A system for encapsulation of single protoplasts within a hydrogel coating

Abstract

A system for encapsulation of protoplasts within a hydrogel coating is described The system comprises reservoir for receiving a source of protoplasts, a microdroplet generator and a crosslinking vessel. The reservoir is configured to receive a source of protoplasts in aqueous suspension, wherein the aqueous suspension comprises a hydrogel precursor, and the reservoir is in fluid communication with the microdroplet generator. The microdroplet generator is configured to generate a plurality of water in oil microdroplets, each microdroplet comprising not more than a single plant protoplast, and each microdroplet further comprising a surfactant coating. The crosslinking vessel comprises first and second liquids that are immiscible, wherein the first liquid is hydrophobic and has a density greater than water and further comprises an amount of a demulsifier, and the second liquid is substantially aqueous and comprises a hydrogel gelation agent. The microdroplet generator is in fluid communication with the crosslinking vessel and delivers the plurality of microdroplets into the first liquid phase.The amount of demulsifier comprised within the first liquid is sufficient to induce an emulsion break proximate to a phase interface between the first and second liquids, such that buoyancy of the plurality of microdroplets facilitates migration of the microdroplets into the second liquid whereupon the exposure to the hydrogel gelation agent results in formation of a plurality of encapsulated protoplasts within a hydrogel coating. Methods for generating encapsulated protoplasts are further described.

Description

[0001] A SYSTEM FOR ENCAPSULATION OF SINGLE PROTOPLASTS WITHIN A HYDROGEL COATING

[0002] Field of the Invention

[0003] The present invention relates to the field of plant engineering, specifically to methods, systems, and apparatus for modifying, selecting, and propagating plant material. In particular, the invention addresses challenges in achieving high single-cell encapsulation efficiency in droplet microfluidics while ensuring the viability and regeneration of single cell protoplasts.

[0004] Background of the Invention

[0005] Droplet-based microfluidics involves the generation of microscale droplets that encapsulate small liquid volumes within immiscible carrier fluids. The technology enables the compartmentalisation of individual components within encapsulated droplets, allowing for precise analysis. Its ability to isolate and study components in discrete droplets has led to significant advancements in fields such as single-cell analysis, drug discovery, synthetic biology, and tissue engineering. However, its application has been predominantly developed for mammalian cells, bacteria, and yeast.

[0006] Several systems rely on the droplet generation process inside the microfluidic chip to separate individual protoplasts. Yu et al. (2018) demonstrate that a single plant protoplast can be encapsulated in aqueous droplets. However, the cell survival rate in this system is low and, as such, the subsequent recovery of viable plants is highly inefficient or even impossible. Grasso et al. (2016) propose a method of encapsulating individual plant cells in hydrogel; however, this method results in a high mortality rate for cells, as it involves the application of high heat for crosslinking the encapsulation.

[0007] Another method is provided in WO2020 / 212713 which describes a system for propagating plant material, in which the protoplasts are injected to a droplet generator, which generates droplets that enclose either a single protoplast or no protoplast by gellifing the encapsulation medium of the droplet. However, this method is directed towards on-chip droplet generation and gellification / crosslinking occurs directly within a channel comprised within a microfluidic device.

[0008] “Microfluidic fabrication of shape-tunable alginate microgels: Effect of size and impact velocity”, (Carbohydrate Polymers 120 (2015) 38-45) describes a capillary-based microfluidic platform for the fabrication of non-spherical sodium alginate microgels wherein sodium alginate droplets are crosslinked off-chip in a mixture of barium acetate and glycerol solution. Morphologies such as teardrop, lamp-like, mushroom-like, double-dimpled and bowl-like microgels are disclosed by controlling the size, impact velocity (at the crosslinking solution / oil interface), and concentration of sodium alginate solution.“Microbead encapsulation of living plant protoplasts: a new tool for the handling of single plant cells” (Applications in Plant Sciences 20164(5): 1500140) discloses wherein living protoplasts obtained from BY-2 tobacco suspension cultures are continuously incorporated into a stream of agarose microdroplets, collected in cooled mineral oil as gelled microbeads, and then transferred into liquid MS medium for culture.

[0009] Combined with downstream processes such as hydrogel formation, droplet sorting or further analysis, which compromises the integrity of droplets, the viability of encapsulated protoplasts is typically reduced in many known methods. Hence, the encapsulation efficiency for the plant protoplast, such as for ‘on-chip’ droplet generation methods described above is limited. Encapsulation efficiency for plant cells is inherently low due to the tendency of plant protoplasts to form clusters, making it harder to ensure each droplet contains only one protoplast. Additionally, the viability of encapsulated protoplasts is often compromised due to shear stresses and mechanical damage during droplet generation, further hindering the survival and recovery of viable protoplasts.

[0010] Therefore, there is a need for a system that efficiently generates a single encapsulated protoplast that is durable, suitable for scalable downstream process such as growing and recovering viable protoplasts. In particular, there is a need for a system that efficiently generates sufficient amounts of single encapsulated protoplasts that are compatible with various downstream applications including high-throughput processes.

[0011] These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

[0012] Summary of the invention

[0013] In a first aspect, the invention provides a system for encapsulation of protoplasts within a hydrogel coating, the system comprising: a reservoir for receiving a source of protoplasts, a microdroplet generator; and a crosslinking vessel. In particular, the reservoir of the system is configured to receive a source of protoplasts in aqueous suspension, the aqueous suspension comprising a hydrogel precursor, and wherein the reservoir is in fluid communication with the microdroplet generator. The microdroplet generator of the system is configured to generate a plurality of water in oil microdroplets, each microdroplet comprising not more than a single plant protoplast, and each microdroplet further comprising a surfactant coating. The crosslinking vessel of the system comprises first and second liquids that are immiscible, wherein the first liquid is hydrophobic and has a density greater than water and further comprises an amount of a demulsifier, and the second liquid is substantially aqueous and comprises a hydrogel gelation agent. The microdroplet generator of the system is in fluid communication with the crosslinking vessel and delivers the plurality of microdroplets into the first liquid phase, and wherein the amount of demulsifier comprised within the first liquid is sufficient to induce an emulsion break proximate to a phase interface between the first and second liquids, such that buoyancy of theplurality of microdroplets facilitates migration of the microdroplets into the second liquid whereupon the exposure to the hydrogel gelation agent results in formation of a plurality of encapsulated protoplasts within a crosslinked hydrogel coating.

[0014] In embodiments, the system additionally comprises a dispersed phase stabiliser region. In further embodiments, the dispersed phase stabiliser region comprises a compression - expansion array, wherein the compression-expansion array comprises a plurality of alternating microfluidic compression and expansion chambers, and optionally wherein the diameter of an expansion chamber is at least 100 pm, at least 250 pm, or at least 400 pm, at most 500 pm, at most 300 pm, or at most 150 pm.

[0015] In embodiments, the microdroplet generator of the system may comprise a plurality of interconnected channels defined by sidewalls, wherein the sidewalls have a radius of curvature greater than zero.

[0016] In embodiments, the system may additionally comprise an outlet that is configured to deliver microdroplets from the microdroplet generator into the first liquid.

[0017] In embodiments, the surfactant coating of the system comprises a perfluoropolyether, polypropylene oxide or polyethylene oxide. In further embodiments, the surfactant coating may comprise PFPE(Perfluoropolyethers)-b-PPO(Polypropylene Oxide)-PEO(Polyethylene Oxide)-PPO-b-PFPE, or hexane, 3-ethoxy-1 ,1 ,1 ,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-.

[0018] In embodiments, the first liquid of the system may comprise a fluorinated oil. In some embodiments, the first liquid may additionally comprise at least one of: FC40 , HFE7500, HFE7100. In embodiments, the first liquid has a density greater than the second liquid, or a density greater than the hydrogel precursor of the microdroplet.

[0019] In embodiments, the demulsifier of the system may comprise a fluorinated alcohol. In some embodiments, the demulsifier comprises at least one of: 1H,1H,2H,2H-Perfluoro-1-octanol or 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluoro-1-octanol.

[0020] Advantageously, in some embodiments, the emulsion break comprises removal of a surfactant coating of a microdroplet followed by direct contact of the hydrogel precursor to the first liquid. In embodiments, the emulsion break occurs without coalescence or flocculation between the microdroplets within the first liquid.

[0021] In embodiments, the hydrogel gelation agent comprises a divalent cation, optionally selected from at least one of: Ca2+, Ba2+, Mg2+, Sr2+, or Co2+, or also Cu2+, Fe2+or Mn2+. The hydrogel gelation agent may promote crosslinking and network formation of the hydrogel precursor molecules. In embodiments, the hydrogel comprises at least one of: an alginate, chitosan, collagen, cellulose derivatives, polyethylene glycol diacrylate (PEGDA), poly vinyl alcohol (PVA), agarose, hyaluronic acid, or gelatin methacryloyl (GelMA).In some embodiments, the diameter of the microdroplet comprising a single protoplast is at least 80pm, at least 110pm, or at least 140pm. In embodiments, the diameter of the microdroplet comprising a single protoplast is at most 150pm, at most 130pm, or at most 100pm.

[0022] Advantageously, in embodiments, the protoplast encapsulated within a hydrogel coating remains viable and fully contained within said hydrogel coating after formation of a micro-callus stage. In embodiments, each of the plurality of encapsulated single protoplasts assumes a teardrop shape.

[0023] In another aspect, a method of generating a plurality of encapsulated protoplasts is provided. The method comprises providing a dispersed phase of protoplasts comprising the protoplasts suspended in an aqueous hydrogel precursor, forming a plurality of microdroplets, wherein each microdroplet comprises not more than a single protoplast encapsulated by aqueous hydrogel precursor, and establishing a surfactant layer around the plurality of microdroplets. The method further comprises providing a fluid dual phase composition comprising a hydrophobic first liquid that has a density greater than water and further comprises an amount of a demulsifier, and a second liquid that is substantially aqueous and comprises a hydrogel gelation agent. The method also comprises the step of introducing the microdroplets to the first liquid, wherein the amount of demulsifier comprised within the first liquid is sufficient to induce an emulsion break proximate to a phase interface between the first and second liquids, such that the buoyancy of the microdroplets without the surfactants facilitates migration of the microdroplets into the second liquid, and forming a plurality of encapsulated protoplasts within a hydrogel coating, wherein the hydrogel coating is formed by exposure of the aqueous hydrogel precursor to the hydrogel gelation agent of the second liquid.

[0024] Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0025] Brief Description of the Drawings

[0026] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0027] Figure 1 is a schematic diagram of a system for encapsulation of protoplasts within a hydrogel coating comprising a reservoir for receiving a source of protoplasts, a microdroplet generator, and a crosslinking vessel.

[0028] Figure 2 is a schematic diagram of an embodiment of a microdroplet generator. The embodiment comprises an inlet and a channel with successive compression-expansion chambers. The design of the microdroplet generator further comprises an inlet for the carrier oil and an outlet for the microdroplets.Figure 3 is a diagram of an example of a microdroplet generator in fluid communication with the crosslinking vessel. In this example, the crosslinking vessel is in the form of 6 well plate. The microdroplet generators is in fluid communication to the first liquid in the crosslinking vessel, to deliver the plurality of microdroplets into the first liquid phase.

[0029] Figure 4 is a schematic diagram of a microdroplet in the crosslinking vessel undergoing emulsion break. The emulsion break process for each microdroplet involves removal of a surfactant coating upon contact with a demulsifier, such that the buoyancy of a microdroplets facilitates migration of the microdroplets into the second liquid. Upon contact with the second liquid, hydrogen gelation agent of the second liquid gellifies / crosslinks the hydrogel precursor of the microdroplets.

[0030] Figure 5 is a microscopic image of a microdroplet formed from a hydrogel precursor, and microdroplets with crossed hydrogel coating generated from the crosslinking vessel. The microdroplets are characterised by a teardrop shape.

[0031] Figure 6 is a microscopic image of a compression-expansion chamber, microdroplets formed from a microdroplet generator comprising a hydrogel precursor each encapsulating single protoplasts, and microdroplets with crossed hydrogel coating generated from the crosslinking vessel each encapsulating single protoplasts.

[0032] Figure 7 is a microscopic image of a microcalli grown from protoplasts. The top row shows microcalli cultivated from protoplasts that are single encapsulated by an alginate hydrogel using the system of an embodiment of the present invention, following several weeks of cell divisions and growth of microcalli from protoplasts. Alginate hydrogel embedded protoplasts without single cell encapsulation (positive control) are on the bottom row. Microcalli formation demonstrated by the single encapsulated protoplasts is in coherence with the microcalli formed in the positive control.

[0033] Figure 8 is a microscopic image of a failed alternative off-chip hydrogel gelation strategy involving administration of an organic acid. Left : Off-chip hydrogel microdroplets without protoplasts. Non-uniform particle size, some degree of aggregation and random coalescence is observed during Ca2+-EDTA and Acetic acid interaction at pH=7.0. Right : Off-chip hydrogel with protoplasts. This is an example of a failed off-chip gelation. Ca2+-EDTA and acetic acid interaction on microparticles encapsulating protoplasts results in significant clustering.

[0034] Figure 9 is a microscopic image of microdroplets produced from the microdroplet generator and a graph summarising microdroplet characterisation. Left- Computer vision-based detection of liquid droplet boundaries. Right: Quantification of key droplet metrics, including average droplet size (120 pm ± 15 pm), average volume (0.9 nL ± 0.12 nL), coefficient of variation (CV), and the total number of droplets detected across all analysed images. The x-axis represents droplet size (pm), while the y-axis indicates the number of occurrences. CV reflects droplet uniformity, with each dot corresponding to a single droplet size detected by the computer vision script.

[0035] Figure 10 is a graph depicting a total number of counted droplets per image when passed through analysis of encapsulation efficiency (left), a graph depicting mean droplet size depicted per image in micrometres (middle), and mean volume of a droplet in nanolitres (right). Each data point in the left a middle image represent the metrics from each image.

[0036] Figure 11 is a graph depicting an expected encapsulation efficiency and an observed encapsulation efficiency of the present invention. Top : Poisson statistics for different values of A (average number of cells per droplet). The maximum single cell encapsulation efficiency achievable without the dispersed phase stabiliser is 28%. Bottom : The experimental observed encapsulation efficiency with the dispersed phase stabiliser. 34% of all crosslinked hydrogel particles encapsulated single protoplast.

[0037] Detailed Description of the Invention

[0038] Although the invention will be described by way of examples, it will be appreciated by a person skilled in the art that the invention could be modified to take many alternative forms without departing from the spirit and scope of the invention as defined in the appended claims.

[0039] All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] According to the invention, there is provided a system comprising a microdroplet generator configured to generate a plurality of water in oil microdroplets, each microdroplet comprising not more than a single plant protoplast, and a crosslinking vessel configured to generate a plurality of encapsulated protoplasts within a hydrogel coating. In the crosslinking vessel, facilitated by buoyant forces on the microdroplets, the plurality of microdroplets migrate to a liquid comprising a hydrogel gelation agent.

[0041] Figure 1 shows schematic diagram of an embodiment of the invention which comprises a system for encapsulation of protoplasts within a hydrogel coating comprising a reservoir for receiving a source of protoplasts, a microdroplet generator, and a crosslinking vessel.

[0042] As used herein, the term “plant protoplasts” (also referred to simply as “protoplasts”, throughout this disclosure) refers to a plant cell that has had its cell wall completely or partially removed. Removal of cell wall can be affected by mechanical, chemical or enzymatic means. In embodiments, protoplasts are obtained from suitable plant material using cell wall digestive enzymes. For example, enzymes such as cellulase, macerozyme, pectinase, hemicellulase, pectolyase, driselase, xylanase and combinations thereof may be suitable for use in the context of the invention. In embodiments, cellulase may be used at a concentration of 1 w% - 1 ,5w%. In embodiments, macerozyme may be used at a concentration of0.2w% - 0.4w%. In embodiments, hemicellulase may be used at a concentration of 2w% - 5w%. In embodiments, pectolyase may be used at a concentration 0.01w% - 0.5w%. In embodiments, driselase may be used at a concentration of 0.5w% - 2w%. Protocols for obtaining protoplasts from plant tissues are known in the art and will not be discussed further here. In embodiments, suitable plant cell material may include root tissue, leaf mesophyll and / or cultured callus. In embodiments, the protoplasts are obtained using the protocol described in Yoo, Cho, & Sheen (2007), which is incorporated herein by reference.

[0043] The term "microdroplet" as used herein refers to the discrete droplets formed from the system of the present invention. In an aspect of an invention, the microdroplet is formed by a microdroplet generator configured to generate a plurality of water in oil microdroplets. A volume of a microdroplet may be at least 100 pl, at least 500 pl, at least 1 nl, at least 10 nl, or at least 100 nl. A volume of a microdroplet may be at most 250 nl, at most 50 nl, at most 5 nl, at most 750 pl, or at most 250 pl.

[0044] In an aspect of an invention, the microdroplet comprise no more than a single plant protoplast encapsulated by an aqueous suspension of a hydrogel precursor, each microdroplet further comprising a surfactant coating.

[0045] As used herein, the term “encapsulation” relates to the process of forming or enclosing a droplet in which the protoplast is protected. In aspects of the invention, the droplet is a microdroplet. One advantage of encapsulation is that it protects a protoplast by forming a layer of protecting medium around the cells that acts like an “artificial cell wall”. As a result, the cell viability of the protoplasts is increased and the efficiency of whole plant recovery from these protoplasts is improved.

[0046] As used herein, the term “single cell encapsulation” refers to the process of encapsulating a single cell. In aspects of the invention, the single cell encapsulation involves encapsulation of a single protoplast. Single cell encapsulation may involve encapsulating cells that are initially randomly dispersed in the aqueous suspension, followed by a droplet formation process. Single cell encapsulation is usually a random event as the distribution of the single cell prior to the droplet formation process is random. The probability of multiple cells entering the same droplet, while dependent on cell concentration and droplet volume, follows random distribution principles. The number of cells and beads inside each droplet can be estimated using the Poisson distribution, in which the probability P(X=x) of finding x cells (or beads) per droplet is given by the equation P(X=x) = e-A[Ax / X!] with A representing the mean number of cells (or beads) in the volume of each droplet. As a result, droplet occupancy can be tuned by changing the cell density in the aqueous phase.

[0047] One benefit of a single cell encapsulation is that it allows separation and independent analysis of each cell, or protoplasts, encapsulated. Each protoplast remains enclosed and protected inside the droplets throughout downstream process such as analytical and sorting steps, which classifies each protoplastaccording to its characteristics. If necessary, encapsulated protoplast can be lysed and intracellular biomolecules assayed.

[0048] Following the encapsulation process, the single encapsulated protoplasts may be manually or automatically placed into a tissue culture system, for example on an agar gel plate containing plant growth media or into a micro well plate containing gel and liquid with plant growth promoting media. In embodiments, the single encapsulated protoplasts can be cultured to generate microcalli (Figure 7), which are small, undifferentiated cell clusters that arise from plant tissue cultures in vitro. In embodiments, a callus inducing media may be used. For example, media such as Gamborg B5, Murashige Skoog or others may be used. As explained above, salts, vitamins and auxins, cytokinins and / or other hormones promoting growth of the single protoplast into a callus may be included in the medium, or the tissue culture plate.

[0049] In embodiments, calli having achieved a predetermined size may be moved into plant growth media with different auxins and cytokinins ratio to induce shoot formation. In embodiments, calli having undergone shoot formation may be moved to yet another plant growth medium to induce root formation. Typically, any manipulation of the calli may be done under sterile conditions. These microcalli can then further regenerate whole plantlets.

[0050] The term "aqueous suspension" encompasses any aqueous-based suspension relevant to the formation of a microdroplet. In an embodiment, aqueous suspensions may include water-based dispersions, colloidal suspensions, and particulate suspensions in aqueous media. In embodiments, the aqueous suspension may comprise one of the followings: sodium alginate; agar; agarose; agar substitute; and gellan. In embodiments, the aqueous suspension may be viscous, wherein the aqueous suspension has high resistance to deformation when subjected to shear stress. In an aspect of the invention, the aqueous suspension comprises a hydrogel precursor. In embodiments, a hydrogel precursor comprises dispersed hydrophilic polymer chains remaining in a liquid state without chemically or physically connected in a network. In embodiments, the aqueous suspension comprising the hydrogel precursor may further comprise protoplasts in a dispersed phase. In embodiments, the hydrogel precursor comprises at least one of: an alginate, chitosan, collagen, cellulose derivatives, polyethylene glycol diacrylate (PEGDA), poly vinyl alcohol (PVA), agarose, hyaluronic acid, or gelatin methacryloyl (GelMA). In embodiments, the aqueous suspension is osmotically balanced, optionally comprising mannitol, sorbitol, or polyethylene glycol (PEG).

[0051] Single protoplast encapsulation process faces multiple challenges. Primarily, the encapsulation efficiency for plant cells is inherently low due to damaged protoplasts often forming clusters, preventing analysis in a single cell scale. In some cases, stress triggers the release of chloroplasts and cytoplasmic fluid into the surrounding hydrogel, leading to the formation of complex aggregates with previously encapsulated cells. This aggregation prevents effective single-cell protoplast encapsulation, ultimately reducing overall encapsulation efficiency.Another challenge is that inherently low viability of the protoplast, due to low stress tolerance, low structural stability, and high osmotic sensitivity of the protoplast, reduces the chance for successful propagation of the plant material throughout further processes including sorting, separation, microcalli growth, and selection, which further reduces the number of viable protoplasts. Therefore, there is a need for a single protoplast encapsulation system that minimises the damage or impact on the cells and allows the encapsulate protoplasts to exhibit high viability metrics, such as cell wall regeneration and division to a micro-callus or callus.

[0052] Additionally, single-cell encapsulation from the cells suspended in an aqueous suspension poses a significant challenge due to the difficulty of controlling viscous liquids for uniform droplet generation and consistent encapsulation. The inherent resistance of viscous liquids to deformation makes it harder to manipulate the hydrogel suspension in droplet-generation systems.

[0053] Advantageously, the system of the present invention comprises an improved microdroplet generator that increase single protoplast encapsulation efficiency by facilitating control of the microdroplets by manipulating inertial lift. In embodiments, the microdroplet generator of the present invention comprises a dispersed phase stabiliser that utilise inertial lift to control the fluid flow, and the distance between each microdroplets. Advantageously, inertial lift achieves cell ordering by the balance of inertial lift forces and shear-induced forces within a microchannel. As cells flow through confined channels, these forces guide them into equilibrium positions, leading to their self-arrangement into well-defined streamlines.

[0054] In an aspect of the invention, a system for encapsulation of protoplasts within a hydrogel coating comprises a reservoir for receiving a source of protoplasts, and a microdroplet generator. The reservoir is configured to receive a source of protoplasts in aqueous suspension, and the reservoir is in fluid communication with the microdroplet generator. In embodiments, the reservoir is directly connected to the microdroplet generator with a continuous channel. In embodiments, the microdroplet generator may be operably connected to the reservoir with an inlet assembly configured to introduce the protoplast in aqueous suspension into the microdroplet generator. The inlet assembly may further comprise one or more fluid flow rate controlling elements optionally comprising a pressure-regulated source. In embodiments, the reservoir is in one-way fluid communication to the microdroplet generator, wherein the protoplasts in aqueous suspension are moved from the reservoir to the microdroplet generator.

[0055] In embodiments, the microdroplet generator may be configured to receive a carrier fluid, such as an oil phase fluid. In further embodiments, the microdroplet generator may be operably connected to an oil phase fluid reservoir with an inlet assembly configured to introduce the oil phase fluid into the microdroplet generator. The inlet assembly may further comprise one or more fluid flow rate controlling elements optionally comprising a pressure-regulated source. In embodiments, the oil phase fluidreservoir is in one-way fluid communication to the microdroplet generator, wherein the oil phase fluid is moved from the reservoir to the microdroplet generator.

[0056] In an aspect of the invention, the microdroplet generator produces a plurality of microdroplets in a form of water-in-oil emulsion. In various embodiments, microdroplet generator may comprise a droplet formation region. As used herein, an “emulsion” refers to a two-phase system, wherein liquid droplets are suspended in another liquid wherein the two liquids are immiscible, meaning that they do not dissolve within another. The microdroplets in a form of water-in-oil emulsion are in “dispersed phase” meaning that the microdroplets are distributed throughout oil in an immiscible manner.

[0057] In embodiments, the droplet formation region is configured to provide protoplasts in aqueous suspension to an immiscible fluid. In embodiments, the immiscible fluid is an oil phase fluid. In embodiments, the droplet formation region may be configured to provide protoplasts in aqueous suspension in discrete amounts to the oil phase fluid that is moving in a steady, continuous manner. In embodiments, the immiscible oil phase fluid serves as a continuous medium that surrounds the microdroplets. In embodiments, the droplet formation region may comprise a structure wherein the inlet assembly configured to introduce the protoplasts in aqueous suspension and the inlet assembly configured to introduce the oil phase fluid are configured to form a junction. Alternatively, the inlet assemblies may be configured so that the fluids to be hydrodynamically focused into a single collection channel.

[0058] In embodiments, the microdroplet generator comprises a dispersed phase stabiliser region. In further embodiments, the dispersed phase stabiliser region comprises a compression - expansion array (Figure 2). In embodiments, the compression-expansion array comprises a plurality of alternating microfluidic compression and expansion chambers, wherein there are at least 5, at least 10, at least 50, or at least 100 compression-expansion chambers. In some embodiments, there are at most 300, at most 150, at most 75, and at most 25 compression-expansion chambers. The diameter of the expansion chamber may be at least 100 pm, at least 300 pm, or at least 500pm. In embodiments, the diameter of the expansion chamber may be at most 800 pm, at most 600 pm, or at most 400 pm. The width of the compression chamber may be at least 50 pm, at least 100 pm, or at least 200 pm. The width of the compression chamber may be at most 300 pm, at most 200 pm, or at most 75pm. In embodiments, the dispersed phase stabiliser region is configured to generate differences in fluid velocity and pressure along the parts of the dispersed phase stabiliser regions. In further embodiments, the fluid velocity of the compression chamber is higher than the fluid velocity in the expansion chamber. In further embodiments, the fluid flow in the expansion chamber separates, wherein some fluid recirculates along the walls of the expansion chamber and some progresses to the adjacent compression chamber. In embodiments, the microdroplets subject to the fluid flow in the expansion chamber experiences drag and / or inertial lift. In further embodiments, the microdroplets subject to the fluid flow in the expansion chamber increase the distance between the droplets. In embodiments, the distance is at least 3x, at least 5x, or at least 10x of the diameter of the microdroplet in spherical form. In embodiments, themicrodroplets subject to the fluid flow in the compression-expansion chamber reduces the variation of the distance between each microdroplets, optionally wherein the coefficient of variation is at most 10%, at most 5% or at most 3%. Overall, the microdroplets generated by the flow characteristic of compression-expansion chamber may increase efficiency in single protoplast encapsulation.

[0059] In embodiments of the present invention, a microdroplet generator may comprise a plurality of interconnected channels defined by sidewalls, wherein the sidewalls have a radius of curvature greater than zero. In embodiments, the angle of the curvature may be at least 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80° or 90°, typically 10° to 45°. An angle of curvature as described reduces the occurrence of sharp corners or edges which can cause damage to protoplasts passing through the channels.

[0060] In a microdroplet generator, the size of the microdroplets may be determined by the rate of flow (fluid velocity) of the protoplasts in aqueous suspension and that of the oil phase fluid. The contact of the protoplast in aqueous suspension and the oil phase fluid creates an interfacial tension between the two fluids. Higher interfacial tension between the two phases may lead to formation of large droplets. According to the embodiments of the invention, an average diameter of the microdroplet is at least 70 pm, at least 80 pm, at least 100 pm, or typically at least 120 pm. In embodiments, the average diameter of the microdroplet is at most 200 pm, at most 175 pm, or at most 160 pm, typically at most 150 pm. Advantageously, in the present invention, use of a dispersed phase stabiliser, a compression-expansion chamber, provides microdroplets with uniform size by providing a consistent shear force, flow rate, and interfacial tension throughout the microdroplet generator. In embodiments, the standard deviation of the microdroplet diameter is at least 1pm, at least 5pm, or at least 10pm. In embodiment, the standard deviation of the microdroplet diameter is at least 20 pm, at least 7pm, or at least 2pm.

[0061] In an aspect of the invention, the oil suspension comprises surfactant molecules configured to provide surfactant coating following the generation of microdroplets. The term “surfactant coating” as used herein refers to a monolayer of amphiphilic molecules adsorbed onto a microdroplet surface. The hydrophilic heads of the surfactant molecules are oriented towards the aqueous phase, while the hydrophobic tails interact with the hydrophobic phase. In an aspect of the invention, the microdroplet is a water-in-oil microdroplet, and the hydrophilic heads of the surfactant molecules orient inward toward the water core, while the hydrophobic tails extend outward into the surrounding oil phase. In further embodiments, the hydrophilic heads form hydrogen bonds with the water molecules. The surfactant molecules effectively reduce interfacial tension of the suspended liquid microdroplets and the surrounding fluid. This way, use of the aqueous suspension comprising surfactant molecules stabilises the structure of the microdroplets. In embodiments, reduction of interfacial tension may promote the formation of smaller and uniformly sized droplets. In further embodiments, the surfactant coating on the microdroplets prevents coalescence of the dispersed microdroplets within the solution. In embodiments, the surfactant coating on the microdroplets prevents coalescence of the microdroplets within the microdroplet generator. In embodiments of the present invention, the surfactant coating comprises a perfluoropolyether, polypropylene oxide or polyethylene oxide. In further embodiments, the surfactantcoating comprises PFPE(Perfluoropolyethers)-b-PPO(Polypropylene Oxide)-PEO(Polyethylene Oxide)-PPO-b-PFPE or Hexane, 3-ethoxy-1 ,1 ,1 ,2,3,4,4,5,5,6,6,6-dodecafluoro-(2-trifluoromethyl)-. A surfactant can be added to the oil solution in concentrations at about 1%, 3%, 5%, 10%, 15%, 20%, about 25% v / v or in the range of any value in between.

[0062] According to the invention, the system for encapsulation of protoplasts within a hydrogel coating further comprises a crosslinking vessel comprising first and second liquids that are immiscible, wherein the first liquid is hydrophobic and has a density greater than water and further comprises an amount of a demulsifier, and the second liquid is substantially aqueous and comprises a hydrogel gelation agent. In embodiments, the second liquid comprises an osmolarity adjusted buffer. In embodiments, the crosslinking vessel may have a flat bottom. In embodiments, the crosslinking vessel may comprise a 6 well plate (Figure 3). In embodiments, the volume of the first and second liquid combined may be at least 4ml. In embodiments, the volume of the first and second liquid combined may be at most 15ml, at most 10ml, or at most 6ml.

[0063] In embodiments, the first liquid comprises at least one of: a fluorinated oil, wherein the fluorinated oil comprises at least one of: FC40, HFE7500, or HFE7100. In embodiments, the first liquid of the crosslinking vessel has a density greater than the second liquid. In embodiments, the first liquid has a density greater than the hydrogel precursor of the microdroplet. In embodiments, the fluorinated oil has a density of at least 1 ,4g / mL, at least 1 ,6g / mL, or at least 1 ,8g / mL. In embodiments, the fluorinated oil has a density of at least 1.9g / mL, at most 1.7g / mL, or at most 1.5g / mL. Fluorinated oils are characterised with highly electronegative fluorine atoms that weaken van der Waals forces. Fluorinated oils are therefore immiscible with both water and hydrocarbons, forming a third "flourous" phase.

[0064] The term “demulsifier” in the context of the invention is a chemical that separates or breaks emulsion. Demulsifier disrupts an emulsion by destabilising the forces that keep the emulsion stable. For instance, in an emulsion wherein microdroplets are stabilised by an electronic repulsion, demulsifiers may work by neutralising surface charges of the microdroplets. Some demulsifiers destabilise an emulsion by disrupting a surfactant or polymeric layer around microdroplets. In an embodiment, the demulsifier is an oil-based demulsifier, wherein the oil-based demulsifier comprises a solvent base and demulsifiers. In various embodiments, the demulsifier of the first liquid comprises a fluorinated alcohol. In an embodiment, a highly localised electron charge of a fluorine atom of the fluorinated alcohol may disrupt the surfactant layer of the microdroplet. In an embodiment, the fluorine atom may disrupt hydrogen bonding between the hydrophilic heads of the surfactant and water molecules. In some embodiments, the demulsifier comprises at least one of: 1H,1H,2H,2H-Perfluoro-1-octanol or 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8,8,8-Tridecafluoro-1 -octanol. In embodiments, the demulsifier is at least 10%, at least 20%, or at least 40% v / v of the first solution. In embodiments, the demulsifier is at most 45%, at most 35%, at most 15% or at most 5% v / v of the first solution. In preferred embodiments, the demulsifier is around 20-30% v / v of the first solution.The term “hydrogel gelation” as used herein is a process in which a hydrogel precursor transitions from a liquid state to a solid or semi-solid gel state. Prior to the gelation, the hydrogel precursor polymer chains are weakly associated and mobile in an aqueous medium.

[0065] The hydrogel gelation process involves crosslinking of polymer chains, which forms a three-dimensional network capable of retaining water. In specific embodiments, the hydrogel is comprised of an alginate.

[0066] Alginic acid and its alkaline metal salts (alginates) are linear polysaccharides of p-D-mannuronate (M) and a-L-guluronate (G) residues arranged in homopolymeric blocks. The M / G sequence varies based on biological source and environmental conditions, affecting alginate properties. Some bacteria (Azotobacter, Pseudomonas) also produce alginates as biofilms. Alginate is water soluble and forms gels in the presence of various divalent cations which act as cross-linkers for polymer chains. Ca2+ions are most commonly used to prepare alginate gels. The metal ions are coordinated by the G blocks due to the1C4 conformation of the G units and the ability of the paired G units to generate molecular cavities in the shape of an “egg-box” that are capable of accommodating ions. Other divalent alkaline-earth ions such as Ba2+, Mg2+and Sr2+have been shown to form alginate gels with stronger material properties. Alginate gels of the type described herein may be used in any of the embodiments of the present invention.

[0067] The term “hydrogel gelation agent” in the context of the invention is an agent that promotes or triggers hydrogel gelation process. In some embodiments, the hydrogel- hydrogel gelation agent interaction involves non-covalent interaction, such as alginate - calcium ion. In embodiments, the hydrogel gelation agent may promote hydrogen bonding to crosslink the hydrogels. In embodiments, the hydrogel gelation agent comprises a divalent cation, such as at least one of: Ca2+, Cu2+, Ba2+, Mg2+, Mn2+, Sr2+, Zn2+, Fe2+or Co2+. In embodiments, the concentration of the hydrogel gelation agent is at least 10mM, at least 50mM, at least 100mM, or at least 300mM. In embodiments, the concentration of the hydrogel gelation agent is at most 500mM, at most 250mM, at most 75mM, or at least 25mM. Typically, the hydrogel gelation agent comprises Ca2+. In other embodiments, the hydrogel-hydrogel gelation agent interaction may involve additional covalent crosslinking, for example, UV-activated acrylate groups in a polyethylene glycol hydrogel.

[0068] Another significant challenge in single-protoplast encapsulation using hydrogel is maintaining protoplast viability during the gelation process. Grasso et al (2016) developed a hydrogel gelation method based on heat-responsive crosslinking of hydrogen however, this method significantly compromises protoplast viability, as protoplasts lack plant wall and are therefore highly sensitive to heat. Similarly, Chen et al (2022) demonstrated a UV-assisted hydrogel gelation method, which also negatively impacts protoplast viability. Other approaches have relied on ion transfer between Ca-EDTA and acetic acid for hydrogel gelation. This method involved a hydrogel gelation agent, Ca2+, chelated with EDTA and dissolved in the hydrogel precursor, while acetic acid was incorporated into the continuous oil phase. The hydrogel precursor crosslinking process required significant exposure time for acetic acid to interact with Ca-EDTA. However, for protoplast encapsulation — where plant cells already lack a protective cell wall —this exposure significantly reduced cell survival. Even brief contact with acetic acid led to decreased protoplast viability. Shao et al. explored an alternative approach, replacing EDTA with nitrilotriacetic acid (NTA), a different chelating agent known to reduce viability stress on protoplasts. However, cells encapsulated using Ca-NTA showed no indications of viability, such as cell division.

[0069] The inventors had also experimented an off-chip gelation method that involves an alginate-based hydrogel precursor comprising Ca-EDTA, and continuous oil phase initially without acetic acid. After collecting sufficient droplets off-chip, acetic acid was added to the collection vial to trigger gelation. However, this approach had two major drawbacks. First, the gelation time varied across droplets due to slight differences in size, leading to inconsistent crosslinking. As a result, hydrogel beads fused into large, inseparable clusters with varying degrees of gelation, making it impossible to encapsulate individual plant cells in distinct hydrogel beads (Figure 8). Second, extending the crosslinking time by allowing the hydrogel solution to interact with the oil phase (containing surfactant and acetic acid) severely impacted cell viability. Prolonged exposure to chemical stress reduced survival rates, and the few surviving cells failed to divide or form callus in later stages.

[0070] Therefore, there is a need for a single protoplast encapsulation and gelation method that ensures protoplast viability and regeneration with high encapsulation efficiency.

[0071] The invention advantageously provides higher encapsulation efficiency for single protoplasts. The system of the invention demonstrates much higher encapsulation efficiency compared to theoretically predicted encapsulation efficiency (Example 11).

[0072] The invention also advantageously provides a buoyancy-based gelation method that does not expose the microdroplets to chemical environments which would lower the viability of the protoplasts. Further, the buoyancy-based gelation method retains the microdroplets encapsulating single protoplasts without coalescence or collapse.

[0073] In an aspect of the invention, the microdroplet generator is in fluid communication with the crosslinking vessel and delivers the plurality of microdroplets into the first liquid phase, and wherein the amount of demulsifier comprised within the first liquid is sufficient to induce an emulsion break proximate to a phase interface between the first and second liquids, such that buoyancy of the plurality of microdroplets facilitates migration of the microdroplets into the second liquid whereupon the exposure to the hydrogel gelation agent results in formation of a plurality of encapsulated protoplasts within a hydrogel coating (Figure 6).

[0074] In embodiments, the microdroplet generator comprises a microdroplet outlet. In embodiments, the outlet is configured to facilitate the controlled release of microdroplets to the first liquid phase of the crosslinking vessel. In embodiments, the outlet may comprise microvalves or nozzles. In embodiments, the outlet tip may be placed within the first liquid.In an aspect of the invention, an emulsion break (or demulsification) involves removal of a surfactant coating of a microdroplet followed by demulsifiers, followed by migration of the microdroplets to the phase interface between the first and second liquids (Figure 4). In an aspect, an emulsion break involves an emulsion break due to buoyancy, wherein the microdroplets migrate from the first to the second liquid. In embodiments, the migration is driven by the difference in density between the microdroplets and the first liquid. In embodiments, the emulsion break occurs without coalescence or flocculation between the microdroplets within the first liquid. In embodiments, the emulsion break of each microdroplet occurs in a serial manner, wherein some microdroplets have their surfactant coating removed and migrate to the second liquid before other microdroplets.

[0075] In some embodiments, the hydrogel coating of the microdroplet is in a teardrop shape (Figure 5). In embodiments, the teardrop shape comprises a round shape on one end and a conical shape at the opposite end. In embodiments, the microdroplet in teardrop shape comprises a single encapsulated protoplast. In embodiments, the round shape portion of the teardrop shape is formed following the migration of the microdroplets to the phase interface between the first and the second liquid. At the phase interface, the surface of the microdroplets is partially exposed to the hydrogel gelation agent of the second liquid, leading to crosslinking of the hydrogel polymer (gelation) and hydrogel coating formation. The surface of the microdroplets without any exposure to the hydrogel gelation agent remains aqueous. Further migration of the microdroplets from the phase interface to the second liquid against the gravity generates a drag of the aqueous portion of the microdroplet, leading to a conical shape.

[0076] The invention is further described with reference to non-limiting examples which follow.

[0077] Examples

[0078] Single cell encapsulation

[0079] 2 ml of aqueous cell suspension with 106 / ml density is taken into a separate vial. Cell suspension is mixed in 1 :1 of 2.4wt% solution of Alginate - Mannitol solution. Mixture is gently mixed to reach a homogeneous state. By use of an appropriate pressurised chamber with 0.45mm internal diameter tubing, cell mixture is brought to the microfluidic chip. The chip contains two inlets, one for the continuous phase (0.6 vol% Bio-Rad surfactant solution in HFE7500, or 0.5 vol% Emulseo Fluo-Surf-O in HFE7500) and second for the cell suspension. Outlet tubing is plugged in and microdroplet formation is carried out. Different pressure range values are adjusted. The microdroplet generated may be collected.

[0080] Microdroplet characterisation

[0081] Microdroplets were characterised by observing and quantifying the liquid droplet boundaries, mean diameter of each droplet, standard deviation (STD) and coefficient of variance (CV) of the diameter, mean volume of each droplet, and the number of droplets observed.Microdroplets were collected from the microdroplet generator in a small vessel. Following the collection, the droplets are loaded in a cavity glass slide covered with a cover glass slides prior to imaging.

[0082] An in-house MATLAB script was developed to detect droplet boundaries using the built-in functions “strel” and “imfindcircles” for efficient identification and overlay of circular regions on a colour image. First, the RGB image was converted to greyscale. The “strel” function was then applied to create a structuring element for morphological operations, which enhanced circular features by smoothing or filling gaps, along with the application of a smoothing filter. The processed image was subsequently converted into a binary format, and the “imfindcircles” function was executed to detect circular regions. To quantify size, a standard length reference was used for length-to-pixel conversion, and the detected circles were overlaid on the original colour image. The computed circle dimensions were scaled back to real-world measurements using the conversion factor, and the resulting data was visualised in a bubble chart. Additionally, statistical parameters such as mean, standard deviation, coefficient of variation, and volume were computed and incorporated into the bubble chart, as illustrated in Figure 9.

[0083] 15-30 images of microdroplets in hydrogel precursors produced from the microdroplet generator hare analysed using the method above (Figure 9, Left). The average droplet size is calculated to be 120um ± 15 urn, and average volume 0.9 nL ± 0.12 nL (Figure 9, Right).

[0084] Predicting encapsulation efficiency

[0085] A random distribution (Poisson distribution) was used to describe the probability distribution of microdroplets that encapsulate a single protoplast, produced from a microdroplet generator without dispersed phase stabiliser. In the equation below, P(k) represents the probability of a droplet containing k cells, k is calculated as 1 in this case. A, the average number of cells per droplet, is determined by multiplying the average droplet volume by the cell density introduced into the aqueous phase inlet of the microfluidic chip.

[0086]

[0087] The prediction gives maximum of single cell encapsulation efficiency achievable in a conventional microdroplet maker considerably lower than 10%. If a conventional drop-maker is used with a conventional droplet generation density (e.g. 200,000 cells per mL), the expected droplet size is approximately 105 pm, corresponding to a volume of 0.61 nL. Probability statistics indicate that, considering a significantly broad cell size distribution of protoplasts ranging from 20 pm to 60 pm, the probability of obtaining single-cell encapsulation in a droplet of 105 pm at a density of 200,000 cells per mL is considerably lower than 10%. This is mainly due to cell clustering and tendency of protoplasts to form doublets and triplets.The prediction is based on the average microdroplet size of 120um ± 15 um, and average volume 0.9 nL ± 0.12 nL of the present system, maximum of single cell encapsulation efficiency achievable in the present system achievable as 28% (Figure 11 , top).

[0088] Experimental encapsulation efficiency quantification of the present system

[0089] To experimentally assess the encapsulation efficiency of the present system that comprises a dispersed phase stabiliser, the number of microdroplets that contain a single protoplast generated from a microdroplet generator of the present system, are counted (Figure 10). Based on these observations, 34% of all observed microdroplets encapsulate single protoplast (Figure 11 , bottom).

[0090] Therefore, the encapsulation efficiency of the present system is significantly higher than those of the routine off-chip gelation method and is higher than the predicted level of encapsulation efficiency based on Poisson distribution.

[0091] Normally, the experimental encapsulation efficiency is usually low or at max equal to the statistical prediction. Therefore, the invention provides an unexpected technical effect that the single protoplast encapsulation higher than the predicted level of encapsulation efficiency based on Poisson distribution.

[0092] Buoyancy based hydrogel gelation (crosslinking) process

[0093] Each well is filled with 2ml of 25 vol% solution of Emulsion break solution (first liquid) at the bottom and a 4ml layer of CaCl2-2H20-Mannitol solution (second solution) is added on the top. Droplet collection tubing end is submerged into the first liquid, once a droplet surrounded by the surfactant oil leaves the tubing and enters the chamber, the surfactant molecules are replaced with the molecules of the first liquid and as the droplet is hydrogel it starts to float (if there is no E-break and droplet are collected in a well, droplets will float on the surface of the oil as oil is more dense than the droplets). As soon as the droplet reaches the oil-water interface instead of floating droplet is pulled into the aqueous phase as the surfactant is no longer present to reduce the interfacial tension. Moreover, as the water phase is Ca solution, hydrogel starts to crosslink instantaneously. As soon as a small region of the hydrogel is crosslinked remaining hydrogel forms a cone shape at the bottom and a tear-drop shaped crosslinked hydrogel is transferred from the water-in-oil emulsion to freely floating hydrogel particle.

[0094] The complete transition time for a plant cell from the liquid culture and alginate mix solution to the microfluidic chip and back into the culture with a hydrogel cladding takes less than 3 minutes. Moreover, no EDTA mixture (with 7.0 pH) and acid environment is present to increase cell mortality.

[0095] References

[0096] Yu Z, Boehm, C R, Hibber J M, Abell C, Haseloff J, Burgess S J & Reyna-Llorens I, Droplet-based microfluidic analysis and screening of single plant cells. bioRxiv, 8 Oct 2017 (doi: https: / / doi.org / 10.1101 / 199992).Yoo, S.-D., Cho, Y.-H., & Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols, 2, 1565. Retrieved from https: / / doi.org / 10.1038 / nprot.2007.199

[0097] Grasso, M.S. and Lintilhac, P.M., 2016. Microbead encapsulation of living plant protoplasts: A new tool for the handling of single plant cells. Applications in Plant Sciences, 4(5), p.1500140

[0098] Chen, M., Aluunmani, R., Bolognesi, G. and Vlad isavlje vic, G.T., 2022. Facile microfluidic fabrication of biocompatible hydrogel microspheres in a novel microfluidic device. Molecules, 27(13), p.4013

[0099] Shao, F., Yu, L., Zhang, Y., An, C., Zhang, H., Zhang, Y., Xiong, Y. and Wang, H., 2020. Microfluidic encapsulation of single cells by alginate microgels using a trigger-gellified strategy. Frontiers in Bioengineering and Biotechnology, 8, p.583065

Claims

Claims:

1. A system for encapsulation of protoplasts within a hydrogel coating, the system comprising:(a) a reservoir for receiving a source of protoplasts;(b) a microdroplet generator; and(c) a crosslinking vessel,wherein the reservoir is configured to receive a source of protoplasts in aqueous suspension, the aqueous suspension comprising a hydrogel precursor, and wherein the reservoir is in fluid communication with the microdroplet generator,the microdroplet generator being configured to generate a plurality of water in oil microdroplets, each microdroplet comprising not more than a single plant protoplast, and each microdroplet further comprising a surfactant coating,the crosslinking vessel comprising first and second liquids that are immiscible, wherein the first liquid is hydrophobic and has a density greater than water and further comprises an amount of a demulsifier, and the second liquid is substantially aqueous and comprises a hydrogel gelation agent, wherein the microdroplet generator is in fluid communication with the crosslinking vessel and delivers the plurality of microdroplets into the first liquid phase, and wherein the amount of demulsifier comprised within the first liquid is sufficient to induce an emulsion break proximate to a phase interface between the first and second liquids, such that buoyancy of the plurality of microdroplets facilitates migration of the microdroplets into the second liquid whereupon the exposure to the hydrogel gelation agent results in formation of a plurality of encapsulated protoplasts within a hydrogel coating.

2. A system of claim 1 , wherein the microdroplet generator comprises a dispersed phase stabiliser region.

3. A system of claim 2, wherein the dispersed phase stabiliser region comprises a compression - expansion array.

4. A system of claim 3, wherein the compression-expansion array comprises a plurality of alternating microfluidic compression and expansion chambers, wherein the diameter of an expansion chamber is at least 100 pm, at least 250 pm, or at least 400 pm, at most 500 pm, at most 300 pm, or at most 150 pm.

5. A system of claim 3, wherein the width of a compression chamber is at least 50 pm or at least 100 pm, at most 200 pm or at most 75 pm.

6. A system of any preceding claim, wherein the microdroplet generator comprises a plurality of interconnected channels defined by sidewalls, wherein the sidewalls have a radius of curvature greater than zero.

7. A system of any preceding claim, wherein the system further comprises an outlet that is configured to deliver microdroplets from the microdroplet generator into the first liquid.

8. A system of any preceding claim, wherein the surfactant coating comprises a perfluoropolyether, polypropylene oxide or polyethylene oxide.

9. A system of claim 7, wherein the surfactant coating comprises PFPE(Perfluoropolyethers)-b- PPO(Polypropylene Oxide)-PEO(Polyethylene Oxide)-PPO-b-PFPE.

10. A system of any preceding claim, wherein the surfactant coating comprises Hexane, 3-ethoxy- 1 ,1 ,1 ,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl.

11. A system of any preceding claim, wherein the first liquid comprises at least one of: a fluorinated oil or a hydrocarbon oil.

12. A system of claim 10, wherein the fluorinated oil comprises at least one of: FC40 , HFE7500, HFE7100.

13. A system of claim 10, wherein the hydrocarbon oil comprises at least one of: Mineral Oil , Silicone Oil, Vegetable Oil, Hexadecane.

14. A system of any preceding claim, wherein the first liquid has a density greater than the second liquid.

15. A system of any preceding claim, wherein the first liquid has a density greater than the hydrogel precursor of the microdroplet.

16. A system of any preceding claim, wherein the demulsifier comprises a fluorinated alcohol.

17. A system of any preceding claim, wherein the demulsifier comprises at least one of:1H,1H,2H,2H-Perfluoro-1-octanol or 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluoro-1-octanol.

18. A system of any preceding claim, wherein the emulsion break comprises removal of a surfactant coating of a microdroplet followed by direct contact of the hydrogel precursor to the first liquid.

19. A system of any preceding claim, wherein the emulsion break occurs without coalescence or flocculation between the microdroplets within the first liquid.

20. A system of any preceding claim, wherein the second liquid comprises an osmolarity adjusted buffer.

21. A system of any preceding claim, wherein the hydrogel gelation agent comprises a divalent cation, optionally at least one of: Ca2+, Cu2+, Ba2+, Mg2+, Mn2+, Sr2+, Zn2+, Fe2+or Co2+.

22. A system of claim 21 , wherein the divalent cation is Ca2+at a concentration in the second liquid of at least 10mM, at least 50mM, at least 10OmM, or at least 300mM.

23. A system of claim 21 , wherein the divalent cation is Ca2+at concentration in the second liquid of at most 500mM, at most 250mM, at most 75mM, or at most 25mM.

24. A system of any preceding claim, wherein the hydrogel gelation agent promotes crosslinking and network formation of the hydrogel precursor molecules.

25. A system of any preceding claim, wherein the hydrogel comprises at least one of: an alginate, chitosan, collagen, cellulose derivatives, polyethylene glycol diacrylate (PEGDA), poly vinyl alcohol (PVA), agarose, hyaluronic acid, or gelatin methacryloyl (GelMA).

26. A system of any preceding claim, wherein the diameter of the microdroplet comprising a single protoplast is at least 80pm, at least 110pm, or at least 140pm.

27. A system of any preceding claim, wherein the diameter of the microdroplet comprising a single protoplast is at most 150pm, at most 130pm, or at least 100pm.

28. A system of any preceding claim, wherein the protoplast encapsulated within a hydrogel coating remains viable and fully contained within said hydrogel coating after formation of a micro-callus stage.

29. A system of any preceding claim, wherein each of the plurality of encapsulated single protoplasts is in a teardrop shape.

30. A method of generating a plurality of encapsulated protoplasts, the method comprising: providing a dispersed phase of protoplasts comprising the protoplasts suspended in an aqueous hydrogel precursor;forming a plurality of microdroplets, wherein each microdroplet comprises not more than a single protoplast encapsulated by aqueous hydrogel precursor;establishing a surfactant layer around the plurality of microdroplets;providing a fluid dual phase composition comprising a hydrophobic first liquid that has a density greater than water and further comprises an amount of a demulsifier, and a second liquid that is substantially aqueous and comprises a hydrogel gelation agent;introducing the microdroplets to the first liquid, wherein the amount of demulsifier comprised within the first liquid is sufficient to induce an emulsion break proximate to a phase interface between the first and second liquids, such that the buoyancy of the microdroplets without the surfactants facilitates migration of the microdroplets into the second liquid; andforming a plurality of encapsulated protoplasts within a hydrogel coating, wherein the hydrogel coating is formed by exposure of the aqueous hydrogel precursor to the hydrogel gelation agent of the second liquid.

31. A method of claim 29, wherein the method further comprises:separating a plurality of encapsulated single protoplasts within a hydrogel coating from the first and the second liquid by centrifugation.

32. A method of claim 29 or 30, wherein the number of single protoplasts encapsulated within a hydrogel coating is at least 20%, at least 25%, or at least 30% of the number of single protoplasts encapsulated by aqueous hydrogel precursor.

33. A method of claim 29, wherein a plurality of microdroplets is formed by providing the dispersed phase protoplasts to a continuous-flow fluid, wherein the dispersed phase and the continuous flow fluid are immiscible.

34. A method of any of claims 29 to 32, wherein the surfactant coating comprises a perfluoropolyether, polypropylene oxide or polyethylene oxide.

35. A method of claim 33, wherein the surfactant coating comprises PFPE(Perfluoropolyethers)- b-PPO(Polypropylene Oxide)-PEO(Polyethylene Oxide)-PPO-b-PFPE.

36. A method of any of claims 29 to 34, wherein the surfactant coating comprises Hexane, 3- ethoxy-1 ,1 ,1 ,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl.

37. A method of any of claims 29 to 35, wherein the first liquid comprises a fluorinated oil or a hydrocarbon oil.

38. A method of claim 36, wherein the fluorinated oil comprises at least one of: FC40 , HFE7500, HFE7100.

39. A method of claim 36, wherein the hydrocarbon oil comprises at least one of: mineral oil , silicone oil, vegetable oil, or hexadecane.

40. A method of any of claims 27 to 34, wherein the first liquid has a density greater than the hydrogel precursor of the microdroplet.

41. A method of any of claims 27 to 35, wherein the demulsifier comprises a fluorinated alcohol.

42. A method of any of claims 27 to 36, wherein the demulsifier comprises 1 H,1 H,2H,2H- Perfluoro-1 -octanol or 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluoro-1-octanol.

43. A method of any of claims 27 to 37, wherein the emulsion break comprises removal of a surfactant coating of a microdroplet followed by direct contact of the hydrogel precursor to the first liquid.

44. A method of any of claims 27 to 38, wherein the emulsion break occurs without coalescence or flocculation between the microdroplets within the first liquid.

45. A method of any of claims 27 to 39, wherein the second liquid comprises an osmolarity adjusted buffer.

46. A method of any of claims 27 to 40, wherein the hydrogel gelation agent comprises Ca2+, Cu2+, Ba2+, Mg2+, Mn2+, Sr2+, Zn2+, Fe2+or Co2+.

47. A method of any of claims 27 to 41 , wherein the concentration of Ca2+in the second liquid is at least 10mM, at least 50mM, at least 100mM, or at least 300mM.

48. A method of any of claims 27 to 42, wherein the concentration of Ca2+in the second liquid is agent is at most 500mM, at most 250mM, at most 75mM, or at least 25mM.

49. A method of any of claims 27 to 43, wherein the hydrogel gelation agent promotes crosslinking and network formation of the hydrogel precursor molecules.

50. A method of any of claims 27 to 44, wherein the hydrogel comprises at least one of: an alginate, chitosan, collagen, or cellulose derivatives, polyethylene glycol diacrylate (PEGDA), poly vinyl alcohol (PVA), agarose, hyaluronic acid, or gelatin methacryloyl (GelMA).

51. The system of any preceding claim, wherein the diameter of the microdroplet comprising a single protoplast is at least 80pm, at least 110pm, or at least 140pm.

52. The system of any preceding claim, wherein the diameter of the microdroplet comprising a single protoplast is at most 150pm, at most 130pm, or at most 100pm.

53. A method of any of claims 27 to 47, wherein the protoplast encapsulated within a hydrogel coating remains viable and fully contained within said hydrogel coating after formation of a micro-callus stage.

54. A method of any of claims 27 to 48, wherein each plurality of encapsulated single protoplasts is in a teardrop shape.

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