Published on 26 December 2024
Compartmentalization is a vital aspect of living cells to orchestrate intracellular processes. Similarly, constructing dynamic and responsive sub-compartments is key to synthetic cell engineering. In recent years, liquid-liquid phase separation (LLPS) via coacervation has offered an innovative avenue for creating membraneless organelles (MOs) within artificial cells. Along with other disordered proteins, elastin-like polypeptides (ELPs), are starting to play an important role in engineering synthetic cells, to design and program ELP-based membraneless organelles (EMOs) within cell-mimicking containers, and to achieve sub-compartmentalization. Thus, showing a triggerable and reversible phase separation of ELP-based coacervates in cell-sized containers can be highly useful to form compartmentalized synthetic cell containers.
Coacervation is a phase separation process in which a homogeneous liquid solution containing solute molecules (polymers or multivalent molecules) separates into two co-existing phases: a dense, solute-rich phase (the coacervate) and a dilute, solute-poor phase. This phenomenon can be triggered by modulating the properties of the solutes (electrostatic charges, polarity, solubility…) or environmental changes (temperature, pH..).
Using a lab-on-a-chip approach and applying water-in-oil-in-water double emulsions (DEs) as synthetic cell models, the aim of this work is to achieve reversible EMO formation and dissolution upon external environmental triggers (such as oscillatory osmotic imbalance) and allow controlled long-term visualization of the resulting dynamics (using a dial-a-wave (DAW) junction together with microfluidic traps).
The particularity of this works resides in the reversibility of ELP coacervation to form dynamic cell organelle models. Owning to their responsiveness to multiple triggers (pH, temperature, salt, and protein concentration), the designed pH-responsive ELPs were used to form condensates (Figure 3a): the variant PRE-h-46 ((GXGVP)60, with X = V/F/E/H [7:8:4:1]; transition pH of 5.1 at 21 °C, and transition temperature of 58°C at pH 7.4; the letters correspond to the amino-acid sequence). The approach aimed to utilize their reversible LLPS behavior to form dynamic and responsive EMOs within DEs functioning as synthetic cell containers.
Figure 3b shows the concept pictorially:
This EMO formation-dissolution cycle can be repeated on demand to compartmentalize the vesicle interior as per the need.
To obtain efficient encapsulation of the ELPs in DEs in a high-throughput manner, DEs were produced in a lab-on-a-chip setting.
Figure 3c shows the second junction of a typical production (also see Supplementary Movies 1 and 2): the inner aqueous containing ELPs (cy3-labeled ELP in this case to facilitate fluorescence visualization) is pinched off at the first junction (not shown) to first form single emulsions which subsequently get transferred into the outer aqueous buffer to form DEs.
The device operates at a production rate between 100–500 Hz, producing approximately 10⁶ DEs in a typical 1-hour production run. The formed DEs demonstrate excellent encapsulation of the inner aqueous phase, as evidenced by the high fluorescent intensity within the DEs and a very low background signal (Fig. 3d). The produced DE population is also monodispersed, with Fig. 3e showing the size distribution of DEs from a single batch, having a mean diameter of 33.8 µm ± 1.92 µm (mean ± standard deviation; coefficient of variation <6%; n = 767). The DEs are collected and stored at 4 °C, where they remain stable for several weeks.
[VIDEO 1] Bright-field video showing on-chip high-throughput DE production.
[VIDEO 2] Fluorescence video showing efficient encapsulation of ELPs during on-chip DE production. The inner aqueous phase contained 25 µM PRE-h-46 (with 4 mol% cy3-PRE-h-46 for fluorescence visualization).
After demonstrating the EMO cycle in bulk settings, the team focused on using an on-chip system that allows for prolonged visualization of DEs and effective triggering of multiple cycles without disturbing the DEs. The idea of the device is sketched in Fig. 4a, and it consists of two modules.
The DAW junction is a microfluidic junction that has two feeding ports to allow for fast switching between the two inputs (see Fig. 4b). These two channels meet at a junction bifurcating into three outlets. The middle outlet feeds into the trap arrays, while the outer two outlets function as waste channels and allow the flow in the middle outlet to vary between carrying one of the two inputs, without getting any backflow into the inactive inlet.
Figure 4 b – Working principle of the DAW junction. The top image depicts a ‘switch-off’ trigger when higher pressure is applied at inlet 2 (PBS), while the bottom image depicts the “switch-on” trigger allowing the feed from inlet 1 (containing 10 µM FITC in PBS) to flow into the main channel containing the traps.
The trapping chip with DAW junction was operated using three solutions:
The microfluidic device is first filled with OA. Then, the loading inlet is connected to a XXS microfluidic reservoir containing the DEs. The DEs are then loaded into the chip by applying 20 and 80 mbar pressures on inlet 1 and loading inlet, respectively. The pressure from inlet 1 is for avoiding the backflow from the DAW junction when loading the DEs. After 10 min of loading, the loading inlet pressure was switched off and the inlet 1 pressure was increased to 50 mbar for washing away the extra DEs. Finally, the tubing connecting inlet 2, now filled with OA, was replaced by a new one for the hypertonic feed solution.
For EMO formation, the pressures applied on inlets 1 and 2 were 10 and 40 mbar, respectively. For EMO dissolution, the pressures were 40 and 10 mbar on inlet 1 and inlet 2 respectively.
Downstream of the DAW junction, a trap array was designed with the traps scaled to accommodate DEs of 40 µm in size (Fig. 4c). Each trap featured a narrow 10 µm opening at the rear, allowing the external solution to pass through while retaining the trapped DEs. This narrow gap at the tail of the U-shaped trap facilitated the flow of DEs into the trap, where they became confined. Once a DE was trapped, it significantly increased the flow resistance within the trap, restricting fluid flow across it.
The idea is as follows: A hypertonic external solution results in an efflux of water from the DEs to balance the osmolarity, and as a consequence, increases the salt and ELP concentration in their lumen. Since the increased concentration of salts (such as phosphates and sodium chloride) and ELPs both lower the transition temperature of ELPs, this triggers the liquid-liquid phase separation (LLPS) process, leading to EMO formation in the DE population, with each vesicle containing a single EMO. Conversely, placing the DEs back into an isotonic environment results in water influx, lowering the salt and ELP concentration, leading to the dissolution of the EMO.
Two feeding channels meet at a junction bifurcating into three outlets. The middle outlet feeds into the trap arrays, while the outer two outlets function as waste channels and allow the flow in the middle outlet to vary between carrying one of the two inputs, without getting any backflow into the inactive inlet. As can be seen in Fig. 4b, the DAW junction is able to provide a switch on/off function by simply adjusting the pressure ratio of the two feeding ports.
[VIDEO 3] Proof-of-principle DAW junction in action: A fluorescent solution (10 µM fluorescein isothiocyanate (FITC) in phosphate buffer saline (PBS)) and a non-fluorescent solution (PBS) was used for the demo.
DAW junction was operated at low pressures (<60 mbar) with a switch time in the order of seconds. Downstream the DAW junction, we designed a trap array to trap the DEs on chip. Figure 3c shows a typical trapping sequence of DEs. Due to the narrow gap at the tail of the U-shaped trap, DEs flow in and get trapped in it. Trapped DEs significantly increase the flow resistance within the trap and restrict the flow across it (see Figure 4d-f). As a result, filled traps redirect the fluid flow to the gaps between the traps and avoid further interaction with the incoming DEs. The DEs thus get localized in separate traps or simply exit the chip.
As demonstrated in static conditions, the reversible LLPS was further conducted in the microfluidic trap array.
In Figure 5a, the red bordered panels depict the dissolved states of EMOs, whereas the blue bordered panels illustrate their condensed states. The decrease in the size of the DEs, as observed in the bright-field panel, occurred after introducing the hypertonic solution, owing to the osmotic imbalance. Concurrently, the emergence of dense, dark spots, colocalizing with intensely fluorescence areas, confirmed the EMO formation.
[VIDEO 4] Time-lapse of a single trapped DE demonstrating EMO multiple formation-dissolution cycles.
Figure 5 a – EMO formation-dissolution cycles within trapped DEs. Time-lapse of bright-field and fluorescence images showing the dynamic process of EMO formation and dissolution in response to repeated hyperosmotic shocks within a trapped DE.
The dynamic changes in the intensity standard deviation (ISD) values were observed for multiple triggers across 4.5 h (Fig. 5b). Each switch-on operation (introduction of the hypertonic solution) led to a concomitant increase in ISD, signifying a local redistribution in the form of coacervation of ELP molecules within the DEs. Conversely, the switch-off operation (introduction of the isotonic solution) resulted in a reduction of ISD, attributed to the dissolution of EMOs. Regardless of the long- or short-time intervals between the triggers, we observed similar EMO formation-dissolution process and its manifestation into ISD values, indicating the stability of the DEs and the on-chip manipulation system.
This successful execution of seven continuous cycles within 4.5 h confirms the stability of DEs within the chip and the precise control over EMO dynamics.
Here, a lab-on-a-chip system is presented to reversibly trigger peptide-based coacervates within cell-mimicking confinements. Double-emulsion droplets (DEs) are used as synthetic cell containers, while pH-responsive elastin-like polypeptides (ELPs) serve as the coacervate system. For controlled long-term visualization and modulation of the external environment, researchers developed an integrated microfluidic device for trapping and environmental stimulation of DEs, with negligible mechanical force, and demonstrated a proof-of-principle osmolyte-based triggering to induce multiple MO formation-dissolution cycles. In conclusion, this work showcases the use of DEs and ELPs in designing membraneless reversible compartmentalization within synthetic cells via physicochemical triggers. Additionally, presented on-chip platform can be applied over a wide range of phase separation and vesicle systems for applications in synthetic cells and beyond.
Elveflow flow control systems provide precise and stable flow control, enabling highly accurate and reproducible experimental conditions essential for reliable data collection and analysis. The inclusion of the XXS microfluidic reservoir further helped our study by minimizing sample volumes, still ensuring precise handling, which enhances the accuracy and consistency in microfluidic experiments.
Siddharth Deshpande is an interdisciplinary scientist fascinated by living systems and keen on understanding how biomolecules self-organize to form functional modules necessary for life. Since 2019, Siddharth leads his EmBioSys Lab at Wageningen University, the Netherlands, embedded within the Physical Chemistry and Soft Matter chair group. The broad vision of his lab is to tackle unsolved biological questions through appropriate bottom-up/biomimetic systems and create soft matter-based materials with biotechnological potential. Current research tackles diverse topics: from shaping synthetic cells and studying protein phase separation, to designing biosensors and making particle-stabilized emulsions. The use and development of on-chip microfluidic technology for controlled experimentation forms an integral part of the lab. Siddharth is always keen on exploring unknown directions and has carried out a variety of collaborative research over the years, from surface-sensing by bacteria and developing antibiotic-screening platforms, to directed evolution of proteins and adhesion mechanism of ticks.
Chang Chen is a PhD student in the EmBioSys group. Chang completed his bachelor’s degree at Harbin Institute of Technology (HIT) from 2012 to 2016. After that, he came to HIT, Shenzhen (HITsz) and got his master’s degree in January 2019. His master project focused on multilayer microfluidic platform development for on-chip cell manipulation. From January 2019, he worked as a research assistant at the Center for Microflows and Nanoflows in HITsz (Jan 2019 to Aug 2020) and Cell Mechanics Laboratory in Peking University (Sep 2020 to May 2021), respectively. Now he is a PhD student at Wageningen University & Research. His PhD project is about bioengineering cell-mimicking vesicles that can undergo shape transformations and primitive migration. The aim is to study the fundamental interplay between biomolecular condensates, aqueous two phases systems, and deformable lipid membranes.
Written and reviewed by Louise Fournier, PhD in Chemistry and Biology Interface. For more content about microfluidics, you can have a look here.
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