The ability of protein polymers derived from elastin to undergo LLPS (Dzuricky et al., 2018), presents a unique potential to create in vitro systems for studying the principles that govern the formation of biomolecular condensates, but also opens the possibility to create innovative synthetic condensates with customized topology, mechanical properties, or chemical nature (Dai et al., 2023b).

The recombinant polymeric nature of ELRs places them at the converging point of synthetic biology and polymer science. Harnessing synthetic biology tools allows the creation of recombinant polymers with precisely tailored phase transitions, facilitating the incorporation of unnatural amino acids and post-translational modifications (Costa et al., 2018; Mozhdehi et al., 2018; Scheibel et al., 2020). Concurrently, the application of polymer science principles enables the rational design of modular self-assembling systems and their bioconjugation with other polymers (Acosta et al., 2020b; Saha et al., 2020; Garanger and Lecommandoux, 2022).

ELRs present an opportunity to uncover the variables controlling the intricate LLPS processes occurring in more complex IDPs. For instance, the generation of microdroplets using polymers via microfluidics allows the creation of cell-free systems that mimic intracellular molecular crowding and offers a means to study phase transitions (Liu et al., 2020). In these in vitro models, it has been observed that diverse molecular assembly patterns can be achieved (Simon et al., 2017). The combination of ELRs with different hydrophobicities, i.e., transition temperatures, allows for the creation of multicompartimentalized microenvironments. It has been observed that the mixing properties of ELRs conform to the Flory-Huggins mean-field theory, enabling the understanding and prediction of the miscibility or immiscibility of ELR-based coacervates based on molecular parameters such as chain length, composition, and interaction with the solvent and between ELRs (Simon et al., 2017).

Microfluidics has enabled the creation of paired emulsions from immiscible polymers, such as dextran and poly (ethylene glycol) (PEG) to simulate crowded molecular environments (Zhao et al., 2020). These synthetic systems have served to investigate phase separation and spatial distribution of ELRs in confined environments with multiple phases, where ELR coacervation preferably occurs at the interface of the two phases (Zhao et al., 2020). This type of synthetic cell-like system allows also to study the dynamic coacervation of ELRs and the combination with other proteins, and can serve as a platform to evaluate interactions of IDPs with other cellular components. Indeed, in a different study, monoblock ELRs were covalently bioconjugated with dextran and PEG to study the mechanisms of co-assembly. Once again, it was demonstrated that ELR coacervates preferably form at the interface of both phases, and that this co-assembly mechanism was reversible (Zhao et al., 2021). Moreover, it has been demonstrated that synthetic condensates based on bioconjugated ELRs exhibit a remarkable capability to enhance the rate of enzymatic reactions. By creating cytomimetic protocellular models using water-in-oil-in-water (W/O/W) double emulsions and employing monoblock ELRs and ELR-bioconjugates with PEG and horse radish peroxidase as a model multicomponent enzymatic system, a study model for the kinetics of enzymatic reactions under different osmotic pressures was established. This approach demonstrated that synthetic condensates significantly improved the rate of enzymatic reactions after hypertonic shock (Schvartzman et al., 2023).

The versatile design of ELRs enables the production of protein polymers with multifaceted functionalities. It is feasible to create in vitro models of biomolecular condensates induced by variables other than temperature, such as pH. ELRs containing charged amino acids in their sequence are responsive to pH changes (Ribeiro et al., 2009; MacKay et al., 2010). By controlling the proportion of charged (i.e., Glu and His) and hydrophobic (i.e., Val, Ile, Phe and Tyr) amino acids in the guest position, de Haas et al. engineered pH-responsive ELRs with the capability to form condensates under narrow pH changes within a physiologically relevant range (i.e., pH 4–7) (de Haas et al., 2023).

Moreover, ELRs can be modified at the sequence level to incorporate other protein domains capable of interacting with other cellular components, thus developing models of multivalent condensates. Since one of the roles of biomolecular condensates is the control of gene expression, the interaction of ELR-based condensates with nucleic acids by introducing nucleic acid-binding domains allows the construction of intricate systems capable of elucidating mechanistic variables in the aggregation of more complex natural ribonucleoproteins (Simon et al., 2019). Thus, opening avenues for designing synthetic condensates applicable to metabolic engineering (Qian et al., 2022).

However, it is noteworthy to consider that synthetic models have limitations, since the study conditions may differ from intracellular conditions. Nevertheless, it is possible to transfect living cells with genes encoding protein polymers and express them directly intracellularly to create even more precise in vitro models. These approaches facilitate the examination of their interactions with intracellular components in both eukaryotic and prokaryotic living cells, thereby establishing more intricate condensates (Dai et al., 2023a). These sophisticated models can serve as potent tools in synthetic biology for cellular engineering applications. In this way, the capacity of amphiphilic block co-ELRs to program self-assembly in living E. coli cells was investigated (Huber et al., 2023). From a large library of building blocks, crucial molecular variables were unveiled for programming self-assembly, including charge, distribution, and hydrophobic strength. This demonstrated that depending on the composition of ELRs, the derived condensates are capable of influencing the distribution and spatial organization of DNA, potentially guiding molecules into synthetic condensates.

The creation of synthetic condensates from ELRs has also served to shed light on other biological processes where LLPS is involved, such as elastogenesis. Elastogenesis, the formation of elastic fibers involves a harmonious orchestration of TE synthesis, coacervation, crosslinking, and deposition on microfibril scaffolds (Yeo et al., 2011). The first step of elastogenesis is the condensation or coacervation of TE (Tu et al., 2010). Understanding this process is crucial for comprehending pathologies related to poor elastic fiber deposition and for engineering functional materials that accurately mimic elastic matrices (Tarakanova et al., 2018).

ELRs inspired by representative TE domains emerged as invaluable tools for in vitro modeling, particularly in the early stages of elastogenesis. Multiple elastin-like polypeptides have been produced, using different TE exons as repeat units, with those located in the hinge region (i.e., exons 20, 21, 23, and 24) being among the most recurrently employed (Muiznieks and Keeley, 2010; Reichheld et al., 2014). These exons, combining hydrophobic and hydrophilic elements, play a crucial role in the structural flexibility of TE (Yeo et al., 2016), presenting a simplified but representative version of the full protein (Reichheld et al., 2017). Through the permutation of these exons, Muiznieks et al. developed ELRs with different coacervation capacities. These models allowed the in vitro study of specific mutations in different ELR domains and evaluated their effect on coacervate formation. The meticulous control of coacervate (condensate) droplet growth and stability stands as a prerequisite for the proper maturation of elastic fibers. Understanding the formation of these coacervates holds significance from both physiological and biomaterial science perspectives. In this way, these models allowed the investigation of coacervation events, aggregation at interfaces, and co-localization with other proteins involved in elastogenesis, such as Fibrillin-1 (Muiznieks et al., 2014). While molecular weight and hydrophobicity of the recombinant polypeptides are crucial modulators of the initial phase separation, other factors influence the maturation of the formed liquid coacervate. Hydrophobic elastin domains initiate phase separation, but the addition of charged lysine residues in crosslinking domains introduces an amphipathic character, influencing droplet stability. These protein polymer approaches can also facilitate the emulation of crosslinking effects in vitro, providing insights into the intricate interplay between coacervation and crosslinking processes (Lau et al., 2022).

Condensate properties can be controlled by sequence changes in the ELRs. While TE can exhibit LLPS, Ceballos et al. have demonstrated that the production of recombinant polymers bioinspired in TE exons 20–21-23–24, together with an increase of the hydrophobic content in their terminal domains, has the potential to alter the condensate maturation. This alteration leads to liquid-solid transitions in saline solutions providing a valuable avenue for modulating elastogenesis processes in vitro (Vidal Ceballos et al., 2022).

Moreover, the creation of models from ELRs enables the monitoring of coacervation processes with enhanced resolution (Reichheld et al., 2017). Thanks to the use of ELRs, it has been experimentally demonstrated, employing solution and solid-state NMR, that the hydrophobic domains of TE are intrinsically disordered, demonstrating their enormous potential to mimic complex in vitro phase transition processes and study them in detail in a controlled manner that allows replication.

Biomineralization is the natural process through which living organisms create minerals by depositing inorganic molecules using organic molecules as scaffolds, thus creating biocomposite materials such as bone and enamel (Aparicio and Ginebra, 2015). Depending on the type of organism, different biohybrid structures can be generated by depositing various minerals, including magnetite, silicates, or hydroxyapatite (Boskey and Villarreal-Ramirez, 2016). Particularly relevant from a pathological and biomaterial science perspective is the deposition of calcium phosphate on macromolecular biological structures. In this context, IDPs play a crucial role, as their structural flexibility allows them to interact with ions in solution, leading to conformational changes that enable them to modulate and inhibit in vivo biomineralization (Boskey and Villarreal-Ramirez, 2016). Even in the case of bones, where collagen type I, a protein with a defined secondary structure, serves as a scaffold for the formation of mineralized tissue with apatite crystals (Beniash, 2011), IDPs control the deposition and maintenance of biomineral (Boskey and Villarreal-Ramirez, 2016). Thus, protein components of the extracellular matrix with high proportions of disordered sequences will play an important role in ECM aging and calcification processes and may in turn prove to be a revolutionary source of inspiration for the creation of innovative biomineralized materials.

Arterial calcification, characterized by the deposition of calcium phosphate (Ca–P) minerals on the ECM of arteries, is a pathological condition associated with increased morbidity in patients with chronic kidney disease, type 2 diabetes, atherosclerosis, and certain genetic disorders (Shanahan et al., 2011; Heinz, 2021; Schmelzer and Duca, 2022). Several studies have analyzed minerals formed in cardiovascular tissues (Danilchenko et al., 2013; Cottignoli et al., 2015), and found that the calcification process may resemble the multistep mechanism observed during the formation of the mineral component of bone (Gourgas et al., 2018). Arterial calcification is marked by the initial adsorption of Ca²⁺ ions, the progressive formation of calcium phosphates, and their subsequent transformation into hydroxyapatite and carbonated hydroxyapatite (Gourgas et al., 2018). However, further studies are necessary to validate these findings.

In vitro models offer a valuable approach to study arterial calcification. Early models, utilizing solubilized elastin fragments, allowed to elucidate the biochemical principles of calcification (Starcher et al., n. d.; Sobel et al., 1966). However, these models had significant limitations, including poor solubility, handling difficulties, processability issues, and the presence of impurities and contaminants that could impact results. In contrast, ELRs have emerged as precise in vitro elastin models for calcification. ELRs address the limitations of earlier models, allowing the creation of systems from highly pure, composition-controlled, and tailor-made materials, allowing high reproducibility. Particularly notable contributions have been made by Cerruti and coworkers, who, using recombinant polymers based on cross-linking and hydrophobic domains TE exon repeats, allow for controlled self-assembly and cross-linking to form biomimetic gels and membranes (Figure 2). These membranes allowed the development of an in vitro model for medial calcification (Gourgas et al., 2019b; 2019a; Parashar et al., 2021; Lau et al., 2022).

The model, employing cross-linked ELR membranes immersed in simulated body fluid (SBF), replicates mineral phase evolution observed in mouse models. This allows an understanding of the topographical factors influencing crystal formation (Gourgas et al., 2019b), the creation of models of calcification adapted to different pathological conditions (Gourgas et al., 2019a), and the study of interactions with calcification inhibitors (Parashar et al., 2021). Although it proves to be a promising model as a potential drug screening platform, future directions involve optimizing coacervation and cross-linking conditions to enhance biomimicry, enabling a more comprehensive understanding of medial calcification processes and the development of potential treatments.

While the ability of elastin to calcify poses a significant medical challenge, it has also been employed to create biohybrid innovative materials with properties mimicking those of natural mineralized tissues that may serve as bone or enamel in vitro models. One critical property for controlling mineral deposition is the degree of structural disorder and order in the proteins of our scaffold and their capacity to form hierarchical assemblies (Elsharkawy and Mata, 2018). The interaction of inorganic ions with the disordered regions of the protein leads to its transition to more ordered structures, and this conformational reorganization will mark the nucleation and growth of the crystal.

In the initial studies aimed at in vitro production of organic-inorganic composites from ELRs, a biomimetic approach was chosen based on incorporating the peptide DDDEEKFLRRIGRFG into the ELR backbone at the sequence level. This peptide corresponds to the SNA15 domain of statherin, a salivary IDP that regulates calcium and phosphate mineralization of enamel (Gil Mur, 2016). However, despite the ability of statherin-derived peptides to promote mineralization in ELR membranes, hydrogels, or coatings (Li Y. et al., 2014; 2015; Tejeda-Montes et al., 2014; Shuturminska et al., 2017), it was observed that the self-assembly capacity of ELRs played a crucial role in promoting crystal nucleation. Studies with ELRs in solution already showed that molecular designs leading to supramolecular aggregates in suspension had a greater capacity to induce crystal nucleation (Prieto et al., 2011; Misbah et al., 2016). The crystal morphology could be guided depending on their ability to self-assemble into defined nanostructures (i.e., micelles) or amorphous coacervates. Later, Aparicio and coworkers studied the mineralization of ELR fibrils in vitro using the polymer-induced liquid-precursor (PILP) process (Li et al., 2017). They compared the ability to achieve intrafibrillar mineralization in ELRs with different sequences and observed two highly relevant events. Firstly, the coacervation of ELRs was necessary to induce mineralization, and secondly, fibers derived from ELRs without bioactive sequences, once mineralized, were much more stable than those presenting different functional motifs, whether cell-adhesive or statherin-derived peptides. This demonstrated that the introduction of charged peptides into the ELR backbone determines its self-assembly and will alter its balance between ordered and disordered structures, directly impacting the in vitro mineralization of the protein material.

Mata and coworkers systematically studied the control of the degree of structural disorder and order in self-assembled ELR membranes through chemical crosslinking (Elsharkawy et al., 2018). Modulating the degree of disordered structures allows controlling and programming the calcium phosphate nucleation and hierarchical growth of hydroxyapatite crystals, similar to what occurs in dental enamel. This way, mimicking its mechanical properties is achieved. Moreover, this process can be applied to different surface topographies, creating customized models of mineralized tissue. The tunability of the elastin-like matrices together with the nanotopography enables precise spatiotemporal control of the directionally guided growth of hierarchically organized mineralized structures over millimeter length scales, which has implications for developing biomimetic models with advanced functionalities for applications in materials science and tissue regeneration (Deng et al., 2021).

Spheroids have been widely used in TERM as in vitro 3D models. These 3D structures present high cell-cell and cell-ECM interactions enhancing cell viability, protein secretion, and stem cell differentiation due to the increased chemical/mechanical interactions (Kim et al., 2020). The culture methods employed for spheroid fabrication maximize cell-cell interactions (Caprio and Burdick, 2023) and can be broadly divided in scaffold-free and scaffold-based approaches. Scaffold-free approaches, such as hanging drop, magnetic levitation, pellet culture, or spinner flask techniques, have been broadly used to construct spheroids (Decarli et al., 2021). However, these methods involve labor-intensive processes, low throughput, and difficulty in spheroid size control. Consequently, scaffold-based approaches using functional biomaterials have been developed to produce high-throughput, homogeneous spheroids (Ryu et al., 2019). Scaffold-based approaches can be roughly divided into hydrogels, where cells are embedded into the scaffold, and solid scaffold, where cells are seeded atop the scaffold (Table 1) (Knight and Przyborski, 2015; Lee and Lee, 2022).

The encapsulation of cells in a hydrogel, a popular option for 3D cultures (Tibbitt and Anseth, 2009), can be designed to support specific types of cell growth and function by encapsulating cells in an artificial ECM environment that promotes spheroid formation (Heywood et al., 2004; Topman et al., 2013). Cells can be encapsulated into the gels through various methods, including self-assembly, radical polymerizations induced by UV exposure, as well as ionic or chemical cross-linking. In contrast, seeding cells onto coated surfaces entails the prior application of biomaterial solid scaffolds, followed by cell deposition atop the scaffold (Knight and Przyborski, 2015). Solid scaffolds may feature specific patterns conducive to spheroid formation in designated areas, promoting tissue-like mass aggregation and enhancing cell-cell interactions over cell-surface interactions (Ursini-Siegel, 2023).

These scaffold-based approaches may consist of natural materials such as fibrin, hyaluronic acid, collagen, or Matrigel, as well as synthetic materials like PEG and poly (-caprolactone) (PCL) (Lee and Lee, 2022). Drawbacks observed in biological matrices like Matrigel (i.e., poor mechanical properties, inherent variability, and high sensitivity to enzymes) or in synthetic polymers (i.e., biofunctionalization requirement, poor biocompatibility, slow water absorption, and poor toughness) underscore the necessity for the development of a biocompatible, reproducible, and cost-effective matrix with control over mechanical and chemical properties (Wu X. et al., 2021; Wu Y. et al., 2021).

The use of ELRs offers multiple advantages over the traditional materials used for designing an optimal matrix for spheroid fabrication in a scaffold-based approach. ELRs allow the construction of an adaptable platform with a high degree of fabrication control over scaffold properties such as porosity, surface polarity, mechanical strength, and rate degradation (Fernández-Colino et al., 2018; Fernández-Colino et al., 2018; Wang et al., 2018; Wang et al., 2018; Flora et al., 2019; Flora et al., 2019; Shayan et al., 2023; Shayan et al., 2023). For instance, although limited diffusion in hydrogels has proven advantageous in fields such as controlled release of drugs and nutrient delivery in agriculture, it can hinder the prolonged survival of cells (Jongpaiboonkit et al., 2008). Therefore, incorporating protease-sensitive sequences into these ECM matrices could expedite their degradation, addressing issues related to extended cell survival (Flora et al., 2019).

Organoids have gained significant attention for their potential applications in modeling tissue development, understanding diseases, and advancing personalized medicine, drug screening, and cell therapy (Hofer and Lutolf, 2021). They are also a promising tool to limit the need for animal experimentation. These scale-down models of organs can be generated for a variety of organs, such as the gut, stomach, kidney, liver, pancreas, mammary glands, prostate, upper and lower airways, thyroid, retina, and brain (Takasato et al., 2015; McCracken et al., 2017; Workman et al., 2017; Hu et al., 2018).

Despite their potential, the translation of organoids to real-life applications remains a significant challenge. One of the biggest obstacles to clinical translation is the ill-defined growth environments of organoids, leading to a high variability and heterogeneity in cellular composition and in the resulting organoid phenotype (Hofer and Lutolf, 2021). Relying only on cell-intrinsic self-organization limits external control over fate and morphogenesis and often lacks stromal, vascular and immunological components (Zhao et al., 2022). Therefore, it is crucial to design environments that not only support stem cell maintenance but also enable precise spatiotemporal modulation of bioactive cues to guide organoid growth leading to a best understanding of organogenesis.

Examples of this approach include the application and combination of hydrogel chemistries and organ-on-a-chip technology. This integration aims to better mimic the stem cell niche by delivering and presenting in vivo-based biochemical cues, as well as incorporating biophysical and topological parameters often lacking in traditional culture systems (Kratochvil et al., 2019; Hofer and Lutolf, 2021; Zhao et al., 2022). Based on these insights, engineered matrices offer the potential for enhanced control over supplied morphogenic signals in both space and time, allowing precise control over stem cell decisions during organoid development (Hofer and Lutolf, 2021).

In the pursuit of organoids as models for organs, numerous researchers seek to address diseases that impact a significant portion of the population, such as type 1 diabetes (Xu et al., 2018). This condition leads to the destruction of pancreatic beta cells, with the transplantation of cadaveric human islets emerging as a promising alternative (Ames et al., 2000; Shapiro et al., 2016). However, the clinical demand surpasses the available supplies (Walker et al., 2022). For this reason, several research groups have explored the production of functional beta-like cells from pluripotent stem cells (Pagliuca et al., n. d.; Walker et al., 2022). Many of these methods rely on the use of Matrigel, but its undefined composition makes it difficult to understand which factors are responsible for differentiation, as well as tuning of its mechanical properties, that are critical in stem cell differentiation (Goldstein et al., 2011). A recent breakthrough in the ability of ELR to support the survival and growth of primary endocrine cells and endocrine progenitor cells in a 3D space was reported by Kozlowski et al. The group introduced a novel method for culturing pancreatic endocrine-like cells without the use of Matrigel (Kozlowski et al., 2023). ELRs containing cell-binding ECM peptides derived from fibronectin (i.e., ELR-FN) (Nakamura et al., 2022) or laminin alpha 3 (i.e., ELR-LAMA3) (Tjin et al., 2014), essential for pancreatic endocrine functions, were engineered. For this study, the researchers chose the 158A mutant to match the stiffness of the Matrigel-methylcellulose medium as closely as possible. They found that 2% solutions of ELR-FN or ELR-LAMA3 form semisolid matrices with elastic moduli comparable to the ideal modulus for human forebrain and human and mouse intestine, and to that of the Matrigel-methylcelluose medium, which is known to support the growth of primary murine pancreatic ductal progenitor cells. (Jiang et al., 2002). These ELRs are flanked by leucine zipper domains from rat cartilage oligomeric matrix protein, forming bundles in solution, leading to the formation of a physically cross-linked hydrogel (Figure 4B right). Both protein hydrogels supported the survival and growth of primary endocrine cells and endocrine progenitor cells in 3D space, compared to the control culture containing Matrigel, which favors ductal cell formation, and to DMEM-F12 medium, where cells remain in suspension and re-aggregate. Pancreatic progenitor cells cultured in ELR-FN or ELR-LAMA3 exhibited several types of colonies termed “grape-like”, “budding ring” and “proto-ring”, more endocrine in nature. However, Matrigel-methylcellulose medium employed in recent studies by this group supported the growth of “MM Ring” colonies, more duct-like and proliferative (Figures 4A,C right). Thus, their study provides a proof-of-concept cell culture matrix for endocrine cell differentiation in young mice without Matrigel.

Despite the utilization of various complex materials in the development of 3D pancreatic organoids (i.e., Matrigel (Huch et al., 2013), decellularized intestinal tissues (Giobbe et al., 2019) and decellularized pancreatic tissues (Sackett et al., 2018)), these results are consistent with the possibility of growing various progenitor cell types in 3D culture without Matrigel. Furthermore, the tunability of the mechanical and chemical properties of the protein elastin-like hydrogels described makes them valuable for conducting mechanistic studies on endocrine cell differentiation.

In an effort to overcome the limited tunability and reproducibility associated with Matrigel (Kozlowski et al., 2021), synthetic matrices have been developed to support the formation of induced pluripotent stem cell (iPSC)-derived intestinal organoids (Gjorevski et al., 2016; Cruz-Acuña et al., 2017; Hushka et al., 2020). Mohimani et al. utilized PEG as a synthetic polymer but faced challenges due to its interaction with the immune system and antibody formation (Mohimani et al., 2017).

Addresing these limitations, Heilshorn’s team introduced a tunable and fully defined matrix termed based on hyaluronan ELR composite protein (HA-ELR). (Hunt et al., 2021). This matrix enables the formation, growth, passaging, and differentiation of human intestinal organoids from dissociated patient-derived intestinal cells (Figure 4A left) Elastin constitutes a significant portion of the intestinal ECM, while HA serves as a crucial glycosaminoglycan supporting normal intestinal growth and interacting with the CD44 receptor responsible for maintaining and proliferating intestinal stem cells. This rationale led them to develop a reproducible hydrogel incorporating these two components. HA-ELR gels were formed through straightworward bioconjugations reactions by modifying hydrazine-bearing ELRs and benzaldehyde-bearing HA, thus leading to the formation of dynamic covalently cross-linked hydrogels (Figure 4B left). Human, patient-derived enteroids embedded within HA-ELR exhibited de novo spheroid formation within 3 days. This contrasts with ELR matrices, which were unable to support enteroid formation, indicating the necessity of HA for robust enteroid formation. Additionally, enteroids formed in HA-ELR exhibited proper intestinal epithelial polarity similar to decellularized matrix derived from Engelbreth-Holm-Swarm mouse sarcoma (e.g., Matrigel). HA-ELR matrices can be enzymatically degraded using elastase and hyaluronidase, facilitating enteroid dissociation into single cells for subsequent passages. Successful new enteroid formation for up to 12 passages without visible change in morphology was achieved. To assess whether HA-ELR could support the differentiation of enteroids, cells embedded in HA-ELR were allowed to form enteroids for 10 days before differentiation into mature organoids over 5 days. This resulted in the formation of undulating lumens, indicative of mature intestinal cell subtypes (Figure 4C left). The study specified matrix stiffness, stress relaxation rate, and integrin-ligand concentration independently and quantitatively, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. This work shows that HA-ELR provides a promising 3D in vitro model for understanding intestinal development and enteric disease, devoid of animal-derived products or synthetic materials with clinical translation issues. It supports enteroid growth and differentiation akin to animal-derived matrices, offering a reproducible, biodegradable, minimal matrix for potential clinical applications.

Although 3D organoid tissue models have an invaluable valor to study several biological processes, several techniques such as immunostaining, mechanosensing, mechanotransduction and optical strategies have been refined over the years for application in 2D cultures (Yousafzai and Hammer, 2023). For this reason, current limitations in processing organoids limit the capacity for higher-throughput analysis. Adapting these techniques to 3D culture models will enhance the advancement of our understanding of cellular behavior and facilitate organoids processing. In this context, Kaplan’s team fabricated smart material hydrogel transfer devices utilizing stimuli-responsive silk-elastin-like recombinamers (SELRs) for use in organoid histological processing (Parker et al., 2020). The procedure involves transferring organoids to SELR hydrogel, followed by subjecting the material to an increase of the temperature (65°C) to induce hydrogel contraction, securing the organoids and enabling the creation of multisample constructs, and allowing placement on a microscope slide. Histological processing (i.e., hematoxylin and eosin staining) and immunostaining of these organoids using SELR hydrogels demonstrated the maintenance of cerebral organoid features compared to controls without the hydrogel carrier system. The utility of SELR devices stems from their optimal design, which not only maximizes material surface area but also enhances throughput and ensures the proper fit of organoids. The future investigation of SELR constructs holds potential for the development of materials on a broader scale, particularly for applications such as clinical diagnostic screening of patient tissue samples.

Organs-on-a-chip (OOAC) are sophisticated systems that incorporate engineered or natural miniature tissues cultivated within microfluidic chips, to replicate the specific functions of living human organs (Leung et al., 2022). These systems offer a 3D environment responsive to mechanical, electrical, and chemical stimuli in small samples, thereby reducing the need for substantial amounts of reagents or extended analysis times (Osório et al., 2021). OOAC represents an animal-free technology with predictive capabilities, ultimately reducing costs and time associated with pre-clinical testing.

Examples of emerging OOAC technologies include heart-on-a-chip, lung-on-a-chip, kidney-on-a-chip, bone-on-a-chip, liver-on-a-chip, and skin-on-a-chip where most of them are based on poly(dimethylsiloxane) (PDMS) chip with a Matrigel matrix.(Leung et al., 2022) However, the materials currently employed in OOAC research face challenges that can compromise the reliability of these systems as pre-clinical models (Danku et al., 2022). A comprehensive review by Luana A. Osório et al. (Osório et al., 2021) discusses the various biomaterials employed in OOAC systems and their associated limitations. ELRs proven properties have a great potential of application to solve the existint limitations of the materials commonly used in OOAC technologies. For instance, PDMS, biocompatible material extensively employed in biofabrication due to its excellent transparency and elasticity, exhibits poor chemical resistance, absorbing certain organic compounds, drugs, and biomolecules (Koyilot et al., 2022). While PDMS is widely used in lung-on-a-chip owing to its flexibility, it remains a non-degradable material that fails to contribute to the formation of a natural ECM (Huh, 2015). The biodegradable properties of the ELRs could help to solve this problem. Another material commonly used is Matrigel, a biocompatible material with similar mechanical properties to natural ECM and used in liver-on-a-chip. However, Matrigel introduces variability in experimental results due to batch-to-batch differences (Jang et al., 2019). Conversely, the recombinant design and bioproduction of ELR provides batch-to-batch consistency and extreme control over composition (Walker, 2009). Collagen, one of the major components of ECM, offers favorable permeability, biocompatibility, and enzymatic degradation, enabling cellular remodeling of the ECM gel. Although widely used in cardiac, hepatic and gut-on-a-chip, collagen lacks mechanical strength and structural stability when hydrated (Arlk et al., 2021). ELR-collagen scaffolds have demonstrated superior mechanical attributes compared to pure collagen scaffolds, thereby influencing cellular morphology and differentiation potential (Amruthwar et al., 2013). Similarly, fibrin, another biocompatible and biodegradable material, allows gel modeling at room temperature but exhibits weak mechanical properties. Fibrin hydrogels have been extensively studied for tissue engineering, but they show rapid degradation and contraction over time and low mechanical properties. To achieve better mechanical properties and elasticity, Stojic et al. incorporated ELRs network into plasma-derived fibrin hydrogels (Stojic et al., 2021). Their results demonstrated that incorporation of ELRs significantly increased mechanical properties, as determined in tension and oscillatory shear, when ELR content is equal or higher than 3%. This superior behavior is also reflected in terms of shrinkage, both in the absence and presence of cells, where an increase of scaffold stability was observed with the increase of ELR content. In contrast, synthetic biomaterials offer regulable mechanical properties but necessitate evaluation of immune responses due to potential cytotoxic effects. ELRs are non-immunogenic (Ibáñez-Fonseca et al., 2020) and the biocompatibility of ELR-based hydrogels formed via physical or chemical crosslinking has been established (Ibáñez-Fonseca et al., 2018).

These challenges, alongside the advantages offered by OOAC in pre-clinical settings, have spurred rapid developments in biomaterials and manufacturing techniques. However, each biomaterial presents limitations in achieving the optimal scaffold for these systems. Consequently, there is a concerted effort towards the development of 3D scaffolds capable of meeting multiple criteria, including biocompatibility, biodegradability, pore size, and suitable mechanical properties. The significant advantages of ELRs highlighted throughout this section underscore the potential of ELRs as a promising material for future applications in this field. These advantages, as discussed, not only showcase the potential of ELRs to address the limitations associated with current biomaterials, but also strongly advocate for their integration into OOAC systems.

3D bioprinting, arising from additive manufacturing, involves the automated and precise deposition of cells, biomaterials, and bioactive molecules, collectively forming a “bioink.” It addresses the limitations of conventional 2D platforms by enabling the creation of scaffolds with tailored structural and biochemical properties (Jain et al., 2022; Lam et al., 2023; Gold et al., 2019; Matai et al., 2020; Ahadian and Khademhosseini, 2018; Agarwal et al., 2020; Noroozi et al., 2023; Aazmi et al., 2024). This capability is pivotal for generating in vitro models that accurately mimic the intricate features of native tissues, facilitating regenerative medical endeavors (Nam et al., 2015; Langhans, 2018; Bédard et al., 2020; Hwang et al., 2021). Recent examples have highlighted the potential of 3D bioprinting in creating in vitro models of human tissues and diseases, including notable applications such as: accurate modeling of the complex tumor microenvironments (Kort-Mascort et al., 2023; Mu et al., 2023; Zhou et al., 2023); physiologically relevant models for the entire respiratory tract (Moura et al., 2023); replication of native skin features for wound healing studies (Norahan et al., 2023); engineered cardiac tissues such as in vitro cardiac models and vascular channels (Lu et al., 2023); or normal and disease hepatic tissue models (Sun et al., 2023).

Various commonly used 3D bioprinting methods such as extrusion, inkjet, and laser-assisted methods contribute to the precise control of spatial cell distribution and the surrounding microenvironment (Filipa Duarte Campos et al., 2016) (Derakhshanfar et al., n. d.) (Li et al., 2020). Furthermore, innovative approaches such as liquid-in-liquid printing (Luo et al., 2019) (Chen et al., n. d.), 3D Embedded Printing (Miller et al., 2012; Kyle and Jessop, 2017; Aazmi et al., 2022; Wu et al., 2022a; Wu et al., 2022b; Li et al., 2022; Budharaju et al., 2024), Freeform Reversible Embedding of Suspended Hydrogels (FRESH) printing (Hinton et al., 2015; Lee et al., 2019), Suspended Layer Additive Manufacturing (SLAM) (Grigoryan et al., 2019; Noor et al., 2019; Senior et al., 2019; Shapira et al., 2020), and light-based vat-polymerization techniques such as Volumetric bioprinting (VAM) (Bernal et al., 2019; 2022; Größbacher et al., 2023; Levato et al., 2023; Ribezzi et al., 2023), have emerged as a potential tool to print soft materials, which is particularly intricate (Puertas-Bartolomé et al., 2020; Budharaju et al., 2024).

However, despite significant advances in bioprinting techniques in the last years, bioprinting faces a significant challenge in the limited repertoire of suitable bioinks compared to the diverse biomaterials used in traditional tissue engineering scaffolds (Levato et al., 2020) (Gungor-Ozkerim et al., n. d.; Hull et al., 2021). Various factors contribute to this limitation, including biocompatibility, rheological properties, crosslinking and gelation characteristics, and mechanical properties. The selection of suitable bioinks is critical for successful bioprinting. On the one side, a bioink must not only be compatible with the printing process but also create a conducive environment for cell survival, growth, and differentiation, as well as specific cues to meet (Ashammakhi et al., 2019; Urciuolo et al., 2023) (Ihalainen et al., n. d.) the unique needs of different cell types, fostering a more accurate representation of the native tissue environment. On the other hand, the bioink should have a specific viscosity and shear-thinning behavior to facilitate extrusion through the printing nozzle, support the layer-by-layer construction, and maintain shape fidelity and structural integrity of the printed construct (Gungor-Ozkerim et al., n. d.; Malda et al., 2013) (Gungor-Ozkerim et al., n. d.; Malda et al., 2013). Additionally, the mechanical properties of bioinks are crucial for mimicking native tissue characteristics (Mandrycky et al., 2016).

Researchers are actively addressing the challenge of the restricted current range of bioinks by exploring novel biomaterials and refining existing formulations to expand the bioink toolkit (Gungor-Ozkerim et al., n. d.) (Gungor-Ozkerim et al., n. d.; Hull et al., 2021) and enable the creation of more complex and functional tissues. Hydrogels are currently the most versatile biomaterials used in bioink formulations. closely resembling the ECM mechanical properties, composition, and achitecture (Gungor-Ozkerim et al., 2018; Valot et al., n. d.; Tibbitt and Anseth, 2009; Gopinathan and Noh, 2018; Li et al., 2020). (Chemistry and 2020, n. d.). Naturally derived bioinks such as alginate, collagen, fibrin, gelatin, silk fibroin, hyaluronic acid, and decellularized extracellular matrix have been often used for the preparation of bioinks. They offer an environment comparable to the extracellular matrix, and essential properties like cell adhesion and proliferation (Gopinathan and Noh, 2018; Li et al., 2020). However, these materials often face challenges such as low batch-to-batch reproducibility due to their natural origin, and relatively low mechanical properties and structural drawbacks (Fedorovich et al., 2007; Gasperini et al., 2014; Ahadian and Khademhosseini, 2018). Furthermore, polysaccharides such as alginate and hyaluronic acid have yet to be functionalized to allow cell adhesion and proteolytic degradation. Bioinks based on synthetic materials like PLGA, pluronic acid, PEG, PLA, and PCL have also been commonly employed due to their superior printability (Gopinathan and Noh, 2018; Li et al., 2020). However, these synthetics struggle to provide an optimal cell environment and must be modified to enable functions such as cell adhesion, proliferation, or proteolytic degradation (Smith et al., 2004; Censi et al., 2011; Chang et al., 2011; Zhang C. et al., 2016). Thus, a potential breakthrough lies in the development of biomimetic inks, merging the biological advantages of natural components with the design flexibility of synthetic polymers.