Spheroid-Hydrogel-Integrated Biomimetic System: A New Frontier in Advanced Three-Dimensional Cell Culture Technology

Three-dimensional (3D) cell culture has been a focus of research for decades, driven by the need to accurately mimic the natural cellular microenvironment in vitro [1‒3]. The potential of successful 3D cell culture to revolutionize our understanding of cell behavior, disease mechanisms, therapeutic strategies, drug screening, and tissue engineering is widely recognized. Traditional two-dimensional (2D) culture systems have shown limitations in reproducing the complexity of in vivo conditions, leading to the development of various 3D culture methods [4]. These 3D models show enhanced cell-cell and cell-matrix interactions, more accurate gene expression profiles, and improved responses to stimuli, providing more reliable platforms for biological research and drug discovery [5‒10].

Among the advances in 3D culture systems, organoid culture using Matrigel, a complex mixture of extracellular matrix (ECM) proteins, has emerged as one of the most advanced and widely adopted methods [11‒14]. Organoids have attracted considerable attention due to their ability to self-organize into structures that mimic the architecture and function of native organs and have found successful applications in developmental biology, disease modeling, and personalized medicine [5, 15, 16]. However, despite these advances, several challenges remain to the widespread adoption of 3D culture systems. Issues such as reproducibility, scalability, and standardization need to be addressed, and the high variability in experimental results due to the complex nature of 3D systems and the heterogeneity of commercial matrices such as Matrigel requires further research into the fundamental aspects of cell-matrix interactions [16‒19].

Although organoids can more closely mimic real tissues, current cell culture technologies and experimental limitations make it difficult to achieve reproducibility, with significant variation observed between individual organoids cultured under identical conditions [16, 20]. In addition, the generation and maintenance of organoids require considerable time and resources. In this context, cell spheroids remain an effective alternative in 3D cell culture, offering distinct advantages in terms of simplicity, reproducibility, and efficiency [21, 22]. They are particularly useful for early-stage research and large-scale screening, providing results that more closely resemble cells cultured in the real environment than 2D cell culture [22‒24]. Cells within spheroids form 3D structures that maintain cell-cell interactions, allowing the reproduction of physiological characteristics that are difficult to observe in 2D culture. For example, tumor spheroids can mimic features of solid tumors such as oxygen and nutrient gradients, proliferation gradients, and necrotic cores, making them widely used in cancer research [25, 26]. In addition, neural spheroids are used to mimic complex brain tissue structures, while liver spheroids are valuable in drug metabolism studies [27, 28].

However, spheroid culture has several notable limitations. Primarily, as a form of matrix-free 3D cell culture, it lacks the cell-matrix interactions that are critical in real tissues. This can significantly affect cell behavior and function as interactions with the matrix regulate important biological processes such as cell polarization, differentiation, and migration [29‒31]. The absence of these interactions can be particularly limiting when studying cell types that are highly dependent on the matrix, such as cancer or mesenchymal cells [32, 33]. In addition, each tissue has unique mechanical properties that are difficult to mimic or control by spheroid culture alone. The limitations in reproducing the stiffness, elasticity, and viscosity of real tissues can limit the study of cell types that are sensitive to mechanical stimuli (e.g., muscle cells, bone cells, cartilage cells) [34‒36]. This may lead to reduced physiological accuracy, with some cellular functions and responses differing from those in vivo.

To overcome these limitations, the combination of spheroids with hybrid systems incorporating matrices such as hydrogels should be considered. Hydrogels, widely used as cell scaffolds for matrix-based 3D cell culture, can greatly enhance cell-matrix interactions by dispersing cells within them, playing a critical role in cell adhesion, proliferation, differentiation, and functional expression [37‒39]. The 3D structure of hydrogels mimics the ECM of real tissues, allowing cells to grow in a more physiologically relevant environment. One of the key advantages of hydrogels is the ability to control their physicochemical properties. By adjusting stiffness, porosity, and degradation rate, different tissue types can be mimicked [37, 38]. In addition, the chemical composition of hydrogels can be modified to incorporate specific biomolecules or growth factors, allowing more precise control of cell behavior [40‒42]. Moreover, hydrogels can be derived from various sources, including natural and synthetic polymers, and formed through diverse crosslinking mechanisms, offering a wide range of properties suitable for different biomedical applications [43‒45]. For instance, soft hydrogels can be used to model brain tissue, while stiffer hydrogels can mimic muscle or bone [46]. Degradable hydrogels can be employed in drug delivery systems or tissue engineering applications where scaffold resorption is desired [47, 48]. Additionally, stimuli-responsive hydrogels can be utilized in smart drug delivery systems or dynamic cell culture models [38, 49, 50]. This versatility allows researchers to create tailored microenvironments that closely replicate specific tissue types or disease states, making hydrogels a crucial component in advancing 3D cell culture technologies like spheroid-hydrogel-integrated biomimetic system (SHIBS).

The restricted growth and migration of cells within hydrogels somewhat limit cell-cell interactions, which can be a limiting factor in mimicking high-density tissues [51‒53]. Therefore, the development of hybrid systems combining cell aggregates with hydrogels is necessary to provide both rich cell-cell and cell-matrix interactions. While Matrigel is currently the most widely used biomimetic hydrogel, its undefined chemical and physical properties limit the reproducibility of cell growth and culture conditions, posing a significant challenge for clinical therapeutic development [17, 19].

Future 3D cell culture research must focus on clearly defining and controlling the chemical and physical properties of the matrix. It is essential to use hydrogel materials with defined chemical and physical properties and to adjust the composition and preparation conditions to control the properties and obtain reproducible results. Research into the use of hydrogels with well-defined chemical compositions rather than decellularized extracellular matrices is actively progressing [17, 19]. These include both synthetic polymer-based hydrogels and certain natural polymer-based hydrogels. Synthetic polymer hydrogels offer advantages in precise control of physicochemical properties and selective introduction of specific biomolecules or growth factors, although they are known to lack biological properties [54, 55]. Meanwhile, some natural polymer-based hydrogels also exhibit low batch-to-batch variability and allow property control based on molecular weight. For example, purified alginate, hyaluronic acid, and gelatin can be used to produce hydrogels with consistent properties by controlling the molecular weight distribution [56‒58].

This review proposes a new 3D cell culture system based on spheroids and chemically defined hydrogels, called the SHIBS. SHIBS aims to provide a more physiologically relevant 3D culture environment by combining the advantages of spheroid culture, which excels in cell-cell interactions, with those of hydrogels, which provide cell-matrix interactions. This is expected to enhance tissue-specific functions, improve accurate prediction of drug responses, and advance disease modeling. By using chemically defined hydrogels, SHIBS can greatly enhance reproducibility and controllability, overcoming the limitations of using matrices with undefined compositions, such as Matrigel, and providing more accurate and consistent 3D culture models.

This review provides a comprehensive analysis of SHIBS, a cutting-edge approach in 3D cell culture technology. This review primarily focuses on studies published since 2020 that integrate spheroid and hydrogel technologies, providing a current perspective on the rapidly evolving field of SHIBS. By identifying key challenges and proposing innovative solutions, this review aims to guide future research and development in the field. Unlike previous reviews that have focused on either spheroids or hydrogels in isolation, this work uniquely integrates these two critical components and provides insights into their synergistic benefits. The review explores the broad applications of SHIBS in various biomedical fields, including drug screening, disease modeling, and regenerative medicine, and highlights their potential to revolutionize the development of more effective and safer therapies. By presenting a roadmap for the advancement of SHIBS, from basic research to clinical applications, this review provides a valuable resource for researchers and clinicians working toward more physiologically relevant in vitro models and personalized medicine approaches.

SHIBS is an advanced 3D cell culture approach developed in response to the need for more physiologically relevant in vitro models. This methodology combines the strengths of cell spheroid culture and hydrogel encapsulation while mitigating their individual limitations. SHIBS integrates two primary 3D cell culture technologies: cell spheroid culture, which is matrix-free, and hydrogel encapsulation, which is matrix-based. Figure 1 illustrates the key components and advantages of SHIBS, highlighting how this system integrates spheroids within a hydrogel matrix to mimic the physiological environment while preserving critical cell-cell and cell-ECM interactions.

Cell spheroid culture excels at mimicking cell-cell interactions and maintaining tissue-specific functions but lacks cell-ECM interactions [29, 30]. On the other hand, single-cell hydrogel encapsulation effectively simulates cell-ECM interactions and allows modulation of mechanical properties but struggles to reproduce high-density cell-cell interactions [59, 60]. SHIBS addresses these limitations by incorporating pre-formed spheroids into chemically defined hydrogels. This approach preserves the high-density cell-cell interactions within the spheroid core, while cells at the spheroid periphery interact with the surrounding ECM. Although not all cells within the spheroid directly interface with the ECM, this arrangement more closely mimics the natural tissue environment where cells experience a gradient of cell-cell and cell-ECM interactions. Thus, SHIBS provides a more physiologically relevant model by combining the benefits of both spheroid culture and hydrogel encapsulation.

The core principles of SHIBS work together to create a culture environment that closely mimics the complexity and functionality of native tissues. This system maintains a high density of cell-cell interactions, a key advantage inherited from spheroid culture, which is critical for preserving tissue-specific cellular organization and function [56, 58, 61, 62]. At the same time, it provides extensive cell-ECM interactions, a benefit derived from hydrogel encapsulation that is essential for replicating the natural cellular microenvironment and supporting proper cell behavior.

In addition, SHIBS offers controllability of the mechanical and biochemical microenvironments, another advantage of hydrogel systems, allowing researchers to fine-tune culture conditions for specific tissue types or disease states. While not all hydrogels inherently mimic native tissue characteristics, synthetic approaches and careful engineering allow the creation of hydrogels that closely approximate the biochemical and biomechanical properties of specific tissues. For example, Park et al. [61] used gelatin methacrylate (GelMA) hydrogels of varying stiffness (5–10%) to encapsulate HepG2 spheroids for improved hepatotoxicity assessment. Similarly, Aung et al. [56] used GelMA hydrogels (7–10%) with varying stiffness to create a tumor-on-a-chip device to study breast cancer-immune cell interaction. These studies highlight how the ability to modulate matrix properties in SHIBS can significantly influence cellular behavior and provide more physiologically relevant models. The integration of these features results in a more accurate replication of complex cellular behaviors and responses, bridging the gap between traditional in vitro cultures and in vivo conditions.

The potential of SHIBS has been demonstrated in several biomedical applications. In drug screening, Park et al. developed a SHIBS-based hepatotoxicity assessment model using HepG2 spheroids encapsulated in GelMA hydrogels of varying stiffness (5–10%) [61]. This model showed increased sensitivity in detecting drug-induced liver injury compared to conventional 2D cultures. In cancer research, Horder et al. [57] used a SHIBS model combining adipose-derived stromal cell spheroids with hyaluronic acid hydrogels to study breast cancer-adipose tissue interactions, observing changes in lipid content and ECM remodeling that mimic in vivo tumor progression. For tissue engineering applications, de Melo et al. [54] created cartilage-like tissue constructs using human mesenchymal stem cell (MSC) spheroids encapsulated in PEGDA-alginate interpenetrating network hydrogels, demonstrating the potential of SHIBS in cartilage regeneration through precise control of mechanical properties while maintaining high cell viability.

SHIBS consists of two key components: cell spheroids and chemically defined hydrogels. The selection and characteristics of these components are critical to the overall performance and applicability of SHIBS and are carefully chosen based on specific research objectives and the type of tissue being mimicked. Table 1 compares the key characteristics of SHIBS with conventional culture methods, highlighting its advantages in combining physiological relevance with controllable mechanical and biochemical properties while maintaining high cell-cell and cell-ECM interactions.

While detailed methods of spheroid fabrication have been extensively discussed in our previous work, it is important to highlight key aspects relevant to SHIBS [7]. Common spheroid generation methods include hanging drop, non-adhesive surface culture, and agitation-based techniques, each offering unique advantages in scalability, uniformity, and ease of use. Controlling spheroid size and shape is crucial for reproducibility in SHIBS, with techniques such as micromolding, microfluidics, and micropatterned substrates allowing for precise control over these parameters. The size of spheroids is a critical consideration; while larger spheroids can better replicate tissues, they may suffer from mass transfer limitations and potential necrotic cores. Additionally, larger spheroids can be challenging to analyze via microscopy and immunostaining due to limited dye penetration. Typically, spheroids with diameters of 100–300 μm are used in SHIBS to balance tissue mimicry with analytical accessibility [7]. The transfer process from spheroid formation to hydrogel encapsulation can present challenges such as spheroid disruption, potentially affecting the integrity and functionality of the resulting SHIBS. To address these issues, methods such as using micro-well plates with non-adhesive surfaces or temperature-sensitive surfaces are commonly employed to retrieve spheroids without damage [61, 63]. These considerations are vital when integrating spheroids with hydrogels in SHIBS as they significantly impact the system’s overall performance and reproducibility.

Cell spheroids play a critical role in SHIBS by reproducing tissue-specific functions and maintaining complex cell-cell interactions. These 3D cell aggregates can be formed from a variety of cell types, each serving a different research purpose. For example, in cancer research, spheroids generated from breast cancer cells (such as MCF-7 and MDA-MB-231), osteosarcoma cells (MG-63), neuroblastoma cells (STA-NB15), and human colorectal cancer cells are widely used in drug response and resistance studies [56, 57, 64‒67]. The work of Aung et al. with breast cancer models illustrates how these spheroids can effectively mimic the heterogeneity and 3D structure of actual tumors, thereby providing more accurate results in drug screening processes [56].

The application of spheroids in SHIBS goes beyond cancer research. In the field of stem cell research, spheroids composed of human MSCs are invaluable for differentiation and regenerative medicine studies [53, 54, 58, 68‒70]. The work of Wu et al. with bone marrow-derived MSCs and dental pulp stem cells demonstrates how spheroid culture allows a more accurate evaluation of stem cell differentiation capabilities and therapeutic potential in a 3D environment [53].

To develop tissue-specific models, hepatocyte spheroids, typically using HepG2 or HepaRG cells, are used in drug metabolism and toxicity studies [61, 71‒73]. These models, as seen in the study of Park et al. [61] using HepG2 cells, preserve liver-specific functions while allowing assessment of long-term drug toxicity. In the field of neural system modeling, spheroids composed of neurons derived from neural stem cells, astrocytes, neural progenitors derived from human induced pluripotent stem cells (hiNPCs), and neuroblastoma cells (SH-SY5Y) are used [55, 62, 74]. The 3D neuronal model developed by Kapr et al. [74] using hiNPCs illustrates the potential of these spheroids to study the pathophysiology of neurodegenerative diseases. It is worth noting that SHIBS can also incorporate co-culture spheroids, combining multiple cell types to more accurately mimic complex tissue environments [75, 76]. These advanced spheroids offer enhanced physiological relevance, particularly in modeling heterogeneous tissues or tumor microenvironments.

Complementing the spheroids in SHIBS are chemically defined hydrogels, which are preferred for their ability to increase the reproducibility and controllability of the system. These hydrogels can be broadly categorized into synthetic polymers and purified natural polymers. Among synthetic polymer hydrogels, polyethylene glycol (PEG)-based hydrogels are widely used due to their high biocompatibility and ease of functionalization [54, 55]. The work of Melo et al. in developing an interpenetrating network hydrogel using PEGDA and alginate demonstrates how PEG can be modified with different functional groups, allowing rapid and uniform gel formation through techniques such as photocrosslinking or click chemistry [54]. This versatility allows for easy tuning of mechanical strength to mimic different tissue types. Other synthetic polymers, such as poly(N-isopropyl acrylamide) (pNIPAAm), are used for their unique properties, such as temperature responsiveness [69]. While synthetic polymer hydrogels offer excellent control over mechanical properties and reproducibility, they often lack the biological cues present in natural extracellular matrices. For instance, PEG-based hydrogels, despite their tunable mechanics, do not inherently support cell adhesion or provide biochemical signals crucial for many cellular functions.

In the category of purified natural polymer hydrogels, materials such as gelatin, hyaluronic acid, alginate, and fibrin are commonly used [53, 56‒58, 64, 65]. GelMA, as used in the Aung et al. [56] study, is particularly favored for its ability to mimic tissue-specific ECM components while allowing photocrosslinking and adjustment of mechanical strength through concentration control. Alginate, as seen in the work of Wu et al. [53], is proving to be particularly suitable for bioprinting applications due to its rapid gelation properties and its utility in forming complex 3D structures when combined with other materials.

The effectiveness of SHIBS largely depends on the complementary selection of spheroids and hydrogels, optimized according to the target tissue or disease model. For example, when modeling hard tissues such as bone, the combination of high-stiffness hydrogels with osteoblast spheroids, as demonstrated in the study by Melo et al. [54], can more accurately reproduce the properties of the real tissue. Conversely, for softer tissues such as the brain, the use of low-stiffness hydrogels in conjunction with neural cell spheroids, as shown in the work of Kapr et al. [74] can more closely mimic the properties of the native tissue.

Through these varied combinations of spheroids and hydrogels, SHIBS offers the potential to more accurately mimic specific tissue types or disease states. This versatility makes SHIBS a valuable tool in several fields, including drug screening, disease mechanism research, and regenerative medicine. As SHIBS technology continues to evolve, it is expected to enable the creation of increasingly complex and sophisticated biomimetic systems, further enhancing its potential in biomedical research and applications. The ongoing refinement of both spheroid culture techniques and hydrogel formulations promises to expand the capabilities of SHIBS, potentially revolutionizing our approach to in vitro modeling and accelerating advances in drug discovery, disease understanding, and regenerative therapies.

The integration of spheroids with hydrogels is a critical factor in determining the performance and application potential of SHIBS. Several methods have been developed, each with its own strengths and limitations, and each suitable for different applications.

Photocrosslinking and ionic crosslinking are two widely used methods, each with unique advantages. Monteiro et al. [64] used photocrosslinking with 10% GelMA (gelatin methacrylate) and Irgacure 2959 photoinitiator to encapsulate MG-63 osteosarcoma spheroid. This method allows for rapid and uniform crosslinking, allowing the formation of complex 3D structures through spatially controlled polymerization. However, potential cytotoxicity from UV exposure and photoinitiators must be carefully managed. In contrast, Wu et al. [53] used ionic crosslinking with alginate-based hydrogels for bone marrow-derived MSC spheroids. This method, which uses calcium ions, has the advantage of crosslinking under physiological conditions, which may preserve cell viability better than photocrosslinking. However, it may provide less control over the final hydrogel structure and properties.

Recent advances have introduced enzyme-mediated crosslinking and thermosensitive hydrogels as promising alternatives. Xu et al. [77] used transglutaminase to crosslink gelatin in their study of cell migration, demonstrating the potential of enzyme-mediated crosslinking to form networks under mild conditions with minimal negative impact on cell viability. This method may be particularly valuable for sensitive cell types or applications that require gradual gelation. Morello et al. developed a temperature-responsive hydrogel using chitosan and pectin that gels at 37°C and remains injectable at room temperature [66]. This approach, used to encapsulate colorectal cancer spheroids, offers exciting possibilities for minimally invasive, injectable systems in regenerative medicine and cancer research. However, careful optimization of gelation kinetics and mechanical properties is critical to ensure proper spheroid distribution and support.

The advance of 3D bioprinting technology has opened new avenues for precise placement of spheroids and hydrogels. Nothdurfter et al. [65] created complex structures with neuroblastoma spheroids using a mixture of GelMA and fibrin hydrogels. This approach allows unprecedented control over the spatial arrangement of multiple cell types and hydrogel compositions, enabling the creation of more physiologically relevant tissue models. However, the complexity of bioprinting systems and the potential shear stress on cells during the printing process present challenges that need to be addressed.

Regardless of the integration method, maintaining the structural integrity of the spheroids during the crosslinking process is critical. Park et al. [61] demonstrated this by carefully adjusting the GelMA concentration (5–10%) when encapsulating HepG2 spheroids, balancing hydrogel stiffness to maintain spheroid structure while providing an appropriate physical environment. Similarly, Wu et al. highlighted the importance of hydrogel viscoelasticity, using alginates of different molecular weights to modulate MSC spheroid migration and fusion [53]. These studies highlight the need for thorough optimization of hydrogel properties for each specific application and cell type. This level of control is particularly important when dealing with the diverse mechanical requirements of different tissue types. For example, brain tissue models may require very soft hydrogels, while bone tissue models may need stiffer matrices. By using chemically defined hydrogels with tunable properties, SHIBS can accommodate this wide range of mechanical needs, allowing for more accurate tissue-specific modeling.

The variety of integration methods available for SHIBS provides researchers with a wide range of tools to create physiologically relevant 3D models. The choice between synthetic and natural hydrogels for spheroid encapsulation involves trade-offs. Synthetic hydrogels may offer better control over crosslinking and mechanical properties but might require additional modification to support cell-matrix interactions. Natural hydrogels inherently provide these interactions but may suffer from batch-to-batch variability and limited mechanical strength. As the field advances, combining these methods or developing hybrid approaches may lead to even more sophisticated and versatile SHIBS platforms. The choice of integration method should be guided by the specific requirements of the target tissue model or application, considering factors such as cell type sensitivity, desired mechanical properties, and the need for spatial control. Future research in this area will likely focus on further refining these methods to improve their biocompatibility, scalability, and ability to recapitulate complex tissue architectures.

Biomimicry in the context of tissue engineering refers to the creation of artificial systems that closely replicate the structural, functional, and biochemical properties of native tissues. SHIBS excels at reproducing the complex microenvironments found in native tissues, offering significant advantages over traditional culture methods. This section will explore how SHIBS enhances biomimicry by replicating tissue-specific cellular organization and function, recreating physiological gradients and zonation, and mimicking ECM composition and mechanical properties. These features collectively contribute to creating more physiologically relevant models for studying cellular behavior and tissue function, bridging the gap between traditional in vitro cultures and in vivo conditions.

Wang et al. [78] demonstrated how chondrocyte spheroids in GelMA/HAMA hydrogels maintained their phenotype and created physiologically relevant hypoxic conditions, mimicking the natural cartilage environment. The study found higher HIF-1α expression in the spheroids, indicating better maintenance of the chondrocyte phenotype. Similarly, Park et al. showed that HepG2 spheroids in gelatin hydrogels exhibited enhanced expression of drug-metabolizing enzymes and more closely mimicked liver function [61].

Of particular note is the ability of the system to recapitulate physiological gradients within 3D structures. Oxygen gradients, nutrient diffusion, and metabolic zonation can be accurately modeled, as seen in the liver spheroid study [61]. This capability is particularly relevant for tissues with complex architectures and metabolic profiles, such as liver and cartilage.

Vahala et al. [79] further demonstrated the ability of the system to mimic the tumor microenvironment by using GelAGE hydrogels with a linear stiffness gradient. They observed that spheroid volume decreased with increasing stiffness, while protein expression (TRPV4, integrin β1, E-cadherin, F-actin) also varied with stiffness, closely mimicking the heterogeneity found in real tumors.

This enhanced biomimicry of tissue microenvironments in SHIBS offers significant advantages for studying complex biological phenomena that are difficult to observe in traditional 2D cultures or even simpler 3D models. By more closely mimicking the spatial organization, cell-cell interactions, and biochemical gradients found in native tissues, SHIBS enables researchers to study subtle aspects of tissue function, such as zone-specific gene expression patterns, cellular responses to localized signaling cues, and the impact of tissue architecture on cell behavior. This level of biomimicry is particularly valuable for understanding tissue-specific disease mechanisms, developmental processes, and cellular responses to various stimuli in a more physiologically relevant context.

SHIBS offers precise control over the mechanical and biochemical properties of the cellular microenvironment. While synthetic hydrogels excel in providing precise control over mechanical properties, they often require additional functionalization to mimic the biochemical complexity of natural tissues. Conversely, purified natural hydrogels offer inherent bioactivity but may present challenges in achieving the same degree of mechanical tunability. This section highlights studies demonstrating control over mechanical properties, biochemical properties, or both, showcasing the versatility of SHIBS in mimicking various tissue types and disease states.

The control of mechanical properties in SHIBS has been demonstrated in various studies, highlighting its importance in cellular behavior. Bruns et al. [80] showed how varying hydrogel stiffness affects glioblastoma spheroids, with softer hydrogels promoting invasion and stiffer hydrogels altering drug sensitivity. Extending this to dynamic systems, Nguyen et al. used viscoelastic stiffening hydrogels to study pancreatic cancer progression, observing that increasing matrix stiffness promoted epithelial-mesenchymal transition and malignant phenotypes [81].

The impact of matrix stiffness on cellular mechanics and behavior was further elucidated by some studies. Taubenberger et al. [82] demonstrated that increased matrix stiffness leads to increased cellular stiffness through ROCK and F-actin-dependent mechanisms, which subsequently affects cell cycle progression. This mechano-regulation of the cell cycle has profound implications for understanding tissue homeostasis and disease progression. The effect of mechanical properties on stem cell fate and differentiation is particularly noteworthy. Yin et al. [83] showed that MSCs in hydrogels of moderate stiffness (∼880 Pa) exhibited higher expression of the stemness markers Sox2, Oct4, and Nanog.

Recent advancements in SHIBS have begun to incorporate control over both mechanical and biochemical properties. Li et al. [84] demonstrated that MSC spheroids combined with collagen and black phosphorus in biodegradable hydrogels showed improved viability and osteogenic differentiation, especially at specific concentrations of black phosphorus. This study exemplifies the potential of SHIBS to provide a more comprehensive mimicry of the in vivo environment.

The precise control of mechanical and biochemical properties offered by SHIBS provides researchers with a powerful tool for dissecting the complex interactions between cells and their microenvironment. This level of control enables the systematic study of how specific environmental cues influence cellular behavior, gene expression, and function. By allowing researchers to isolate and manipulate individual parameters, SHIBS facilitates the identification of key factors that drive cellular processes in both normal and pathological conditions.

Dynamic modeling in the context of SHIBS refers to the ability to reproduce and study time-dependent changes in the cellular microenvironment that occur during physiological and pathological processes. This approach allows researchers to observe and analyze how cells respond to changing conditions over time, mimicking the dynamic nature of living tissues. By incorporating elements that can change over time, such as matrix stiffness, biochemical gradients, or cellular composition, SHIBS enables the study of complex biological phenomena that are difficult to capture in static models. This capability is particularly valuable for understanding developmental processes, disease progression, and cellular adaptation to changing environments.

The ability of SHIBS to model dynamic physiological processes is particularly evident in cancer research and tissue engineering applications. Major et al. developed a composite hydrogel using adipose-derived ECM and silk fibroin that progressively stiffened over 3 weeks, mimicking the evolving mechanical landscape of breast tissue during tumor progression [85]. They observed that MCF-7 breast cancer cells exhibited growth arrest and phenotypic changes in response to increasing matrix stiffness, highlighting the importance of temporal mechanical cues in cancer models. Building on this concept, Cai et al. [86] investigated how matrix confinement influences cell sorting and collective migration in 3D spheroids. By dynamically altering collagen-alginate hydrogel stiffness using calcium and calcium chelators, they found that high confinement triggered cell sorting while reducing confinement facilitated collective cell invasion in sorted spheroids. This work demonstrates how SHIBS can be used to study complex cellular behaviors that depend on both intercellular adhesion and matrix properties, providing insights into tumor progression and metastasis.

In the field of tissue engineering, SHIBS has shown promise in modeling the development of vascularized tissues. Kwon et al. [87] used co-cultured spheroids of human adipose-derived stem cells and human umbilical vein cells encapsulated in gelatin methacrylate hydrogels to create pre-vascularized 3D tissues. By optimizing parameters such as co-culture ratio, hydrogel strength, and pre-vascularization time, they were able to engineer tissues that formed complex vascular networks in vitro and successfully integrated with host blood vessels upon transplantation. This study highlights the potential of SHIBS in creating dynamic models that can transition from in vitro to in vivo applications.

The versatility of SHIBS in modeling various physiological processes is further exemplified by the work of Chen et al., who developed β-cyclodextrin-crosslinked polyacrylamide hydrogels for 3D tumor model construction [88]. By tuning the hydrogel's mechanical properties and β-cyclodextrin density, they created a platform capable of capturing cells and promoting spheroid formation. Importantly, their system allowed for the co-culture of tumor cells with fibroblasts, mimicking the complexity of the tumor microenvironment and demonstrating the potential of SHIBS in creating more realistic disease models.

The work of Thai et al. [89] on degradable PEG hydrogels demonstrated how matrix degradation affects cell spreading and metabolic activity, providing insight into wound healing processes. They found that degradable hydrogels increased MMP-2 secretion, which promoted cell spreading and metabolic activity, which is critical for wound healing processes. This study showcases how SHIBS can be used to model dynamic cellular responses to changing matrix properties, a key aspect of many physiological and pathological processes. Zhang et al. used temperature-responsive hydrogels to study the differentiation of stem cell spheroids [90]. They found that dynamic mechanical stimulation by temperature changes promoted osteogenesis over adipogenesis, demonstrating how SHIBS can be used to study the effects of dynamic mechanical cues on stem cell fate. This application of SHIBS in stem cell research opens up new possibilities for understanding and controlling cellular differentiation in regenerative medicine.

The ability of SHIBS to model dynamic physiological processes represents a significant advance in our understanding of complex biological systems. By capturing the temporal aspects of cellular responses to changing environments, SHIBS provides unique insights into the adaptive mechanisms of cells and tissues that are often overlooked in static models. This dynamic modeling capability is particularly valuable for studying processes that unfold over time, such as tissue regeneration, disease progression, and cellular adaptation to stress. As SHIBS technology continues to evolve, it promises to provide increasingly sophisticated models of physiological processes, bridging the gap between in vitro experiments and in vivo reality.

SHIBS offers unique advantages in modeling complex multi-cellular and multi-tissue systems, allowing the study of cell-cell interactions and tissue-tissue interfaces in a more physiologically relevant context. This capability is critical for understanding the intricate dynamics of organ systems where multiple cell types interact within a structured 3D environment. By enabling the co-culture of different cell types within a controllable hydrogel matrix, SHIBS provides a platform for investigating how cellular heterogeneity contributes to tissue function and disease progression.

The power of SHIBS to reconstruct complex tissue architectures is exemplified by the work of Daly et al. [91] in cardiac microtissue. In their study, they observed organized gap junction formation through connexin-43 expression, a key feature of functional cardiac tissue. This ability to create structured, multi-cellular environments opens the possibility of modeling complex organ systems where the spatial organization of different cell types is critical for proper function. Such models can provide insights into developmental processes, disease mechanisms, and potential therapeutic interventions that are difficult to obtain from simpler in vitro systems or animal models.

The versatility of SHIBS extends to the study of complex biological processes such as angiogenesis, which involves the coordinated action of multiple cell types. Vorwald et al. [92] demonstrated this by investigating the role of Notch3 signaling in promoting blood vessel formation using mixed spheroids of endothelial and mesenchymal stromal cells. Their findings that mixed spheroids formed more consistent vascular networks than shell spheroids, with higher Notch3 expression in mixed spheroids, highlight the importance of cellular organization and signaling in tissue development. This type of study demonstrates how SHIBS can be used to dissect complex cellular interactions and signaling pathways in a controlled, 3D environment.

SHIBS also holds great promise for creating more realistic models of the tumor microenvironment, which is characterized by complex interactions between cancer cells and surrounding stromal cells. Horder et al. used SHIBS to create a 3D breast cancer-adipose tissue model by bioprinting adipose-derived stromal cell spheroids with breast cancer cells [57]. This approach allowed them to observe intricate cell-cell and cell-matrix interactions, including cancer cell-induced lipid depletion and ECM remodeling in adipose tissue. Such models provide a more accurate representation of the tumor microenvironment than traditional 2D cultures or single-cell 3D cultures and offer new insights into cancer progression and potential therapeutic targets.

Further exemplifying the versatility of SHIBS in modeling complex tissue environments, Lazzari et al. developed a triple co-culture system to mimic pancreatic tumor complexity [76]. Their model incorporated pancreatic cancer cells, fibroblasts, and endothelial cells in a single spheroid, successfully reproducing key aspects of the pancreatic tumor microenvironment, including its characteristic fibrosis and aberrant vasculature. This sophisticated SHIBS model demonstrated increased resistance to anticancer treatments compared to simpler models, highlighting the critical role of the tumor microenvironment in drug response.

The versatility of SHIBS in multi-cellular and multi-tissue modeling represents a significant leap forward in our ability to replicate and study complex biological systems in vitro. This capability allows researchers to study intricate cellular interactions and communication networks that are critical to tissue function and disease progression but are often lost in simpler models. By enabling the co-culture of multiple cell types in physiologically relevant spatial arrangements, SHIBS provides a platform for studying how different cell populations influence each other's behavior, metabolism, and gene expression.

SHIBS significantly improves the predictive power of in vitro models for drug development and toxicology studies, potentially reducing the need for animal testing and improving the efficiency of drug discovery pipelines. Park et al. demonstrated that 3D HepG2 spheroid models in SHIBS showed increased expression of cytochrome P450 family proteins compared to 2D cultures, indicating superior predictive power for drug metabolism and hepatotoxicity assessment [61]. They observed significant differences in drug response, particularly for acetaminophen, with IC₅₀ values varying widely between 2D and 3D cultures.

Hong et al. developed a 3D-printed HepG2 liver spheroid model for high-content in situ quantification of drug-induced liver toxicity [71]. This approach provided a platform for more reliable biological information through high-content monitoring, demonstrating the potential of SHIBS in advanced drug screening.

The system also allows for more accurate prediction of drug response heterogeneity. Kim et al. [93] used a microfluidic system to create cell aggregates of different shapes and demonstrated improved viability and gene expression in certain configurations. Rosary-shaped aggregates showed higher neurogenic gene expression, including NeuroD1, highlighting the importance of 3D structure in cellular function and drug response.

Bayat et al. used SHIBS to study the apoptotic effect of atorvastatin on glioblastoma spheroids [94]. They found that the 3D model provided more physiologically relevant drug responses, with atorvastatin inducing apoptosis through caspase-8 and caspase-3 pathways in a dose-dependent manner.

In summary, SHIBS represents a paradigm shift in 3D cell culture technology, offering unparalleled advantages in biomimicry, control, dynamic modeling, versatility, and predictive power. These advantages synergistically enhance our ability to model complex biological systems and processes, pushing the boundaries of what’s possible in in vitro research.

As we look to the future, the true potential of SHIBS lies in its ability to bridge the gap between simplified in vitro models and the complexity of in vivo systems. By providing a more physiologically relevant platform, SHIBS has the potential to accelerate drug discovery, improve our understanding of disease mechanisms and pave the way for personalized medicine approaches.

The diverse potential applications of SHIBS technology across various fields of biomedical research are summarized in Figure 2, demonstrating its broad impact from drug discovery to regenerative medicine. SHIBS has the potential to revolutionize the drug discovery and development process by providing more physiologically relevant models for drug screening and testing. Park et al. [61] demonstrated that HepG2 spheroids in gelatin hydrogels exhibited enhanced expression of drug-metabolizing enzymes and more closely mimicked liver function. This improved biomimicry could lead to more accurate predictions of drug efficacy and toxicity in the early stages of drug development.

Building on this foundation, Antunes et al. [75] demonstrated the potential of SHIBS in modeling complex disease states. They developed a 3D co-culture system using prostate cancer cells and human osteoblasts in photocrosslinkable hydrogels, mimicking prostate cancer-to-bone metastasis. This co-culture SHIBS model showed increased resistance to cisplatin compared to traditional spheroid models, emphasizing the importance of including ECM components in drug screening platforms. Such advancements in multi-cellular SHIBS models could enable more accurate predictions of drug efficacy and toxicity, particularly in complex tissue contexts.

These advancements in SHIBS technology pave the way for more personalized approaches to drug discovery. By creating SHIBS models that incorporate a patient’s own cells, researchers could predict individual drug responses, paving the way for truly personalized medicine approaches. In addition, the ability of SHIBS to maintain long-term cultures could enable the study of chronic drug effects and toxicity, addressing a significant gap in current drug development processes.

The complex, multi-cellular nature of SHIBS also opens up the possibility of studying drug interactions in more realistic tissue contexts. This could be particularly valuable for developing combination therapies or understanding drug effects in the context of specific disease microenvironments, potentially leading to more effective and safer therapeutic strategies.

SHIBS offers unique advantages for modeling complex diseases, especially those involving multiple cell types and tissue structures. Horder et al. [57] used SHIBS to create a 3D breast cancer-adipose tissue model and observed complex cell-cell and cell-matrix interactions, including cancer cell-induced lipid depletion and ECM remodeling in adipose tissue. This type of model provides insights into tumor-stroma interactions that are difficult to achieve with simpler in vitro systems.

Looking to the future, SHIBS could be used to create even more sophisticated disease models. For example, in neurodegenerative disease research, SHIBS could be used to replicate the complex cellular interactions in the brain, including neurons, glia, and vascular cells. This could provide new insights into diseases such as Alzheimer’s or Parkinson’s, potentially revealing new therapeutic targets or allowing neuroprotective strategies to be tested in a more realistic context.

In the field of infectious diseases, SHIBS could be used to model the interaction between pathogens and human tissues. By incorporating immune cells into these models, researchers could study the dynamics of infection and immune response in a controlled, physiologically relevant environment. This could be particularly valuable for understanding and developing treatments for emerging infectious diseases.

The ability of SHIBS to maintain complex tissue structures and cellular functions for extended periods of time makes it an ideal platform for regenerative medicine and tissue engineering applications. Li et al. [84] demonstrated that MSC spheroids combined with collagen and black phosphorus in biodegradable hydrogels showed enhanced viability and osteogenic differentiation, highlighting the potential of SHIBS in bone tissue engineering.

Building on these findings, future applications of SHIBS in regenerative medicine could include the development of more complex, functional tissue constructs for transplantation. For example, researchers could use SHIBS to create vascularized tissue constructs, addressing one of the major challenges in tissue engineering. By incorporating endothelial cells and supporting cell types in specific spatial arrangements, it may be possible to guide the formation of blood vessel networks within engineered tissues.

SHIBS could also play a critical role in optimizing stem cell-based therapies. By providing a more physiologically relevant environment for studying stem cell behavior, differentiation, and integration into host tissues, SHIBS could help researchers develop more effective protocols for stem cell expansion and differentiation. This could lead to improved outcomes in a wide range of regenerative medicine applications, from treating spinal cord injuries to repairing damaged heart tissue.

SHIBS provides a powerful platform for basic biological research, allowing scientists to study cellular behavior and tissue formation processes in more physiologically relevant contexts. Zhang et al. [37] used temperature-responsive hydrogels to study the differentiation of stem cell spheroids, demonstrating how dynamic mechanical cues influence stem cell fate decisions.

In the future, SHIBS could be used to explore even more complex biological questions. For example, in developmental biology, SHIBS could be used to model key stages of embryonic development, providing insights into the mechanisms of tissue formation and organ development. This could lead to a deeper understanding of congenital disorders and potentially inform new strategies for prenatal intervention.

In the field of cancer biology, SHIBS could be used to study the entire process of metastasis, from the initial escape of cancer cells from a primary tumor to their colonization of distant tissues. By creating SHIBS models that incorporate multiple tissue types, researchers could track cancer cell behavior and identify factors that promote or inhibit metastasis at each stage of the process.

In addition, SHIBS could play a critical role in the study of complex cellular communication networks. By allowing the co-culture of multiple cell types in physiologically relevant spatial arrangements, SHIBS could help uncover new mechanisms of intercellular signaling and tissue homeostasis. This could have far-reaching implications for our understanding of both normal physiology and disease processes.

While SHIBS offers significant advantages in modeling complex biological systems, it also faces several challenges and limitations that must be addressed for its wider adoption and development. Recognizing and addressing these challenges is critical to the continued advancement of SHIBS technology. Table 2 outlines the principal challenges currently faced in SHIBS technology development and presents potential solutions for each, providing a roadmap for future research and technological advancements in this field.

One of the major challenges of SHIBS is its increased complexity compared to traditional 2D cell culture systems. As demonstrated by Nothdurfter et al. [65] in their study of vascularized neuroblastoma tumor environments, SHIBS involves several complicated steps, including spheroid formation, hydrogel preparation, and integration of these components. This complexity can lead to variability in results and challenges in reproducibility. For example, small differences in spheroid size or hydrogel properties can significantly affect experimental results. However, this complexity is a necessary trade-off for the enhanced biological relevance that SHIBS provides and can be managed through appropriate standardization and optimization protocols.

The transition from well-established 2D culture systems to complex 3D systems such as SHIBS represents a significant barrier to widespread adoption. Morello et al. [66] highlighted this challenge in their work on thermosensitive chitosan/pectin hydrogels for tumor spheroid culture. The behavior of cells in 3D SHIBS models may differ significantly from 2D cultures, requiring a reevaluation of established protocols and interpretations. This transition requires time, investment, and a paradigm shift in both academia and industry.

Scalability and adaptability to high-throughput screening remain major challenges for SHIBS. While studies such as Hong et al. [71] demonstrate the potential of SHIBS for assessing drug-induced liver toxicity, current SHIBS fabrication methods are often better suited for small-scale experiments. Adapting SHIBS for high-throughput screening, as required in the pharmaceutical industry, presents technical and logistical challenges that need to be addressed.

Analytical and imaging techniques also present technical difficulties in SHIBS. The 3D structure and often opaque nature of hydrogels can limit high-resolution imaging and real-time monitoring that is easily achieved in 2D cultures. This can make it difficult to observe cellular behavior or drug responses within the spheroid, especially in its core. Advanced imaging and analysis techniques need to be developed to realize the full potential of SHIBS.

To further enhance the physiological relevance of SHIBS, the incorporation of complex tissue structures such as vascularization is necessary. This challenge was highlighted by Kang et al. in their work to establish a 3D in vitro liver model [72]. While SHIBS can model certain aspects of tissue complexity, the creation of fully functional vascular networks remains a challenge. This limitation may affect long-term culture and the development of larger tissue models due to limitations in oxygen and nutrient diffusion. Another challenge in SHIBS is balancing the mechanical properties of hydrogels with the needs of spheroid growth and analysis. While stiff hydrogels like PEG-based systems offer excellent stability and control, they can restrict spheroid growth and make retrieval difficult. Conversely, soft hydrogels like pure collagen may not provide sufficient mechanical support. Ongoing research is focused on developing hydrogel systems that can dynamically adapt their properties over time, potentially allowing for initial stability followed by controlled degradation or softening to accommodate spheroid growth and facilitate analysis.

Regulatory considerations also present challenges for the clinical application of SHIBS. While 2D culture systems have well-established validation procedures recognized by regulatory agencies, standardization and validation guidelines for complex 3D models such as SHIBS are still evolving. This may limit the use of SHIBS in preclinical studies or drug approval processes, as highlighted by Ip et al. in their development of a human liver microphysiological co-culture system [73].

Finally, the high level of technical expertise and cost associated with SHIBS can be limiting factors. Compared to 2D cultures, the complex manufacturing processes and analytical methods require specialized personnel and expensive equipment, which may limit the widespread adoption of SHIBS. This was evident in the work of Kapr et al. [74] on human induced pluripotent stem cell-derived neural progenitors, which required sophisticated bioengineering techniques.

Despite these challenges, SHIBS continues to hold great promise. The transition from 2D to 3D models represents a necessary evolution in biomedical research. As evidenced by the diverse applications demonstrated in studies by Jury et al. [55] on bioorthogonally crosslinked hydrogels for 3D neuronal cell culture and Xu et al. [77] on biopolymer-based bicontinuous hydrogels for 3D cell migration, SHIBS has the potential to revolutionize various fields of biomedical research and drug development [55, 77]. It will be critical to overcome current limitations through continued research, technological development, and collaboration between academia and industry. This multidisciplinary approach will be essential to establish SHIBS as a key tool in future biomedical research and drug development, bridging the gap between in vitro models and in vivo reality.

The development of SHIBS represents a critical step in the advancement of in vitro modeling technologies, paving the way for more sophisticated systems in the future. Future developments in SHIBS may focus on creating advanced hybrid systems that combine the mechanical tunability of synthetic hydrogels with the bioactivity of natural materials, potentially offering the best of both worlds for more accurate tissue modeling. While the potential transition to organoid-hydrogel-integrated biomimetic systems is on the horizon, the immediate focus is on the rigorous validation and standardization of SHIBS, coupled with the integration of emerging technologies to enhance its capabilities.

The incorporation of microfluidic technology into SHIBS holds great promise for revolutionizing its functionality. As demonstrated by Li et al. [62] in their study of neurospheroid brain-like co-culture constructs, microfluidic systems can facilitate continuous nutrient supply and removal of metabolic waste, enabling long-term culture and providing a more physiologically relevant environment. This integration could greatly expand SHIBS applications, particularly in modeling complex, dynamic biological processes.

Advances in 3D bioprinting technologies will complement the development of SHIBS. The work of Melo et al. [54] on 3D-printed cartilage-like tissue constructs with spatially controlled mechanical properties demonstrates the potential of this technology. By enabling the precise spatial arrangement of multiple cell types and hydrogel compositions, 3D bioprinting could increase the complexity and accuracy of tissue models created using SHIBS.

The future of SHIBS is closely tied to advances in personalized medicine. Patient-derived SHIBS models could revolutionize drug response prediction and personalized treatment development. This approach could be particularly impactful in oncology and rare disease research, where individual variations significantly affect treatment outcomes.

Integration with organ-on-a-chip systems is another exciting direction for SHIBS development. By combining SHIBS with these platforms, as suggested by the work of Kang et al. [72] on 3D in vitro liver models, researchers could develop multi-organ models capable of studying complex interorgan interactions. This integration could dramatically improve our ability to predict systemic drug effects and toxicity.

The application of artificial intelligence (AI) and machine learning to SHIBS data analysis is poised to significantly enhance its predictive capabilities. By analyzing large data sets generated by SHIBS experiments, AI algorithms could identify complex biological patterns and predict drug responses with unprecedented accuracy, potentially streamlining the drug screening process and providing new biological insights.

However, the realization of these advances depends on the thorough validation and standardization of current SHIBS technology. The relative simplicity and reproducibility of spheroids make them ideal models for standardizing hydrogel-integration systems. As demonstrated by Jury et al. [55] in their work on bioorthogonally crosslinked hydrogels for 3D neuronal cell culture, establishing basic parameters for hydrogel-integrated systems via SHIBS will provide a critical foundation for future developments.

The standardization process should include establishing protocols for different tissue types and disease models, validating reproducibility and predictive power through large data sets, confirming consistency of results through multi-center studies, and working with regulatory agencies to establish data acceptance criteria. Figure 3 outlines the projected evolution of SHIBS technology, from current basic models to advanced systems integrating microfluidics and 3D bioprinting, ultimately aiming at fully functional tissue/organ models for personalized medicine applications.

In summary, SHIBS is a critical stepping stone to more advanced in vitro modeling systems. Its thorough validation, standardization, and integration with emerging technologies will provide a robust foundation for the development of increasingly complex and physiologically relevant in vitro models. These advances are expected to improve the efficiency of drug development processes, accelerate the progress of personalized medicine, and open new frontiers in biomedical research and medical technology.

SHIBS represents a significant advancement in 3D cell culture technology, bridging the gap between traditional in vitro models and the complexity of in vivo systems. This review provides a comprehensive overview of the principles, applications, current challenges, and future perspectives of SHIBS. SHIBS combines the strengths of spheroid culture and hydrogel technology to overcome the limitations of conventional 2D and 3D culture systems. By simultaneously providing enhanced cell-cell and cell-ECM interactions, SHIBS provides a more accurate mimicry of the in vivo environment. The system’s ability to precisely control mechanical and biochemical properties makes it adaptable for modeling different tissue types and disease states.

Applications of SHIBS span a wide range of fields, including drug discovery and development, disease modeling, regenerative medicine, and basic biological research. SHIBS has proven particularly valuable in improving the accuracy of drug screening, recapitulating the tumor microenvironment, and advancing stem cell research. However, challenges remain for the widespread adoption of SHIBS. These include ensuring reproducibility, scaling up production, developing high-resolution imaging techniques, and addressing regulatory considerations. Overcoming these hurdles will require continued collaboration between academia and industry.

Looking to the future, SHIBS is expected to serve as a critical foundation for the development of a more complex and physiologically relevant organoid-hydrogel-integrated biomimetic system. Integration with cutting-edge technologies such as microfluidics, 3D bioprinting, and AI promises to further expand the capabilities of SHIBS. Standardization and optimization of SHIBS remain key challenges for its future development. As reliability and predictive power improve through data accumulation and validation, SHIBS has the potential to revolutionize biomedical research and drug development processes.

In conclusion, SHIBS is the forefront of 3D cell culture technology, significantly enhancing the physiological relevance of in vitro models. With ongoing research and technological advancements, SHIBS is poised to become more sophisticated and widely applicable, ultimately contributing to the development of more effective and safer therapies, improved disease understanding, and the advancement of personalized medicine. The continued development and application of SHIBS are expected to open new frontiers in biomedical research and medical technology innovation.

The author has no conflicts of interest to declare.

This study was supported by the National Research Foundation (NRF) (Grant No. NRF-2021R1A6A1A03038996) and the Basic Science Research Capacity Enhancement Project through the Korea Basic Science Institute (National Research Facilities and Equipment Center) (Grant No. 2019R1A6C1010016) and funded by the Ministry of Science and ICT (MSIT), and the Gachon University Research Fund of 2024 (GCU-202400910001).

S.Y. contributed to the revisions of the manuscript. H.J.L. contributed to the conception of the manuscript and the original draft preparation which includes the collection of information from identified databases, constructing the evidence tables, analyzing the results, and drawing diagrams.

The author declares that all data supporting the findings of this study are available within the manuscript or are available from the corresponding author upon request.