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Hyaluronic acid (HA), a major glycosaminoglycan of the brain extracellular matrix, modulates cell behaviors through binding its receptor, Cd44. In this study, we assessed the influence of HA on high-grade brain tumors in vitro. The model comprised cell cultures derived from six rodent carcinogen-induced brain tumors, forming 3D spheroids prone to spontaneous fusion. Supplementation of the standard culture medium with 0.25% HA significantly inhibited the fusion rates, preserving the shape and size uniformity of spheroids. The 3D cultures were assigned to two groups; a Cd44lo group had a tenfold decreased relative expression of Cd44 than another (Cd44hi) group. In addition, these two groups differed by expression levels of Sox2 transcription factor; the correlation analysis revealed a tight negative association for Cd44 and Sox2. Transcriptomic responses of spheroids to HA exposure also depended on Cd44 expression levels, from subtle in Cd44lo to more pronounced and specific in Cd44hi, involving cell cycle progression, PI3K/AKT/mTOR pathway activation, and multidrug resistance genes. The potential HA-induced increase in brain tumor 3D models' resistance to anticancer drug therapy should be taken into account when designing preclinical studies using HA scaffold-based models. The property of HA to prevent the fusion of brain-derived spheroids can be employed in CNS regenerative medicine and experimental oncology to ensure the production of uniform, controllably fusing neurospheres when creating more accurate in vitro brain models. [ABSTRACT FROM AUTHOR]

Hyaluronic acid (HA), a major glycosaminoglycan of the brain extracellular matrix, modulates cell behaviors through binding its receptor, Cd44. In this study, we assessed the influence of HA on high-grade brain tumors in vitro. The model comprised cell cultures derived from six rodent carcinogen-induced brain tumors, forming 3D spheroids prone to spontaneous fusion. Supplementation of the standard culture medium with 0.25% HA significantly inhibited the fusion rates, preserving the shape and size uniformity of spheroids. The 3D cultures were assigned to two groups; a Cd44lo group had a tenfold decreased relative expression of Cd44 than another (Cd44hi) group. In addition, these two groups differed by expression levels of Sox2 transcription factor; the correlation analysis revealed a tight negative association for Cd44 and Sox2. Transcriptomic responses of spheroids to HA exposure also depended on Cd44 expression levels, from subtle in Cd44lo to more pronounced and specific in Cd44hi, involving cell cycle progression, PI3K/AKT/mTOR pathway activation, and multidrug resistance genes. The potential HA-induced increase in brain tumor 3D models’ resistance to anticancer drug therapy should be taken into account when designing preclinical studies using HA scaffold-based models. The property of HA to prevent the fusion of brain-derived spheroids can be employed in CNS regenerative medicine and experimental oncology to ensure the production of uniform, controllably fusing neurospheres when creating more accurate in vitro brain models.

Cells with various structures and proteins naturally come together to cooperate in vivo. This study used cell spheroids cultured in agarose micro-wells as a 3D model to study the movement of cells or spheroids toward other spheroids. The formation dynamics of tumor spheroids and the interactions of two batches of cells in the agarose micro-wells were studied. The results showed that a concave bottom micro-well (diameter: 2 mm, depth: 2 mm) prepared from 3% agarose could be used to study the interaction of two batches of cells. The initial tumor cell numbers from 5 × 103 cells/well to 6 × 104 cells/well all could form 3D spheroids after 3 days of incubation. Adding the second batch of DU 145 cells to the existing DU 145 spheroid resulted in the formation of satellite cell spheroids around the existing parental tumor spheroid. Complete fusion of two generation cell spheroids was observed when the parental spheroids were formed from 1 × 104 and 2 × 104 cells, and the second batch of cells was 5 × 103 per well. A higher amount of the second batch of cells (1 × 104 cell/well) led to the formation of independent satellite spheroids after 48 h of co-culture, suggesting the behavior of the second batch of cells towards existing parental spheroids depended on various factors, such as the volume of the parental spheroids and the number of the second batch cells. The interactions between the tumor spheroids and Human Umbilical Vein Endothelial Cells (HUVECs) were modeled on concave agarose micro-wells. The HUVECs (3 × 103 cell/well) were observed to gather around the parental tumor spheroids formed from 1 × 104, 2 × 104, and 3 × 104 cells per well rather than aggregate on their own to form HUVEC spheroids. This study highlights the importance of analyzing the biological properties of cells before designing experimental procedures for the sequential fusion of cell spheroids. The study further emphasizes the significant roles that cell density and the volume of the spheroids play in determining the location and movement of cells. [ABSTRACT FROM AUTHOR]

3D self-assembly of biological tissues is essential for organ development, tissue engineering, transplantation, and organ regeneration. Although multicellular spheroids have been broadly used as 3D tissue models, it is challenging to precisely manipulate their interaction, limiting their potential in understanding and modeling complex tissue self-assembly. Here, we present a novel acoustofluidic device for the controllable fusion of multicellular spheroids. By introducing standing sound waves into a microfabricated chamber, our acoustofluidic device could achieve the trapping and assembling of multicellular spheroids in a highly biocompatible, label-free, and contact-free manner. After incorporating with a time-lapse imaging and in situ culture approach, our acoustofluidic system could manipulate and track the dynamic interaction of the multicellular spheroids. Moreover, the interaction dynamics of homotypic and heterotypic spheroid pairs were quantitatively characterized, and the adhesion molecules of paired spheroids were analyzed. Together, we developed a unique engineering tool, which is contactless, versatile, and biocompatible, all of which make it useful for 3D biological self-assembly and translational applications in regenerative medicine. [ABSTRACT FROM AUTHOR]

While decades of research have enriched the knowledge of how to grow cells into mature tissues, little is yet known about the next phase: fusing of these engineered tissues into larger functional structures. The specific effect of multicellular interfaces on tissue fusion remains largely unexplored. Here, a facile 3D‐bioassembly platform is introduced to primarily study fusion of cartilage–cartilage interfaces using spheroids formed from human mesenchymal stromal cells (hMSCs) and articular chondrocytes (hACs). 3D‐bioassembly of two adjacent hMSCs spheroids displays coordinated migration and noteworthy matrix deposition while the interface between two hAC tissues lacks both cells and type‐II collagen. Cocultures contribute to increased phenotypic stability in the fusion region while close initial contact between hMSCs and hACs (mixed) yields superior hyaline differentiation over more distant, indirect cocultures. This reduced ability of potent hMSCs to fuse with mature hAC tissue further underlines the major clinical challenge that is integration. Together, this data offer the first proof of an in vitro 3D‐model to reliably study lateral fusion mechanisms between multicellular spheroids and mature cartilage tissues. Ultimately, this high‐throughput 3D‐bioassembly model provides a bridge between understanding cellular differentiation and tissue fusion and offers the potential to probe fundamental biological mechanisms that underpin organogenesis. [ABSTRACT FROM AUTHOR]

Abstract While decades of research have enriched the knowledge of how to grow cells into mature tissues, little is yet known about the next phase: fusing of these engineered tissues into larger functional structures. The specific effect of multicellular interfaces on tissue fusion remains largely unexplored. Here, a facile 3D‐bioassembly platform is introduced to primarily study fusion of cartilage–cartilage interfaces using spheroids formed from human mesenchymal stromal cells (hMSCs) and articular chondrocytes (hACs). 3D‐bioassembly of two adjacent hMSCs spheroids displays coordinated migration and noteworthy matrix deposition while the interface between two hAC tissues lacks both cells and type‐II collagen. Cocultures contribute to increased phenotypic stability in the fusion region while close initial contact between hMSCs and hACs (mixed) yields superior hyaline differentiation over more distant, indirect cocultures. This reduced ability of potent hMSCs to fuse with mature hAC tissue further underlines the major clinical challenge that is integration. Together, this data offer the first proof of an in vitro 3D‐model to reliably study lateral fusion mechanisms between multicellular spheroids and mature cartilage tissues. Ultimately, this high‐throughput 3D‐bioassembly model provides a bridge between understanding cellular differentiation and tissue fusion and offers the potential to probe fundamental biological mechanisms that underpin organogenesis.

At long time scales, tissue spheroids may flow or appear solid depending on their capacity to reorganize their internal structure. Understanding the relationship between intrinsic mechanical properties at the single cell level, and the tissue spheroids dynamics at the long-time scale is key for artificial tissue constructs, which are assembled from multiple tissue spheroids that over time fuse to form coherent structures. The dynamics of this fusion process are frequently analyzed in the framework of liquid theory, wherein the time scale of coalescence of two droplets is governed by its radius, viscosity and surface tension. In this work, we extend this framework to glassy or jammed cell behavior which can be observed in spheroid fusion. Using simulations of an individual-cell based model, we demonstrate how the spheroid fusion process can be steered from liquid to arrested by varying active cell motility and repulsive energy as established by cortical tension. The divergence of visco-elastic relaxation times indicates glassy relaxation near the transition toward arrested coalescence. Finally, we investigate the role of cell growth in spheroid fusion dynamics. We show that the presence of cell division introduces plasticity in the material and thereby increases coalescence during fusion.

Reconstruction of large 3D tissues based on assembly of micro-sized multi-cellular spheroids has gained attention in tissue engineering. However, formation of 3D adipose tissue from spheroids has been challenging due to the limited adhesion capability and restricted cell mobility of adipocytes in culture media. In this study, we addressed this problem by developing adipo-inductive nanofibers enabling dual delivery of indomethacin and insulin. These nanofibers were introduced into composite spheroids comprising human adipose-derived stem cells (hADSCs). This approach led to a significant enhancement in the formation of uniform lipid droplets, as evidenced by the significantly increased Oil red O-stained area in spheroids incorporating indomethacin and insulin dual delivery nanofibers (56.9 ± 4.6%) compared to the control (15.6 ± 3.5%) with significantly greater gene expression associated with adipogenesis ( C/EBPA, PPARG, FABP4 , and adiponectin) of hADSCs. Furthermore, we investigated the influence of culture media on the migration and merging of spheroids and observed significant decrease in migration and merging of spheroids in adipogenic differentiation media. Conversely, the presence of adipo-inductive nanofibers promoted spheroid fusion, allowing the formation of macroscopic 3D adipose tissue in the absence of adipogenic supplements while facilitating homogeneous adipogenesis of hADSCs. The approach described here holds promise for the generation of 3D adipose tissue constructs by scaffold-free assembly of stem cell spheroids with potential applications in clinical and organ models., (© 2024 IOP Publishing Ltd.)

Three-dimensional (3D) cell culture has been used in many fields of biology because of its unique advantages. As a representative of the 3D systems, 3D spheroids are used as building blocks for tissue construction. Larger tumor aggregates can be assembled by manipulating or stacking the tumor spheroids. The motivation of this study is to investigate the behavior of the cells distributed at different locations of the spheroids in the fusion process and the mechanism behind it. To this aim, spheroids with varying grades of maturity or age were generated for fusion to assemble micro-tumor tissues. The dynamics of the fusion process, the motility of the cells distributed in different heterogeneous architecture sites, and their reactive oxygen species profiles were studied. We found that the larger the spheroid necrotic core, the slower the fusion rate of the spheroid. The cells that move were mainly distributed on the spheroid's surface during fusion. In addition to dense microfilament distribution and low microtubule content, the reactive oxygen content was high in the fusion site, while the non-fusion site was the opposite. Last, multi-spheroids with different maturities were fused to complex micro-tissues to mimic solid tumors and evaluate Doxorubicin's anti-tumor efficacy., (© 2024 IOP Publishing Ltd.)

Tissue engineering-based therapies rely on the delivery of monolayered fibroblasts on two-dimensional polystyrenecoated and extracellular matrix (ECM) surfaces to regenerate connective tissues. However, this approach may fail to mimic their three-dimensional (3D) native architecture and function. We hypothesize that ECM fibrous proteins, which direct the migration of cells in vivo, may attach and guide polystyrene- and Matrigel TM-ECM (M-ECM)-adherent fibroblasts to rearrangement into large multicellular macrostructures with the ability to proliferate. Gingival monolayered fibroblasts and their derived spheroids were added and adhered to tissue culture polystyrene and M-ECM surfaces. The cells were covered with a layer of collagen1 hydrogel combined with vitronectin, fibronectin or fibrin, or 10% M-ECM. The development of 3D cell constructs was characterized by epifluorescence and confocal scanning microscope image analysis. The ECM turnover and the proliferative capabilities of the fibroblasts were determined via gene expression profiling of collagen1, fibronectin, matrix metalloproteinase/metallopeptidase 2, Nanog, and SRY (sex-determining region Y)- box2 (Sox2). Expression of the Sox2 protein was followed by immunostaining. The collagen1 protein had the strongest effect on monolayered and spheroid cell rearrangements, forming large spherical shapes and fused 3D macroconstructs. The addition of fibrin protein was typically required to achieve a similar effect on M-ECM-adherent monolayered fibroblasts. The spheroid fusion process was followed by an increase in cell density and the formation of tight clusters. The fused spheroids continued to maintain their intracellular ECM turnover and proliferation capacities. Collagen1 is a valuable component in the rearrangement of adherent fibroblast monolayers and spheroids. Fibroblast spheroids should preferably be used as basic building blocks to assemble multicellular connective tissue-like macrostructures. [ABSTRACT FROM AUTHOR]

Aspiration-assisted freeform bioprinting (AAfB) has emerged as a promising technique for precise placement of tissue spheroids in three-dimensional (3D) space enabling tissue fabrication. To achieve success in embedded bioprinting using AAfB, an ideal support bath should possess shear-thinning behavior and yield-stress to facilitate tight fusion and assembly of bioprinted spheroids forming tissues. Several studies have demonstrated support baths for embedded bioprinting in the past few years, yet a majority of these materials poses challenges due to their low biocompatibility, opaqueness, complex and prolonged preparation procedures, and limited spheroid fusion efficacy. In this study, to circumvent the aforementioned limitations, we present the feasibility of AAfB of human mesenchymal stem cell (hMSC) spheroids in alginate microgels as a support bath. Alginate microgels were first prepared with different particle sizes modulated by blending time and concentration, followed by determination of the optimal bioprinting conditions by the assessment of rheological properties, bioprintability, and spheroid fusion efficiency. The bioprinted and consequently self-assembled tissue structures made of hMSC spheroids were osteogenically induced for bone tissue formation. Alongside, we investigated the effects of peripheral blood monocyte-derived osteoclast incorporation into the hMSC spheroids in heterotypic bone tissue formation. We demonstrated that alginate microgels enabled unprecedented positional accuracy (∼5%), transparency for visualization, and improved fusion efficiency (∼97%) of bioprinted hMSC spheroids for bone fabrication. This study demonstrates the potential of using alginate microgels as a support bath for many different applications including but not limited to freeform bioprinting of spheroids, cell-laden hydrogels, and fugitive inks to form viable tissue constructs., (© 2022 IOP Publishing Ltd.)

Bioengineering of 3D microtissues from cell spheroids is demonstrated by employing the vibration of acoustic standing waves and its hydrodynamic effect at the bottom of a liquid-carrier chamber. A large number of cell spheroids (>10(4) ) are assembled in seconds into a closely packed structure in a scaffold-free fashion under nodal pattern of the standing waves in a fluidic environment., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)