of 34 transferred droplet networks, allowing for patterned gene expression in bacterial cells by the mask layers rather than the reservoir layers. Next, we investigated the number of mask layers that were necessary to consistently induce patterned gene expression. We printed droplet networks composed of 1, 2, 3 and 4-layered masks comprising a stripe-like pattern and an 8-layered reservoir on top (33 mM arabinose and 50 pg mU1aHU). Accordingly, 4 connected mask layers were necessary to induce stripe-like gene expression in bacterial cells (Figure 3(d)-Figure 3(g)). Finally, we compared gene expression patterns induced by arabinose release from droplet networks with and withoutreservoirs. Droplet networks with reservoirs consisted of 4-layered masks encoding for a singledroplet pathway and 4-layered reservoirs on top, whereas droplet networks without reservoirs consisted of 8-layered masks encoding for a single -droplet pathway. Using both types of droplet networks (with and without reservoirs), single-droplet gene expression patterns could be induced. Further, we found that there was no significant difference between droplet network type regarding PF and mean mCherry expression, confirming that droplet networks composed of masks and reservoirs are not only more efficient in terms of printing time and occupied space but also induced gene expression at similar spatial resolution compared to droplet networks comprised completely of mask layers.

[0436] The work leading to this invention has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation Programme (Horizon 2014-2020) / ERC grant agreement n° 833792.

Claims

CLAIMS1. A process for reducing the size of droplets in a synthetic droplet assembly,wherein the synthetic droplet assembly comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,and wherein the process comprises removing water from the aqueous medium of droplets in the droplet assembly.

2. A process according to claim 1 wherein removing water from the aqueous medium of droplets in the droplet assembly comprises evaporating said water.

3. A process according to claim 2 wherein evaporating said water comprises heating the droplet assembly.

4. A process according to claim 3 wherein heating the droplet assembly comprises exposing the droplet assembly to a temperature of at least 30 °C,optionally wherein heating the droplet assembly comprises exposing the droplet assembly to a temperature of from 30 °C to 90 °C, preferably from 30 °C to 70 °C, more preferably to a temperature of from 30 °C to 50 °C, for instance to a temperature of from 35 °C to 45 °C.

5. A process according to claim 4 wherein heating the droplet assembly comprises exposing the droplet assembly to said temperature for a duration of at least 5 minutes, optionally for a duration of at least 20 minutes, or for a duration of at least 1 hour, for instance for a duration of from 1 to 4 hours.

6. A process according to any one of the preceding claims wherein the droplet assembly is in a hydrophobic medium, preferably wherein the hydrophobic medium is an oil.

7. A process according to claim 6 wherein removing water from the aqueous medium of droplets in the droplet assembly comprises evaporating said water, wherein evaporating said water comprises heating the droplet assembly by heating the hydrophobic medium, optionally wherein the hydrophobic medium is heated to a temperature as defined in claim 4 and optionally for a duration as defined in claim 5.

8. A process according to claim 1 wherein removing water from the aqueous medium of droplets in the droplet assembly comprises removing said water by osmosis.

9. A process according to claim 8 wherein removing said water by osmosis comprises:contacting the droplet assembly with an external aqueous medium, wherein the external aqueous medium has an osmolarity which is greater than the osmolarity of the aqueous medium in the droplets, optionally wherein said contacting comprises (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has an osmolarity which is greater than the osmolarity of the aqueous medium of the droplets, and (iii) maintaining contact between the droplet assembly and the external aqueous medium.

10. A process according to claim 8 wherein the aqueous medium in the droplets of the droplet assembly has a first osmolarity, and removing said water by osmosis comprises:contacting the droplet assembly with an external aqueous medium which has a second osmolarity, wherein the second osmolarity is greater than the first osmolarity.optionally wherein said contacting comprises (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has said second osmolarity, and (iii) maintaining contact between the droplet assembly and the external aqueous medium.

11. A process according to claim 10 wherein the first osmolarity is from 0 osm to 7 osm and the second osmolarity is from 0.001 osm to 7.001 osm.

12. A process according to any one of claims 9 to 11 wherein removing said water by osmosis comprises contacting the droplet assembly with said external aqueous medium for a duration of at least 0.5 h and up to 7 h.

13. A process according to any one of the preceding claims wherein removing water from the aqueous medium of droplets in the droplet assembly reduces the mean diameter of the droplets in the droplet assembly from a first droplet diameter to a second droplet diameter, wherein:the first droplet diameter is at least 10 µm, optionally at least 60 µm; and / orthe second droplet diameter is less than 60 µm, optionally less than 10 µm, for instance less than 5 µm.

14. A process according to any one of the preceding claims wherein removing water from the aqueous medium of droplets in the droplet assembly reduces the mean volume of the droplets in the droplet assembly from a first droplet volume to a second droplet volume, optionally wherein:the first droplet volume is at least 0.5 picolitres (pL), optionally at least 113 pL; and / or the second droplet volume is less than 113 pL, optionally less than 0.5 pL, for instance less than 65 femtolitres (fL).

15. A process according to any one of the preceding claims wherein removing water from the aqueous medium of droplets in the droplet assembly reduces the volume of the droplet assembly from a first droplet assembly volume to a second droplet assembly volume, wherein the second droplet assembly volume is from 0.5 % to 99% of the first droplet assembly volume, optionally from 1 % to 70 % of the first droplet assembly volume, for instance from 2 % to 50 % of the first droplet assembly volume, or from 5 % to 40 % of the first droplet assembly volume, for instance from 10 % to 40 % of the first droplet assembly volume.

16. A process for increasing the size of droplets in a synthetic droplet assembly, wherein the synthetic droplet assembly comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets,and wherein the process comprises adding water to the aqueous medium of droplets in the droplet assembly.

17. A process according to claim 16 wherein adding water to the aqueous medium of droplets in the droplet assembly comprises adding said water by osmosis.

18. A process according to claim 17 wherein adding said water by osmosis comprises contacting the droplet assembly with an external aqueous medium, wherein the external aqueous medium has an osmolarity which is less than the osmolarity of the aqueous medium in the droplets,optionally wherein said contacting comprises (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has said osmolarity which is less than the osmolarity of the aqueous medium of the droplets, and (iii) maintaining contact between the droplet assembly and the external aqueous medium.

19. A process according to claim 18 wherein the aqueous medium in the droplets of the droplet assembly has a first osmolarity, and adding said water by osmosis comprises contacting the droplet assembly with an external aqueous medium which has a second osmolarity, wherein the second osmolarity is less than the first osmolarity,optionally wherein said contacting comprises: (i) bringing the droplet assembly into contact with an external aqueous medium, (ii) adjusting the osmolarity of the external aqueous medium so that the external aqueous medium has said second osmolarity, and (iii) maintaining contact between the droplet assembly and the external aqueous medium,optionally wherein the first osmolarity is from 0.001 osm to 7.001 osm and the second osmolarity is from 0 osm to 7 osm.

20. A process according to claim 18 or claim 19 wherein adding said water by osmosis comprises contacting the droplet assembly with said external aqueous medium for a duration of at least 0.5 hours and up to 7 hours.

21. A process according to any one of claims 16 to 20 wherein adding water to the aqueous medium of droplets in the droplet assembly increases the mean diameter of the droplets in the droplet assembly from a first droplet diameter to a second droplet diameter, wherein:the first droplet diameter is less than 60 µm, optionally less than 10 µm, for instance less than 5 µm; and / orthe second droplet diameter is at least 10 µm, optionally at least 60 µm.

22. A process according to any one of claims 16 to 21 wherein adding water to the aqueous medium of droplets in the droplet assembly increases the mean volume of the droplets in the droplet assembly from a first droplet volume to a second droplet volume, wherein:the first droplet volume is less than 113 pL, optionally less than 0.5 pL, for instance less than 65 femtolitres (fL); and / orthe second droplet volume is at least 0.5 picolitres (pL), optionally at least 113 pL.

23. A process according to any one of claims 16 to 22 wherein adding water to the aqueous medium of droplets in the droplet assembly increases the volume of the droplet assembly from a first droplet assembly volume to a second droplet assembly volume wherein the second droplet assembly volume is greater than the first droplet assembly volume, wherein the second droplet assembly volume is at least double the first droplet assembly volume,optionally wherein the second droplet assembly volume is 2 to 50 times the first droplet assembly volume, for instance from 3 to 20 times the first droplet assembly volume, or from 5 to 10 times the first droplet assembly volume.

24. A synthetic droplet assembly which comprises a plurality of droplets, wherein each of said droplets comprises (i) an aqueous medium, and (ii) an outer layer of amphipathic molecules around the surface of the aqueous medium, and each of said droplets contacts another of said droplets to form a layer of said amphipathic molecules as an interface between the contacting droplets, wherein:• the mean diameter of the droplets in the droplet assembly is less than 10 µm; and / or• the mean volume of the droplets in the droplet assembly is less than 0.5 pL.

25. A synthetic droplet assembly according to claim 24 wherein the number of droplets in the droplet assembly is at least 64 and:• the mean diameter of the droplets in the droplet assembly is less than 5 µm; and / or• the mean volume of the droplets in the droplet assembly is less than 65 femtolitres (fL).

Images