Exploring the potential of all-aqueous immiscible systems for preparing complex biomaterials and cellular constructs

Raquel C. Gonçalves , Mariana B. Oliveira * and João F. Mano *
Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. E-mail: mboliveira@ua.pt; jmano@ua.pt

First published on 1st July 2024

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Raquel C. Gonçalves

Raquel C. Gonçalves is a PhD Student in Medical Biotechnology at the University of Aveiro, Portugal, in the Associate Laboratory CICECO – Aveiro Institute of Materials, since 2021. Her research is focused on the development of straightforward and all-aqueous processing methods for the fabrication of freeform shape-versatile soft compartments for tissue regeneration purposes.

Mariana B. Oliveira

Mariana B. Oliveira is an Assistant Researcher at CICECO – Aveiro Institute of Materials, of the University of Aveiro, Portugal. Her research is focused on the development of biomaterials mostly focused on immunomodulation and tissue regeneration. In the last few years, she has been mostly dedicated to the exploration of cells as building blocks of functional biomaterials, as well as to the development of green technologies to process shape-versatile biomedical devices.

João F. Mano

João F. Mano is a Full Professor at the Chemistry Department of the University of Aveiro, Portugal, and the vice-director of the Associate Laboratory CICECO – Aveiro Instituto of Materials, where he is directing the COMPASS Research Group. He has been using biomimetic and nano/micro-technology approaches to develop polymer-based biomaterials and surfaces for the creation of biomedical devices with enhanced structural and multi-functional properties. He also designs microenvironments to regulate cell behavior and organization, with the goal of clinically applying these technologies in advanced therapies and drug screening, or in the bioengineering of disease models. He serves as the Editor-in-Chief of Materials Today Bio (Elsevier).

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Wider impact This work aims to provide a comprehensive, critical and insightful review on the recent application of all-aqueous immiscible systems in the processing of biomaterials and cell-rich constructs. The main properties of immiscible phases and dynamics of aqueous interfaces are discussed in a biomaterial perspective, while addressing current limitations of the use of aqueous two-phase systems, based on their intrinsic low interfacial tension. Strategies for the fabrication of biomaterials of different shapes and complexities, as well as cell micropatterning and aggregation methods in all-aqueous environments, are further reviewed. Importantly, key processing requirements are critically evaluated in both biomaterial and cell-based approaches, while emphasizing the main advantages and challenges in the use of these systems, and the application of the produced biomaterials in different areas such as drug delivery, regenerative medicine and tissue engineering. Finally, potential new research directions are addressed to stimulate the development of materials with improved functionality. This may be helpful to researchers who aim at expanding the application and complexity of their systems, or at starting using aqueous-based systems to process new materials, targeting a broad interested scientific community and industry stakeholders with different backgrounds such as materials and biomedical engineering, biotechnology, medicine, and pharmaceutical sciences, among others.

1. Introduction

Aqueous two-phase systems (ATPSs), also known as aqueous biphasic systems, are characterized by the presence of two water-rich immiscible phases that are formed when two incompatible components (e.g., certain combinations of polymers and salts) are mixed above critical concentrations, resulting in liquid–liquid phase separation.¹ From a thermodynamic perspective, the phenomenon of phase separation in ATPSs occurs when the enthalpic contribution caused by incompatible interactions between the polymers overcomes the entropic driving force for mixing.^1–3

Many hydrophilic components have been used to generate ATPSs, most commonly polymer/polymer and polymer/salt combinations. Dextran and polyethylene glycol (PEG) are examples of incompatible polymers, which when mixed in water-based solvents above critical concentrations spontaneously separate into a dextran-rich phase (bottom) and a PEG-rich phase (top). These systems, along with polymer/salt systems, have been extensively studied for more than six decades since Albertsson's first works in the 1950s,⁴ with most wide application in areas of bioseparation and biotechnological processing. Nevertheless, in order to extend the range of applications of ATPSs, several alternative biphasic systems containing specific salts as ionic liquids, and a diversity of polysaccharides, proteins or surfactants have been proposed.¹ The mild environment and high-water content provided by these all-aqueous systems are revealed to be great assets for greener and safer separation and recovery of biological products such as proteins, nucleic acids, antibodies and cells, while preserving their properties.^5–7 However, more recently, these unique features have attracted the attention from other fields of science including biomedicine and tissue engineering. In this regard, the potential of all-aqueous structures as templates for the fabrication of biocompatible materials, bioinspired cellular models, bioreactors for (bio)chemical reactions, or as platforms for advanced cell culture techniques has been explored.^3,8–10

In the scope of biomaterial fabrication, the behavior of ATPSs has been frequently compared to that of oil/water-based systems, primarily due to their shared characteristics of phase immiscibility and their ability to provide an interface for the assembly of agents of interest. However, the use of non-organic solvents provides higher biocompatibility and a wider range of possible water-soluble solutes able to be incorporated into the system, offering unique application advantages. In fact, hydrogels, particles, fibers or capsules fabricated using all-aqueous immiscible phases and/or interfaces have already shown potential in fields that require very mild processing conditions, such as drug delivery, cell encapsulation and tissue regeneration. Furthermore, the possibility to add more phase-forming components (to produce more than two phases, generating AT+nPS) enables manipulation of the system's phase separation behavior and interface dynamics, or the alignment with 3D printing technology opens up great possibilities for processing more complex biomaterials with intricate architectures.

The compatibility of some polymer-based ATPSs with cell culture conditions, most commonly the ones prepared with polymers, makes them promising platforms for applications involving the confinement of cells for patterning purposes. Those are interesting for studying cell behaviors such as differentiation and migration, or for self-assembly into 3D cell constructs, typically in a spherical shape, that better recapitulate cell–cell interactions and the native tissue microenvironment. Moreover, the interfacial tension of ATPSs can be tailored to induce the assembly of cells at the interface of the aqueous template, or the interface of bulk-separated phases that can be used to produce cell mono-, multi- or hybrid layers, with potential for application in regenerative medicine.

This review will highlight some of the main features and properties of different ATPSs, as well as focus on recent approaches that use ATPSs for the generation of biomaterials, for patterning cells in 2D and engineering of cellular constructs. The exploration of these aspects may guide and inspire scientists interested in understanding the potential as well as the main challenges of using these aqueous systems in a variety of emerging applications.

2. ATPSs: general properties and characterization

To determine the potential range of concentrations of phase-forming components necessary to generate an ATPS, phase diagrams are created for a set of conditions such as temperature, pH, and salt concentration. In general, ATPSs are ternary systems composed of water and two components; however, phase diagrams are typically denoted in more simplistic two-axis orthogonal representations where the concentration of water is absent (Fig. 1A).¹ All the mixtures above the binodal curve undergo liquid–liquid phase separation resulting in a two-phase system, where each of the phases, frequently represented as top and bottom phases, is typically enriched in one of the components (segregative system). Concentrations at or below the curve give a homogenous monophasic system.^11,12 The concentration of the bottom phase component is represented on the horizontal axis (abscissa), while the top phase concentration is plotted on the ordinate. When applying ATPSs, it is important to understand that systems near critical conditions, such as the critical point – where the composition and volume of the two phases are identical – and points along the binodal curve are more unstable and sensitive to environmental variations.³ The lines that connect two points on the binodal curve named tie-lines (TLs) are determined to give the total composition and volume ratio of the phases under equilibrium conditions, and the end points (or nodes) of the TLs represent the final composition of the two phases after complete separation (Fig. 1A).^1,11,12 The length of the TLs and slope can be calculated in order to understand the phase diagram and help designing other TLs to determine other compositions of the system, since they are parallel to each other.¹

The determination of the binodal curve has been most commonly achieved using traditional titration turbidimetric and cloud-point methods.¹ Titration methods rely on the mixture of stock solutions of known concentrations of the different phase-forming components, until visual observation of a cloudy solution (cloud-point method) or until an increase in turbidity of the mixture measured through a light detector (turbidimetric method), which is indicative of phase immiscibility.^19,20 Then water is added to the mixture until it becomes clear, and the mass difference is used to re-calculate the final concentrations of the phase components, which define a point in the binodal curve. Although turbidity measurement is more sensitive and less prone to subjectivity compared to cloud-point methods, both titration strategies are quite time-consuming and consume large amounts of reagents. Therefore, high-throughput screening approaches based on the use of microfluidic devices or 96-well plates have been proposed.^21,22 These strategies rely on the observation under an optical microscope of emulsion micro-droplets or aqueous interfaces, indicative of phase immiscibility and two-phase formation.

2.1. An overview of environmental and physicochemical factors influencing ATPSs

A fundamental key to apply and explore ATPSs may reside in the understanding of their main properties, and how different environmental and physicochemical factors may influence their formation and stability. The phase separation process and thermodynamic equilibrium of ATPSs are influenced not only by temperature and pH but also by osmolarity, as well as other physicochemical properties of the phases that depend on the chemical nature of the phase-forming components, such as hydrophilicity, viscosity and density. In the biomedical field, various studies have taken advantage of this dependence on external stimuli and physicochemical properties of the phases to control the formation of all-aqueous structures.

2.2. Interface properties and dynamics

ATPSs behave in a similar way to emulsions of water and oil presenting an interface between the two bulk phases with an associated interfacial tension (γ). However, compared to water/oil interfaces (γ = 1–40 mN m⁻¹), the interfacial tension of ATPSs is very low, typically within the range of 10⁻⁴ to 10⁻¹ mN m⁻¹.⁵⁶ The interfacial tension of ATPSs depends on several factors including temperature, polymer and/or salt concentration, and polymer molecular weight, in the way that the larger the ability of phases to separate and the far above the binodal curve, the higher is the interfacial tension.^3,5 Different types of ATPSs also often feature distinct ranges of interfacial tensions. For example, the range of values is relatively higher for polymer/salt systems (e.g., PEG/salt: 10⁻¹–1 mN m^−157,58) than for polymer/polymer systems (e.g., PEG/dextran: 10⁻³–10⁻¹ mN m^−13,14) (Fig. 1B).

The characteristic ultralow interfacial tension makes ATPSs very sensitive to external hydrodynamic perturbances induced by shaking, stirring or gravity, making emulsion structures, such as droplets and jets, highly prone to deformation which hinders size and morphology uniformization. At the laboratory scale, microfluidic setups have helped standardize these structures by precisely controlling droplet formation, as well as solidification processes through gelation of one or both phases. However, this vulnerability to external fluctuations may result in periodic structures with unique morphologies at the water/water interface, when using, for example, mechanical vibrations.^59,60

As abovementioned, many ATPS-based micro-scale applications involving the formation of jets and droplets are using microfluidic systems. When co-flowing aqueous solutions of incompatible components, the ultralow interfacial tension of water/water emulsions favors jet formation rather than droplets, due to the slow growth of the Rayleigh–Plateau (RP) instability associated with the breakup of liquid jets into multiple droplets.⁶¹ The growth rate (ω) of the instability of an aqueous jet dispersed in a continuous outer fluid in microfluidic devices can be simplified by eqn (1) which depends on the interfacial tension between the two phases (γ), viscosity of the outer fluid (μ₀), size of the channel (R), perturbation wavenumber (k), radius of the jet (x), and the viscosity ratio (λ).⁶²

	(1)

According to this equation, the lower interfacial tension induces slower growth rates. Thus, in all-aqueous jets, the growth rate of the instability is much smaller compared to that of water–oil jets, which have higher interfacial tensions. This results in a significantly lower breakup into droplets and consequent formation of long liquid jets in microfluidic setups under co-flow. Indeed, it is very challenging to control the efficiency of droplet formation as well as their size and uniformity. For this reason, when the goal is to create uniform droplets in microfluidic channels, active and passive methods have been employed. The active approach involves the application of external forces such as pulse, mechanical vibrations or piezo-electric perturbations to the jetting process, in order to disrupt the streaming and control droplet formation.⁸ On the other hand, passive methods rely on the spontaneous breakup of the jet by taking advantage of the microfluidic geometry and flow dynamics, such as gravity-driven hydrostatic pressure applied to the flowing phase, to control the interface instability and produce microfluidic droplets.^61,63 Moreover, an oil phase can be added to the microfluidic setup to facilitate the breakup of aqueous flow into droplets by oil pinching,⁶⁴ or another possibility would be using an ATPS with increased interfacial tension. For example, Mastiani et al. observed that using a PEG/salt system having a relatively higher range of interfacial tension compared with a PEG/dextran system, uniform droplets could be generated without applying external perturbations.⁵⁷ Even if the interfacial tension of the PEG/salt system is still very low compared to that of water/oil systems, the faster growth rate of interfacial instability and jet breakup prevented the formation of long jets and non-uniform droplets as was observed in the PEG/dextran system.

In the context of applying ATPSs to extrusion 3D printing methods, where one aqueous ink phase is printed within another aqueous bath phase, the presence of interfacial tension-related RP instability causes an undesirable breakup of extruded jets into droplets. The timescale (τ) at which the jets break depends on the interfacial tension and viscosity of the solutions, as defined by eqn (2):

	(2)

where η is the viscosity of the bath phase, r is the thickness of extruded jet, γ the interfacial tension between two liquid phases, and α is a constant that depends on the viscosity ratio between the two fluids.⁴⁶ Considering this, in systems with higher interfacial tension such as water/oil, a faster breakup of the printed filament into droplets occurs compared to ATPSs. Moreover, the equation suggests that the phenomenon can be delayed by increasing the viscosity of the bath phase, creating more stable jets. However, the effective suppression of RP instabilities can be challenging, resulting in the need to promote the solidification of the phases or interfacial interactions to prevent the breakup and stabilize the printed jets.^13,46Fig. 1B summarizes the different ranges of interfacial tension and viscosity of immiscible systems composed of oil/water, aqueous polymer/polymer and polymer/salt, according to the literature.^13–18 The effect of these variables in the stability of liquid structures such as droplet or filaments, as well as on cell viability is also evaluated.

Another distinctive feature of all-aqueous interfaces is the larger thickness compared to their oil-based counterparts, with an estimated difference of at least one order of magnitude (in the order of tens to hundreds of nanometers).⁶⁵ This feature is particularly important when considering typical emulsion stabilization methods using surfactant molecules or nanoparticles of several of nanometers.⁸ These agents can easily stabilize water/oil interfaces but are ineffective for stabilizing all-aqueous emulsion droplets or jets, since they are too small to settle at the thicker water/water interface.⁶⁵ Also, the ultralow interfacial tension reduces the adsorption energy of small particles, hampering their spontaneous adsorption at the aqueous–aqueous interface.⁹ Nevertheless, larger particles have been demonstrating effectiveness in adsorbing at all-aqueous interfaces allowing the stabilization of aqueous emulsions, as will be explored in more detail in Section 3.2.1.

3. Application of ATPSs for the engineering and processing of biomaterials

All-aqueous and generally mild conditions provided by ATPSs facilitate the generation of cytocompatible materials. Most explored strategies consist of dispensing one phase in the other, typically in a jetting or dripping mode,⁶⁶ allowing the formation of emulsion-like jets or droplets delineated by the interface of the two aqueous phases (Fig. 2A and B). Macroscale structures can be achieved using for example manual dropwise addition and 3D printing, and micro-sized droplets or jets through microfluidic, or electrospray/spinning technologies that allow a more precise control over the structure size, dispersity and configuration compared to typical emulsifying techniques. The generated droplets or jets can be further converted into hydrogel particles or fibers, or their interface can be used to assemble materials. Also, materials can be generated by the bulk mixture of both immiscible phases and further solidification or interfacial assembly, allowing the generation of both spherical particles/capsules or porous and compartmentalized hydrogels (Fig. 2C and D). Fig. 2 summarizes the different approaches discussed in this review to produce different biomaterials based on aqueous templates.

3.1. All-aqueous phase solidification-based biomaterials

3.2. All-aqueous interface-based biomaterials

3.3. Emulsions and biomaterials with increased complexity

ATPSs can be manipulated to produce biomaterials with complex shapes rather than spherical such as Janus or Cerberus emulsions,¹²⁶ or with multiple compartments inside. This section will provide an overview of different examples demonstrating the formation of multiple emulsions and biomaterials with increased complexity either by exploring phase separation processes driven by temperature or osmotic gradients, or the addition of a third hydrophilic incompatible polymer to the processing method, or introduction of an oil phase.

3.4. Potential and key considerations: ATPSs in biomaterial-based applications

Overall, resorting on aqueous immiscible phases or interfaces has shown great promise in producing biocompatible hydrogel or membrane-based materials, with controllable features, improved stability, and capacity to encapsulate functional biological agents. For a general perspective, Table 1 summarizes several biomaterials with different architectures that have been prepared using all-aqueous immiscible phases, as well as their crosslinking and fabrication strategy, and applications. One of the main advantages relies on the ability to create more complex architectures including core–shell, spiky, multicompartment, porous and heterogenous structures in a simpler manner, that in other strategies would require either the use of removable solid templates, or other multi-step and time-consuming techniques such as layer-by-layer. This can be achieved in all-aqueous environments by the simple bulk mixture of the immiscible phase, the addition of other immiscible phases or phase-forming component to the processing method, or adjustment of temperature or osmolarity to induce phase separation processes.

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ATPSs are compatible with techniques such as microfluidics and electrospray that allow high throughput and reproducible generation of spherical and fiber-shaped materials, as well as control over size and uniformity. Moreover, the 3D bioprinting technology combined with ATPSs has enabled the generation of free-standing structures or porous and perfusable hydrogels with high precision and spatiotemporal control. Nevertheless, it is important to note that certain characteristics of the ATPSs may hamper the direct implementation of the system in the technique, necessitating the introduction of external actuators, or manipulation of physicochemical properties of the phases. For example, the ultralow interfacial tension hinders the generation of droplets in microfluidic co-flowing systems, and large viscosity and density differences between the phases compromise the stability of printed filaments in liquid-in-liquid bioprinting approaches. Therefore, depending on the desired final structure, it is important to confront possible challenges that might arise when employing different ATPSs, playing with concentrations or molecular weights, or varying the composition of the phases with the addition of other components. This is essential for selecting fluid properties that are suitable for the targeted application.

Interfacial stability is particularly important in strategies relying on the assembly of polymers or particles at the water/water interface. This stability may depend on whether phases from a pre-equilibrium process are used or not, meaning, for example, equilibrated PEG-rich and dextran-rich phases, or concentrated solutions of PEG and dextran, sometimes referred to as prototypical ATPSs. When the interface is not equilibrated, the components of the system, including water and the polymers, move across the interface through diffusion and osmosis to reach equilibrium, which may interfere with the assembly process. However, it is technically simpler to prepare prototypical phases without involving a pre-equilibration step, and the use of non-equilibrated interfaces has been shown as a useful strategy to induce higher levels of complexity in all-aqueous structures through diffusion mechanisms. Hence, the strategy of phase preparation for biomaterial generation is another aspect to consider depending on the purpose.

While for interfacial-templated biomaterials the most widely used system is the PEG/dextran, probably due to its well-studied features related to phase density, viscosity, and interfacial tension, other ATPSs have been explored in the fabrication of phase-templated materials. In fact, the use of phase-forming polymers that have the ability to be photocrosslinked through UV irradiation, including PEGDA or GelMA, is particularly interesting. This enables better control and precision over the material fabrication process, without depending on environmental factors, such as temperature, that can be more difficult to regulate. Furthermore, the utilization of gelatin or other naturally derived polymers provides an opportunity to create materials with improved cytocompatibility, making them particularly valuable in strategies involving cell encapsulation for tissue engineering and regenerative applications. Both hydrogel-based materials and capsules formed through the interfacial complexation of oppositely charged PEs of natural origin, such as alginate and chitosan, have shown ability to encapsulate cells and promote the formation of functional microtissue aggregates.^120,121 Also, all-aqueous environments are shown to be adequate for the encapsulation and release of functional molecules, such as proteins and enzymes, in capsules fabricated through the interfacial complexation of synthetic PEs.^117,118 On the other hand, the application of polymer/salt systems in biomaterial fabrication has not been extensively reported. These systems can provide a relatively higher interfacial tension and lower phase viscosity compared to typical polymer/polymer systems, which might be of interest in microfluidic droplet formation. However, many cells and biomolecules are sensitive to environments with elevated salt concentrations and osmolarity, which may be required to produce biphasic conditions.

4. Application of ATPSs in scaffold-free cell-based approaches

As outlined in some examples within this review, the cytocompatibility of polymer-based biomaterials, such as hydrogels or membrane-based capsules, fabricated using aqueous immiscible systems, has been verified, demonstrating their ability to maintain the viability, proliferation, and function of encapsulated cells. However, the use of immiscible all-aqueous phases has also shown great promise in other scaffold-free cell-based approaches. Those involve the gentle confinement of cells at a low-tension interface-separated phase for the precise positioning in space of different cells under 2D cell culture conditions, or for further allowing their aggregation and producing spherical or interfacial microtissues.

Typically, in these kinds of applications, PEG/dextran systems are employed, where the polymers are directly dissolved in the appropriate cell culture medium to form the two immiscible phases. The cell partition behavior between the two phases and their interface is an important parameter to consider. The partition of cells can be influenced not only by the physicochemical properties of the ATPSs such as temperature, pH, ionic composition of the separation medium, molecular weight of phase-forming polymers, and interfacial tension, but also by the surface properties of the cells such as charge, area and wettability.¹⁴⁵ If we neglect the electrostatic potential between the phases, when both non-ionic PEG and dextran polymers are dissolved in the same culture media, the partition coefficient (K) of cells, here defined as the ratio of cells in the respective bottom and top phases, can be described as a function of the interfacial tension (γ) according to eqn (3):¹⁴⁶

−logK ∝ αγ	(3)

where α is a constant number. Considering this relation, to increase the K value and promote the partition of cells to the dextran phase, the interfacial tension should be decreased.¹⁴⁶ In fact, Tavana et al. demonstrated that in lower tension PEG/dextran systems, the cells were preferably located in the dextran-rich bottom phase, and increasing the interfacial tension by increasing polymer's concentration caused the partition of the cells towards the interface.¹⁴⁵ Moreover, while maintaining the interfacial tension, the partition behavior of human cancer cells and mouse mesenchymal stem cells was different due to variations in cell surface hydrophilicity, corroborating the role of the cell-type specific surface properties in their partition.¹⁴⁵

4.1. 2D cell micropatterning

In the context of cell biology and tissue engineering, the ability to spatially organize homo- or heterotypic cultures of cells in 2D in vitro environments can be useful to study different cell behaviors such as cell growth, differentiation, migration and cell–cell interactions, as well as study physio/pathological processes such as tissue formation or tumor progression.¹⁴⁷ In the last few years, several patterning and printing techniques have been explored to create different 2D cell patterns. A group of techniques, such as soft lithography (or microcontact printing), photolithography or microfluidics, consist of the functionalization of the surface of substrates with proteins or adhesion-promoting molecules at discriminate places, in order to precisely control the positioning of cells at those adhesion patterns whereas the other areas are left acellular.^147,148 Other approaches are based on the direct spatial deposition of cells using inkjet bioprinters that use thermal or piezoelectric mechanisms to precisely deposit cell droplets in a spatiotemporally controlled manner, or through laser-assisted bioprinting.¹⁴⁸

Although these techniques offer the possibility to spatially organize cell droplets or lines with high resolution, they either use multi-step and time-consuming procedures or need specialized energy requirements. On the other hand, cell micropatterning using ATPSs relies on the inherent ability to confine cells into desired configurations within interface-separated aqueous phases in an easily implementable manner using simple pipetting methods.³ Moreover, contrary to lithographic strategies that rely on the physical contact of a stamp or a mask with the dry substrate, this method can position cells in a non-contact manner (as the bioprinting techniques, but in all-aqueous environments), allowing the patterning in delicate substrates, such as cell monolayers, without causing significant damage.^146,149

Usually, nano- or microliter dextran droplets or lines are dispensed in a substrate covered with PEG solution, with aqueous phases deriving from a pre-equilibrated system using cell culture media as solvents. Low concentrations of phase-forming polymers are used to maintain the partition of the cells in the dextran phase used for patterning. Both circular and linear patterns can be created using this technology. However, to maintain the stability of linear or user-defined configurations and prevent them from breaking-up into droplets, the surface type where the cells are being patterned is very important. In fact, it should be noted that the interaction between the dextran phase and biological molecules on a surface is crucial for the linear patterns to remain stable, and therefore, cell monolayers and decellularized matrices are suitable surfaces for this purpose.¹⁵⁰ When using a cell monolayer as the surface, the ultralow interfacial tension of the ATPS, the affinity of the cell surface to the dextran phase, the roughness of the cell monolayer, and the equilibrium between all associated interface tensions (PEG–dextran–cell monolayer) are all factors that contribute to the stability of the cell patterns.¹⁴⁶ Indeed, linear patterns were shown to be stable for extended periods of at least a day.¹⁴⁹ In addition, the resolution of printed cell lines is influenced by several parameters such as the printing speed, the loaded volume on the tip and the tip diameter (Fig. 6A1).¹⁵⁰

Hossain Tavana and colleagues have widely explored the potential of ATPS-based cell micropatterning for several applications. They developed a method that allowed patterning of uniform-sized droplets with defined interspacing, using slot pins that load and dispense nanoliters of the dextran-rich phase containing the cells or bioreagents in a surface (culture plates, hydrogels or cell monolayers) covered by the PEG-rich phase. With this, they demonstrated the possibility not only to deliver plasmids and lentivirus vectors to specific places on a cell monolayer, allowing the localized transfection of discrete groups of cells,¹⁴⁹ but also to promote the neuronal differentiation of clusters of stem cells patterned on top of stromal cell monolayers (Fig. 6A2).^151,155,156 On the other hand, they also verified the possibility to spatially promote the differentiation of cells that were randomly seeded on top of patterned pre-formed clusters of different cell types.¹⁵⁷ Besides this or patterning cells on top of cell monolayers, the establishment of heterotypic cell cultures using ATPSs can also be achieved by adding different cell types to the immiscible phases, separately. Fang et al. were able to study surface receptor/ligand interactions between neighboring cells, specifically involved in breast cancer metastasis, by patterning breast cancer cells and human embryonic kidney cells, mixed in the dextran and PEG phases, respectively.¹⁵⁸

Additionally, the ATPS-based patterning technology enables the creation of cell-excluded gaps that may serve as the migration space for adhered cells (Fig. 6A3).^152,159 In this case, a droplet of the denser dextran phase is patterned on culture dishes or microwells and dehydrated. Then, a cell-containing PEG phase is added to rehydrate the dextran pattern forming a circular droplet-templated cell-excluded area. The increased interfacial tension during the rehydration process prevented the cells from crossing the interface, allowing them to settle down and adhere on the boundaries of the dextran gap. This technology could be adapted for the high-throughput screening of anti-migration compounds, including anti-cancer chemotherapeutic agents.^152,159

4.2. Micro/macro-tissue formation

In vitro tissue models made of cells and extracellular matrix (ECM) organized in a 3D environment can recapitulate more closely the architecture and cellular microenvironment of the in vivo scenario, improving tissue functionality. Although scaffolding materials such as hydrogels have been successfully supporting the achievement of 3D-organized engineered tissues, approaches based on the self-assembly of cells into multicellular aggregates without using exogenous materials may offer several advantages, such as increased proximity between the cells endorsing better cell–cell communication and ability to produce their own ECM during tissue formation.¹⁶⁰

Spheroids are considered the most versatile 3D cellular model due to their spherical shape, being currently the most widely used cell structures in the fields of tissue engineering and disease modeling for drug screening.¹⁶¹ In particular, spheroids made of cancer cells can recapitulate certain features of solid tumors such as the presence of a necrotic core where hypoxia and nutrient depletion occur in the central area due to diffusional limitations. Spheroids have been produced by stimulating cell aggregation in hanging drops, multi-well plates with non-adherent polymer coatings (commercially available ultra-low attachment plates), or in bioreactors with mechanical agitation.¹⁶¹ However, these techniques present several limitations including the evaporation of media from drops, challenges in handling plates during culture, the need for specialized and expensive equipment, exposure of cells to non-physiological shear stress, or the generation of non-uniform spheroids. The generation of spheroids using an ATPS can prevent some of these shortcomings. Recently, heterotypic spheroids made of endothelial and hepatic cells have been produced using a PEG/dextran emulsion, stabilized with whey protein particles.¹⁶² However, due to size variability between emulsion droplets, randomly sized spheroids are formed using this strategy. Nevertheless, dispensing techniques that better control droplet size and uniformity can be employed to produce spheroids using all-aqueous systems. Han et al. demonstrated that in cell-laden dextran droplets dispensed in normal wells containing PEG, spheroids could be formed if the appropriate cell type is used (i.e., spheroid-forming cells), and if the buoyancy force (from the density difference between the phases) and interfacial tension are sufficient to maintain the cells at the interface and promote their aggregation at the apex of the dextran drop meniscus.¹⁶³ Otherwise, cells would form loose aggregates at the interface or adhere to the surface of the plate. On the other hand, non-adherent wells containing the PEG phase can be used to produce uniform spheroids in a reproducible manner. Cells are confined in the dextran droplet in close proximity with each other and, within 24 h, compact spheroids are formed. Afterwards, the ATPS can be diluted with fresh culture medium to retrieve the cell aggregate.¹⁶⁴ In the absence of the ATPS droplets, only loose aggregates are formed under the same conditions. The size of the spheroids depends not only on the cell density, but also on the volume of the dispensed droplets.^164,165 Additionally, in this case, if cells tend to partition to the interface of the droplet in response to a higher interfacial tension, this will disrupt the self-assembly process into spheroids, leading to the formation of several small, loosely aggregated interfacial structures.¹⁴⁵

Based on this, tumor-mimetic spheroids produced using ATPSs have shown important features, including proliferative and growth capacity after 7 days of culture,¹⁶⁵ and production of ECM proteins after 3 days namely collagen I, laminin and fibronectin.¹⁵³ Additionally, they exhibit a necrotic core characterized by hypoxia and lack of cell proliferative activity,¹⁵³ and show higher resistance to drugs compared with 2D cell cultures (Fig. 6B1).^153,164,165 In fact, the group of Tavana demonstrated the compatibility of this technology with high-throughput drug screening by using a simultaneous robotic pipetting method in 96- or 384-well plates.^153,165 With this, they were able to analyze dose-dependent responses to different drugs and perform combined or cyclical drug treatments in colorectal cancer spheroids.^166,167

Besides spherical aggregates, cells can also assemble into sheets or monolayers, if they attach to a surface for proliferation and ECM production, and are further released. Conventional approaches to produce these cell-rich constructs are based on the culture of cells in thermo-responsive substrates modified with PNIPAM, allowing cell adhesion at physiological temperatures (37 °C) and promoting their release by lowering the temperature below 32 °C to induce the swelling of PNIPAM chains.¹⁶⁰ However, this method is quite expensive, and presents some practical issues regarding the manipulation of the cell sheets. Alternatively, magnetic fields can be used to release cell sheets when using magnetic nanoparticles internalized within the cells.¹⁶⁸ However, besides the complexity of this strategy and reliance on the effectiveness of cellular uptake, there are some concerns related to toxicity.

The formation of cell sheets in a substrate-free environment at the interface of a macrophase-separated ATPS has been shown as a simpler, faster, and cost-effective approach than the ones dependent on thermal or magnetic stimuli. This involves loading a tube with the dextran phase dissolved in medium, followed by overlaying the PEG medium solution containing the cells. Within 15 minutes, the cells start settling at the interface. In fact, Frampton et al. observed that 2 h was enough to produce planar cell sheets with sufficiently strong cell–cell interactions, with ability to be handled, washed, and transported.¹⁵⁴ Macroscopic cell mono- and multi-layers are retrieved after gently pouring the mixture in culture dishes with fresh culture medium. However, the formation of robust and stable tissue-like constructs depends on the phase-forming polymer concentrations as well as the cell type used (Fig. 6B2).¹⁵⁴ Moreover, Hung et al. recently demonstrated that the interfacial self-assembling process is dependent on the initial spatial distribution of the cells within the PEG solution.¹⁶⁹ If the cells are not homogeneously dispersed, their distribution at the interface will not be uniform, leading to fragmented structures. To overcome this, they used aqueous immiscible phases derived from a pre-equilibrated system (PEG-rich and dextran-rich) to provide a more stable interface for cells to assemble, and added a centrifugation step after layering the cell–laden PEG-rich phase, to evenly distribute the cells at the interface. Using these methodologies, skin-like¹⁵⁴ and corneal-like¹⁶⁹ tissue constructs were fabricated, with potential applications in tissue engineering and regenerative medicine. Another approach based on withdrawal processes of thin films at all-aqueous interfaces was used by Chan et al. to assemble hybrid cell monolayers.¹⁷⁰ Their methodology consisted of the addition of a droplet of a denser dextran solution containing the cells in a PEG/dextran bulk-phase separated system. As the droplet passes the interface, a PEG-rich thin film is formed around the droplet, and is eventually withdrawn, supporting the formation of a monolayer of cells at the bulk interface. To achieve this assembly process, a force balance between the droplet weight and the interfacial tension of the PEG/dextran system is needed. Therefore, the volume of the dispensed droplet, the concentration of dextran in the droplet, and concentrations of PEG and dextran to produce the bulk phase separation that influence the interfacial tension of the system are all parameters that should be controlled. Importantly, this method allowed the formation of monolayers consisting of different cell types, cell aggregates, or even hybrid cell-microparticle aggregates with functional properties such as response to magnetic fields.¹⁷⁰

4.3. Potential and key considerations: ATPSs in scaffold-free applications

As the examples described demonstrate, the use of all-aqueous immiscible phases for patterning cells under 2D culture conditions, or producing cellular constructs, including spheroids and planar cell sheets, can offer several advantages in terms of cost-effectiveness, user-friendliness and implementability, over conventional techniques that have been currently applied. Moreover, although the application of these systems in this area is relatively recent, some procedures have been already robotized and optimized to allow higher reproducibility and generate high throughput screening platforms. Nevertheless, there are important aspects that need to be addressed when employing ATPSs in cell-based applications. First, the concentration of the phase-forming polymers should be as low as possible to minimize adverse effects on cell viability and perturbations of medium composition (since the polymers are dissolved in the cell culture media).^149,152,154 For example, Tavana et al. demonstrated that cells cultured in a medium supplemented with dextran (500 kDa) and PEG (8 kDa) at concentrations of 5% and 4%, respectively, were 95% viable after a period of 24 h.¹⁴⁹ At the same time, the concentrations used need to guarantee the working conditions in the biphasic region of the system, which means above the critical conditions, to allow proper cell confinement. On the other hand, since the interfacial tension of the system, that is directly related to the polymer's concentration, affects the partition of the cells, it becomes a crucial parameter to account depending on the application. Hence, before starting working with different cells or ATPSs for this kind of application, it is advisable to characterize the aqueous system and verify its compatibility with the cell types, considering possible variability in the conditions necessary for cell culture, differences in the cell surface properties, and concerns related to viability.

It is also important to note that the addition of polymers as PEG and dextran to cell culture medium can impact certain biological aspects of cellular response due to their macromolecular crowding properties and ability to change medium viscosity. The addition of macromolecular crowders such as Ficoll or dextran sulphate in culture medium has been shown to significantly increase the deposition of ECM proteins including collagen and fibronectin by human fibroblasts, osteoblasts, tenocytes and mesenchymal stem cells (hMSCs) in vitro, even at low serum concentration.^171–173 This enhancement of ECM deposition is helpful in the generation of more dense and cohesive living cell sheets, with fibrillar patterns and tissue-like organization.^172,173 Moreover, the adipogenic and chondrogenic differentiation of hMSCs was enhanced in the presence of molecularly crowding environments.^174,175 With respect to the influence of viscosity as a biophysical cue on cellular functions, this mechanism has been investigated mostly in 2D substrates and scaffolds with different viscoelastic properties, as reviewed elsewhere.¹⁷⁶ On the other hand, research has been carried out on understanding the effect of adding polymers such as PEG, dextran or methylcellulose to manipulate media viscosities, mostly because in vivo extracellular fluid viscosity may vary under physio and pathological conditions. For example, increased viscosities of cell culture medium (8 mPa s) obtained by the addition of methylcellulose have been associated with increased breast cancer cell migration and induced changes in the cell morphology in microfluidic channels.¹⁷⁷ In other examples, it has been noticed that PEG-supplemented viscous media affected the aggregation of bovine chondrocytes in a 3D environment, and the differentiation of hMSCs under cell morphology-controlled conditions.^178,179 Very high medium viscosities (approximately 680 mPa s) retarded cell–cell interactions due to higher resistance to 3D cell motility, promoting a slower formation of cell aggregates.¹⁷⁸ Moreover, higher viscosities of induction medium have shown to promote osteogenic differentiation and inhibit adipogenic differentiation of large and elongated hMSCs.¹⁷⁹ Altogether, these studies demonstrate that incorporating phase-forming polymers into the medium can indeed impact cell behavior. Therefore, if higher molecular weight polymers or increased polymer concentrations are used, there might be a need to consider the evaluation of possible effects at the cell molecular and morphological level, depending on the purpose.

Another critical consideration is whether to employ phases derived from a pre-equilibrium process, or concentrated polymer phases (prototypical). In micropatterning approaches, most studies applied equilibrium-derived PEG-rich and dextran-rich phases. In the case of interfacial cell layers formed in a bulk phase-separated ATPS, it was observed that a pre-equilibrium is in fact essential to provide stable interfaces for the assembly of cells and generation of more robust constructs. When the bulk interface is derived from prototypical PEG and dextran phases, the diffusion of water and the polymers destabilizes the interface, which may compromise the cell assembly process.¹⁶⁹

PEG and dextran are undoubtedly the most widely selected phase-forming polymers to produce immiscible phases and aqueous interfaces for cell culture applications. This is mainly due to compatibility of the system with a wide range of temperatures, including physiological temperatures (37 °C) needed for cell incubation, and ability to produce phases in appropriate cell culture solvents, ensuring poor cytotoxicity.¹⁰ Nonetheless, for example, Tavana's group demonstrated that other polymer/polymer ATPSs such as PVA/dextran or polyacrylamide/PEG enabled the formation of spheroids.¹⁶⁵ By contrast, the application of polymer/salt ATPSs might not be appropriate for cell micropatterning or construction of scaffold-free cell structures, due to detrimental effects of high salt concentration environments on the culture media osmolarity and cell viability.¹⁶⁹ Furthermore, it was demonstrated that interfaces derived from oil/water systems negatively impacted the formation of cell sheets,^169,170 which was most probably related to the higher interfacial tension and hydrophobic nature of the interface that interfered with the cell assembly process and viability of the cells. In fact, although some particles, polymers, proteins and lipids have been shown to self-assemble at oil/water interfaces for emulsion stabilization purposes, the environment at these interfaces can lead to the denaturation and changes in the conformation of proteins resulting in loss of their native function, and also to lipid oxidation, thereby compromising the cell structure and ability to assemble and interact with neighboring cells.^180,181 On the other hand, recently, bulk interfaces from a non-cytotoxic ionic liquid-based ATPS were used to culture mammalian cells. hMSCs were able to attach and spread at the interface due to the adsorption of proteins from cell culture medium and formation of an interfacial protein nanolayer, in which the protein structure and adsorbed amount depended on the chemical structure of the ionic liquid.¹⁸²

5. Conclusions and future perspectives

Over the last decade, all-aqueous immiscible systems have been explored as simpler, cost-effective, and biocompatible platforms for the generation of materials ranging from spherical and fiber-shaped structures (mostly micro-scaled) to bulk hydrogels with porous or heterogeneous properties. The main strategies involve the generation of all-aqueous templates by dispensing one phase in the other, or through the bulk mixture of both phases, and further conversion of the phases or phase of interest into hydrogel materials, or the use of the interface for the assembly of the materials. Despite some major advances having been made, a complete fundamental understanding of phase separation behavior and ATPS properties, particularly at the interface level, is still lacking, which compromises the development of new strategies to engineer novel interfacial-based assemblies beyond the widely studied electrostatic polyelectrolyte/particle complexation.⁸ Once a more widespread understanding of these and more data related to interface properties and dynamics is available for a wide range of aqueous phase systems, one would propose the creation of computational models that could help predict the movement and interaction of material-forming components in the system.

The generation of more complex all-aqueous structures has been explored using different approaches, including the induction of phase separation inside aqueous or oil-based droplets through osmotic pressure or temperature, or the inclusion of another incompatible component in the system. The exploration of multiphase systems with four or five phases and multiple interfacial dynamics could enable the generation of even more intricate structures, with different compartments containing different partition properties, interesting for drug delivery and other biomedical applications. Furthermore, progress in the application of ATPSs in the emerging 3D printing technology has enabled the generation of both soft membrane-based and hydrogel-based materials, with interesting complex geometries and branched architectures. A potential avenue for future exploration may involve the fabrication of highly hierarchical structures and hybrid materials, which may be of interest for the development of engineered tissues with increased levels of complexity, biomimicry, and improved functionality.

Given the high-water content, compatibility with physiologically relevant conditions, and compartmentalization capacity, materials produced in ATPS-enabled environments and architectures have potentially suitable applications in clinical therapeutics, such as drug delivery, especially biopharmaceuticals like nucleic acids and proteins, and regenerative medicine. Although some studies in the scope of drug delivery have shown efficiency in the loading and delivery capacity of molecules in vitro, further studies regarding mechanical stability, interaction with tissues, and drug bioavailability are necessary to prove their effective performance in vivo.¹⁸³ The potential of ATPS-based materials in the field of tissue regeneration as well as translation to the pre-clinical in vivo scenario, has been explored particularly in wound repair and regeneration, with the development of strategies that enabled personalized defect-filling properties such as injectability or in situ crosslinking. Moreover, there are promising results demonstrating the feasibility of the application of cell clusters developed within ATPS-based capsules or particles in the field of pancreatic islet transplantation for the treatment of type I diabetes;^80,120,121 however, the evaluation of their in vivo efficacy still needs to be examined.

Regarding cell-based approaches, ATPSs offer the possibility of positioning cells in space in a gentle and controlled manner, free of contact with solids, both in circular and linear patterns. This enables the generation of cellular microenvironments useful for studying homo- and heterotypic interactions and cell behavior in response to external stimuli. This can also be interesting for tissue engineering strategies involving the formation of highly structured heterotypic microenvironments, which could promote the alignment or spatial organization of different cell types in order to recapitulate the architecture of native tissues. However, in vivo cells reside in a 3D microenvironment composed of an ECM of proteins and biomolecules, which provides them with structural and biochemical support. The ATPS confinement ability under cell culture conditions enables the formation of microtissue aggregates in a faster and reproducible manner, not only in the dispensed phase but also at the interface of the aqueous template. The bulk interface of ATPSs can also be used for the assembly of cell sheets that can be easily retrieved and have already shown potential in tissue regeneration of in vivo and ex vivo animal models. Moreover, if cell aggregates are formed for sufficient culture times and ECM-secreting cells are used, microtissue structures with endogenous ECM are produced. Therefore, it would be interesting to combine micropatterning techniques, cell assembly processes, and possibly 3D printing technologies, for the construction of tissues with a higher degree of complexity that can better recapitulate the in vivo environment. The addition of gel-forming polymers with cell adhesive moieties such as collagen,¹⁸⁴ or the use of other ATPSs with gelation properties such as PEG/gelatin, could also be advantageous to provide additional structural support and improve tissue formation.

On the other hand, the ATPS-generated simplistic spherical cellular aggregates that can recapitulate certain aspects of solid tumors are useful as in vitro disease models for drug screening. In the past few years, there has been growing interest in the area of organoids, which consist of more complex cell-based in vitro models derived from the self-organization of human stem or progenitor cells that recapitulate physiological structures and functions of specific organs.¹⁸⁵ Organoids have offered potential applications in the biomedical field, particularly in drug discovery, tissue regeneration, and personalized medicine. The process of organoid formation requires very controlled environments composed of soluble factors that are presented to cells in a spatio-temporally controlled manner in order to promote cell differentiation into organ-specific cell types.¹⁸⁵ The compatibility of ATPSs with cell culture conditions allied with the ability to promote the aggregation of cells and growth of microtissues in confined environments may potentially demonstrate the possibility of applying these systems for engineering organoids. However, it would be necessary to evaluate potential adverse effects on the viability of these cells and their fate, as well as compatibility with the supplementation needed on the cell culture medium when in the presence of polymeric phases, as well as additional physical cues that may be required.

Overall, the examples outlined in this review highlight the broad potential of all-aqueous systems within the featured fields; nonetheless, it is crucial to consider specific factors related to ATPS properties, namely, low interfacial tension, phase viscosity and density, concentration of polymer/salt, and equilibration of the interface, depending on the envisioned application. As the field evolves and more studies are performed to address concerns about some properties and mechanisms of phase separation and compatibility with other kinds of materials or cell culture environments, these systems could find more widespread applications. Moreover, although different polymers such as PEG, dextran, gelatin, and their methacrylated forms have been widely explored in this field, it could be potentially interesting to explore other types of phase-forming biopolymers such as proteins derived from recombinant processes to improve functionality of the produced biomaterials.

Author contributions

RC Gonçalves: conceptualization, information collection, visualization, and writing (original draft and editing). MB Oliveira: conceptualization, writing, and supervision. JF Mano: conceptualization, supervision, and project administration.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the European Research Council grant agreement ERC-2019-ADG-883370 (project REBORN, DOI: 10.3030/883370). It was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 (DOI: 10.54499/UIDB/50011/2020; DOI: 10.54499/UIDP/50011/2020), and LA/P/0006/2020 (DOI: 10.54499/LA/P/0006/2020), financed by national funds through the FCT/MCTES (PIDDAC).  Mariana B. Oliveira acknowledges national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Scientific Employment Stimulus – Institutional Call – CEECINST/00013/2021 (DOI: 10.54499/CEECINST/00013/2021/CP2779/CT0010). Raquel C. Gonçalves also acknowledges financial support received from FCT through individual grant 2021.07435.BD (DOI: 10.54499/2021.07435.BD).

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