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Three-dimensional cell cultures, or spheroids, are important model systems for cancer research because they recapitulate chemical and phenotypic aspects of in vivo tumors. Spheroids develop radially symmetric chemical gradients, resulting in distinct cellular populations. Stable isotopic labeling by amino acids in cell culture (SILAC) is a well-established approach to quantify protein expression and has previously been used in a pulse-chase format to evaluate temporal changes. In this article, we demonstrate that distinct isotopic signatures can be introduced into discrete spatial cellular populations, effectively tracking proteins to original locations in the spheroid, using a platform that we refer to as spatial SILAC. Spheroid populations were grown with light, medium, and heavy isotopic media, and the concentric shells of cells were harvested by serial trypsinization. Proteins were quantitatively analyzed by ultraperformance liquid chromatography–tandem mass spectrometry. The isotopic signatures correlated with the spatial location and the isotope position do not significantly impact the proteome of each individual layer. Spatial SILAC can be used to examine the proteomic changes in the different layers of the spheroid and to identify protein biomarkers throughout the structure. We show that SILAC labels can be discretely pulsed to discrete positions, without altering the spheroid’s proteome, promising future combined pharmacodynamic and pharmacokinetic studies.

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRASG12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRASG12V-expressing cells. This represents an applicable approach to explore protein–protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

Chemotherapy for glioblastoma multiforme (GBM) remains ineffective due to insufficient penetration of therapeutic agents across the blood–brain barrier (BBB) and into the GBM tumor. Herein, is described, the optimization of the lipid composition and fabrication conditions for a BBB- and tumor penetrating terpolymer-lipid-hybrid nanoparticle (TPLN) for delivering doxorubicin (DOX) to GBM. The composition of TPLNs was first screened using different lipids based on nanoparticle properties and in vitro cytotoxicity by using 23 full factorial experimental design. The leading DOX loaded TPLNs (DOX-TPLN) were prepared by further optimization of conditions and used to study cellular uptake mechanisms, in vitro cytotoxicity, three-dimensional (3D) glioma spheroid penetration, and in vivo biodistribution in a murine orthotopic GBM model. Among various lipids studied, ethyl arachidate (EA) was found to provide excellent nanoparticle properties e.g., size, polydispersity index (PDI), zeta potential, encapsulation efficiency, drug loading, and colloidal stability, and highest anticancer efficacy for DOX-TPLN. Further optimized EA-based TPLNs were prepared with an optimal particle size (103.8 ± 33.4 nm) and PDI (0.208 ± 0.02). The resultant DOX-TPLNs showed ~ sevenfold higher efficacy than free DOX against human GBM U87-MG-RED-FLuc cells in vitro. The interaction between the TPLNs and the low-density lipoprotein receptors also facilitated cellular uptake, deep penetration into 3D glioma spheroids, and accumulation into the in vivo brain tumor regions of DOX-TPLNs. This work demonstrated that the TPLN system can be optimized by rational selection of lipid type, lipid content, and preparation conditions to obtain DOX-TPLN with enhanced anticancer efficacy and GBM penetration and accumulation.

Purpose Multicellular tumor spheroids are realistic in-vitro systems used in radiation biology research to study the effect of anticancer drugs or to evaluate the resistance of cancer cells under specific conditions. When combining the modeling of spheroids together with the simulation of radiation using Monte Carlo methods, one could estimate cell and DNA damage to be compared with experimental data. We developed a Cell Population (CPOP) modeler combined to Geant4 simulations in order to tackle how energy depositions are allocated to cells, especially when enhancing radiation outcomes using high-Z nanoparticles. CPOP manages to model large three-dimensional cell populations with independent deformable cells described with their nucleus, cytoplasm and membranes together with force law systems to manage cell–cell interactions. Methods CPOP is an opensource platform written in C++. It is divided into two main libraries: a “Modeler” library, for cell geometry modeling using meshes, and a Multi Agent System (MAS) library, simulating all agent (cell) interactions among the population. CPOP is fully interfaced with the Geant4 Monte Carlo toolkit and is able to directly launch Geant4 simulations after compilation. We modeled a full and realistic 3D cell population from SK-MEL28 melanoma cell population cultured experimentally. The spheroid diameter of 550 ± 40 µm corresponds to a population of approximately 1000 cells having a diameter of 17.2 ± 2.5 µm and a nucleus diameter of 11.2 ± 2.0 µm. We decided to reproduce cell irradiations performed with a X-RAD 320 Biological Irradiator (Precision XRay Inc., North Branford, CT). Results We simulated the energy spectrum of secondary particles generated in the vicinity of the spheroid and plotted the different energy spectra recovered internally to the spheroid. We evaluated also the impact of AGuIX (Gadolinium) nanoparticles modeled into the spheroid with their corresponding secondary energy spectra. Conclusions We succeeded into modeling cell populations and combined them with Geant4 simulations. The next step will be to integrate DNA geometrical models into cell nuclei and to use the Geant4-DNA physics and radiolysis modeling capabilities in order to evaluate early strand breaks induced on DNA.

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.

Purpose The differential response of normal and tumor tissues to ultrahigh-dose-rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study was to investigate these effects within hypoxic multicellular tumor spheroids through simulations and experiments. Methods and Materials Physicobiological equations were derived to model (1) the diffusion and metabolism of oxygen within spheroids; (2) its depletion through reactions involving radiation-induced radicals; and (3) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored postradiation. Clonogenic survival of 2 other cell lines was also investigated. Results The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroids compared with conventional irradiation, but no significant difference was observed for well-oxygenated 2-dimensional cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy. Conclusions Tumor spheroids can be used as a model to study FLASH irradiation in vitro. The improved survival of tumor spheroids receiving FLASH radiation confirms that ultrafast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect.

Photochemical internalization (PCI) is a modified form of photodynamic therapy (PDT) that enhances the efficacy of therapeutic agents in a site and temporal specific manner in both in vitro and in vivo publications. The purpose of the study reported here was to evaluate the benefits of a modified PCI protocol in a 3D rat glioma spheroid model. In the modified protocol, F98 glioma cells were incubated with photosensitizer (AlPcS2a) prior to spheroid generation, as opposed to post-spheroid formation photosensitizer exposure commonly used in conventional protocols. The efficacy of both bleomycin and doxorubicin PCI was evaluated using either the conventional or modified protocols. The formed spheroids were then exposed to light treatment from a diode laser, λ= 670 nm. Spheroid growth was monitored for a period of 14 days. The results of spheroid growth assays showed that there was no statistically significant difference in PCI efficacy between the conventional and modified protocols for both of the drugs tested. The direct PDT effect was significantly reduced using the modified protocol. Therefore, due to its several advantages, the modified protocol is recommended for evaluating the efficacy of PCI in tumor spheroid models.

Background: Chronic respiratory diseases (CRD) are a major public health problem worldwide. In the current epidemiological context, CRD have received much interest when considering their correlation with greater susceptibility to SARS-Cov-2 and severe disease (COVID-19). Increasingly more studies have investigated pathophysiological interactions between CRD and COVID-19. Area covered: Animal experimentation has decisively contributed to advancing our knowledge of CRD. Considering the increase in ethical restrictions in animal experimentation, researchers must focus on new experimental alternatives. Two-dimensional (2D) cell cultures have complemented animal models and significantly contributed to advancing research in the life sciences. However, 2D cell cultures have several limitations in studies of cellular interactions. Three-dimensional (3D) cell cultures represent a new and robust platform for studying complex biological processes and are a promising alternative in regenerative and translational medicine. Expert opinion: Three-dimensional cell cultures are obtained by combining several types of cells in integrated and self-organized systems in a 3D structure. These 3D cell culture systems represent an efficient methodological approach in studies of pathophysiology and lung therapy. More recently, complex 3D culture systems, such as lung-on-a-chip, seek to mimic the physiology of a lung in vivo through a microsystem that simulates alveolar-capillary interactions and exposure to air. The present review introduces and discusses 3D lung cultures as robust platforms for studies of the pathophysiology of CRD and COVID-19 and the mechanisms that underlie interactions between CRD and COVID-19.

Cell fate and morphologies are influenced by the mechanical property of matrix. However, the relevant works about the dynamic adjustable of matrix mechanical property is rare and most of them need extra stimulation, such as the controllable of the degradation. In this study, double crosslinking (DC) hydrogels are fabricated by sequential covalent crosslinking and electrostatic interactions between hyaluronic acid and poly-lysine. Without any extra stimulation or treatment, the compressive stress of DC-hydrogels increases from 22.4 ± 9.4 kPa to 320.1 ± 6.6 kPa with the elongation of incubation time in DMEM solution. The change of compressive stress of matrix induced the morphology of L929 fibroblast cells adjusted from the distributed round shape to spheroid cell clusters and finally to spread shape. RNA sequence analysis also demonstrated that the differentially gene expression and GO enrichment between the cells seeded on the DC-hydrogel with different incubation time. In addition, by increasing the electrostatic interactions ratio of the hydrogel, the biodegradation, compressive stress and energy dissipation of the DC-hydrogels were also significantly improved. Therefore, our study provides new and critical insights into the design strategy to achieve DC-hydrogels which can in situ alter cells morphology and open up a new avenue for the application of disease therapy.

There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automated organoid assay system (the Pu·MA System) combined with microfluidic-based flowchips that can facilitate 3D cell-based assays. The flowchip is composed of sample wells, which contain organoids, connected to additional multiple wells that can hold various assay reagents. Organoids are positioned in a protected chamber in sample wells, and fluids are exchanged from side reservoirs using pressure-driven flow. Media exchange, sample staining, wash steps, and other processes can be performed without disruption to or loss of 3D sample. The bottom of the sample chamber is thin, optically clear plastic compatible with high-content imaging (HCI). The whole system can be kept in an incubator, allowing long-term cellular assays to be performed. We present two examples of use of the system for biological research. In the first example, cytotoxicity effects of anticancer drugs were evaluated on HeLa and HepG2 spheroids using HCI and vascular endothelial growth factor expression. In the second application, the flowchip system was used for the functional evaluation of Ca2+ oscillations in neurospheroids. Neurospheres were incubated with neuroactive compounds, and neuronal activity was assessed using Ca2+-sensitive dyes and fast kinetic fluorescence imaging. This novel assay system using microfluidics enables automation of 3D cell-based cultures that mimic in vivo conditions, performs multidosing protocols and multiple media exchanges, provides gentle handling of spheroids and organoids, and allows a wide range of assay detection modalities.

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. Human iPSC (hiPSC)-derived MN spheroids in one chamber elongated their axons into microtunnels, which reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

Cell-based high-throughput screening is a key step in the current disease-based research, drug development, and precision medicine. However, it is challenging to establish a rapid culture and screening platform for rare cells (patient-derived) due to the obvious differences between the traditional 2D cell model and the tumor microenvironment, as well as the lack of a low-consumption screening platform for low numbers of cells. Here, we developed an acoustic drop-assisted superhydrophilic-superhydrophobic microarray platform for the rapid culture and screening of a few cells. By employing hydrophilic and hydrophobic microarrays, we can automatically distribute the cell suspension into uniform droplets, and these cells can spontaneously form compact 3D cell spheroids within 36 h (similar to the microenvironment of tumors in vivo). By using the acoustic droplet ejection device, we can accurately inject a drug solution with a volume of ∼pL to ∼nL into the droplet, and the whole process can be completed within 20 ms (one print). By using three different cell lines (Caco-2, MCF-7, and HeLa) to optimize the platform, the culture and screening of five patients' colon cancer were subsequently realized. Using three conventional chemotherapeutics (5-fluorouracil, cetuximab, and panitumumab) of various concentrations, the best treatment was screened out and compared with the actual treatment effect of the patients, and the results were extremely similar. As a proof-of-concept application, we have proved that our platform can quickly cultivate patient samples and effectively screen the best treatment methods, highlighting its wide application in precision medicine, basic tumor research, and drug development.

Objective: Lipopolysaccharide (LPS) from Gram-negative bacteria causes poor uterine receptivity by inducing excessive inflammation at the maternal-fetal interface. This study aimed to investigate the detrimental effects of LPS on the attachment and outgrowth of various types of trophoblastic spheroids on endometrial epithelial cells (ECC-1 cells) in an in vitro model of implantation.Methods: Three types of spheroids with JAr, JEG-3, and JAr mixed JEG-3 (JmJ) cells were used to evaluate the effect of LPS on early implantation events. ECC-1 cells were treated with LPS to mimic endometrial infection, and the expression of inflammatory cytokines and adhesion molecules was analyzed by quantitative real-time polymerase chain reaction and western blotting. The attachment rates and outgrowth areas were evaluated in the various trophoblastic spheroids and ECC-1 cells treated with LPS.Results: LPS treatment significantly increased the mRNA expression of inflammatory cytokines (CXCL1, IL-8, and IL-33) and decreased the protein expression of adhesion molecules (ITGβ3 and ITGβ5) in ECC-1 cells. The attachment rates of JAr and JmJ spheroids on ECC-1 cells significantly decreased after treating the ECC-1 cells with 1 and 10 μg/mL LPS. In the outgrowth assay, JAr spheroids did not show any outgrowth areas. However, the outgrowth areas of JEG-3 spheroids were similar regardless of LPS treatment. LPS treatment of JmJ spheroids significantly decreased the outgrowth area after 72 hours of coincubation.Conclusion: An in vitro implantation model using novel JmJ spheroids was established, and the inhibitory effects of LPS on ECC-1 endometrial epithelial cells were confirmed in the early implantation process.

Summary Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis., Graphical abstract, Highlights • High-throughput platform to encapsulate human PSCs in agarose microgels • Human PSCs self-organize into epiblast spheroids and undergo lumen expansion • Lumen expansion increases in primed versus naive human PSCs • Monodisperse co-culture of embryonic and extraembryonic stem cells, In this article, Boroviak and colleagues present a high-throughput platform to encapsulate human PSCs into agarose microgels. Human PSCs undergo lumenogenesis and self-organize into rosette-like structures, similar to the human peri-implantation embryo. Their microgel suspension culture regime provides a foundation for the controlled assembly of stem cell-based embryo models in our own species.

Three-dimensional (3D) spheroid cell cultures are excellent models used in cancer biology research and drug screening. The objective of this study was to develop a lung carcinoma spheroid based microfluidic platform with perfusion function to mimic lung cancer pathology and investigate the effect of a potential drug molecule, panaxatriol. Spheroids were successfully formed on agar microtissue molds at the end of 10 days, reaching an average diameter of about 317.18 ± 4.05 ?m and subsequently transferred to 3D dynamic microfluidic system with perfusion function. While the size of the 3D spheroids embedded in the Matrigel matrix in the platform had gradually increased both in the static and dynamic control groups, the size of the spheroids were reduced and fragmented in the drug treated groups. Cell viability results showed that panaxatriol exhibited higher cytotoxic effect on cancer cells than healthy cells and the IC50 value was determined as 61.55 µM. Furthermore, panaxatriol has been more effective on single cells around the spheroid structure, whereas less in 3D spheroid tissues with a compact structure in static conditions compared to dynamic systems, where a flow rate of 2 µL/min leading to a shear stress of 0.002 dyne/cm2 was applied. Application of such dynamic systems will contribute to advancing basic research and increasing the predictive accuracy of potential drug molecules, which may accelerate the translation of novel therapeutics to the clinic, possibly decreasing the use of animal models. Graphic abstract: [Figure not available: see fulltext.] © 2021, The Author(s), under exclusive licence to Springer Nature B.V., 113M050 Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK, The financial support provided by the Scientific and Technological Research Council of Turkey (TUBITAK) through 113M050 Grant is highly appreciated.

Background Fabrication of three-dimensional stem cell spheroids have been studied to improve stem cell function, but the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid were challenges. The incorporation of polymers such as silica and gelatin in spheroids resulted in relatively relaxed assembly of composite spheroids, and enhancing transport of nutrient and biological gas. However, because of the low surface area between cells and since the polymers were heterogeneously distributed throughout the spheroid, these polymers cannot increase the cell to extracellular matrix interactions needed to support differentiation. Methods We developed the stem cell spheroids that incorporate poly(ι-lactic acid) single-segmented fibers synthesized by electrospinning and physical and chemical fragmentation. The proper mixing ratio was 2000 cells/μg fibers (average length of the fibers was 50 μm - 100 μm). The SFs were coated with polydopamine to increase cell binding affinity and to synthesize various-sized spheroids. The function of spheroids was investigated by in vitro analysis depending on their sizes. For statistical analysis, Graphpad Prism 5 software (San Diego, CA, USA) was used to perform one-way analysis of variance ANOVA with Tukey’s honest significant difference test and a Student’s t-test (for two variables) (P Results Spheroids of different sizes were created by modulating the amount of cells and fibers (0.063 mm2–0.322 mm2). The fibers in the spheroid were homogenously distributed and increased cell viability, while cell-only spheroids showed a loss of DNA contents, internal degradation, and many apoptotic signals. Furthermore, we investigated stemness and various functions of various-sized fiber-incorporated spheroids. In conclusion, the spheroid with the largest size showed the greatest release of angiogenic factors (released VEGF: 0.111 ± 0.004 pg/ng DNA), while the smallest size showed greater effects of osteogenic differentiation (mineralized calcium: 18.099 ± 0.271 ng/ng DNA). Conclusion The spheroids incorporating polydopamine coated single-segmented fibers showed enhanced viability regardless of sizes and increased their functionality by regulating the size of spheroids which may be used for various tissue reconstruction and therapeutic applications.

Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.

Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.

Inhibin‐α, a member of transforming growth factor‐β, is elevated in multiple tumors, but its specific roles are poorly understood. Here, we examined the feature of inhibin‐α‐expressing cells in ovarian tumors. Immunohistochemically, inhibin‐α‐expressing tumor cells were detected only in ovarian clear cell carcinoma (OCCC) among various types of ovarian tumors. By comparing the expression of inhibin‐α and Ki‐67, inhibin‐α‐expressing tumor cells were revealed to be less proliferative. When spheroids and chemoresistant cells were derived from OCCC cell lines, the expression level of inhibin‐α was elevated, and that of an immature marker, aldehyde dehydrogenase, was also elevated. In consistent with this, inhibin‐α expression was correlated with other immature markers, such as OCT3/4 and SOX2, and inversely correlated with proliferative marker MKI67 in public database on OCCC. Knockdown of inhibin‐α in OCCC cell decreased chemoresistance. Moreover, prognostic analysis with 69 surgically resected OCCC cases revealed that the increased inhibin‐α expression was an independent unfavorable prognostic factor. These findings suggested that inhibin‐α‐expressing subpopulation of OCCC tumor cells appeared to be less proliferative, immature, and angiogenic and to be related to acceleration of malignant progression., Inhibin‐α‐expressing subpopulation was less proliferative and upregulated in the recurrent site, spheroids and chemoresistant cells. Moreover, those cells had immature properties and high angiogenic potential, leading to unfavorable prognosis

Introduction: Cell aggregation of three-dimensional (3D) culture systems (the so-called spheroids) are designed as in vitro platform to represent more accurately the in vivo environment for drug discovery by using semi-solid media. The uniform multicellular tumor spheroids can be generated based on the interaction of cells with extracellular matrix (ECM) macromolecules such as collagen and integrin. This study aimed to investigate the possible interactions between the cellulose family and collagen using both in vitro and in silico approaches. Methods: The 3D microtissue of JIMT-1 cells was generated using hanging drop method to study the effects of charge and viscosity of the medium containing cellulose family. To determine the mode of interaction between cellulose derivatives (CDs) and collagen-integrin, docking analysis and molecular simulation were further performed using open source web servers and chemical simulations (GROMACS), respectively. Results: The results confirmed that the addition of CDs into the 3D medium can promote the formation of solid spheroids, where methylcellulose (MC) yielded uniform spheroids compared to carboxymethyl cellulose (CMC). Moreover, the computational analysis showed that MC interacted with both integrin and collagen, while sodium carboxymethyl cellulose (NaCMC) only interacted with collagen residues. The stated different behaviors in the 3D culture formation and collagen interaction were found in the physicochemical properties of CDs. Conclusion: Based on in vitro and in silico findings, MC is suggested as an important ECM-mimicking entity that can support the semi-solid medium and promote the formation of the uniform spheroid in the 3D culture.

A stabilized cartilage construct without signs of hypertrophy in chondrocytes is still a challenge. Suspensions of adipose stem/stromal cells (ASCs) and cartilage progenitor cells (CPCs) were seeded into micromolded nonadhesive hydrogel to produce spheroids (scaffold- and serum-free method) characterized by size, immunohistochemistry, fusion, and biomechanical properties. After cell dissociation, they were characterized for mesenchymal cell surface markers, cell viability, and quantitative real-time polymerase chain reaction. Both targeted and nontargeted (shotgun mass spectrometry) analyses were conducted on the culture supernatants. Induced ASC spheroids (ø = 350 μm) showed high cell viability and CD73 downregulation contrasting to CD90. The transforming growth factor (TGF)-β3/TGF-β1 ratio and

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)‐based therapeutic strategies against CSCs. Here, in an in vitro model using the HT‐29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC‐enriched colonospheres (CSCenr‐EXOs) as an antigen source in activating CSC‐specific T‐cell responses. HT‐29 lysate, HT‐29‐EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr‐EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen‐pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr‐EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr‐EXOs significantly increased the IL‐12/IL‐10 ratio in supernatants of mature DCs. CSCenr‐EXO‐loaded DCs effectively promoted T‐cell proliferation. Importantly, T cells stimulated with CSCenr‐EXOs disrupted spheroids' structure. Thus, CSCenr‐EXOs present a novel and promising antigen source that in combination with conventional tumour bulk‐derived antigens should be further explored in pre‐clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.

Visual Abstract, Highlights • hiPSCs are differentiated into CMs with large-scale 2-dimensional culture system, and refined by metabolic purification. • hiPSC-derived CMs are developed into CSs in special microwell plates. • Intramyocardial transplantation of CSs and GH improves cardiac function in small and large animal models. • Engraftment of CMs and angiogenesis are mechanisms for improvement of cardiac function. • Intramyocardial transplantation of CSs with a transplant injection device is a safe, effective, and feasible strategy for the treatment of HF., Summary The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.

We performed a comparative study of the proliferative potential of human mesenchymal stromal cells (MSC) from three sources (tooth pulp, adipose tissue, and Wharton’s jelly) in spheroid culture; human chondroblasts served as the positive control. Histological examination revealed signs of chondrogenic differentiation in all studied cell cultures and the differences in the volume and composition of the extracellular matrix. Spheroids formed by MSC from the tooth pulp and Wharton’s jelly were characterized by low content of extracellular matrix and glycosaminoglycans. Spheroids from adipose tissue MSC contained maximum amount of the extracellular matrix and high content of glycosaminoglycans. Chondrocytes produced glycosaminoglycan-enriched matrix. Type II collagen was produced by chondrocytes (to a greater extent) and adipose tissue MSC (to a lesser extent). The results of our study demonstrate that MSC from the adipose tissue under conditions of spheroid culturing exhibited maximum chondrogenic potential.

Along with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

The surface-modified paclitaxel nanoparticles were prepared to observe its inhibitory effect on the intimal and mediator proliferation and the improvement of vascular remodeling after rabbit abdominal aortic injury. First, paclitaxel nanoparticles were prepared by ultrasonic emulsification solvent evaporation method. The surface of paclitaxel nanoparticles was modified by physical adsorption, the nanoparticles were characterized and the encapsulation efficiency was evaluated. Secondly, the endometrial thickness was measured by hematoxylin and eosin staining, and a spheroid with a smooth surface was observed under a scanning electron microscope. Finally, compared with the control group, local infusion of paclitaxel nanoparticles could effectively inhibit the proliferation of vascular endothelium in a dose-dependent manner. Local intravascular infusion of paclitaxel nanoparticle suspension can effectively inhibit the proliferation of vascular endothelial cells, and its inhibitory effect increases with the increase of the concentration of nanoparticle suspension. The paclitaxel nanoparticle suspension at a concentration of 30 mg/ml can play a good inhibitory role.

Cells of all tissues in human body interact with their neighboring cells and components of the extracellular matrix thereby creating a unique 3D microenvironment. These interactions are realized through a complex network of biochemical and mechanical signals that are important in maintaining normal cellular homeostasis. Numerous attempts have been undertaken during the last two decades to develop 3D models for studying their properties and understanding the mechanisms of regulation of cell microenvironment in vivo. Cardiac spheroids (cardiospheres) are one these models of cardiac microenvironment. In this study we demonstrate that unique microenvironment formed in cardiospheres consists of stem/progenitor and mesenchymal cells surrounded by extracellular matrix proteins synthesized by these cells. TGF-β1 participates in the regulation of contraction of cells forming cardiospheres, promotes activation of the epithelial-mesenchymal transition and self-organization of cells, which leads to the formation of larger spheroids. Thereby, the effect of TGF-β1 on the cells of cardiospheres can serve as a model for studying the mechanisms of regulation of cardiac microenvironment.

Background and objectives The objective of this study was to investigate whether conditioned medium from photobiomodulation (PBM) irradiated adipose-derived stromal cell (ASC) spheroids prior to implanting could stimulate angiogenesis and tissue regeneration to improve functional recovery of skin tissue in an animal skin wound model. Methods and results ASC were split and seeded on chitosan-coated 24 well plate at a density of 7.5×104 cells/cm2, and allowed to adhere at 37°C. Within 3 days of culture, ASC formed spheroids by PBM irradiation. Conditioned medium (CM) fractions were collected from the PBM-ASC to yield nor adipose-derived stromal cell spheroid (spheroid) and PBM-spheroid, respectively, centrifuged at 13,000 g at 4℃ for 10 min, and stored prior to use for ELISA, protein assay, or in vivo wound-healing assays. Phosphate-buffered saline, cultured CM from ASCs, PBM irradiation prior to implanting conditioned medium from ASC, cultured CM from ASC spheroid, and PBM-spheroid-CM (PSC) were transplanted into a wound bed in athymic mice to evaluate therapeutic effects of PSC in vivo. PSC enhanced wound closure in a skin injury model compared to PBS, CM, PBM-CM, and spheroid-CM. The density of vascular formations increased as a result of angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. Conclusions These results indicate that implant of PSC can significantly improve functional recovery compared to PBS, CM, PBM-CM, or spheroid-CM treatment. Implant of PSC may be an effective form of paracrine mediated therapy for treating a wound bed.

Although menopausal hormone therapy (MHT) is the most effective approach to managing the loss of ovarian activity, serious side effects have been reported. Cell-based therapy is a promising alternative for MHT. This study constructed engineered ovarian cell spheroids and investigated their endocrine function. Theca and granulosa cells were isolated from ovaries of 10-week-old rats. Two types of engineered ovarian cell spheroids were fabricated through forced aggregation in microwells, multilayered spheroids with centralized granulosa aggregates surrounded by an outer layer of theca cells and mixed ovarian spheroids lacking spatial rearrangement. The ovarian cell spheroids were encapsulated into a collagen gel. Non-aggregated ovarian cells served as controls. The endocrine function of the engineered ovarian spheroids was assessed over 30 days. The structure of the spheroids was well maintained during culture. The secretion of 17β-estradiol from both types of engineered ovarian cell spheroids was higher than in the control group and increased continuously in a time-dependent manner. Secretion of 17β-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of secreting sex steroid hormones, indicating that the hormone secreting function of ovaries can be recapitulated ex vivo and potentially adapted for MHT.

Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG-2 and RTS-11. The RTG-2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host-pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens.

Assessing phenotypic changes in both cancer cells and surrounding cells, which construct the tumour microenvironment, is essential for understanding the role of bi-directional communication among cells in the tumorigenic process. Here, a 3D in vitro cancer-stroma co-culture system, a co-culture disc, was reported for the spatiotemporal image analysis of cancer-stromal cell interactions. Due to their centre-open disc structure, the lung cancer A549 spheroids could be co-cultured with a high concentration of fibroblasts, without gel shrinkage in the long term (>1 month). In the co-culture disc, some populations of applied normal human lung fibroblasts showed morphological and phenotypic changes into activated myofibroblasts (AMFs) with high expression of myo-fibrotic α-smooth muscle actin fibre in the cell, which is a well-known feature of cancer-associated fibroblasts. The AMFs appeared heterogeneously at the boundary of cancer spheroids, which could not be detected by standard mass analysis using a quantitative RT-qPCR system, and they led to A549 cancer cell migration. In addition, the effects of oncogenic or medicinal additives were quantitatively assessed by combining co-culture discs with image analysis. This system provides a new potential technique to analyse the complicated crosstalk among cancer tissue-constructing cells during the tumorigenic process and provides insight into applications for the quantitative evaluation of substances inducing tumorigenesis as well as drugs to prevent and inhibit cancer progression.

We present a quantitative study of the metabolic activity of a single spheroid culture of human cancer cells. NMR (nuclear magnetic resonance) spectroscopy is an ideal tool for observation of live systems due to its non-invasive nature. However, limited sensitivity has so far hindered its application in microfluidic culture systems. We have used an optimised micro-NMR platform to observe metabolic changes from a single spheroid. NMR spectra were obtained by directly inserting microfluidic devices containing spheroids ranging from 150 $$\upmu$$ μ m to 300 $$\upmu$$ μ m in diameter in 2.5 $$\upmu$$ μ L of culture medium into a dedicated NMR probe. Metabolite concentrations were found to change linearly with time, with rates approximately proportional to the number of cells in the spheroid. The results demonstrate that quantitative monitoring of a single spheroid with $$\le$$ ≤ 2500 cells is possible. A change in spheroid size by 600 cells leads to a clearly detectable change in the l-Lactic acid production rate ($$p=3.5\times 10^{-3}$$ p = 3.5 × 10 - 3 ). The consumption of d-Glucose and production of l-Lactic acid were approximately 2.5 times slower in spheroids compared to monolayer culture of the same number of cells. Moreover, while cells in monolayer culture were found to produce l-Alanine and l-Glutamine, spheroids showed slight consumption in both cases.

Organotypic micrometre-size 3D aggregates of skin cells (multicellular spheroids) have emerged as a promising in vitro model that can be utilized as an alternative of animal models to test active ingredients (AIs) of skincare products; however, a reliable dermal spheroid-based microfluidic (MF) model with a goal of in vitro AI screening is yet to be developed. Here, we report a MF platform for the growth of massive arrays of dermal fibroblast spheroids (DFSs) in a biomimetic hydrogel under close-to-physiological flow conditions and with the capability of screening AIs for skincare products. The DFSs formed after two days of on-chip culture and, in a case study, were used in a time-efficient manner for screening the effect of vitamin C on the synthesis of collagen type I and fibronectin. The computational simulation showed that the uptake of vitamin C was dominated by the advection flux. The results of screening the benchmark AI, vitamin C, proved that DFSs can serve as a reliable in vitro dermal model. The proposed DFS-based MF platform offers a high screening capacity for AIs of skincare products, as well as drug discovery and development in dermatology.

3D spheroid cultures are attractive candidates for application in in vitro drug-induced hepatotoxicity testing models to improve the reliability of biological information obtainable from a simple 2D culture model. Various 3D spheroid culture models exist for hepatotoxicity screening, but quantitative assays of spheroid response in situ are still challenging to achieve with the current 3D liver toxicity platforms. In this study, we developed a 3D printing-based HepG2 liver spheroid culture model for in situ quantitative evaluation and high-content monitoring of drug-induced hepatotoxicity. HepG2 liver spheroids grown in mini-fabricated hydrogel constructs using a 3D bioprinter were used to obtain the EC50 values and to measure the multi-parametric hepatotoxic effects, including mitochondrial permeability transition (MPT), cytosolic calcium levels, and apoptosis. Interestingly, the average fluorescence intensities of apoptotic and cell death markers, calculated for out-of-focus and in-focus spheroids, increased proportionally as a function of the drug concentration, allowing for the determination of the EC50 values. In addition, 3D HepG2 spheroids were more resistant to nefazodone-induced MPT than 2D HepG2 cells, indicating that the gelatin/alginate hydrogel culture system provides enhanced resistance to hepatotoxic drugs. The drug response of HepG2 liver spheroids was also found to be unrelated to the spheroid size. These results demonstrate that the present 3D cell-printing-based embedded HepG2 liver spheroid platform is a promising approach for screening and characterizing drug-induced hepatotoxicity.

Microwells are used in studies to mimic the in vivo environment through an in vitro environment by generating three-dimensional cell spheroids. These microwells have been fabricated in various shapes using different methods according to the research purpose. However, because all microwells up to now have an open top, it has been difficult to culture spheroids of floating cells due to their low density, such as human adipose-derived stem cells (hASCs) that differentiate into adipocytes. Therefore, the labor-intensive hanging droplet method has been mainly used for the study of adipocytes. Here, we introduce a sigma-well, which is a microwell in the shape of the Greek letter sigma (σ) with a roof. Because of its unique shape, the sigma-well is advantageous for the culture of floating cells by reducing cell loss and external interference. The sigma-well was fabricated using the principle of surface tension of polydimethylsiloxane as well as air trapping and thermal expansion. Unlike conventional microwells, because the center of the bottom surface and the inlet of the sigma-well are not located on the same line and have a difference of approximately 218 μm, the spheroids are cultured more stably and may not escape the cavity. In this study, hASC and adipocyte spheroids differentiated using these sigma-wells were successfully cultured. In addition, through cytokine diffusion simulation, it was confirmed that the diffusion and mass transfer in the sigma-well was lower than that in the conventional microwell. It is expected that the morphological features of the sigma-well, which cannot be easily obtained by other methods, can be beneficial for the study of buoyant cell types such as adipocytes.

A spheroid is an aggregation of single cells with structural and functional characteristics similar to those of 3D native tissues, and it has been utilized as one of the typical in vitro three-dimensional (3D) cell models. Scaffold-free spheroids provide outstanding reflection of tissue complexity in a 3D in vivo-like environment, but they can neither fabricate realistic macroscale 3D complex structures without avoiding necrosis nor receive direct external stimuli (i.e., stimuli from mechanical or topographical cues). Here, we propose a spheroid-laden electrospinning process to obtain in vitro model achieved using the synergistic effect of the unique bioactive components provided by the spheroids and stimulating effects provided by the aligned nanofibers. Methods: To show the functional activity of the spheroid-laden structures, we used myoblast-spheroids to obtain skeletal muscle, comprising highly aligned myotubes, utilizing an uniaxially arranged topographical cue. The spheroid-electrospinning was used to align spheroids directly by embedding them in aligned alginate nanofibers, which were controlled with various materials and processing parameters. Results: The spheroids laden in the alginate nanofibers showed high cell viability (>90%) and was compared with that of a cell-laden alginate nanofiber that was electrospun with single cells. Consequently, the spheroids laden in the aligned nanofibers showed a significantly higher degree of myotube formation and maturation. Conclusion: Results suggested that the in vitro model using electrospun spheroids could potentially be employed to understand myogenic responses for various in vitro drug tests.

Background Here, we examined the tumoricidal effect of Gemini surfactant nanoparticles enriched with curcumin on 3D spheroid HT-29 cells. The delivery of curcumin and other phytocompounds to the tumor niche is an important challenge. Methods Spheroid HT-29 cells were generated by using a conventional hanging drop method and exposed to different concentrations of Gemini-curcumin nanoparticles. The changes in spheroid integrity and cell viability were evaluated by measuring the spheroid diameter and LDH release, respectively. The uptake of Gemini-curcumin nanoparticles was detected by flow cytometry assay. Flow cytometric of Rhodamine 123 efflux was also performed. Migration capacity was analyzed using a Transwell insert assay. By using real-time PCR analysis and Western blotting, we studied the expression level of MMP-2, -9, Vimentin, and E-cadherin genes. Results Gemini-curcumin nanoparticles had the potential to disintegrate spheroids and decrease central density compared to the control group (p p p p Conclusion Our data confirmed that Gemini curcumin has the potential to suppress cell proliferation and inhibit metastasis in 3D spheroid HT-29 cells in vitro.

The fabrication of functional tissue is important for tissue engineering, regenerative medicine, and biological research. However, current 3D bioprinting technologies mean it is hard to precisely arrange bioinks (composed of cells and materials) in a high-fidelity 3D structure and print cells of multiple types with sufficient concentrations and superior viabilities; this can severely constrain cell growth, interactions, and functions. Here, an acoustic droplet printing method is introduced to solve these problems in 3D bioprinting. Being nozzle-free, the acoustic printer stably enables high-concentration cells, or even cell spheroids, to be printed without clogging. Cell viability (>94%) using post acoustic printing is higher than those obtained with currently used inkjet-based (>85%) and extrusion-based (40-80%) bioprinting methods. Also, this method involves a small printer that can be flexibly integrated, allowing different kinds of bioinks to be printed. Moreover, the limited printability of low-concentration gelatin methacryloyl (5% (w/v) GelMA) materials is overcome by determining the positioning, fluidity (e.g., spreading), and 3D morphology of the GelMA droplets; therefore, high-fidelity 3D constructs can be fabricated. As a proof of concept, a tumor microenvironment consisting of one tumor spheroid surrounded by a high concentration of cancer-associated fibroblasts (CAFs) was constructed; this was able to establish a dynamic tumor invasion function modulated by reciprocal tumor cell-CAF interactions. The nozzle-free, contact-free, and low cell-damage merits of this method will advance bioprinting, allowing the creation of more functional native tissues, organoids, or disease models.

Music is known for reducing stress, anxiety and depression, improving cognitive performance, and enhancing oestrogen levels. However, its effect on non-auditory mammalian cell system and the molecular events leading to higher oestrogen levels is less explored. Therefore, the present study targeted to know the direct effects of a peaceful Vedic music on 3D cultured buffalo granulosa cell spheroids. The spheroids were daily exposed to the Mahamrityunjaya mantra, a kind of Vedic chants, for 1.5 hr for 6 days. After 6 days, the music effect was analysed by the expression analysis of steroidogenic (CYP19A1, STAR and HSD17β1) and proliferative marker (PCNA) genes. Interestingly, the CYP19A1 gene expression was significantly upregulated by 3.464 ± 0.15 folds in the music exposed spheroids than the non-exposed spheroids. However, the expression of other steroidogenic and proliferative genes was unaltered. These observations provided a transcriptional clue for higher estradiol levels by the music and a scope to use Vedic chants for increasing the CYP19A1 expression to help tackle some pathophysiological conditions.

Sustained proliferation is a significant driver of cancer progression. Cell-cycle advancement is coupled with cell size, but it remains unclear how multiple cells interact to control their volume in 3D clusters. In this study, we propose a mechano-osmotic model to investigate the evolution of volume dynamics within multicellular systems. Volume control depends on an interplay between multiple cellular constituents, including gap junctions, mechanosensitive ion channels, energy-consuming ion pumps, and the actomyosin cortex, that coordinate to manipulate cellular osmolarity. In connected cells, we show that mechanical loading leads to the emergence of osmotic pressure gradients between cells with consequent increases in cellular ion concentrations driving swelling. We identify how gap junctions can amplify spatial variations in cell volume within multicellular spheroids and, further, describe how the process depends on proliferation-induced solid stress. Our model may provide new insight into the role of gap junctions in breast cancer progression., Cell proliferation is regulated by cell volume, but it is unclear how individual cancer cells coordinate to regulate cell volumes in 3D clusters. Here the authors propose a mechano-osmotic model to analyse the exchange of fluid and ions between connected cells and their environment in response to proliferation-induced solid stress.

Introduction Primary cultured hepatocytes are an important model for early safety evaluations of newly developed drugs. Many factors, however, hinder the wider applications of this technology, especially the difficulty to maintain these cells in long-term culture. To date, creating a stable supply of human or animal hepatocytes with proper hepatic function in vitro has not been achieved. Furthermore, frequently harvesting hepatocytes from living donors for use in culture is highly invasive and simply not feasible. We have previously reported that canine bone marrow-derived mesenchymal stem cells (cBMSCs) can be effectively converted into induced hepatocyte-like cells (iHep cells); however, these cells had reduced function in comparison to mature hepatocytes. In recent studies, spheroid formation-based three-dimensional (3D) culture has been noted to greatly increase hepatocyte function; nevertheless, no reports have described the use of this technology for culturing canine hepatocytes. Therefore, in this study, we aimed to establish a 3D spheroid culture using converted canine iHep cells to investigate their function as hepatocytes. Methods The iHep cells were prepared by introducing two genes, namely, the Forkhead box A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α), into cBMSCs seeded onto an ultra-low attachment microplate to induce spheroid formation. Thereafter, the hepatic functions of these spheroids were evaluated using immunocytochemistry, as well as qualitative and quantitative PCR. Results Notably, albumin was observed in the iHep spheroids and the expression of hepatic genes, such as albumin and drug metabolism CYP genes, could also be detected. Another interesting finding was evident upon further comparing the quantified albumin gene and CYP2E1 gene expressions in the two-dimensional and three-dimensional culture systems; notably, a 100- to 200-fold increase in gene expression levels was observed in the three-dimensional spheroids when compared to those in conventional monolayers. Conclusions Upon incorporating three-dimensional technology, we managed to achieve iHep spheroids that are closer in gene expression to living liver tissue compared to conventional monolayer cultures. Thus, we are one step closer to creating a sustainable in vitro hepatocyte model. Furthermore, we believe that this system is capable of maintaining the stable drug metabolizing capacity of canine hepatocytes in vitro, which might be useful in improving current drug assessment studies.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cells and reaggregated into smaller spheroids using the microfabricated vessels. This reaggregation process raised WNT5A and WNT11 expression levels and improved the quality of cardiomyocyte population compared to that in a control group in which dissociation and reaggregation were not performed., Highlights • Microfabricated culture vessels were used to form large number of hiPSC spheroids. • High purity cardiomyocytes were obtained by reaggregation of hiPSC-derived spheroids. • Maturation of cardiomyocyte was promoted by the reaggregation process. • WNTs were also increased the reaggregation process during cardiac differentiation.

Spheroid culture provides cells with a three-dimensional environment that can better mimic physiological conditions compared to monolayer culture. Technologies involved in the generation of cell spheroids are continuously being innovated to produce spheroids with enhanced properties. In this paper, we review the manufacturing capabilities of current cell spheroid generation technologies. We propose that spheroid generation technologies should enable tight and robust process controls to produce spheroids of consistent and repeatable quality. Future technology development for the generation of cell spheroids should look into improvement in process control, standardization, scalability and monitoring, in addition to advanced methods of spheroid transfer and characterization.

Agents that increase tumor radiosensitivity are of interest in improving outcomes in radiotherapy (XRT). DNA-PK inhibitors radiosensitize and alter cell adhesion proteins. We investigated combination radiation and a DNA-PK inhibitor in monolayers vs spheroids.Using HER2 positive mammary carcinoma cells, we investigated the impact of NU7441, a DNA-PK inhibitor, on irradiated monolayer and spheroid cultures. Colony formation assays were performed with monolayer culture cells and spheroids after irradiation with/without NU7441 (5 μM).In monolayer culture cells, α/β increased from 3.0 ± 0.2 Gy (XRT alone) to 6.9 ± 0.2 Gy (XRT+NU7441). Corresponding α/β values for cells obtained by disaggregating treated spheroids were 3.6 ± 0.7 Gy (XRT alone) and 3.5 ± 0.2 Gy (XRT+NU7441). However, spheroid survival was highly sensitive to NU7441 incubation. After 4 Gy XRT alone 75% of the irradiated spheroids remained intact; when NU7441 treatment was involved, 13% remained intact. No spheroids survived to 3 weeks at 6 Gy or more. The discrepancy between the minimal change in α/β from cells derived from spheroids and the spheroid growth response was not related to poor penetration of NU7441.DNA-PK inhibitor NU7441 radiosensitized monolayer cells but not cells obtained from spheroids. NU7441 and radiation increased spheroid fragmentation.

Background High fatality in ovarian cancer is attributed to metastasis, propagated by the release of multi-cellular aggregates/spheroids into the peritoneal cavity and their subsequent mesothelial invasion of peritoneal organs. Spheroids are therefore a common and clinically relevant in vitro model for ovarian cancer research. Spheroids in patients vary significantly in size and shape and display enhanced resistance to anti-cancer drugs compared to monolayers. However, there is no consensus on how spheroid size and shape affect drug resistance. Moreover, existing data regarding the influence of epithelial-to-mesenchymal transition (EMT) profile on spheroid shape and migration is inconclusive. Methods We formed spheroids with OVCAR-3 and OVCAR-8 cells, chosen for their established genetic similarity to the patient tumor samples. We monitored their morphology using confocal microscope with dipping objective and fluorescent microscope. We characterized important EMT biomarkers; E-cadherin, Vimentin and Slug through western blotting in monolayers and spheroids. We treated these spheroids with Taxol and Cisplatin and investigated their migratory profile based on their morphology. Results We report two distinct multicellular structures: loose aggregates (OVCAR-3) and compact spheroids (OVCAR-8). We attribute these different morphologies to the expression of the EMT biomarkers, and their changes upon spheroid formation. Importantly, we did not observe a difference in resistance to the anti-cancer drugs as a function of spheroid size and shape. However, migration capacity of compact spheroid (OVCAR-8) was 15-fold higher compared to that of loose aggregates (OVCAR-3). Conclusions These results highlight the importance of spheroid size and shape on anti-cancer drug resistance and migration profiles. The results of this study can, therefore, help to elucidate general rules for ovarian cancer studies based on 3D samples.

Quantitative determination of cytokines, chemokines, growth factors, and other soluble protein molecules in various biological fluids is a routine task of modern diagnostics and biomedicine; however, currently existing methods of analysis include several stages and take a long time. This paper presents a protocol that allows determination of an analyte concentration in a sample within a few minutes using biomolecule layer interferometry. Conditioned media in which mouse mesenchymal stem cells were grown under normoxia and hypoxia in the form of monolayer, 3D cell sheets, or 3D spheroids were used as a model system, in which the concentration of VEGF-A (vascular endothelial growth factor) was determined. The method showed high sensitivity (at least 0.10 ng/mL of VEGF-A). The obtained values of VEGF-A concentrations in mesenchymal stem cell conditioned media were close to the values determined by enzyme immunoassay. Using biomolecule layer interferometry, a significant 2.5–3.0 fold increase in VEGF-A production by spheroids and cell sheets of mesenchymal stem cells was found in comparison with the cells grown in a monolayer. Thus, the developed protocol made it possible to adapt the method of biomolecule layer interferometry for rapid quantitative analysis of growth factors in the conditioned media.

Three-dimensional in vitro spheroids are a reliable model to study tumor biology and drug toxicity. However, inconsistencies exist in terms of seeding cell density that governs spheroid size and shape, influencing the experimental outcome. We investigated the effect of varying cell densities using glioblastoma cells on tumorsphere formation and their responsiveness to drug treatment. Our results demonstrated that in comparison with spheroids formed with lower cell density, spheroids formed with higher cell density were not only larger in size but also had a larger necrotic core surrounded by a higher number of quiescent cells and were irresponsive to drug treatment. Our study highlights the importance of predetermination of cell density to obtain desired/appropriate spheroid size to produce consistent and reliable data on drug toxicity studies in tumor cells.