Three-dimensional Multicellular Spheroids Research Articles

Polymeric Hybrid Nanomicelles for Cancer Theranostics: An Efficient and Precise Anticancer Strategy for the Codelivery of Doxorubicin/miR-34a and Magnetic Resonance Imaging.

To realize precise tumor therapy, a versatile oncotherapy nanoplatform integrating both diagnostic and therapeutic functions is necessary. Herein, we fabricated a hybrid micelle (HM) utilizing two amphiphilic diblock copolymers, polyethylenimine-polycaprolactone (PEI-PCL) and diethylenetriaminepentaacetic acid gadolinium(III) (Gd-DTPA)-conjugated polyethyleneglycol-polycaprolactone (Gd-PEG-PCL), to codeliver the small-molecule chemotherapy drugs doxorubicin (Dox) and microRNA-34a (miR-34a), denoted as Gd-HM-Dox/34a. Conjugating Gd-DTPA on the surface of hybrid micelles, leading the relaxation rate of Gd-DTPA increased more than 1.4 times (13.6 mM-1 S-1). Furthermore, hybrid micelles enhanced the ability of miR-34a to escape from lysosomes/endosomes and Dox release to the nucleus. In addition, the released miR-34a subsequently downregulates Bcl-2, cyclin D1, CDK6, and Bax expression and inhibits proliferation and migration of MDA-MB-231 breast cancer cells. Moreover, the suitable micelle size improved the penetration of Dox into three-dimensional (3D) multicellular spheroids compared with Gd-HM-Dox and Free Dox, generating efficient cell killing in the 3D multicellular spheroids. Furthermore, the Gd-HM-Dox/34a exhibited augmented accumulation in the tumor tissue, which improved the magnetic resonance (MR) imaging contrast of solid tumors and enhanced the combined efficiency of chemotherapeutic drugs Dox and therapeutic gene miR-34a in suppressing tumor growth on MDA-MB-231 tumor-bearing mice. Therefore, we established a hybrid micelle to offer a promising theranostic approach that inhibits tumor growth and enhances MR imaging.

Oxygen-Sensing Probes and Bandage for Optical Detection of Inflammation.

Small-molecular and macromolecular optical probes based on an iridium complex chromophore were designed for detecting hypoxic inflammation in this work. The optical probes had a large conjugated ligand that could extend its phosphorescence emission to the red-wavelength region, thus increasing the tissue penetration depth of the optical signal. Due to good water solubility, the macromolecular optical probe could image both monolayer cells and three-dimensional multicellular spheroids. Moreover, this macromolecular probe was able to effectively image inflammation and distinguish healthy and inflammatory regions in a mouse model of lipopolysaccharide (LPS)-induced inflammation with low background interference. Furthermore, we integrated the hydrophobic small-molecular probe into the polyurethane thin film, and the resulting film successfully monitored the inflammation of chronic wounds in a mouse model. This work demonstrated the great potential of the iridium complex in optical imaging hypoxia and hypoxia-associated inflammation and will have significant impact on the design of high-sensitivity sensors for the detection of hypoxia.

The HSP90 inhibitor onalespib potentiates 177Lu‑DOTATATE therapy in neuroendocrine tumor cells.

177Lu-DOTATATE was recently approved for the treatment of somatostatin receptor (SSTR)-positive neuroen-docrine tumors (NETs). However, despite impressive response rates, complete responses are rare. Heat shock protein 90 (HSP90) inhibitors have been suggested as suitable therapeutic agents for NETs, as well as a potential radiosensitizers. Consequently, the aim of this study was to investigate whether the HSP90-inhibitor onalespib could reduce NET cell growth and act as a radiosensitizer when used in combination with 177Lu-DOTATATE. The NET cell lines BON, NCI-H727 and NCI-H460, were first characterized with regards to 177Lu-DOTATATE uptake and sensitivity to onalespib treatment in monolayer cell assays. The growth inhibitory effects of the monotherapies and combination treatments were then examined in three-dimensional multicellular tumor spheroids. Lastly, the molecular effects of the treatments were assessed. 177Lu-DOTATATE uptake was observed in the BON and NCI-H727 cells, while the NCI-H460 cells exhibited no detectable uptake. Accordingly, 177Lu-DOTATATE reduced the growth of BON and NCI-H727 spheroids, while no effect was observed in the NCI-H460 spheroids. Onalespib reduced cell viability and spheroid growth in all three cell lines. Furthermore, the combination of onalespib and 177Lu-DOTATATE exerted synergistic therapeutic effects on the BON and NCI-H727 spheroids. Western blot analysis of BON spheroids revealed the downregulation of epidermal growth factor receptor (EGFR) and the upregulation of γ H2A histone family member X (γH2AX) following combined treatment with onalespib and 177Lu-DOTATATE. Moreover, flow cytometric analyses revealed a two-fold increase in caspase 3/7 activity in the combination group. In conclusion, the findings of this study demonstrate that onalespib exerts antitumorigenic effects on NET cells and may thus be a feasible treatment option for NETs. Furthermore, onalespib was able to synergistically potentiate 177Lu-DOTATATE treatment in a SSTR-specific manner. The radiosensitizing mechanisms of onalespib involved the downregulation of EGFR expression and the induction of apoptosis. Consequently, the combination of onalespib and 177Lu-DOTATATE may prove to be a promising strategy with which to improve therapeutic responses in patients with NETs. Further studies investigating this strategy in vivo regarding the therapeutic effects and potential toxicities are warranted to expand these promising findings.

Ratiometric Luminescent Nanoprobes Based on Ruthenium and Terbium-Containing Metallopolymers for Intracellular Oxygen Sensing.

A collection of luminescent metal complexes have been widely used as oxygen probes in the biomedical field. However, single intensity-based detection approach usually suffered from errors caused by the signal heterogeneity or fluctuation of the optoelectronic system. In this work, respective ruthenium (II) and terbium (III) complexes were chosen to coordinate a bipyridine-branched copolymer, so that to produce oxygen-sensitive metallopolymer (Ru-Poly) and oxygen-insensitive metallopolymer (Tb-Poly). Based on the hydrophobic Ru-Poly and Tb-Poly, a ratiometric luminescent oxygen nanoprobe was facilely prepared by a nanoprecipitation method. The nanoprobes have a typical size of ~100 nm in aqueous solution, exhibiting a green-red dual-wavelength emission under the excitation of 300 nm and 460 nm, respectively. The red emission is strongly quenched by dissolved oxygen while the green one is rather stable, and the ratiometric luminescence was well fitted by a linear Stern–Volmer equation. Using the ratiometric biocompatible nanoprobes, the distribution of intracellular oxygen within three-dimensional multi-cellular tumor spheroids was successfully imaged.

Uptake and efflux of FL118 and two FL118 derivatives in 3D cell model.

Drug uptake and efflux are two of the critical factors required in order to be able to define drug efficacy. This study aims to investigate cytotoxicity and uptake mechanisms of two FL118 analogues (7-Q20 and val-FL118) in parallel with FL118 in three-dimensional multi-cellular spheroids model. The influence of compound concentration, time, temperature, cell lines, and the inhibitors of P-gp, BCRP and LAT1 on drug uptake and efflux were investigated. In vitro cytotoxicity studies revealed that FL118, 7-Q20 and val-FL118 exhibited sensitive cytotoxicity to the HCT-116 cell line and the water-soluble compound 7-Q20 showed the lowest IC50. Cellular uptake and efflux of FL118 was independent of efflux pump proteins. Uptake and efflux of 7-Q20 were affected by P-gp, which was one of reasons that caused a lower uptake at 37°C than at 4°C. The carrier protein LAT1 played a role in the cellular intakes of val-FL118. These findings provided basic information for FL118 and the two novel FL118 derivatives for further development.

Acid-labile hyperbranched poly(ortho ester amido amine) as efficient gene carriers: Preparation, characterization, and in vitro evaluation.

Cationic polymers have become one of the most promising synthetic vectors for gene transfection, however, their translation to the clinic has been severely hampered because of their high cytotoxicity and polyplex instability in serum. Herein, we report the design and synthesis of a new type of pH-sensitive hyperbranched poly(ortho ester amido amine) (HPOEAMAM) by the use of ortho ester bonds to give a non-viral vector with high efficiency and safety in two-dimensional (2D) cells and three-dimensional (3D) multicellular tumor spheroids. The pH-sensitive HPOEAMAM forms strong interactions with DNA and completely condense DNA at the weight ratio of 8. Furthermore, ortho ester in HPOEAMAM can be hydrolysed in response to endo/lysosome pHs to facilitate DNA release post internalization and diminish the cytotoxicity. Our findings provide a valuable insight into the construction of pH-sensitive hyperbranched cationic polymers which is an attractive strategy for producing effective gene vectors for clinically viable gene therapy.

Tumor-targeted drug delivery and sensitization by MMP2-responsive polymeric micelles

Low tumor specificity and multidrug resistance (MDR) remain challenging for many anticancer drugs. In this study, the micelles assembled by a matrix metalloproteinase 2 (MMP2)-sensitive self-assembling efflux inhibitor (PEG2k-pp-PE) were developed and evaluated in various cancer models. In vitro, the PEG2k-pp-PE micelles enhanced the cellular uptake and tissue penetration and sensitized the cancers to drug treatments in MDR cancer cells and their three-dimensional multicellular spheroids. Their efflux inhibitory capability was comparable to those of the well-known small-molecule P-glycoprotein (P-gp) inhibitor and polymeric P-gp inhibitor. In vivo, the PEG2k-pp-PE micelles could specifically and effectively deliver the loaded cargoes to the tumor, as evidenced by the enhanced drug accumulation and prolonged drug retention in the tumor tissue, resulting in the improved anticancer activity. Our results suggest that the PEG2k-pp-PE micelles may have great potential to be a simple but multifunctional nanocarrier for concurrent tumor-targeted drug delivery and sensitization of resistant cancers.

Widespread gene transfer to malignant gliomas with In vitro-to-In vivo correlation

Gene therapy of malignant gliomas has shown a lack of clinical success to date due in part to inability of conventional gene vectors to achieve widespread gene transfer throughout highly disseminated tumor areas within the brain. Here, we demonstrate that newly engineered polymer-based DNA-loaded nanoparticles (DNA-NP) possessing small particle diameters (~50 nm) and non-adhesive surface polyethylene glycol (PEG) coatings efficiently penetrate brain tumor tissue as well as healthy brain parenchyma. Specifically, this brain-penetrating nanoparticle (BPN), following intracranial administration via convection enhanced delivery (CED), provides widespread transgene expression in heathy rodent striatum and an aggressive brain tumor tissue established orthotopically in rats. The ability of BPN to efficiently traverse both tissues is of great importance as the highly invasive glioma cells infiltrated into normal brain tissue are responsible for tumor recurrence. Of note, the transgene expression within the orthotopic tumor tissue occurred preferentially in glioma cells over microglial cells. We also show that three-dimensional (3D) multicellular spheroids established with malignant glioma cells, unlike conventional two-dimensional (2D) cell cultures, serve as an excellent in vitro model reliably predicting gene vector behaviors in vivo. Briefly, DNA-NP possessing greater surface PEG coverage exhibited more uniform and higher-level transgene expression both in the 3D model and in vivo, whereas the trend was opposite in 2D culture. The finding here alerts that gene transfer studies based primarily on 2D cultures should be interpreted with caution and underscores the relevance of 3D models for screening newly engineered gene vectors prior to their in vivo evaluation.

Redox-sensitive micelles based on retinoic acid modified chitosan conjugate for intracellular drug delivery and smart drug release in cancer therapy

Novel chitosan-cystamine-retinoic acid conjugate (CS-SS-RA) was synthesized and could self-assemble into redox-sensitive micelles in aqueous environment with low critical micelle concentration value. CS-SS-RA micelles were characterized with spherical shape, desirable particle size, negative zeta potential, high paclitaxel (PTX) loading and encapsulation efficiency and redox-sensitivity. Hemolysis and cytotoxicity studies proved the safety of CS-SS-RA micelles for intravenous administration. Cytotoxicity study against HepG2 cells and the growth inhibition study on three-dimensional multicellular tumor spheroids (MCTSs) revealed that PTX-loaded CS-SS-RA micelles exhibited higher antitumor activity than free PTX. The in vitro cellular uptake profiles of FITC-labeled CS-SS-RA micelles evaluated via confocal laser scanning microscopy and flow cytometry indicated that CS-SS-RA micelles could enhance cellular uptake efficiency of PTX, and their internalization by HepG2 cells were mediated by clathrin-mediated endocytosis and macropinocytosis. These results demonstrated that CS-SS-RA micelles could be developed as a promising platform for intracellular delivery of hydrophobic antitumor agents.

Expression of bladder cancer-associated glycans in murine tumor cell lines.

The characterization of murine cell lines is of great importance in order to identify preclinical models that could resemble human diseases. Aberrant glycosylation includes the loss, excessive or novel expression of glycans and the appearance of truncated structures. MB49 and MB49-I are currently the only two murine cell lines available for the development of preclinical bladder cancer models. The glycans Lewis X (LeX), Sialyl lewis X (SLeX) and Sialyl Tn (STn) have previously been associated with aggressiveness, dissemination and poor prognosis in human bladder cancer, additionally N-glycolyl GM3 (NGcGM3) is a neo-antigen expressed in many types of tumors; however, to the best of our knowledge, its expression has not previously been assessed in this type of cancer. Taking into account the relevance of glycans in tumor biology and considering that they can act as targets of therapies and biomarkers, the present study evaluated the expression of LeX, SLeX, STn and NGcGM3 in MB49 and MB49-I cells, in different growth conditions such as monolayer cultures, three-dimensional multicellular spheroids and mouse heterotopic and orthotopic tumors. The expression of LeX was not detected in either cell line, whereas SLeX was expressed in monolayers, spheroids and orthotopic tumors of both cell lines. STn was only identified in MB49 monolayers and spheroids. There are no reports concerning the expression of NGcGM3 in human or murine bladder cancer. In our hands, MB49 and MB49-I expressed this ganglioside in all the growth conditions evaluated. The assessment of its expression in cancer cell lines and patient tumors is of great importance, considering the relevance of this ganglioside in tumor biology. The data obtained by the present study demonstrates that glycan expression may be substantially altered depending on the growth conditions, highlighting the importance of the characterization of murine cancer models. To the best of our knowledge, the present study is the first to examine the expression of cancer-associated glycans, in the two murine cell lines available for the development of preclinical studies in bladder cancer.

Phenylboronic acid-functionalized ultra-pH-sensitive micelles for enhanced tumor penetration and inhibition in vitro

In this work, the tumor-targeted ultra-pH-responsive conjugates (PBA/Dex-g-OE) and nontargeted conjugates (Dex-g-OE) were successfully prepared and could easily self-assemble into stable micelles with lower CMC values in neutral aqueous solution. Transmission electron microscopy and dynamic light scattering measurement indicated that the resulting micelles have desirable size distribution and regular spherical shape. The PBA/Dex-g-OE micelles possessed high stability in physiological condition and were pH sensitive to both extracellular and intracellular acidic conditions. Doxorubicin (DOX) was efficiently loaded to give the DOX-loaded micelles (PBA/Dex-g-OE-DOX and Dex-g-OE-DOX) with the desirable drug loading contents. In vitro cellular uptake and growth inhibition assays suggested that PBA/Dex-g-OE-DOX micelles were more efficiently internalized by monolayer tumor cells and three-dimensional multicellular tumor spheroids (MCTS) than nontargeted micelles (Dex-g-OE-DOX), leading to the fast and complete destruction of MCTS in vitro.

Dual-stimuli-sensitive poly(ortho ester disulfide urethanes)-based nanospheres with rapid intracellular drug release for enhanced chemotherapy

Herein, new poly(ortho ester disulfide urethanes) (POEDU) and poly(ortho ester urethanes) (POEU) were successfully synthesized via polycondensation between active esters of 1,6-hexandiol (HD) and dual-stimuli-sensitive ortho ester disulfide diamine or pH-senstive ortho ester diamine. The corresponding POEDU and POEU nanospheres were easily fabricated using an oil-in-water emulsion technique. In vitro degradation experiments indicated that POEDU nanospheres degraded faster than POEU nanospheres in mildly acidic and reductive environments. Doxorubicin (DOX) as a model antitumor drug was successfully incorporated into these nanospheres to give DOX-loaded nanoparticles (POEDU-DOX and POEU-DOX). In vitro drug release studies showed that release of DOX from dual-stimuli-sensitive POEDU-DOX was accelerated compared with release from the pH-sensitive POEU-DOX under DL-dithiothreitol (DTT) and mildly acidic conditions. In addition, in vitro uptake and cytotoxicity assays revealed that POEDU-DOX exhibited more efficient antitumor effect than POEU-DOX did against both two-dimensional (2D) cells and three-dimensional (3D) multicellular tumor spheroids (MCTS). Finally, in a mice H22 tumor model, POEDU-DOX exhibited preferable antitumor capability. In conclusion, the pH and redox dual-stimuli-sensitive POEDU nanospheres can be superior drug carriers for cancer treatment.

GSH-Activated Light-Up Near-Infrared Fluorescent Probe with High Affinity to αvβ3 Integrin for Precise Early Tumor Identification.

The development of tumor-associated, stimuli-driven, turn-on near-infrared (NIR) fluorophores requires urgent attention because of their potential in selective and precise tumor diagnosis. Herein, we describe a NIR fluorescent probe (CyA-cRGD) comprised of a fluorescence reporting unit (a cyanine dye) linked with a GSH-responsive unit (nitroazo aryl ether group) and a tumor-targeting unit (cRGD). The NIR fluorescence of CyA-cRGD with sensitive and selective response to GSH can act as a direct off-on signal reporter for GSH monitoring. Notably, CyA-cRGD possesses improved biocompatibility compared with CyA, which is highly desirable for in vivo fluorescence tracking of cancer. Confocal fluorescence imaging confirmed the tumor-targeting capability and GSH detection ability of CyA-cRGD in tumor cells, normal cells, and coincubated tumor /normal cells and in the three-dimensional multicellular tumor spheroid. Furthermore, it was validated that CyA-cRGD could detect tumor precisely in GSH and integrin αvβ 3 high-expressed tumor-bearing mouse models. Importantly, it was confirmed that CyA-cRGD possessed high efficiency for early-stage tumor imaging in mouse models with tumor cells implanted within 72 h. This method provided significant advances toward more in-depth understanding and exploration of tumor imaging, which may potentially be applied for clinical early tumor diagnosis.

Abstract 260: Apigenin nanoparticle suppresses sphere formation in CD133+/ALDH1high prostate cancer stem cells through downregulation of stem cell markers

Abstract The recurrence of prostate cancer after definitive treatment(s) is thought to be due to the potential of rare cancer stem cells (CSCs) to withstand therapy and initiate tumor formation. CSCs are a subpopulation of cancer cells capable of self-renewal and plastic adaptation mediating tumorigenesis, metastasis, and therapy resistance. CSCs are associated with cancer progression and clinical outcome in cancer patients. In the present study, we isolated CSCs from various human prostate cancer 22Rv1, DU145 and PC-3 cells by applying selection of cluster of differentiation 133 cell surface marker and high ALDH1 activity (CD133+/ALDH1high). Next we aimed to identify CD133+/ALDH1high CSCs isolated from human prostate cancer cells and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the CSC population continued to grow in the three-dimensional multicellular spheroids. Differentiation investigated with stem cell-related genomic characteristics exhibited significant increase in genes viz. CD44, Nanog, β-catenin, c-Myc, SOX2 and Oct4 in CSCs at the message level. In further studies we determine the effect of natural plant flavone apigenin on CSC population. Apigenin is abundantly present in common fruits and vegetables and possess remarkable anticancer, anti-angiogenic and anti-metastatic properties. Apigenin loaded polyethylene glycol nanoparticles (Ap-NP) was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of Ap-NP were assessed by transmission electron microscopy. Treatment of CD133+/ALDH1high CSCs with Ap-NP inhibited cell viability and sphere formation at higher extent in time dependent manner, compared to the non-CSCs counterparts. Furthermore, Ap-NP exposure resulted in the downregulation of Nanog, β-catenin, c-Myc, SOX2 and Oct4 in the CSCs. Taken together, our study demonstrate overexpression of potential CSC markers and the ability to target these CSCs by the use of non-toxic agent apigenin killing this rare population that may lead to the total abolition of tumor recurrence and/or metastasis. Citation Format: Rajnee Kanwal, Sanjeev Shukla, Agata A. Exner, Andreas G. Tzakos, Sanjay Gupta. Apigenin nanoparticle suppresses sphere formation in CD133+/ALDH1high prostate cancer stem cells through downregulation of stem cell markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 260.

A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy.

Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 μmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. Mol Cancer Ther; 17(7); 1405-15. ©2018 AACR.

A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity.

The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1α). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo-like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

A novel approach to producing uniform 3-D tumor spheroid constructs using ultrasound treatment

Producing three-dimensional (3-D) multicellular tumor spheroids (TSs) is valuable for characterizing anticancer drugs since they provide a more representative model of the 3-D in vivo tumor than conventional two-dimensional (2-D) monolayer culture. The interaction of tumor cells with the extracellular matrix (ECM) in a 3-D culture environment is more similar to a tumor in vivo than in a 2-D environment; cell-cell and cell-ECM interaction can influence cell behaviour, such as in response to drug treatment. In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. Cell-laden hydrogel droplets are formed using a flow focusing process wherein the hydrogel precursors should be a homogeneous mixture. During gelation of the droplets into beads, the alginate acts as a fast gelling component helping to maintain the spherical shape of beads and preventing coalescence as the temperature-sensitive collagen I component gels more slowly. To produce uniform hydrogel droplets using the microfluidic flow focusing system, the mixtures must be homogeneous. However, collagen's sensitivity to temperature can lead to formation of chunks of collagen gel inside of the mixture, causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure can contribute to a delay in cell proliferation. Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen type I through ultrasonication. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, collagen can form small sized fibrils to thick fibers. Here, the mixtures containing collagen are assessed for morphology of collagen fibers/fibrils, cell viability, and proliferation. Human source Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment (UST) provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not inhibiting cell proliferation.

Prediction of Necrotic Core and Hypoxic Zone of Multicellular Spheroids in a Microbioreactor with a U-Shaped Barrier.

Microfluidic devices have been widely used for biological and cellular studies. Microbioreactors for three-dimensional (3D) multicellular spheroid culture are now considered as the next generation in in vitro diagnostic tools. The feasibility of using 3D cell aggregates to form multicellular spheroids in a microbioreactor with U-shaped barriers has been demonstrated experimentally. A barrier array is an alternative to commonly used microwell traps. The present study investigates oxygen and glucose concentration distributions as key parameters in a U-shaped array microbioreactor using finite element simulation. The effect of spheroid diameter, inlet concentration and flow rate of the medium are systematically studied. In all cases, the channel walls are considered to be permeable to oxygen. Necrotic and hypoxic or quiescent regions corresponding to both oxygen and glucose concentration distributions are identified for various conditions. The results show that the entire quiescent and necrotic regions become larger with increasing spheroid diameter and decreasing inlet and wall concentration. The shear stress (0.5–9 mPa) imposed on the spheroid surface by the fluid flow was compared with the critical values to predict possible damage to the cells. Finally, optimum range of medium inlet concentration (0.13–0.2 mM for oxygen and 3–11 mM for glucose) and flow rate (5–20 μL/min) are found to form the largest possible multicellular spheroid (500 μm), without any quiescent and necrotic regions with an acceptable shear stress. The effect of cell-trap types on the oxygen and glucose concentration inside the spheroid was also investigated. The levels of oxygen and glucose concentration for the microwell are much lower than those for the other two traps. The U-shaped barrier created with microposts allows for a continuous flow of culture medium, and so improves the glucose concentration compared to that in the integrated U-shaped barrier. Oxygen concentration for both types of U-shaped barriers is nearly the same. Due to the advantage of using U-shaped barriers to culture multicellular spheroids, the results of this paper can help to choose the experimental and design parameters of the microbioreactor.

Evaluation of sulfane sulfur bioeffects via a mitochondria-targeting selenium-containing near-infrared fluorescent probe

As a crucial member in antioxidant regulatory systems, sulfane sulfur plays essential roles in cytoprotective mechanisms by directly eliminating ROS and altering ROS-mediated redox signaling. Despite the rising interests in sulfane sulfur, there only a few bio-compatible methods are available for its direct detection. Moreover, most of the existing methods cannot meet the requirements of real-time detection due to the reactive and labile chemical properties of sulfane sulfur. Therefore, we strive to clarify the mutual relationship between mitochondria sulfane sulfur and ROS under hypoxia stress. Herein, we report a near-infrared fluorescent probe Mito-SeH for the selective imaging of mitochondrial sulfane sulfur in cells and in vivo under hypoxia stress. Mito-SeH includes three moieties: a selenol group (SeH) as the stronger sulfur-acceptor; a near-infrared azo-BODIPY fluorophore as the fluorescent modulator; a lipophilic alkyltriphenylphosphonium cation as the mitochondrial delivery. Mito-SeH exhibits excellent selectivity and sensitivity towards the detection of mitochondria sulfane sulfur. The hypoxia response behavior of Mito-SeH is evaluated in monolayer cell and three-dimensional multicellular spheroid to clarify the relationship between sulfane sulfur and hypoxia. We confirm that sulfane sulfur protection mechanism against hypoxia is to inhibition of caspase-dependent apoptosis through directly scavenging ROS pathway. The probe is also applied to measurement of sulfane sulfur in ex vivo-dissected organs of hypoxic mouse model, as well as the probe is successfully used for real-time monitoring the changes of sulfane sulfur and ROS in acute ischemia mice model. We suggest that sulfane sulfur may be a novel therapeutic agent for hypoxia-induced injury.

Efficient and cost-effective generation of hepatocyte-like cells through microparticle-mediated delivery of growth factors in a 3D culture of human pluripotent stem cells

Biomedical application of human pluripotent stem cell-derived hepatocyte-like cells (hPSC-HLCs) relies on efficient large-scale differentiation, which is commonly performed by a suspension culture of three-dimensional (3D) multicellular spheroids in bioreactors. However, this approach requires large amounts of growth factors (GFs) and the need to overcome limited diffusional transport posed by the inherent 3D structure of hPSC spheroids. Here, we have hypothesized that localized delivery of GFs by incorporation of GF-laden degradable polymeric microparticles (MPs) within the hPSC spheroids would circumvent such limitations. In this study, GFs for hepatocytic differentiation were encapsulated in gelatin-coated poly (l-lactic acid)/poly (DL-lactic-co-glycolic acid) (PLLA/PLGA) MPs which were subsequently incorporated into the hPSC spheroids. Gene expression analyses demonstrated that MP delivery of the GFs resulted in similar expression levels of hepatocytic markers despite the use of 10-fold less total GFs. The differentiated HLCs in the MP group exhibited ultrastructure and functional characteristics comparable with the conventional soluble GF group. The generated HLCs in the MP group were successfully engrafted in an acute liver injury mouse model and maintained hepatocytic function after implantation. These results suggested that sustained and localized delivery of GFs using MPs might offer a novel approach towards scalable technologies for hepatocytic differentiation and engineer a better 3D microenvironment for cells.