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Aim: Lack of adequate mechanical strength and progressive shrinkage over time remain challenges in scaffold-free microtissue-based dental pulp regeneration. Surface collagen cross-linking holds the promise to enhance the mechanical stability of microtissue constructs and trigger biological regulations. In this study, we proposed a novel strategy for surface preconditioning microtissues using a natural collagen cross-linker, proanthocyanidin (PA). We evaluated its effects on cell viability, tissue integrity, and biomineralization of dental pulp stem cell (DPSCs)-derived 3D cell spheroids., Methodology: Microtissue and macrotissue spheroids were fabricated from DPSCs and incubated with PA solution for surface collagen cross-linking. Microtissue viability was examined by live/dead staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with transverse dimension change monitored. Microtissue surface stiffness was measured by an atomic force microscope (AFM). PA-preconditioned microtissues and macrotissues were cultured under basal or osteogenic conditions. Immunofluorescence staining of PA-preconditioned microtissues was performed to detect dentin sialophosphoprotein (DSPP) and F-actin expressions. PA-preconditioned macrotissues were subjected to histological analysis, including haematoxylin-eosin (HE), alizarin red, and Masson trichrome staining. Immunohistochemistry staining was used to detect alkaline phosphatase (ALP) and dentin matrix acidic phosphoprotein 1 (DMP-1) expressions., Results: PA preconditioning had no adverse effects on microtissue spheroid viability and increased surface stiffness. It reduced dimensional shrinkage for over 7 days in microtissues and induced a larger transverse-section area in the macrotissue. PA preconditioning enhanced collagen formation, mineralized nodule formation, and elevated ALP and DMP-1 expressions in macrotissues. Additionally, PA preconditioning induced higher F-actin and DSPP expression in microtissues, while inhibition of F-actin activity by cytochalasin B attenuated PA-induced dimensional change and DSPP upregulation., Conclusion: PA surface preconditioning of DPSCs spheroids demonstrates excellent biocompatibility while effectively enhancing tissue structure stability and promoting biomineralization. This strategy strengthens tissue integrity in DPSC-derived spheroids and amplifies osteogenic differentiation potential, advancing scaffold-free tissue engineering applications in regenerative dentistry., (© 2024 The Author(s). International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

Purpose: The insulin-like growth factor (IGF) system includes IGF-I, IGF-II insulin and their membrane receptors. IGF system also includes a family of proteins namely insulin-like growth factor-binding proteins (IGFBPs) composed for six major members (IGFBP-1 to IGFBP6), which capture, transport and prolonging half-life of IGFs. However, it has been described that IGFBPs can also have other functions., Methods: IGFBP5 expression was inhibited by shRNAs, migration was analyzed by scratch-wound assays, invasion assays were performed by the Boyden chamber method, spheroids formation assays were performed on ultra-low attachment surfaces, expression and phosphorylation of proteins were analyzed by Western blot., Results: IGFBP5 is a repressor of IGF-IR expression, but it is not a repressor of IR in MCF-7 breast cancer cells. In addition, IGFBP5 is a suppressor of migration and MMP-9 secretion induced by IGF-I and insulin, but it does not regulate invasion in MCF-7 cells. IGFBP5 also is a repressor of MCF-7 spheroids formation. However treatment with 340 nM rescues the inhibitory effect of IGFBP in the MCF-7 spheroids formation., Conclusion: IGFBP5 regulates IGF-IR expression, migration and MMP-9 secretion induced by IGF-I and/or insulin, and the spheroids formation in MCF-7 breast cancer cells., (© 2024. The Author(s).)

Background: In vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8-16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%)., Methods: Bovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7-8. Expanded blastocysts on days 7-8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing., Results: Under 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted., Conclusion: OES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development., (© 2024. The Author(s).)

Traditional microelectrode arrays (MEAs) are limited to measuring electrophysiological activity in two dimensions, failing to capture the complexity of three-dimensional (3D) tissues such as neural organoids and spheroids. Here, we introduce a flower-shaped MEA (e-Flower) that can envelop submillimeter brain spheroids following actuation by the sole addition of the cell culture medium. Inspired by soft microgrippers, its actuation mechanism leverages the swelling properties of a polyacrylic acid hydrogel grafted to a polyimide substrate hosting the electrical interconnects. Compatible with standard electrophysiology recording systems, the e-Flower does not require additional equipment or solvents and is ready to use with preformed 3D tissues. We designed an e-Flower achieving a curvature as low as 300 micrometers within minutes, a value tunable by the choice of reswelling media and hydrogel cross-linker concentration. Furthermore, we demonstrate the ability of the e-Flower to detect spontaneous neural activity across the spheroid surface, demonstrating its potential for comprehensive neural signal recording.

Background and Objectives: Techniques for imaging the mechanical properties of cells are needed to study how cell mechanics influence cell function and disease progression. Mechano-microscopy (a high-resolution variant of compression optical coherence elastography) generates elasticity images of a sample undergoing compression from the phase difference between optical coherence microscopy (OCM) B-scans. However, the existing mechano-microscopy signal processing chain (referred to as the algebraic method) assumes the sample stress is uniaxial and axially uniform, such that violation of these assumptions reduces the accuracy and precision of elasticity images. Furthermore, it does not account for prior information regarding the sample geometry or mechanical property distribution. In this study, we investigate the feasibility of training a conditional generative adversarial network (cGAN) to generate elasticity images from phase difference images of samples containing a cell spheroid embedded in a hydrogel., Methods: To construct the cGAN training and simulated test sets, we generated 30,000 artificial elasticity images using a parametric model and computed the corresponding phase difference images using finite element analysis to simulate compression applied to the artificial samples. We also imaged real MCF7 breast tumor spheroids embedded in hydrogel using mechano-microscopy to construct the experimental test set and evaluated the cGAN using the algebraic elasticity images and co-registered OCM and confocal fluorescence microscopy (CFM) images., Results: Comparison with the simulated test set ground truth elasticity images shows the cGAN produces a lower root mean square error (median: 3.47 kPa, 95 % confidence interval (CI) [3.41, 3.52]) than the algebraic method (median: 4.91 kPa, 95 % CI [4.85, 4.97]). For the experimental test set, the cGAN elasticity images contain features resembling stiff nuclei at locations corresponding to nuclei seen in the algebraic elasticity, OCM, and CFM images. Furthermore, the cGAN elasticity images are higher resolution and more robust to noise than the algebraic elasticity images., Conclusions: The cGAN elasticity images exhibit better accuracy, spatial resolution, sensitivity, and robustness to noise than the algebraic elasticity images for both simulated and real experimental data., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: BFK reports a relationship with OncoRes Medical Pty Ltd that includes equity or stocks. All other authors declare that there are no conflicts of interest related to this article., (Copyright © 2024. Published by Elsevier B.V.)

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications., (© 2024 The Authors. Small Methods published by Wiley‐VCH GmbH.)

Objective: This study aims to investigate the morphological and histological characteristics of three-dimensional cell spheroids derived from the uveal melanoma (UM) cell line C918 and assess the impact of luteolin on their cell viability., Methods: C918 cells were cultured in ultra-low adsorption 96-well plates, and morphological changes in C918 three-dimensional cell spheroids were observed over varying time intervals. Histological features of C918 multicellular spheroids cultured in ultra-low adsorption 6-well plates were examined using both HE staining and immunohistochemical staining. The CCK8 reagent was employed to measure the optical density at a 450 nm wavelength after 72-h treatments with varying luteolin concentrations in both two-dimensional and three-dimensional cultured C918 cells. The IC50 values were compared between the two culture conditions., Results: Over time in culture, the volume of C918 three-dimensional cell spheroids gradually increased, and an ischemic- and hypoxic-like region became evident within the spheroids on days 4 to 6 of culture. Histological staining demonstrated positive expression of cell viability marker antibodies (Ki67) and melanoma marker antibodies (MelanA, HMB45, S-100) in the multicellular spheroids from three-dimensional culture. CCK-8 experiments revealed that the IC50 values for luteolin in C918 cells were 183.50 μmol/L in three-dimensional culture and 16.19 μmol/L in two-dimensional culture after 72 h. Three-dimensional cultured C918 cells, treated with varying luteolin concentrations for 72 h, were observed under a microscope. The maximum cross-sectional area showed no statistically significant differences between the groups, but it was reduced in comparison to the control group., Conclusion: Three-dimensional cultured C918 cell spheroids exhibit histological characteristics similar to real tumors and are less responsive to luteolin than their two-dimensional counterparts. They offer a valuable model for anti-tumor drug screening., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Traumatic brain injury (TBI) is a major cause of disability and mortality worldwide. However, existing treatments still face numerous clinical challenges. Building on our prior research showing peripheral nerve-derived stem cell (PNSC) spheroids with Schwann cell-like phenotypes can secrete neurotrophic factors to aid in neural tissue regeneration, we hypothesized that repeated intrathecal injections of PNSC spheroids would improve the delivery of neurotrophic factors, thereby facilitating the restoration of neurological function and brain tissue repair post-TBI., Methods: We generated PNSC spheroids from human peripheral nerve tissue using suspension culture techniques. These spheroids were characterized using flow cytometry, immunofluorescence, and reverse-transcription polymerase chain reaction. The conditioned media were evaluated in SH-SY5Y and RAW264.7 cell lines to assess their effects on neurogenesis and inflammation. To simulate TBI, we established a controlled cortical impact (CCI) model in rats. The animals were administered intrathecal injections of PNSC spheroids on three occasions, with each injection spaced at a 3-day interval. Recovery of sensory and motor function was assessed using the modified neurological severity score (mNSS) and rotarod tests, while histological (hematoxylin and eosin, Luxol fast blue staining) and T2-weighted magnetic resonance imaging analyses, alongside immunofluorescence, were conducted to evaluate the recovery of neural structures and pathophysiology., Results: PNSC spheroids expressed high levels of Schwann cell markers and neurotrophic factors, such as neurotrophin-3 and Ephrin B3. Their conditioned medium was found to promote neurite outgrowth, reduce reactive oxygen species-mediated cell death and inflammation, and influence M1-M2 macrophage polarization. In the CCI rat model, rats receiving repeated triple intrathecal injections of PNSC spheroids showed significant improvements in sensory and motor function, with considerable neural tissue recovery in damaged areas. Notably, this treatment promoted nerve regeneration, axon regrowth, and remyelination. It also reduced glial scar formation and inflammation, while encouraging angiogenesis., Conclusion: Our findings suggest that repeated intrathecal injections of PNSC spheroids can significantly enhance neural recovery after TBI. This effect is mediated by the diverse neurotrophic factors secreted by PNSC spheroids. Thus, the strategy of combining therapeutic cell delivery with multiple intrathecal injections holds promise as a novel clinical treatment for TBI recovery., (© 2024. The Author(s).)

The field of bioelectronics is developing exponentially. There is now a drive to interface electronics with biology for the development of new technologies to improve our understanding of electrical forces in biology. This builds on our recently published work in which we show wireless electrochemistry could be used to grow bioelectronic functional circuitry in 2D cell layers. To date our ability to merge electronics with in situ with biology is 3D limited. In this study, we aimed to further develop the wireless electrochemical approach for the self-assembly of microwires in situ with custom-designed and fabricated 3D cancer spheroids. Unlike traditional electrochemical methods that rely on direct electrical connections to induce currents, our technique utilises bipolar electrodes that operate independently of physical wired connections. These electrodes enable redox reactions through the application of an external electric field. Specifically, feeder electrodes connected to a power supply generate an electric field, while the bipolar electrodes, not physically connected to the feeder electrodes, facilitate the reduction of silver ions from the solution. This process occurs upon applying a voltage across the feeder electrodes, resulting in the formation of self-assembled microwires between the cancer spheroids.Thereby, creating interlinked bioelectronic circuitry with cancer spheroids. We demonstrate that a direct current was needed to stimulate the growth of conductive microwires in the presence of cell spheroids. Microwire growth was successful when using 50 V (0.5 kV/cm) of DC applied to a single spheroid of approximately 800 µm in diameter but could not be achieved with alternating currents. This represents the first proof of the concept of using wireless electrochemistry to grow conductive structures with 3D mammalian cell spheroids., (© 2024. The Author(s).)

This study describes the formation of single-chain polymer dots (Pdots) via ultrasonic emulsification of nonionic donor-acceptor-donor type (D-A-D) alkoxy thiophene-benzobisthiadiazole-based conjugated polymers (Poly BT) with amphiphilic cetyltrimethylammonium bromide (CTAB). The methodology yields Pdots with a high cationic surface charge (+56.5 mV ± 9.5) and average hydrodynamic radius of 12 nm. Optical characterization reveals that these Pdots emit near-infrared (NIR) light at a maximum wavelength of 860 nm owing to their conjugated polymer backbone consisting of D-A-D monomers. Both colloidal and optical properties of these Pdots make them promising fluorescence emissive probes for bioimaging applications. The significant advantage of positively charged Pdots is demonstrated in diffusion-limited mediums such as tissues, utilizing human epithelial breast adenocarcinoma, ATCC HTB-22 (MCF-7), human bone marrow neuroblastoma, ATCC CRL-2266 (SH-SY5Y), and rat adrenal gland pheochromocytoma, CRL-1721 (PC-12) tumor spheroid models. Fluorescence microscopy analysis of tumor spheroids from MCF-7, SH-SY5Y, and PC-12 cell lines reveals the intensity profile of Pdots, confirming extensive penetration into the central regions of the models. Moreover, a comparison with mitochondria staining dye reveals an overlap between the regions stained by Pdots and the dye in all three tumor spheroid models. These results suggest that single-chain D-A-D type Pdots, cationized via CTAB, exhibit long-range mean free path of penetration (≈1 µm) in dense mediums and tumors., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Parenteral nutrition (PN) is typically administered to individuals with gastrointestinal dysfunction, a contraindication for enteral feeding, and a need for nutritional therapy. When PN is the only energy source in patients, it is defined as total parenteral nutrition (TPN). TPN is a life-saving approach for different patient populations, both in infants and adults. However, despite numerous benefits, TPN can cause adverse effects, including metabolic disorders and liver injury. TPN-associated liver injury, known as intestinal failure-associated liver disease (IFALD), represents a significant problem affecting up to 90% of individuals receiving TPN. IFALD pathogenesis is complex, depending on the TPN components as well as on the patient's medical conditions. Despite numerous animal studies and clinical observations, the molecular mechanisms driving IFALD remain largely unknown. The present study was set up to elucidate the mechanisms underlying IFALD. For this purpose, human liver spheroid co-cultures were treated with a TPN mixture, followed by RNA sequencing analysis. Subsequently, following exposure to TPN and its single nutritional components, several key events of liver injury, including mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, apoptosis, and lipid accumulation (steatosis), were studied using various techniques. It was found that prolonged exposure to TPN substantially changes the transcriptome profile of liver spheroids and affects multiple metabolic and signaling pathways contributing to liver injury. Moreover, TPN and its main components, especially lipid emulsion, induce changes in all key events measured and trigger steatosis., (© 2024. The Author(s).)

Water-in-water (W/W) emulsions provide bio-compatible all-aqueous compartments for artificial patterning and assembly of living cells. Successful entrapment of cells within a W/W emulsion via the formation of semipermeable capsules is a prerequisite for regulating on the size, shape, and architecture of cell aggregates. However, the high permeability and instability of the W/W interface, restricting the assembly of stable capsules, pose a fundamental challenge for cell entrapment. The current study addresses this problem by synthesizing multi-armed protein fibrils and controlling their assembly at the W/W interface. The multi-armed protein fibrils, also known as 'fibril clusters', were prepared by cross-linking lysozyme fibrils with multi-arm polyethylene glycol (PEG) via click chemistry. Compared to linear-structured fibrils, fibril clusters are strongly adsorbed at the W/W interface, forming an interconnected meshwork that better stabilizes the W/W emulsion. Moreover, when fibril clusters are complexed with alginate, the hybrid microcapsules demonstrate excellent mechanical robustness, semi-permeability, cytocompatibility and biodegradability. These advantages enable the encapsulation, entrapment and long-term culture of tumor spheroids, with great promise for applications for anti-cancer drug screening, tumor disease modeling, and tissue repair engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pancreatic islet transplantation is an effective treatment for type I diabetes mellitus. However, many problems associated with pancreatic islet engraftment remain unresolved. In this study, we developed a hydrogel microwell device for islet implantation, fabricated by crosslinking gelatin-methacryloyl (GelMA) and 2-hydroxyethyl methacrylate (HEMA) in appropriate proportions. The fabricated hydrogel microwell device could be freeze-dried and restored by immersion in the culture medium at any time, allowing long-term storage and transport of the device for ready-to-use applications. In addition, due to its non-swelling properties, the shape of the wells of the device was maintained. Thus, the device allowed pancreatic β cell lines to form spheroids and increase insulin secretion. Intraperitoneal implantation of the β cell line-seeded GelMA/HEMA hydrogel microwell device reduced blood glucose levels in diabetic mice. In addition, they were easy to handle during transplantation and were removed from the transplant site without peritoneal adhesions or infiltration by inflammatory cells. These results suggest that the GelMA/HEMA hydrogel microwell device can go from spheroid and/or organoid fabrication to transplantation in a single step.

The high incidence of malignant melanoma highlights the need for in vitro models that accurately represent the tumour microenvironment, enabling developments in melanoma therapy and drug screening. Despite several advancements in 3D cell culture models, appropriate melanoma models for evaluating drug efficacy are still in high demand. The 3D pneumatic extrusion-based bioprinting technology offers numerous benefits, including the ability to achieve high-throughput capabilities. However, there is a lack of research that combines pneumatic extrusion-based bioprinting with analytical assays to enable efficient drug screening in 3D melanoma models. To address this gap, this study developed a simple and highly reproducible approach to fabricate a 3D A375 melanoma cell culture model using the pneumatic extrusion-based bioprinting technology. To optimise this method, the bioprinting parameters for producing 3D cell cultures in a 96-well plate were adjusted to improve reproducibility while maintaining the desired droplet size and a cell viability of 92.13 ± 6.02%. The cross-linking method was optimised by evaluating cell viability and proliferation of the 3D bioprinted cells in three different concentrations of calcium chloride. The lower concentration of 50 mM resulted in higher cell viability and increased cell proliferation after 9 d of incubation. The A375 cells exhibited a steadier proliferation rate in the 3D bioprinted cell cultures, and tended to aggregate into spheroids, whereas the 2D cell cultures generally formed monolayered cell sheets. In addition, we evaluated the drug responses of four different anti-cancer drugs on the A375 cells in both the 2D and 3D cell cultures. The 3D cell cultures exhibited higher levels of drug resistance in all four tested anti-cancer drugs. This method presents a simple and cost-effective method of producing and analysing 3D cell culture models that do not add additional complexity to current assays and shows considerable potential for advancing 3D cell culture models' drug efficacy evaluations., (Creative Commons Attribution license.)

Introduction: Overactive bladder (OAB) is a highly prevalent condition with limited treatment options due to poor efficacy, side effects, and patient compliance. Novel drug delivery systems that can target the bladder wall may improve OAB therapy., Methods: We explored a polydopamine (PDA)-coated lactobacillus platform as a potential carrier for localized OAB treatment. Urinary microbiome profiling was performed to identify the presence of lactobacillus in healthy and OAB groups. Lactobacillus-PDA nanoparticles were synthesized and characterized by electron microscopy and spectrophotometry. A rat bladder perfusion model and human bladder smooth muscle cell spheroids were used to assess the distribution and penetration of the nanoparticles. The efficacy of the Lactobacillus-PDA system (LPS) for delivering the antimuscarinic drug solifenacin was evaluated in an OAB rat model., Results: Urinary microbiome profiling revealed lactobacillus as a dominant genus in both healthy and OAB groups. The synthesized Lactobacillus-PDA nanoparticles exhibited uniform size and optical properties. In the rat bladder perfusion model, the nanoparticles distributed throughout the bladder wall and smooth muscle without toxicity. The nanoparticles also penetrated human bladder smooth muscle cell spheroids. In the OAB rat model, LPS facilitated the delivery of solifenacin and improved treatment efficacy., Discussion: The results highlight LPS as a promising drug carrier for targeted OAB therapy via penetration into bladder tissues. This bacteriotherapy approach may overcome limitations of current systemic OAB medications. Lactobacillus, a probiotic bacterium present in the urinary tract microbiome, was hypothesized to adhere to and penetrate the bladder wall when coated with PDA nanoparticles, making it a suitable candidate for localized drug delivery., Competing Interests: All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Wang et al.)

Three-dimensional (3D) spheroid models are crucial for cancer research, offering more accurate insights into tumour biology and drug responses than traditional 2D cell cultures. However, inconsistent and low-throughput spheroid production has hindered their application in drug screening. Here, we present an automated high-throughput platform for a spheroid selection, fabrication, and sorting system (SFSS) to produce uniform gelatine-encapsulated spheroids (GESs) with high efficiency. SFSS integrates advanced imaging, analysis, photo-triggered fabrication, and microfluidic sorting to precisely control spheroid size, shape, and viability. Our data demonstrate that our SFSS can produce over 50 GESs with consistent size and circularity in 30 min with over 97% sorting accuracy while maintaining cell viability and structural integrity. We demonstrated that the GESs can be used for drug screening and potentially for various assays. Thus, the SFSS could significantly enhance the efficiency of generating uniform spheroids, facilitating their application in drug development to investigate complex biological systems and drug responses in a more physiologically relevant context.

Late-onset Alzheimer's disease (LOAD) is the most common form of Alzheimer's disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-β (Aβ) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)-based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aβ-dependent neurodegeneration, and treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aβ deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

Indocyanine green (ICG) is an FDA-approved medical diagnostic agent that is widely used as a near-infrared (NIR) fluorescent imaging molecular probe. However, ICG tends to aggregate to form dimers or H-aggregates in water and lacks physical and optical stability, which greatly decreases its absorbance and fluorescence intensity in various applications. Additionally, ICG has no tissue- or tumor-targeting properties, and its structure is not easy to modify, which has further limited its application in cancer diagnosis. In this study, we addressed these challenges by developing a supramolecular colloidal carrier system that targets tumor cells. To this end, we synthesized a water-soluble β-cyclodextrin (β-CD) polymer conjugated with folate (FA), denoted PCD-FA, which is capable of forming inclusion complexes with ICG in water through host-guest interactions between the β-CD moieties and ICG molecules. The inclusion complexes formed by PCD-FA and ICG, called ICG@PCD-FA, dispersed stably in solution as colloidal nanoparticles, greatly improving the physical and optical properties of ICG by preventing ICG dimer formation, where ICG appeared as monomers and even J-aggregates. This resulted in stronger and more stable absorption at a longer wavelength of 900 nm, which may allow for deeper tissue penetration and imaging with reduced interference from biological tissues' autofluorescence. Moreover, ICG@PCD-FA showed a targeting effect on folate receptor-positive (FR+) tumor cells, which specifically highlighted FR+ cells via NIR endoscopic imaging. Notably, ICG@PCD-FA further improved permeation and accumulation in FR+ 3D tumor spheroids. Therefore, this ICG@PCD-FA supramolecular colloidal system may have a great potential for use in tumor NIR imaging and diagnostic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Small-cell lung cancer (SCLC) accounts for 15% of lung cancers and has a dismal prognosis due to early dissemination and acquired chemoresistance. The initial good response to chemotherapy is followed by refractory relapses within 1-2 years. Mechanisms leading to chemoresistance are not clear and progress is poor., Areas Covered: This article reviews the current evidence of the resistance of SCLCs at the cellular level including alteration of key proteins and the possible presence of cancer stem cells (CSCs). Without compelling evidence for cellular mechanisms and clinical failures of novel approaches, the study of SCLC has advanced to the role of 3D tumor cell aggregates in chemoresistance., Expert Opinion: The scarcity of viable tumor specimen from relapsed SCLC patients has hampered the investigations of acquired chemoresistance but a panel of nine SCLC circulating tumor cell (CTC) cell lines have revealed characteristics of SCLC in the advanced refractory states. The chemoresistance of relapsed SCLC seems to be linked to the spontaneous formation of large spheroids, termed tumorospheres, which contain resistant quiescent and hypoxic cells shielded by a physical barrier. So far, drugs to tackle large tumor spheroids are in preclinical and early clinical development.

Engineering functional tissues and organs remains a fundamental pursuit in bio-fabrication. However, the accurate constitution of complex shapes and internal anatomical features of specific organs, including their intricate blood vessels and nerves, remains a significant challenge. Inspired by the Matryoshka doll, here a new method called "Intra-Embedded Bioprinting (IEB)" is introduced building upon existing embedded bioprinting methods. a xanthan gum-based material is used which served a dual role as both a bioprintable ink and a support bath, due to its unique shear-thinning and self-healing properties. IEB's capabilities in organ modeling, creating a miniaturized replica of a pancreas using a photocrosslinkable silicone composite is demonstrated. Further, a head phantom and a Matryoshka doll are 3D printed, exemplifying IEB's capability to manufacture intricate, nested structures. Toward the use case of IEB and employing an innovative coupling strategy between extrusion-based and aspiration-assisted bioprinting, a breast tumor model that included a central channel mimicking a blood vessel, with tumor spheroids bioprinted in proximity is developed. Validation using a clinically-available chemotherapeutic drug illustrated its efficacy in reducing the tumor volume via perfusion over time. This method opens a new way of bioprinting enabling the creation of complex-shaped organs with internal anatomical features., (© 2023 The Authors. Small Methods published by Wiley‐VCH GmbH.)

In Brief: This study reveals that orthotopic transplantation of 3D hUC-MSC spheroids is more effective than monolayer-cultured hUC-MSCs in improving POF and distinctly reducing oxidative stress through the paracrine effect, thereby preventing apoptosis and autophagy of GCs., Abstract: Premature ovarian failure (POF) is a common reproductive disease in women younger than 40 years old, and studies have demonstrated that the application of human umbilical cord mesenchymal stem cells (hUC-MSCs) is a promising therapy strategy for POF. Given the previously established therapeutic advantages of 3D MSC spheroids, and to evaluate their effectiveness, both 3D hUC-MSC spheroids and monolayer-cultured hUC-MSCs were employed to treat a cyclophosphamide-induced POF rat model through orthotopic transplantation. The effects of these two forms on POF were subsequently assessed by examining apoptosis, autophagy, and oxidative damage in ovarian granulosa cells (GCs). The results indicated that hUC-MSC spheroids exhibited superior treatment effects on resisting autophagy, apoptosis, and oxidative damage in GCs compared to monolayer-cultured hUC-MSCs. To further elucidate the impact of hUC-MSC spheroids in vitro, a H2O2-induced KGN cells model was established and co-cultured with both forms of hUC-MSCs. As expected, the hUC-MSC spheroids also exhibited superior effects in resisting apoptosis and autophagy caused by oxidative damage. Therefore, this study demonstrates that 3D hUC-MSC spheroids have potential advantages in POF therapy; however, the detailed mechanisms need to be further investigated. Furthermore, this study will provide a reference for the clinical treatment strategy of POF.

Liver cancer, characterized as a kind of malignant tumor within the digestive system, poses great health harm, and immune escape stands out as an important reason for its occurrence and development. Chemokines, pivotal in guiding immune cells' migration, is necessary to initiate and deliver an effective anti-tumor immune response. Therefore, understanding the chemotactic environment and identifying chemokines that regulate recruitment of immune cells to the tumor microenvironment (TME) are critical to improve current immunotherapy interventions. Herein, we report a well-defined inverse opal scaffold generated with a microfluidic emulsion template for the construction of a vascularized liver tumor model, offering insights into immune cells' recruitment. Due to the excellent 3D porous morphology of the inverse opal scaffold, human hepatocellular carcinoma cells can aggregate in the pores of the scaffold to form uniform multicellular tumor spheroids. More attractively, the vascularized liver tumor model can be achieved by constructing a 3D co-culture system involving endothelial cells and hepatocellular carcinoma cells. The results demonstrate that the 3D co-cultured tumor cells increase the neutrophil chemokines remarkably and recruit neutrophils to tumor tissues, then promote tumor progression. This approach opens a feasible avenue for realizing a vascularized liver tumor model with a reliable immune microenvironment close to that of a solid tumor of liver cancer.

Recent advances in single-cell and multicellular microfluidics technology have provided powerful tools for studying cancer biology and immunology. The ability to create controlled microenvironments, perform high-throughput screenings, and monitor cellular interactions at the single-cell level has significantly advanced our understanding of tumor biology and immune responses. We discuss cutting-edge multicellular and single-cell microfluidic technologies and methodologies utilized to investigate cancer-immune cell interactions and assess the effectiveness of immunotherapies. We explore the advantages and limitations of the wide range of 3D spheroid and single-cell microfluidic models recently developed, highlighting the various approaches in device generation and applications in immunotherapy screening for potential opportunities for point-of-care approaches.

Background: The field of tissue engineering has remarkably progressed through the integration of nanotechnology and the widespread use of magnetic nanoparticles. These nanoparticles have resulted in innovative methods for three-dimensional (3D) cell culture platforms, including the generation of spheroids, organoids, and tissue-mimetic cultures, where they play a pivotal role. Notably, iron oxide nanoparticles and superparamagnetic iron oxide nanoparticles have emerged as indispensable tools for non-contact manipulation of cells within these 3D environments. The variety and modification of the physical and chemical properties of magnetic nanoparticles have profound impacts on cellular mechanisms, metabolic processes, and overall biological function. This review article focuses on the applications of magnetic nanoparticles, elucidating their advantages and potential pitfalls when integrated into 3D cell culture systems. This review aims to shed light on the transformative potential of magnetic nanoparticles in terms of tissue engineering and their capacity to improve the cultivation and manipulation of cells in 3D environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Pancreatic islets are important in nutrient homeostasis and improved cellular models of clonal origin may very useful especially in view of relatively scarce primary material. Close 3D contact and coupling between β-cells are a hallmark of physiological function improving signal/noise ratios. Extracellular electrophysiology using micro-electrode arrays (MEA) is technically far more accessible than single cell patch clamp, enables dynamic monitoring of electrical activity in 3D organoids and recorded multicellular slow potentials (SP) provide unbiased insight in cell-cell coupling., Objective: We have therefore asked whether 3D spheroids enhance clonal β-cell function such as electrical activity and hormone secretion using human EndoC-βH1, EndoC-βH5 and rodent INS-1 832/13 cells., Methods: Spheroids were formed either by hanging drop or proprietary devices. Extracellular electrophysiology was conducted using multi-electrode arrays with appropriate signal extraction and hormone secretion measured by ELISA., Results: EndoC-βH1 spheroids exhibited increased signals in terms of SP frequency and especially amplitude as compared to monolayers and even single cell action potentials (AP) were quantifiable. Enhanced electrical signature in spheroids was accompanied by an increase in the glucose stimulated insulin secretion index. EndoC-βH5 monolayers and spheroids gave electrophysiological profiles similar to EndoC-βH1, except for a higher electrical activity at 3 mM glucose, and exhibited moreover a biphasic profile. Again, physiological concentrations of GLP-1 increased AP frequency. Spheroids also exhibited a higher secretion index. INS-1 cells did not form stable spheroids, but overexpression of connexin 36, required for cell-cell coupling, increased glucose responsiveness, dampened basal activity and consequently augmented the stimulation index., Conclusion: In conclusion, spheroid formation enhances physiological function of the human clonal β-cell lines and these models may provide surrogates for primary islets in extracellular electrophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Puginier, Leal-Fischer, Gaitan, Lallouet, Scotti, Raoux and Lang.)

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches., (© 2024. The Author(s).)

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

This paper introduces an innovative method for the fabrication and infusion of microwell arrays based on digital light processing (DLP) 3D printing. A low-cost DLP 3D printer is employed to fabricate microstructures rapidly with a broad dynamic range while maintaining high precision and fidelity. We constructed microwell arrays with varying diameters, from 200 to 2000 μm and multiple aspect ratios, in addition to microchannels with widths ranging from 45 to 1000 μm, proving the potential and flexibility of this fabrication method. The superimposition of parallel microchannels onto the microwell array, facilitated by positive or negative pressure, enabled the transfer of liquid to the microwells. Upon removal of the microchannel chip, a dispensed microdroplet array was obtained. This array can be modulated by adjusting the volume of the microwells and the inflow fluid. The filled microwell array allows chip-to-chip dispensing to the microreactor array through binding and centrifugation, facilitating multistep and multireagent assays. The 3D printing approach also enables the fabrication of intricate cavity designs, such as micropyramid arrays, which can be integrated with parallel microchannels to generate spheroid flowcells. This device demonstrated the ability to generate spheroids and manipulate their environment. We have successfully utilized precise modulation of spheroids size and performed parallel drug dose-response assays to evaluate its effectiveness. Furthermore, we managed to execute dynamic drug combinations based on a compact spheroids array, utilizing two orthogonal parallel microchannels. Our findings suggest that both the combination and temporal sequence of drug administration have a significant impact on therapeutic outcomes.

Uncontrolled growth of tumor cells in confined spaces leads to the accumulation of compressive stress within the tumor. Although the effects of tension within 3D extracellular matrices (ECMs) on tumor growth and invasion are well established, the role of compression in tumor mechanics and invasion is largely unexplored. In this study, we modified a Transwell assay such that it provides constant compressive loads to spheroids embedded within a collagen matrix. We used microscopic imaging to follow the single cell dynamics of the cells within the spheroids, as well as invasion into the 3D ECMs. Our experimental results showed that malignant breast tumor (MDA-MB-231) and non-tumorigenic epithelial (MCF10A) spheroids responded differently to a constant compression. Cells within the malignant spheroids became more motile within the spheroids and invaded more into the ECM under compression; whereas cells within non-tumorigenic MCF10A spheroids became less motile within the spheroids and did not display apparent detachment from the spheroids under compression. These findings suggest that compression may play differential roles in healthy and pathogenic epithelial tissues and highlight the importance of tumor mechanics and invasion., (Creative Commons Attribution license.)

The metabolite transport inhibition of tumor cells holds promise to achieve anti-tumor efficacy. Herein, we presented an innovative strategy to hinder the delivery of metabolites through the in-situ besieging tumor cells with polyphenolic polymers that strongly adhere to the cytomembrane of tumor cells. Simultaneously, these polymers underwent self-crosslinking under the induction of tumor oxidative stress microenvironment to form an adhesive coating on the surface of the tumor cells. This polyphenol coating effectively obstructed glucose uptake, reducing metabolic products such as lactic acid, glutathione, and adenosine triphosphate, while also causing reactive oxygen species to accumulate in the tumor cells. The investigation of various tumor models, including 2D cells, 3D multicellular tumor spheroids, and xenograft tumors, demonstrated that the polyphenolic polymers effectively inhibited the growth of tumor cells by blocking key metabolite transport processes. Moreover, this highly adhesive coating could bind tumor cells to suppress their metastasis and invasion. This work identified polyphenolic polymers as a promising anticancer candidate with a mechanism by impeding the mass transport of tumor cells., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a few µ m to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates., (Creative Commons Attribution license.)

Three-dimensional (3D) cell assemblies, such as multicellular spheroids, can be powerful biological tools to closely mimic the complexity of cell-cell and cell-matrix interactions in a native-like microenvironment. However, potential applications of large spheroids are limited by the insufficient diffusion of oxygen and nutrients through the spheroids and, thus, result in the formation of a necrotic core. To overcome this drawback, we present a new strategy based on nanoparticle-coated microparticles. In this study, microparticles function as synthetic centers to regulate the diffusion of small molecules, such as oxygen and nutrients, within human mesenchymal stem cell (hMSC) spheroids. The nanoparticle coating on the microparticle surface acts as a nutrient reservoir to release glucose locally within the spheroids. We first coated the surface of the poly(lactic- co -glycolic acid) (PLGA) microparticles with mesoporous silica nanoparticles (MSNs) based on electrostatic interactions and then formed cell-nanofunctionalized microparticle spheroids. Next, we investigated the stability of the MSN coating on the microparticles' surface during 14 days of incubation in cell culture medium at 37 °C. Then, we evaluated the influence of MSN-coated PLGA microparticles on spheroid aggregation and cell viability. Our results showed the formation of homogeneous spheroids with good cell viability. As a proof of concept, fluorescently labeled glucose (2-NBD glucose) was loaded into the MSNs at different concentrations, and the release behavior was monitored. For cell culture studies, glucose was loaded into the MSNs coated onto the PLGA microparticles to sustain local nutrient release within the hMSC spheroids. In vitro results demonstrated that the local delivery of glucose from MSNs enhanced the cell viability in spheroids during a short-term hypoxic culture. Taken together, the newly developed nanofunctionalized microparticle-based delivery system may offer a versatile platform for local delivery of small molecules within 3D cellular assemblies and, thus, improve cell viability in spheroids.

Examining tumor heterogeneity is essential for selecting an appropriate anticancer treatment for an individual. This study aimed to distinguish low- and high-aggressive tumor cells by analyzing the formation patterns of spheroids. The droplet-based microfluidic system was employed for the formation of each spheroid from four different subtypes of breast tumor cells. Additionally, heterotypic spheroids with T lymphocytes and cancer-associated fibroblasts (CAFs) were produced, and distinctions between low- and high-aggressive tumor cells were explored through the analysis of formation patterns using circularity, convexity, and cell distributions. In both homotypic spheroids and heterotypic spheroids with T lymphocytes, spheroids formed from low-aggressive tumor cells exhibited high circularity and convexity. On the other hand, spheroids formed from high-aggressive tumor cells had relatively low circularity and convexity. In the case of heterotypic spheroids with CAFs, circularity and convexity did not exhibit clear differences between low- and high-aggressive tumor cells, but distinct variations were observed in cell distributions. CAFs and low-aggressive tumor cells were evenly distributed, whereas the CAFs were predominantly located in the inner layer, and high-aggressive tumor cells were primarily located in the outer layer. This finding can offer valuable insights into predicting the aggressiveness of unknown tumor cells.

Background: Lung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies., Methods: To overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy., Results: We showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level., Conclusions: This study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment., (© 2024. The Author(s).)

While 3D tumor models have greatly evolved over the past years, there is still a strong requirement for more biosimilar models which are capable of recapitulating cellular crosstalk within the tumor microenvironment while equally displaying representative levels of tumor aggressiveness and invasion. Herein, we disclose an assembloid melanoma model based on the fusion of individual stromal multicellular spheroids (MCSs). In contrast to more traditional tumor models, we show that it is possible to develop self-organizing, heterotypic melanoma models where tumor cells present stem-cell like features like up-regulated pluripotency master regulators SOX2, POU5F1 and NANOG. Additionally, these assembloids display high levels of invasiveness while embedded in 3D matrices as evidenced by stromal cell promotion of melanoma cell invasion via metalloproteinase production. Furthermore, sensitivity to anticancer drug doxorubicin was demonstrated for the melanoma assembloid model. These findings suggest that melanoma assembloids may play a significant role in the field of 3D cancer models as they more closely mimic the tumor microenvironment when compared to more traditional MCSs, opening the doors to a better understanding of the role of tumor microenvironment in supporting tumor progression. STATEMENT OF SIGNIFICANCE: The development of complex 3D tumor models that better recapitulate the tumor microenvironment is crucial for both an improved comprehension of intercellular crosstalk and for more efficient drug screening. We have herein developed a self-organizing heterotypic assembloid-based melanoma model capable of closely mimicking the tumor microenvironment. Key features recapitulated were the preservation of cancer cell stemness, sensitivity to anti-cancer agents and tumor cell invasion promoted by stromal cells. The approach of pre-establishing distinct stromal domains for subsequent combination into more complex tumor constructs provides a route for developing superior tumor models with a higher degree of similarity to native cancer tissues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Many cell types require direct cell-cell interactions for differentiation and function; yet, this can be challenging to incorporate into 3-dimensional (3D) structures for the engineering of tissues. Here, a new approach is introduced that combines aggregates of cells (spheroids) with similarly-sized hydrogel particles (microgels) to form granular composites that are injectable, undergo interparticle crosslinking via light for initial stabilization, permit cell-cell contacts for cell signaling, and allow spheroid fusion and growth. One area where this is important is in cartilage tissue engineering, as cell-cell contacts are crucial to chondrogenesis and are missing in many tissue engineering approaches. To address this, granular composites are developed from adult porcine mesenchymal stromal cell (MSC) spheroids and hyaluronic acid microgels and simulations and experimental analyses are used to establish the importance of initial MSC spheroid to microgel volume ratios to balance mechanical support with tissue growth. Long-term chondrogenic cultures of granular composites produce engineered cartilage tissue with extensive matrix deposition and mechanical properties within the range of cartilage, as well as integration with native tissue. Altogether, a new strategy of injectable granular composites is developed that leverages the benefits of cell-cell interactions through spheroids with the mechanical stabilization afforded with engineered hydrogels., (© 2024 Wiley‐VCH GmbH.)

3D cell culture is becoming increasingly important for mimicking physiological tissue structures in areas such as drug discovery and personalized medicine. To enable reproducibility on a large scale, automation technologies for standardized handling are still a challenge. Here, a novel method for fully automated size classification and handling of cell aggregates like spheroids and organoids is presented. Using microfluidic flow generated by a piezoelectric droplet generator, aggregates are aspirated from a reservoir on one side of a thin capillary and deposited on the other side, encapsulated in free-flying nanoliter droplets to a target. The platform has aggregate aspiration and plating efficiencies of 98.1% and 98.4%, respectively, at a processing throughput of up to 21 aggregates per minute. Cytocompatibility of the method is thoroughly assessed with MCF7, LNCaP, A549 spheroids and colon organoids, revealing no adverse effects on cell aggregates as shear stress is reduced compared to manual pipetting. Further, generic size-selective handling of heterogeneous organoid samples, single-aggregate-dispensing efficiencies of up to 100% and the successful embedding of spheroids or organoids in a hydrogel with subsequent proliferation is demonstrated. This platform is a powerful tool for standardized 3D in vitro research., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or instead utilize existing extracellular matrix microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3-dimensional migration, few recapitulate these natural migration paths. Here, we develop a biopolymer-based bicontinuous hydrogel system that comprises a covalent hydrogel of enzymatically crosslinked gelatin and a physical hydrogel of guest and host moieties bonded to hyaluronic acid. Bicontinuous hydrogels form through controlled solution immiscibility, and their continuous subdomains and high micro-interfacial surface area enable rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior is mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which is shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a design that leverages important local interfaces to guide rapid cell migration., (© 2024. The Author(s).)

Thyroid cancer (TC) is the prevalent endocrine tumor with a rising incidence, particularly in higher-income countries, leading to an increased interest in its management and treatment. While overall, survival rates for TC are usually favorable, advanced cases, especially with metastasis and specific histotypes, pose challenges with poorer outcomes, advocating the need of systemic treatments. Targeted therapies have shown efficacy in both preclinical models and clinical trials but face issues of resistance, since they usually induce partial and transient response. These resistance phenomena are currently only partially addressed by traditional preclinical models. This review explores the limitations of traditional preclinical models and emphasizes the potential of three-dimensional (3D) models, such as transwell assays, spheroids, organoids, and organ-on-chip technology in providing a more comprehensive understanding of TC pathogenesis and treatment responses. We reviewed their use in the TC field, highlighting how they can produce new interesting insights. Finally, the advent of organ-on-chip technology is currently revolutionizing preclinical research, offering dynamic, multi-cellular systems that replicate the complexity of human organs and cancer-host interactions.

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling., (Creative Commons Attribution license.)

Docetaxel (DTX) is one of the chemotherapeutic drugs indicated as a first-line treatment against metastatic prostate cancer (mPCa). This study aimed to compare the impact of DTX on mPCa (DU-145) tumor cells cultured as 2D monolayers and 3D multicellular tumor spheroids (MCTS) in vitro . The cells were treated with DTX (1-96 µM) at 24, 48, or 72 hr in cell viability assays (resazurin, phosphatase acid, and lactate dehydrogenase). Cell death was assessed with fluorescent markers and proliferation by clonogenic assay (2D) and morphology, volume, and integrity assay (3D). The cell invasion was determined using transwell (2D) and extracellular matrix (ECM) (3D). Results showed that DTX decreased cell viability in both culture models. In 2D, the IC 50 (72 hr) values were 11.06 μM and 14.23 μM for resazurin and phosphatase assays, respectively. In MCTS, the IC 50 values for the same assays were 114.9 μM and 163.7 μM, approximately 10-fold higher than in the 2D model. The % of viable cells decreased, while the apoptotic cell number was elevated compared to the control in 2D. In 3D spheroids, only DTX 24 μM induced apoptosis. DTX (≥24 μM at 216 hr) lowered the volume, and DTX 96 μM completely disintegrated the MCTS. DTX reduced the invasion of mPCa cells to matrigel (2D) and migration from MCTS to the ECM. Data demonstrated significant differences in drug response between 2D and 3D cell culture models using mPCa DU-145 tumor cells. MCTS resembles the early stages of solid tumors in vivo and needs to be considered in conjunction with 2D cultures when searching for new therapeutic targets.