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1
LIN, SUNG-JAN, WEN-CHU HSIAO, CHIH-JUNG HSU, JAU-SHIUH CHEN, SHIOU-HWA JEE, TSEN-FANG TSAI, HSIN-SU YU, and TAI-HORNG YOUNG. "THE EFFECT OF SERUM CONCENTRATION ON THE SPHEROID FORMING ACTIVITY AND CELL GROWTH OF HUMAN MELANOCYTES ON CHITOSAN SURFACE." Biomedical Engineering: Applications, Basis and Communications 18, no. 01 (February 25, 2006): 42–46. http://dx.doi.org/10.4015/s1016237206000099.
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We have developed a chitosan-based melanocyte patch to improve the drawbacks encountered in autologous melanocyte transplantation for vitiligo treatment by use of melanocyte suspension. Melanocytes spontaneously grow into three dimensional spheroids on chitosan-coated surface and melanocyte spheroids show a better survival in growth factor and serum-deprived condition. Hence, preculturing melanocytes into spheroids on chitosan surface can provide melanocytes a survival advantage in stringent conditions. Up to date, the mechanism of the melanocyte spheroid formation is not clear. It has been shown that certain factors in serum are crucial for multicellular spheroid formation in cancer cell lines. In this work, we investigate the effect of serum concentration on the melanocyte spheroid formation. The trend for melanocyte spheroid formation is gradually decreased when serum concentration is serially increased from 0% to 20%. Our result shows that melanocyte spheroid formation is not inhibited in the absence of serum and serum deprivation accelerates melanocyte spheroid formation. However, melanocyte growth is greatly reduced when serum is completely removed. Our results suggest that melanocyte spheroid formation on chitosan is not dependent on serum factors. However, the presence of serum contributes to the cell growth on chitosan-coated surface. This result is important in the future development of serum free culture system for spheroidal melanocyte patch.
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Smith, J. Torquil, and H. Frank Morrison. "Approximating spheroid inductive responses using spheres." GEOPHYSICS 71, no. 2 (March 2006): G21—G25. http://dx.doi.org/10.1190/1.2187738.
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Spheroid responses are important as limiting cases when modeling inductive responses of isolated metallic objects such as unexploded military ordnance. The response of high-permeability ([Formula: see text] ≥ 50) conductive spheroids of moderate aspect ratios (0.25–4) to excitation by uniform magnetic fields in the axial or transverse direction is approximated by the response of spheres of appropriate diameters, of the same conductivity and permeability, with magnitude rescaled based on the differing volumes, dc magnetizations, and high-frequency limit responses of the spheres and modelled spheroids. In the frequency domain, the scaled sphere responses agree within 5% of complex magnitudes for prolate spheroids and within 7% for oblate spheroids. The approximation is more accurate for source magnetic fields in the spheroid's shorter direction than in the spheroid's longer direction. In the time domain, the approximation describes spheroid responses over five decades of time after transmitter shutoff, with a maximum discrepancy of 20%.
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Saad, E. I. "Translation and rotation of a porous spheroid in a spheroidal container." Canadian Journal of Physics 88, no. 9 (September 2010): 689–700. http://dx.doi.org/10.1139/p10-040.
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The flow problem of an incompressible axisymmetrical quasisteady translation and steady rotation of a porous spheroid in a concentric spheroidal container are studied analytically. The same small departure from a sphere is considered for each spheroidal surface. In the limit of small Reynolds number, the Brinkman equation for the flow inside the porous region and the Stokes equation for the outside region in their stream functions formulations and velocity components, which are proportional to the translational and angular velocities, respectively, are used. Explicit expressions are obtained for both inside and outside flow fields to the first order in a small parameter characterizing the deformation of the spheroidal surface from the spherical shape. The hydrodynamic drag force and couple exerted on the porous spheroid are obtained for the special cases of prolate and oblate spheroids in closed forms. The dependence of the normalized wall-corrected translational and rotational mobilities on permeability for a porous spheroid in an unbounded medium and for a solid spheroid in a cell on the particle volume fraction is discussed numerically and graphically for various values of the deformation parameter. In the limiting cases, the analytical solutions describing the drag force and torque or mobilities for a porous spheroid in the spheroidal vessel reduce to those for a solid sphere and for a porous sphere in a spherical cell.
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Iyengar, T., and T. Radhika. "Stokes flow of an incompressible micropolar fluid past a porous spheroidal shell." Bulletin of the Polish Academy of Sciences: Technical Sciences 59, no. 1 (March 1, 2011): 63–74. http://dx.doi.org/10.2478/v10175-011-0010-5.
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Stokes flow of an incompressible micropolar fluid past a porous spheroidal shellConsider a pair of confocal prolate spheroids S0and S1where S0is within S1. Let the spheroid S0be a solid and the annular region between S0and S1be porous. The present investigation deals with a flow of an incompressible micropolar fluid past S1with a uniform stream at infinity along the common axis of symmetry of the spheroids. The flow outside the spheroid S1is assumed to follow the linearized version of Eringen's micropolar fluid flow equations and the flow within the porous region is assumed to be governed by the classical Darcy's law. The fluid flow variables within the porous and free regions are determined in terms of Legendre functions, prolate spheroidal radial and angular wave functions and a formula for the drag on the spheroid is obtained. Numerical work is undertaken to study the variation of the drag with respect to the geometric parameter, material parameter and the permeability parameter of the porous region. An interesting feature of the investigation deals with the presentation of the streamline pattern.
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Bridges, Michael A., David C. Walker, Robert A. Harris, Bruce R. Wilson, and A. George F. Davidson. "Cultured human nasal epithelial multicellular spheroids: polar cyst-like model tissues." Biochemistry and Cell Biology 69, no. 2-3 (February 1, 1991): 102–8. http://dx.doi.org/10.1139/o91-016.
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We report here a new readily cultured nonadherent hollow spheroidal epithelial tissue model: human nasal epithelial multicellular spheroids, prepared from brushings of human nasal epithelium in vivo. Although cultured cyst-like epithelial models developed from embryonic, transformed, or polypoid tissues have been reported previously, human nasal epithelial multicellular spheroids are derived from normal mature nontransformed human airway epithelial cells. In our studies, spheroids ranged in size from 50 to 700 μm diameter (averaging approximately 250 μm). Cells of the spheroid displayed morphological polarity and formed junctional complexes. Transcellular electrolyte transport may underlie the increase in spheroid size which occurred in culture. The ease and simplicity of the brushing and culture procedures reported here render normal and diseased human cell populations more readily accessible to investigation. We believe human nasal epithelial multicellular spheroids may have important applications in the study of electrolyte and fluid transport processes, ciliary motility, epithelial polarity, cellular metabolism, and drug cytotoxicity in normal and pathophysiological states of the human respiratory tract (e.g., cystic fibrosis).Key words: cultured airway epithelial cells, electrolyte and fluid transport, spheroid, cyst, cystic fibrosis.
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Nurhazirah Mohamad Yunos, Taufiq Khairi Ahmad Khairuddin, Nor Muhainiah Mohd Ali, and Suzarina Ahmed Sukri. "Spheroidal First Order Polarization Tensor (SFOPT) Toolkit." Semarak International Journal of Fundamental and Applied Mathematics 1, no. 1 (April 3, 2024): 24–37. http://dx.doi.org/10.37934/sijfam.1.1.2437.
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The polarization tensor (PT) has been utilized in numerous applications involving electric and electromagnetic fields, such as metal detection, landmine detection, and electrical imaging. In these applications, the PT is implemented in the process of identifying object, where the object can be represented in the form of the first order PT. Thus, it is crucial to have an application that provides easy access to calculate the first order PT for the object. However, the existing application in the literature has some limitations, focusing solely on computing the first order PT for a prolate spheroid with semi axes and an oblate spheroid with semi axes . Therefore, the purpose of this study is to invent a graphical user interface (GUI) for the Spheroidal First Order Polarization Tensor (SFOPT) Toolkit which facilitates efficient computations and visualizations related to spheroids. The SFOPT Toolkit will be developed by using App Designer in MATLAB. The SFOPT interface integrates four essential functions: computation of the first order PT for a spheroid, classification of spheroidal types, three-dimensional visualization of spheroids, and determination of semi axes from the computed first order PT. Through illustrative examples, we demonstrate the effectiveness and versatility of the SFOPT framework, offering insights into its practical utility and potential applications in diverse fields. The reliability of the toolkit is also presented, and the findings shows the error of computations are small. The toolkit is a user-friendly application since the users can easily access it by downloading the application instead of running the coding themselves. Moreover, this toolkit can be a reference for researchers to compute the first order PT for a spheroid and determine the semi axes (size) of the spheroid from the given first order PT.
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Cesarz, Zoe, and Kenichi Tamama. "Spheroid Culture of Mesenchymal Stem Cells." Stem Cells International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/9176357.
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Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.
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Okumura, Tsuyoshi, Toshikazu Masuya, Yoshimi Takao, and Kouichi Sawada. "Acoustic scattering by an arbitrarily shaped body: an application of the boundary-element method." ICES Journal of Marine Science 60, no. 3 (January 1, 2003): 563–70. http://dx.doi.org/10.1016/s1054-3139(03)00060-2.
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Abstract The boundary-element method (BEM) is applied to calculate target strength (TS) and extinction cross-section of a scatterer. The scattering amplitudes of four types of prolate spheroid, namely, the vacant, rigid, liquid-filled, and gas-filled, are calculated using the BEM. Comparison between the results of the BEM and the prolate-spheroid model (PSM), which is a numerical calculation using spheroidal-wave functions, shows excellent agreement for the vacant, rigid, and liquid-filled spheroids. The TS of a gas-filled prolate spheroid, calculated by the BEM, shows resonance at a low frequency. The lowest resonant frequency, the peak ratio, and the quality factor (Q) of this resonance are compared with those given by the T-matrix and an analytical solution. Again the results show excellent agreement.
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Jang, JunHwee, and Eun-Jung Lee. "Rapid Formation of Stem Cell Spheroids Using Two-Dimensional MXene Particles." Processes 9, no. 6 (May 28, 2021): 957. http://dx.doi.org/10.3390/pr9060957.
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Cell spheroids have been studied as a biomimic medicine for tissue healing using cell sources. Rapid cell spheroid production increases cell survival and activity as well as the efficiency of mass production by reducing processing time. In this study, two-dimensional MXene (Ti3C2) particles were used to form mesenchymal stem cell spheroids, and the optimal MXene concentration, spheroid-production times, and bioactivity levels of spheroid cells during this process were assessed. A MXene concentration range of 1 to 10 μg/mL induced spheroid formation within 6 h. The MXene-induced spheroids exhibited osteogenic-differentiation behavior, with the highest activity levels at a concentration of 5 μg/mL. We report a novel and effective method for the rapid formation of stem cell spheroids using MXene.
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Park, In-Su, Phil-Sang Chung, and Jin Chul Ahn. "Angiogenic Synergistic Effect of Adipose-Derived Stromal Cell Spheroids with Low-Level Light Therapy in a Model of Acute Skin Flap Ischemia." Cells Tissues Organs 202, no. 5-6 (2016): 307–18. http://dx.doi.org/10.1159/000445710.
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Human adipose-derived mesenchymal stem cells (hASCs) are an attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted hASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted spheroid hASCs in skin flaps of mice. hASCs were cultured in monolayers or spheroids. LLLT, hASCs, spheroids and spheroids transplanted with LLLT were applied to the skin flaps. Healing of the skin flaps was assessed by gross evaluation and by hematoxylin and eosin staining and elastin van Gieson staining. Compared with the spheroid group, skin flap healing was enhanced in the spheroid + LLLT group, including the neovascularization and regeneration of skin appendages. The survival of hASCs was enhanced by decreased apoptosis of hASCs in the skin flaps of the spheroid + LLLT group. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASC and spheroid groups. These data suggest that LLLT was an effective biostimulator of spheroid hASCs in the skin flaps, enhancing the survival of hASCs and stimulating the secretion of growth factors.
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Pevná, Viktória, Mariana Máčajová, Andrej Hovan, Gregor Bánó, Majlinda Meta, Boris Bilčík, Júlia Palková, and Veronika Huntošová. "Spheroidal Model of SKBR3 and U87MG Cancer Cells for Live Imaging of Caspase-3 during Apoptosis Induced by Singlet Oxygen in Photodynamic Therapy." Biomedicines 10, no. 9 (August 31, 2022): 2141. http://dx.doi.org/10.3390/biomedicines10092141.
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Aspects related to the response of cells to photodynamic therapy (PDT) have been well studied in cell cultures, which often grow in monolayers. In this work, we propose a spheroidal model of U87MG and SKBR3 cells designed to mimic superficial tumor tissue, small spheroids (<500 µm) suitable for confocal fluorescence microscopy, and larger spheroids (>500 µm) that can be xenografted onto quail chorioallantoic membrane (CAM) to study the effects of PDT in real time. Hypericin was used as a model molecule for a hydrophobic photosensitizer that can produce singlet oxygen (1O2). 1O2 production by hypericin was detected in SKBR3 and U87MG spheroid models using a label-free technique. Vital fluorescence microscopy and flow cytometry revealed the heterogeneity of caspase-3 distribution in the cells of the spheroids. The levels of caspase-3 and apoptosis increased in the cells of spheroids 24 h after PDT. Lactate dehydrogenase activity was evaluated in the spheroids as the most reliable assay to detect differences in phototoxicity. Finally, we demonstrated the applicability of U87MG spheroids on CAM in photodiagnostics. Overall, the variability and applicability of the prepared spheroid models were demonstrated in the PDT study.
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Lewis, Natasha S., Emily EL Lewis, Margaret Mullin, Helen Wheadon, Matthew J. Dalby, and Catherine C. Berry. "Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence." Journal of Tissue Engineering 8 (January 1, 2017): 204173141770442. http://dx.doi.org/10.1177/2041731417704428.
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Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies.
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Mattapally, Saidulu, Wuqiang Zhu, Vladimir G. Fast, Ling Gao, Chelsea Worley, Ramaswamy Kannappan, Anton V. Borovjagin, and Jianyi Zhang. "Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice." American Journal of Physiology-Heart and Circulatory Physiology 315, no. 2 (August 1, 2018): H327—H339. http://dx.doi.org/10.1152/ajpheart.00688.2017.
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The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca2+ transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1– 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1– 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.
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Das, Viswanath, Tomáš Fürst, Soňa Gurská, Petr Džubák, and Marián Hajdúch. "Reproducibility of Uniform Spheroid Formation in 384-Well Plates." Journal of Biomolecular Screening 21, no. 9 (July 10, 2016): 923–30. http://dx.doi.org/10.1177/1087057116651867.
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Spheroid cultures of cancer cells reproduce the spatial dimension–induced in vivo tumor traits more effectively than the conventional two-dimensional cell cultures. With growing interest in spheroids for high-throughput screening (HTS) assays, there is an increasing demand for cost-effective miniaturization of reproducible spheroids in microtiter plates (MPs). However, well-to-well variability in spheroid size, shape, and growth is a frequently encountered problem with almost every culture method that has prevented the transfer of spheroids to the HTS platform. This variability partly arises due to increased susceptibility of MPs to edge effects and evaporation-induced changes in the growth of spheroids. In this study, we examined the effect of evaporation on the reproducibility of spheroids of tumor and nontumor cell lines in 384-well plates, and show that culture conditions that prevent evaporation-induced medium loss result in the formation of uniform spheroids across the plate. Additionally, we also present a few technical improvements to increase the scalability of the liquid-overlay spheroid culturing technique in MPs, together with a simple software routine for the quantification of spheroid size. We believe that these cost-effective improvements will aid in further improvement of spheroid cultures for HTS drug discovery.
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Bell, Jordan, Shilpaa Mukundan, Matthew Teryek, Bo Lin, Biju Parekkadan, and Leo Chan. "Abstract 184: High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry." Cancer Research 82, no. 12_Supplement (June 15, 2022): 184. http://dx.doi.org/10.1158/1538-7445.am2022-184.
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Abstract Three-dimensional cancer models have gained popularity for in vitro studies of chemotherapeutic compounds by providing a more physiologically relevant analog of gas, nutrient, and drug diffusion throughout the tumor microenvironment. Some 3D assays are performed to study individual spheroids over time, where a majority of these assays rely on maintaining a single spheroid in each well of a 96-well round-bottom ultra-low attachment plate, limiting the number of spheroids in a study. Other assays may gather population-level data from large ensembles of spheroids grown together, but the information about individual differences amongst the spheroids is lost. Important kinetic information may also be lost for destructive endpoint assays such as MTS or MTT. Here, we describe the development of a 3D image cytometry assay that is capable of generating kinetic data for thousands of breast cancer spheroids at the individual level. T47D spheroids are grown and maintained in a 24-well Aggrewell࣪400 plate and imaged using the Celigo image cytometer. Each well contains more than 1000 subwells that both aid in spheroid formation and constrain each spheroid to a specific location. Using the spheroid location data, we are able to track and monitor the growth of each spheroid over 7 days. Furthermore, we investigate the dose-dependent effects on spheroid viability of 6 anti-cancer drugs (Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen) using calcein AM and propidium iodide (PI). To validate the viability measurement results, we utilize the CellTiter96® MTS assay as an orthogonal method to compare the dose-dependent trends using both the calcein AM and PI fluorescence intensities as well as the spheroid sizes. This work may lay a foundation for the investigation of other spheroids, organoids, or tissue samples, significantly increasing the number of spheroids analyzed per condition, improving the statistical analysis, and adding more parameters to further analyze the spheroids. These improvements may be especially helpful for spheroids grown from patient-derived or otherwise heterogeneous cell populations Citation Format: Jordan Bell, Shilpaa Mukundan, Matthew Teryek, Bo Lin, Biju Parekkadan, Leo Chan. High-throughput chemotherapeutic drug screening of tumor spheroids with individual spheroid results using image cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 184.
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Roh, Hyewon, Hwisoo Kim, and Je-Kyun Park. "Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip." Biosensors 11, no. 12 (December 9, 2021): 506. http://dx.doi.org/10.3390/bios11120506.
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Spheroid, a 3D aggregate of tumor cells in a spherical shape, has overcome the limitations of conventional 3D cell models to accurately mimic the in-vivo environment of a human body. The spheroids are cultured with other primary cells and embedded in collagen drops using hang drop plates and low-attachment well plates to construct a spheroid–hydrogel model that better mimics the cell–cell and cell–extracellular matrix (ECM) interactions. However, the conventional methods of culturing and embedding spheroids into ECM have several shortcomings. The procedure of transferring a single spheroid at a time by manual pipetting results in well-to-well variation and even loss or damage of the spheroid. Based on the previously introduced droplet contact-based spheroid transfer technique, we present a poly(dimethylsiloxane) and resin-based drop array chip and a pillar array chip with alignment stoppers, which enhances the alignment between the chips for uniform placement of spheroids. This method allows the facile and stable transfer of the spheroid array and even eliminates the need for a stereomicroscope while handling the cell models. The novel platform demonstrates a homogeneous and time-efficient construction and diverse analysis of an array of fibroblast-associated glioblastoma multiforme spheroids that are embedded in collagen.
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Kim, Young-Su, Arun Asif, Abdul Rahim Chethikkattuveli Salih, Jae-Wook Lee, Ki-Nam Hyun, and Kyung-Hyun Choi. "Gravity-Based Flow Efficient Perfusion Culture System for Spheroids Mimicking Liver Inflammation." Biomedicines 9, no. 10 (October 1, 2021): 1369. http://dx.doi.org/10.3390/biomedicines9101369.
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The spheroid culture system provides an efficient method to emulate organ-specific pathophysiology, overcoming the traditional two-dimensional (2D) cell culture limitations. The intervention of microfluidics in the spheroid culture platform has the potential to enhance the capacity of in vitro microphysiological tissues for disease modeling. Conventionally, spheroid culture is carried out in static conditions, making the media nutrient-deficient around the spheroid periphery. The current approach tries to enhance the capacity of the spheroid culture platform by integrating the perfusion channel for dynamic culture conditions. A pro-inflammatory hepatic model was emulated using a coculture of HepG2 cell line, fibroblasts, and endothelial cells for validating the spheroid culture plate with a perfusable channel across the spheroid well. Enhanced proliferation and metabolic capacity of the microphysiological model were observed and further validated by metabolic assays. A comparative analysis of static and dynamic conditions validated the advantage of spheroid culture with dynamic media flow. Hepatic spheroids were found to have improved proliferation in dynamic flow conditions as compared to the static culture platform. The perfusable culture system for spheroids is more physiologically relevant as compared to the static spheroid culture system for disease and drug analysis.
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Cao, Viet Phuong, Sera Hong, and Joon Myong Song. "3D bioprinting-based single liver tumor spheroid analysis for aflatoxin B1-induced drug-resistant cancer cell." International Journal of Bioprinting 9, no. 6 (August 18, 2023): 0985. http://dx.doi.org/10.36922/ijb.0985.
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Aflatoxin B1, found in a variety of foods, is a mycotoxin known to cause cancer. Therefore, humans may be exposed to it through their daily diet. In this study, a three-dimensional (3D) tumor spheroid model was developed via 3D bioprinting to examine whether exposure of HepG2 liver tumor spheroids to aflatoxin B1 can increase the population of drug-resistant liver cancer cells in a single tumor spheroid. Two biomarkers, CD133 (prominin-1) and aldehyde dehydrogenase 1 (ALDH1), were used to identify drug-resistant cancer cells formed in the single liver tumor spheroids. The induction of drug-resistant cancer cells in the single tumor spheroids was examined through single spheroid imaging and fluorescence-activated cell sorting (FACS). The increase of drug-resistant cancer cells, which was caused by aflatoxin B1 in a dose-dependent manner, was quantitatively monitored at the single tumor spheroid level using both methods. 3D bioprinting-fabricated single liver tumor spheroid model successfully determined drug-resistant liver cancer cells caused by aflatoxin B1
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Tenschert, Esther, Johann Kern, Annette Affolter, Nicole Rotter, and Anne Lammert. "Optimisation of Conditions for the Formation of Spheroids of Head and Neck Squamous Cell Carcinoma Cell Lines for Use as Animal Alternatives." Alternatives to Laboratory Animals 50, no. 6 (October 20, 2022): 414–22. http://dx.doi.org/10.1177/02611929221135042.
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The use of in vitro 3-D cell culture models in cancer research has yielded substantial gains in knowledge on various aspects of tumour biology. Such cell culture models could be useful in the study of head and neck squamous cell carcinoma (HNSCC), where mimicking intratumoral and intertumoral heterogeneity is especially challenging. Our research aims to establish 3-D spheroid models for HNSCC that reproduce in vitro the connections between tumour cells and the surrounding microenvironment. The aims of this study were to determine the optimal conditions for the culture and use of spheroids from HNSCC cell lines and optimal timepoint for using the spheroids obtained, to evaluate the effects of coculture with tumour-specific fibroblasts on spheroid formation, and to investigate spheroid responses to cisplatin treatment. Four HNSCC cell lines (UMSCC-11A, UMSCC-11B, UMSCC-22B and UD-SCC-01) were seeded in flat or round bottom well ultra-low attachment spheroid plates, and spheroid formation was evaluated. The HNSCC cell lines were then cocultured with stromal cells of the tumour microenvironment, producing an accelerated formation of dense spheroids. The viability of cells within the spheroids was assessed during cell culture by using a fluorescent dye. Our results suggest that: three out of the four cell lines tested could form usable spheroids with acceptable viability; the addition of stromal cells did not improve the number of viable cells; and the use of round bottom well plates supported the formation of a single spheroid, whereas flat bottom well plates led to the formation of multiple spheroids of different sizes.
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Choi, Jae Won, Sang-Yun Lee, and Dong Woo Lee. "A Cancer Spheroid Array Chip for Selecting Effective Drug." Micromachines 10, no. 10 (October 12, 2019): 688. http://dx.doi.org/10.3390/mi10100688.
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A cancer spheroid array chip was developed by modifying a micropillar and microwell structure to improve the evaluation of drugs targeting specific mutations such as phosphor-epidermal growth factor receptor (p-EGFR). The chip encapsulated cells in alginate and allowed cancer cells to grow for over seven days to form cancer spheroids. However, reagents or media used to screen drugs in a high-density spheroid array had to be replaced very carefully, and this was a tedious task. Particularly, the immunostaining of cancer spheroids required numerous steps to replace many of the reagents used for drug evaluation. To solve this problem, we adapted a micropillar and microwell structure to a spheroid array. Thus, culturing cancer spheroids in alginate spots attached to the micropillar allowed us to replace the reagents in the microwell chip with a single fill of fresh medium, without damaging the cancer spheroids. In this study, a cancer spheroid array was made from a p-EGFR-overexpressing cell line (A549 lung cancer cell line). In a 12 by 36 column array chip (25 mm by 75 mm), the spheroid over 100 µm in diameter started to form at day seven and p-EGFR was also considerably overexpressed. The array was used for p-EGFR inhibition and cell viability measurement against seventy drugs, including ten EGFR-targeting drugs. By comparing drug response in the spheroid array (spheroid model) with that in the single-cell model, we demonstrated that the two models showed different responses and that the spheroid model might be more resistant to some drugs, thus narrowing the choice of drug candidates.
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Shimoto, Takeshi, Xiu-Ying Zhang, Shizuka Akieda, Atsushi Ishikawa, Hidehiko Higaki, and Koichi Nakayama. "Analysis of Cell Spheroid Morphological Characteristics Using the Spheroid Morphology Evaluation System." Journal of Robotics and Mechatronics 30, no. 5 (October 20, 2018): 819–26. http://dx.doi.org/10.20965/jrm.2018.p0819.
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Our research group established a technology for forming three-dimensional cell constructs to regenerate osteochondro cells without scaffolding. The established technology employed spheroids to form cell constructs. We also developed a method for arranging spheroids in arbitrary positions to form cell constructs in complex shapes. However, we could only form cell constructs as expected when the formed spheroids were the appropriate sizes. This study, therefore, aimed to chronologically analyze the spheroid morphological characteristics of rabbit mechanical stem cells using the developed spheroid morphological evaluation system. We set the numbers of cells/wells as 2 × 104, 3 × 104, 4 × 104, 5 × 104, 6 × 104, and 7 × 104 and the passage number as 7. Further, we observed the cultured spheroids every 24 hours after seeding for five days. The analysis enabled us to specify an optimal range for the numbers of cells required to form spheroids with high degrees of circularity. We could also control the formed spheroid sizes by adjusting the cell count and culturing time.
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Srisongkram, Tarapong, Natthida Weerapreeyakul, and Kanjana Thumanu. "Evaluation of Melanoma (SK-MEL-2) Cell Growth between Three-Dimensional (3D) and Two-Dimensional (2D) Cell Cultures with Fourier Transform Infrared (FTIR) Microspectroscopy." International Journal of Molecular Sciences 21, no. 11 (June 10, 2020): 4141. http://dx.doi.org/10.3390/ijms21114141.
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Fourier transform infrared (FTIR) microspectroscopy was used to evaluate the growth of human melanoma cells (SK-MEL-2) in two-dimensional (2D) versus three-dimensional (3D) spheroid culture systems. FTIR microspectroscopy, coupled with multivariate analysis, could be used to monitor the variability of spheroid morphologies prepared from different cell densities. The characteristic shift in absorbance bands of the 2D cells were different from the spectra of cells from 3D spheroids. FTIR microspectroscopy can also be used to monitor cell death similar to fluorescence cell staining in 3D spheroids. A change in the secondary structure of protein was observed in cells from the 3D spheroid versus the 2D culture system. FTIR microspectroscopy can detect specific alterations in the biological components inside the spheroid, which cannot be detected using fluorescence cell death staining. In the cells from 3D spheroids, the respective lipid, DNA, and RNA region content represent specific markers directly proportional to the spheroid size and central area of necrotic cell death, which can be confirmed using unsupervised PCA and hierarchical cluster analysis. FTIR microspectroscopy could be used as an alternative tool for spheroid cell culture discrimination, and validation of the usual biochemical technique.
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Zieger, Viktoria, Ellen Woehr, Stefan Zimmermann, Daniel Frejek, Peter Koltay, Roland Zengerle, and Sabrina Kartmann. "Automated Nanodroplet Dispensing for Large-Scale Spheroid Generation via Hanging Drop and Parallelized Lossless Spheroid Harvesting." Micromachines 15, no. 2 (January 31, 2024): 231. http://dx.doi.org/10.3390/mi15020231.
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Creating model systems that replicate in vivo tissues is crucial for understanding complex biological pathways like drug response and disease progression. Three-dimensional (3D) in vitro models, especially multicellular spheroids (MCSs), offer valuable insights into physiological processes. However, generating MCSs at scale with consistent properties and efficiently recovering them pose challenges. We introduce a workflow that automates large-scale spheroid production and enables parallel harvesting into individual wells of a microtiter plate. Our method, based on the hanging-drop technique, utilizes a non-contact dispenser for dispensing nanoliter droplets of a uniformly mixed-cell suspension. The setup allows for extended processing times of up to 45 min without compromising spheroid quality. As a proof of concept, we achieved a 99.3% spheroid generation efficiency and maintained highly consistent spheroid sizes, with a coefficient of variance below 8% for MCF7 spheroids. Our centrifugation-based drop transfer for spheroid harvesting achieved a sample recovery of 100%. We successfully transferred HT29 spheroids from hanging drops to individual wells preloaded with collagen matrices, where they continued to proliferate. This high-throughput workflow opens new possibilities for prolonged spheroid cultivation, advanced downstream assays, and increased hands-off time in complex 3D cell culture protocols.
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Ikeda-Motonakano, Reiko, Fumika Hirabayashi-Nishimuta, Naomi Yada, Ryota Yamasaki, Yoshie Nagai-Yoshioka, Michihiko Usui, Kohji Nakazawa, Daigo Yoshiga, Izumi Yoshioka, and Wataru Ariyoshi. "Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated Device." Cancers 15, no. 21 (October 26, 2023): 5162. http://dx.doi.org/10.3390/cancers15215162.
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Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24–48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.
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Goisnard, Antoine, Clémence Dubois, Pierre Daumar, Corinne Aubel, Marie Depresle, Jean Gauthier, Bernard Vidalinc, Frédérique Penault-Llorca, Emmanuelle Mounetou, and Mahchid Bamdad. "The New Serum-Free OptiPASS® Medium in Cold and Oxygen-Free Conditions: An Innovative Conservation Method for the Preservation of MDA-MB-231 Triple Negative Breast Cancer Spheroids." Cancers 13, no. 8 (April 18, 2021): 1945. http://dx.doi.org/10.3390/cancers13081945.
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Cancer spheroids are very effective preclinical models to improve anticancer drug screening. In order to optimize and extend the use of spheroid models, these works were focused on the development of a new storage concept to maintain these models in the longer term using the Triple-Negative Breast Cancer MDA-MB-231 spheroid models. The results highlight that the combination of a temperature of 4 °C and oxygen-free conditions allowed the spheroid characteristics of OptiPASS® serum-free culture medium to preserve the spheroid characteristics during 3-, 5- or 7-day-long storage. Indeed, after storage they were returned to normal culture conditions, with recovered spheroids presenting similar growth rates (recovery = 96.2%), viability (Live/Dead® profiles) and metabolic activities (recovery = 90.4%) compared to nonstored control spheroids. Likewise, both recovered spheroids (after storage) and nonstored controls presented the same response profiles as two conventional drugs, i.e., epirubicin and cisplatin, and two anti-PARP1 targeted drugs—i.e., olaparib and veliparib. This new original storage concept seems to induce a temporary stop in spheroid growth while maintaining their principal characteristics for further use. In this way, this innovative and simple storage concept may instigate future biological sample preservation strategies.
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Kitano, Otome, and Kohji Nakazawa. "Neuronal Differentiation of NT2 Cells in Monolayer and Spheroid Cultures." MATEC Web of Conferences 333 (2021): 07008. http://dx.doi.org/10.1051/matecconf/202133307008.
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Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.
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Kitano, Otome, and Kohji Nakazawa. "Neuronal Differentiation of NT2 Cells in Monolayer and Spheroid Cultures." MATEC Web of Conferences 333 (2021): 07008. http://dx.doi.org/10.1051/matecconf/202133307008.
Full textAbstract:
Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.
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Nie, Yan, Xun Xu, Weiwei Wang, Nan Ma, and Andreas Lendlein. "Spheroid formation of human keratinocyte: Balancing between cell-substrate and cell-cell interaction." Clinical Hemorheology and Microcirculation 76, no. 2 (October 30, 2020): 329–40. http://dx.doi.org/10.3233/ch-209217.
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BACKGROUND: The formation of spheroids is tightly regulated by intrinsic cell-cell and cell-substrate interactions. OBJECTIVE: The chitosan (CS)-coating was applied to investigate the driven force directed the spheroid formation. METHODS: The effects of CS on cell functions were studied. Atomic force microscopy was employed to measure the cell- biomaterial interplay at single cell level. RESULTS: HaCaT cells shifted from their flattened sheet to a compact 3D spheroidal morphology when increasing CS-coating concentration. The proliferative capacity of HaCaT was preserved in the spheroid. The expression and activation of integrin β1 (ITGB1) were enhanced on CS modified surfaces, while the active to total ratio of ITGB1 was decreased. The adhesive force of a single HaCaT cell to the tissue culture plate (TCP) was 4.84±0.72 nN. It decreased on CS-coated surfaces as CS concentration increased, from 2.16±0.26 nN to 0.96±0.17 nN. The adhesive force between the single HaCaT cell to its neighbor cell increased as CS concentration increased, from 1.15±0.09 nN to 2.60±0.51 nN. CONCLUSIONS: Conclusively, the decreased cell- substrate adhesion was the main driven force in the spheroid formation. This finding might serve as a design criterion for biomaterials facilitating the formation of epithelial spheroids.
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Murphy, Kaitlin C., Ben P. Hung, Stephen Browne-Bourne, Dejie Zhou, Jessica Yeung, Damian C. Genetos, and J. Kent Leach. "Measurement of oxygen tension within mesenchymal stem cell spheroids." Journal of The Royal Society Interface 14, no. 127 (February 2017): 20160851. http://dx.doi.org/10.1098/rsif.2016.0851.
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Spheroids formed of mesenchymal stem cells (MSCs) exhibit increased cell survival and trophic factor secretion compared with dissociated MSCs, making them therapeutically advantageous for cell therapy. Presently, there is no consensus for the mechanism of action. Many hypothesize that spheroid formation potentiates cell function by generating a hypoxic core within spheroids of sufficiently large diameters. The purpose of this study was to experimentally determine whether a hypoxic core is generated in MSC spheroids by measuring oxygen tension in aggregates of increasing diameter and correlating oxygen tension values with cell function. MSC spheroids were formed with 15 000, 30 000 or 60 000 cells per spheroid, resulting in radii of 176 ± 8 µm, 251 ± 12 µm and 353 ± 18 µm, respectively. Oxygen tension values coupled with mathematical modelling revealed a gradient that varied less than 10% from the outer diameter within the largest spheroids. Despite the modest radial variance in oxygen tension, cellular metabolism from spheroids significantly decreased as the number of cells and resultant spheroid size increased. This may be due to adaptive reductions in matrix deposition and packing density with increases in spheroid diameter, enabling spheroids to avoid the formation of a hypoxic core. Overall, these data provide evidence that the enhanced function of MSC spheroids is not oxygen mediated.
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St-Georges-Robillard, Amélie, Maxime Cahuzac, Benjamin Péant, Hubert Fleury, Muhammad Abdul Lateef, Alexis Ricard, Alexandre Sauriol, Frédéric Leblond, Anne-Marie Mes-Masson, and Thomas Gervais. "Long-term fluorescence hyperspectral imaging of on-chip treated co-culture tumour spheroids to follow clonal evolution." Integrative Biology 11, no. 4 (April 1, 2019): 130–41. http://dx.doi.org/10.1093/intbio/zyz012.
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Abstract Multicellular tumour spheroids are an ideal in vitro tumour model to study clonal heterogeneity and drug resistance in cancer research because different cell types can be mixed at will. However, measuring the individual response of each cell population over time is challenging: current methods are either destructive, such as flow cytometry, or cannot image throughout a spheroid, such as confocal microscopy. Our group previously developed a wide-field fluorescence hyperspectral imaging system to study spheroids formed and cultured in microfluidic chips. In the present study, two subclones of a single parental ovarian cancer cell line transfected to express different fluorophores were produced and co-culture spheroids were formed on-chip using ratios forming highly asymmetric subpopulations. We performed a 3D proliferation assay on each cell population forming the spheroids that matched the 2D growth behaviour. Response assays to PARP inhibitors and platinum-based drugs were also performed to follow the clonal evolution of mixed populations. Our experiments show that hyperspectral imaging can detect spheroid response before observing a decrease in spheroid diameter. Hyperspectral imaging and microfluidic-based spheroid assays provide a versatile solution to study clonal heterogeneity, able to measure response in subpopulations presenting as little as 10% of the initial spheroid.
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Ivers, D. J. "Kinematic dynamos in spheroidal geometries." Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences 473, no. 2206 (October 2017): 20170432. http://dx.doi.org/10.1098/rspa.2017.0432.
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The kinematic dynamo problem is solved numerically for a spheroidal conducting fluid of possibly large aspect ratio with an insulating exterior. The solution method uses solenoidal representations of the magnetic field and the velocity by spheroidal toroidal and poloidal fields in a non-orthogonal coordinate system. Scaling of coordinates and fields to a spherical geometry leads to a modified form of the kinematic dynamo problem with a geometric anisotropic diffusion and an anisotropic current-free condition in the exterior, which is solved explicitly. The scaling allows the use of well-developed spherical harmonic techniques in angle. Dynamo solutions are found for three axisymmetric flows in oblate spheroids with semi-axis ratios 1≤ a / c ≤25. For larger aspect ratios strong magnetic fields may occur in any region of the spheroid, depending on the flow, but the external fields for all three flows are weak and concentrated near the axis or periphery of the spheroid.
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Ota, Hiroki, and Norihisa Miki. "Parallel Formation of Three-Dimensional Spheroid Using Microrotational Flow." Journal of Robotics and Mechatronics 22, no. 5 (October 20, 2010): 587–93. http://dx.doi.org/10.20965/jrm.2010.p0587.
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We propose three-dimensional (3D) spheroid formation involving perfusion and a lab-on-a-chip containing spheroid-forming chamber arrays. Cells are collected forming a spheroid in the chamber in microrotation. We previously reported a single chamber form hepatic spheroids 130 to 430 µm in diameter, controlling size by varying chamber diameter and cell density. Here, we scaled the system up by a factor of 10 while maintaining size control of 180±30 µm in diameter. Results were comparable to those using a single-chamber device. Long-term culture confirmed that cells in the spheroid maintained viability and diameters did not change after 24 hours. The system is readily applicable for creating size-controlled spheroids ensuring reliable, predictable in vitro data for drug screening and biological research.
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Dang, Anju, and Brad Larson. "Brightfield and Fluorescence Imaging using 3D PrimeSurface® Ultra-Low Attachment Microplates." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 178.35. http://dx.doi.org/10.4049/jimmunol.200.supp.178.35.
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Abstract Culturing cells in three-dimensions (3D) has become a well established approach as it is more representative of the in vivo environment than traditional two-dimensional (2D) cultures. Allowing cells to interact with each other in a spheroid creates a micro-environment which mimics in vivo tissue and is a better model for examining the effect of drugs in cancer. Developing uniform spheroids becomes especially important as it forms the basis for robust and reliable assays. S-BIO PrimeSurface® cultureware are ultra low attachment (ULA) dishes and plates that promote scaffold free, self assembly spheroid formation. The plates are pre-coated with a proprietary hydrophilic polymer that enables spontaneous spheroid formation of uniform size. PrimeSurface 96 and 384 ULA plates have good optical clarity making them highly suitable for brightfield and fluorescent imaging. Imaging technologies such as the BioTek Cytation™ 5 allows researchers to study not only spheroid proliferation through brightfield imaging, but also phenotypic events such as hypoxia, apoptosis, or necrosis induction through the use of fluorescent probes and fluorescence imaging. Incorporation of z-stacking and projection techniques in the Gen5™ Microplate Reader and Imager Software create in-focus images of spheroidal cells, allowing accurate, robust, and repeatable determination of the effect of test molecules or conditions. In this app note, we present data generated with BioTek Cytation 5 using PrimeSurface ULA plates to develop simple robust spheroid assays for brightfield and fluorescence imaging.
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Kasamkattil, Jesil, Anna Gryadunova, Ivan Martin, Andrea Barbero, Stefan Schären, Olga Krupkova, and Arne Mehrkens. "Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc." International Journal of Molecular Sciences 23, no. 5 (February 25, 2022): 2530. http://dx.doi.org/10.3390/ijms23052530.
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Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.
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Barrow, Rhiannon, Joseph Wilkinson, Yichen He, Martin Callaghan, Anke Brüning-Richardson, Mark Dunning, and Lucy Stead. "SpheroidAnalyseR—an online platform for analyzing data from 3D spheroids or organoids grown in 96-well plates." Journal of Biological Methods 9, no. 4 (November 23, 2022): e163. http://dx.doi.org/10.14440/jbm.2022.388.
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Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumor compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Spheroids are often only formed from a single cell type which does not represent the situation in vivo. However, despite this, both spheroids and organoids can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course. To address this, we have developed an R Shiny app called SpheroidAnalyseR: a simple, fast, effective open-source app that allows the analysis of spheroid or organoid size data generated in a 96-well format. SpheroidAnalyseR processes and analyzes datasets of image measurements that can be obtained via a bespoke software, described herein, that automates spheroid imaging and quantification using the Nikon A1R Confocal Laser Scanning Microscope. However, templates are provided to enable users to input spheroid image measurements obtained by user-preferred methods. SpheroidAnalyseR facilitates outlier identification and removal followed by graphical visualization of spheroid measurements across multiple predefined parameters such as time, cell-type and treatment(s). Spheroid imaging and analysis can, thus, be reduced from hours to minutes, removing the requirement for substantial manual data manipulation in a spreadsheet application. The combination of spheroid generation in 96-well ultra-low attachment microplates, imaging using our bespoke software, and analysis using SpheroidAnalyseR toolkit allows high throughput, longitudinal quantification of 3D spheroid growth whilst minimizing user input and significantly improving the efficiency and reproducibility of data analysis. Our bespoke imaging software is available from https://github.com/GliomaGenomics. SpheroidAnalyseR is available at https://spheroidanalyser.leeds.ac.uk, and the source code found at https://github.com/GliomaGenomics.
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Vella, Nathan, Diogo Estêvão, Tânia Cruz, Maria José Oliveira, Anthony George Fenech, and Vanessa Petroni Magri. "Abstract LB176: Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids." Cancer Research 84, no. 7_Supplement (April 5, 2024): LB176. http://dx.doi.org/10.1158/1538-7445.am2024-lb176.
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Abstract A major barrier in lung cancer pre-clinical research is the lack of simple, replicative and reproducible cell culture models. Most studies are still focused on 2D, completely ignoring cell-to-cell interactions, in vivo tissue architecture and the surrounding microenvironment. To address this void in cancer research, we aimed to develop a biomimetic 3D lung cancer spheroid model that can be used as a more reliable tool for pharmacological studies. Therefore, 3D spheroids of A549 (adenocarcinoma), H460 (large cell carcinoma) and H520 (squamous cell carcinoma) non-small cell lung cancer (NSCLC) cells were optimized and consequently submitted to AZD2014 treatment, a dual mTOR inhibitor targeting mTORC1 and mTORC2, members of the PI3K/Akt/mTOR pathway, which is commonly deregulated in lung cancer. Each cell line was seeded at 2000, 5000 or 10000 cells/spheroid in agarose micro-molds for eight days. The 3D NSCLC spheroids were further characterized for their tumor features, namely 3D dynamics and morphology, including diameter, circularity and compactness, viability properties, as well as proliferative and necrotic areas. Based on this characterization, the 2000 cells/spheroid condition was selected for further treatments and spheroid treatment time-points were selected. For each cell line, spheroids were treated with AZD2014, using concentrations based on the IC30 values (sensitization concentration) defined under 2D conditions: 10 μM, 10 μM and 0.2 μM for A549, H460 and H520 spheroids, respectively. Treated and untreated spheroids were followed during six days post-treatment, and their morphological characteristics, cell viability, necrotic core formation and proliferative areas, as well as ATP production, were assessed. Spheroids of all cell lines exhibited a significant inhibition of spheroid growth with AZD2014 treatment, which was maintained over the six days. Moreover, a reduction in spheroid intracellular ATP was observed at days three and six post-treatment. An additional treatment regimen was studied by re-treating spheroids with the same concentration of AZD2014 after 48hrs from the first treatment. Spheroids exposed to this dual treatment regimen exhibited a significant inhibition of spheroid growth, while the respective controls exhibited intensified growth. While AZD2014 did not induce significant reductions in 3D cell viability, a clear inhibition of spheroid growth was observed. Having a more intricate cellular organization, this study alleviates the increased complexity of pharmacological responses in 3D spheroids when compared to 2D models. Further studies are being carried out to define the enhanced biomimetic ability of these NSCLC spheroids and the underlying mechanisms involved when targeting the PI3K/Akt/mTOR pathway using AZD2014 as mono-therapy or in combination with two novel anti-sense oligonucleotides (ASO). Citation Format: Nathan Vella, Diogo Estêvão, Tânia Cruz, Maria José Oliveira, Anthony George Fenech, Vanessa Petroni Magri. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB176.
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Rengganaten, Vimalan, Chiu-Jung Huang, Ping-Hsing Tsai, Mong-Lien Wang, Yi-Ping Yang, Yuan-Tzu Lan, Wen-Liang Fang, et al. "Mapping a Circular RNA–microRNA–mRNA-Signaling Regulatory Axis that Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells." International Journal of Molecular Sciences 21, no. 21 (October 23, 2020): 7864. http://dx.doi.org/10.3390/ijms21217864.
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Spheroidal cancer cell cultures have been used to enrich cancer stem cells (CSC), which are thought to contribute to important clinical features of tumors. This study aimed to map the regulatory networks driven by circular RNAs (circRNAs) in CSC-enriched colorectal cancer (CRC) spheroid cells. The spheroid cells established from two CRC cell lines acquired stemness properties in pluripotency gene expression and multi-lineage differentiation capacity. Genome-wide sequencing identified 1503 and 636 circRNAs specific to the CRC parental and spheroid cells, respectively. In the CRC spheroids, algorithmic analyses unveiled a core network of mRNAs involved in modulating stemness-associated signaling pathways, driven by a circRNA–microRNA (miRNA)–mRNA axis. The two major circRNAs, hsa_circ_0066631 and hsa_circ_0082096, in this network were significantly up-regulated in expression levels in the spheroid cells. The two circRNAs were predicted to target and were experimentally shown to down-regulate miR-140-3p, miR-224, miR-382, miR-548c-3p and miR-579, confirming circRNA sponging of the targeted miRNAs. Furthermore, the affected miRNAs were demonstrated to inhibit degradation of six mRNA targets, viz. ACVR1C/ALK7, FZD3, IL6ST/GP130, SKIL/SNON, SMAD2 and WNT5, in the CRC spheroid cells. These mRNAs encode proteins that are reported to variously regulate the GP130/Stat, Activin/Nodal, TGF-β/SMAD or Wnt/β-catenin signaling pathways in controlling various aspects of CSC stemness. Using the CRC spheroid cell model, the novel circRNA–miRNA–mRNA axis mapped in this work forms the foundation for the elucidation of the molecular mechanisms of the complex cellular and biochemical processes that determine CSC stemness properties of cancer cells, and possibly for designing therapeutic strategies for CRC treatment by targeting CSC.
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Boo, Lily, Wan Yong Ho, Norlaily Mohd Ali, Swee Keong Yeap, Huynh Ky, Kok Gan Chan, Wai Fong Yin, et al. "Phenotypic and microRNA transcriptomic profiling of the MDA-MB-231 spheroid-enriched CSCs with comparison of MCF-7 microRNA profiling dataset." PeerJ 5 (July 13, 2017): e3551. http://dx.doi.org/10.7717/peerj.3551.
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Breast cancer spheroids have been widely used as in vitro models of cancer stem cells (CSCs), yet little is known about their phenotypic characteristics and microRNAs (miRNAs) expression profiles. The objectives of this research were to evaluate the phenotypic characteristics of MDA-MB-231 spheroid-enriched cells for their CSCs properties and also to determine their miRNAs expression profile. Similar to our previously published MCF-7 spheroid, MDA-MB-231 spheroid also showed typical CSCs characteristics namely self-renewability, expression of putative CSCs-related surface markers and enhancement of drug resistance. From the miRNA profile, miR-15b, miR-34a, miR-148a, miR-628 and miR-196b were shown to be involved in CSCs-associated signalling pathways in both models of spheroids, which highlights the involvement of these miRNAs in maintaining the CSCs features. In addition, unique clusters of miRNAs namely miR-205, miR-181a and miR-204 were found in basal-like spheroid whereas miR-125, miR-760, miR-30c and miR-136 were identified in luminal-like spheroid. Our results highlight the roles of miRNAs as well as novel perspectives of the relevant pathways underlying spheroid-enriched CSCs in breast cancer.
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Boylan, Kristin L. M., Rory D. Manion, Heena Shah, Keith M. Skubitz, and Amy P. N. Skubitz. "Inhibition of Ovarian Cancer Cell Spheroid Formation by Synthetic Peptides Derived from Nectin-4." International Journal of Molecular Sciences 21, no. 13 (June 30, 2020): 4637. http://dx.doi.org/10.3390/ijms21134637.
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The formation of 3D multicellular spheroids in the ascites fluid of ovarian cancer patients is an understudied component of the disease progression. Spheroids are less sensitive to chemotherapy, in part due to the protection afforded by their structure, but also due to their slower proliferation rate. Previous studies suggest that the cell adhesion molecule Nectin-4 plays a key role in the formation of ovarian cancer spheroids. In this study, we further examined the role of Nectin-4 at early time points in spheroid formation using real-time digital photography. Human NIH:OVCAR5 ovarian cancer cells formed aggregates within 8 h, which further contracted into compact spheroids over 24 h. In contrast, Nectin-4 knockdown cells did not form tightly compacted spheroids. Synthetic peptides derived from Nectin-4 were tested for their ability to alter spheroid formation in two ovarian cancer cell lines. Nectin-4 peptide 10 (N4-P10) had an immediate effect on disrupting ovarian cancer spheroid formation, which continued for over 24 h, while a scrambled version of the peptide had no effect. N4-P10 inhibited spheroid formation in a concentration-dependent manner and was not cytotoxic; suggesting that N4-P10 treatment could maintain the cancer cells as single cells which may be more sensitive to chemotherapy.
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Mao, Wenbin, and Alexander Alexeev. "Motion of spheroid particles in shear flow with inertia." Journal of Fluid Mechanics 749 (May 14, 2014): 145–66. http://dx.doi.org/10.1017/jfm.2014.224.
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AbstractIn this article, we investigate the motion of a solid spheroid particle in a simple shear flow. Using a lattice Boltzmann method, we examine individual effects of fluid inertia and particle rotary inertia as well as their combination on the dynamics and trajectory of spheroid particles at low and moderate Reynolds numbers. The motion of a single spheroid is shown to be dependent on the particle Reynolds number, particle aspect ratio, particle initial orientation and the Stokes number. Spheroids with random initial orientations are found to drift to stable orbits influenced by fluid inertia and/or particle inertia. Specifically, prolate spheroids drift towards the tumbling mode of motion, whereas oblate spheroids drift to the rolling mode. The rotation period and the variation of angular velocity of tumbling spheroids decrease as Stokes number increases. With increasing Reynolds number, both the maximum and minimum values of angular velocity decrease, whereas the particle rotation period increases. We show that particle inertia does not affect the hydrodynamic torque on the particle. We also demonstrate that superposition can be used to estimate the combined effect of fluid inertia and particle inertia on the dynamics of spheroid particles at sufficiently low Reynolds numbers.
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Royo, Felix, Clara Garcia-Vallicrosa, Maria Azparren-Angulo, Guillermo Bordanaba-Florit, Silvia Lopez-Sarrio, and Juan Manuel Falcon-Perez. "Three-Dimensional Hepatocyte Spheroids: Model for Assessing Chemotherapy in Hepatocellular Carcinoma." Biomedicines 12, no. 6 (May 28, 2024): 1200. http://dx.doi.org/10.3390/biomedicines12061200.
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BACKGROUND: Three-dimensional cellular models provide a more comprehensive representation of in vivo cell properties, encompassing physiological characteristics and drug susceptibility. METHODS: Primary hepatocytes were seeded in ultra-low attachment plates to form spheroids, with or without tumoral cells. Spheroid structure, cell proliferation, and apoptosis were analyzed using histological staining techniques. In addition, extracellular vesicles were isolated from conditioned media by differential ultracentrifugation. Spheroids were exposed to cytotoxic drugs, and both spheroid growth and cell death were measured by microscopic imaging and flow cytometry with vital staining, respectively. RESULTS: Concerning spheroid structure, an active outer layer forms a boundary with the media, while the inner core comprises a mass of cell debris. Hepatocyte-formed spheroids release vesicles into the extracellular media, and a decrease in the concentration of vesicles in the culture media can be observed over time. When co-cultured with tumoral cells, a distinct distribution pattern emerges over the primary hepatocytes, resulting in different spheroid conformations. Tumoral cell growth was compromised upon antitumoral drug challenges. CONCLUSION: Treatment of mixed spheroids with different cytotoxic drugs enables the characterization of drug effects on both hepatocytes and tumoral cells, determining drug specificity effects on these cell types.
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Perini, Giordano, Enrico Rosa, Ginevra Friggeri, Lorena Di Pietro, Marta Barba, Ornella Parolini, Gabriele Ciasca, et al. "INSIDIA 2.0 High-Throughput Analysis of 3D Cancer Models: Multiparametric Quantification of Graphene Quantum Dots Photothermal Therapy for Glioblastoma and Pancreatic Cancer." International Journal of Molecular Sciences 23, no. 6 (March 16, 2022): 3217. http://dx.doi.org/10.3390/ijms23063217.
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Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids’ death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.
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Petry, Florian, and Denise Salzig. "Large-Scale Production of Size-Adjusted β-Cell Spheroids in a Fully Controlled Stirred-Tank Reactor." Processes 10, no. 5 (April 27, 2022): 861. http://dx.doi.org/10.3390/pr10050861.
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For β-cell replacement therapies, one challenge is the manufacturing of enough β-cells (Edmonton protocol for islet transplantation requires 0.5–1 × 106 islet equivalents). To maintain their functionality, β-cells should be manufactured as 3D constructs, known as spheroids. In this study, we investigated whether β-cell spheroid manufacturing can be addressed by a stirred-tank bioreactor (STR) process. STRs are fully controlled bioreactor systems, which allow the establishment of robust, larger-scale manufacturing processes. Using the INS-1 β-cell line as a model for process development, we investigated the dynamic agglomeration of β-cells to determine minimal seeding densities, spheroid strength, and the influence of turbulent shear stress. We established a correlation to exploit shear forces within the turbulent flow regime, in order to generate spheroids of a defined size, and to predict the spheroid size in an STR by using the determined spheroid strength. Finally, we transferred the dynamic agglomeration process from shaking flasks to a fully controlled and monitored STR, and tested the influence of three different stirrer types on spheroid formation. We achieved the shear stress-guided production of up to 22 × 106 ± 2 × 106 viable and functional β-cell spheroids per liter of culture medium, which is sufficient for β-cell therapy applications.
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Preda, Petruța, Ana-Maria Enciu, Cristiana Tanase, Maria Dudau, Lucian Albulescu, Monica-Elisabeta Maxim, Raluca Nicoleta Darie-Niță, Oana Brincoveanu, and Marioara Avram. "Assessing Polysaccharides/Aloe Vera–Based Hydrogels for Tumor Spheroid Formation." Gels 9, no. 1 (January 7, 2023): 51. http://dx.doi.org/10.3390/gels9010051.
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In vitro tumor spheroids have proven to be useful 3D tumor culture models for drug testing, and determining the molecular mechanism of tumor progression and cellular interactions. Therefore, there is a continuous search for their industrial scalability and routine preparation. Considering that hydrogels are promising systems that can favor the formation of tumor spheroids, our study aimed to investigate and develop less expensive and easy-to-use amorphous and crosslinked hydrogels, based on natural compounds such as sodium alginate (NaAlg), aloe vera (AV) gel powder, and chitosan (CS) for tumor spheroid formation. The ability of the developed hydrogels to be a potential spheroid-forming system was evaluated using MDA-MB-231 and U87MG cancer cells. Spheroid abilities were influenced by pH, viscosity, and crosslinking of the hydrogel. Addition of either AV or chitosan to sodium alginate increased the viscosity at pH 5, resulting in amorphous hydrogels with a strong gel texture, as shown by rheologic analysis. Only the chitosan-based gel allowed formation of spheroids at pH 5. Among the variants of AV-based amorphous hydrogels tested, only hydrogels at pH 12 and with low viscosity promoted the formation of spheroids. The crosslinked NaAlg/AV, NaAlg/AV/glucose, and NaAlg/CS hydrogel variants favored more efficient spheroid formation. Additional studies would be needed to use AV in other physical forms and other formulations of hydrogels, as the current study is an initiation, in evaluating the potential use of AV gel in tumor spheroid formation systems.
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Shimoto, Takeshi, Chihiro Teshima, Toshiki Watanabe, Xiu-Ying Zhang, Atsushi Ishikawa, Hidehiko Higaki, and and Koichi Nakayama. "Study on Pipetting Motion Optimization of Automatic Spheroid Culture System for Spheroid Formation." Journal of Robotics and Mechatronics 33, no. 1 (February 20, 2021): 78–87. http://dx.doi.org/10.20965/jrm.2021.p0078.
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This research group has established a technology for producing a three-dimensional cell constructed using only the cell itself. This technology uses a property in which the spheroids fuse with each other. We developed a system that automates the spheroid production process to obtain reproducible spheroids and suppress variation factors that occur from human operation. However, it has become clear that the dispersion occurs in the diameter depending on the number of cells of the spheroid even if the cells are handled in the same manner. The purpose of this research is to examine an appropriate pipetting motion in accordance with the number of cells of the spheroid to be produced. Rabbit mesenchymal stem cells (rMSCs) are used as the objects. The number of cells was set to 2×104, 3×104, and 4×104 cells/well, and the passage number as 7. The appearance of spheroids cultured using the motion programmed in accordance with each number of cells was observed every 24 hours for 5 days after seeding. The results of the analysis indicate that the optimum motion in each number of cells has been successfully specified, and reproducible spheroids have been successfully produced.
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Chen, Li-Chi, Hsin-Wen Wang, and Chieh-Cheng Huang. "Modulation of Inherent Niches in 3D Multicellular MSC Spheroids Reconfigures Metabolism and Enhances Therapeutic Potential." Cells 10, no. 10 (October 14, 2021): 2747. http://dx.doi.org/10.3390/cells10102747.
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Multicellular spheroids show three-dimensional (3D) organization with extensive cell–cell and cell–extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine.
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Carchman, Evie, Susan Payne, Cheri Pasch, Kristina Matkowskyj, Sam Joseph Lubner, Michael F. Bassetti, Noelle K. LoConte, Daniel Mulkerin, and Dustin A. Deming. "Translational investigations of gastrointestinal malignancies using spheroid cultures." Journal of Clinical Oncology 34, no. 4_suppl (February 1, 2016): 620. http://dx.doi.org/10.1200/jco.2016.34.4_suppl.620.
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620 Background: Spheroid cultures are now being used to investigate the biology of multiple types of cancer. This technique allows for a cost-effective, efficient and reliable means to culture tissues of interest. Recent investigations have demonstrated that spheroids maintain high mutational concordance with the cancers they are derived. We sought to determine if spheroid techniques could be utilized to perform translational investigations into subtypes of gastrointestinal malignancies. Methods: Transgenic mice carrying conditional mutations in key genes known to have important roles in colorectal cancer (CRC) tumorigenesis and progression were treated with a Cre-expressing adenovirus to initiate tumorigenesis. The mutant genes of interest included Apc, Trp53, KRAS, Pik3ca, and/or BRAF. Biopsies of these cancers were obtained with the murine endoscope and the tissue processed for spheroid culture. In addition, transgenic E6 and E7 mice were treated with DMBA and the resulting anal squamous cell carcinomas were harvested for spheroid culture. Cultured spheroids were imaged to characterize growth rate and changes in architecture. In addition, studies assessing response to therapeutics were performed and correlated with in vivo response. Human operative specimens were also obtained for culturing. Results: Multiple lines of murine CRC spheroids were able to be generated with a greater than 90% efficiency rate. These cultures maintained phenotypic characteristics of the cancers from which they were derived. Bright field imaging pre- and post-treatment was able to be utilized for quantification of treatment response. The median relative change in spheroid diameter was compared across treatment groups and correlated with response in vivo. Heterogeneity in the response of individual spheroids to treatment strategies was detected with this method. Murine squamous cell anal carcinoma was spheroid lines were generated similarly. Human CRC and anal cancers were also generated with high efficiency. Conclusions: Spheroid cultures from CRC and anal cancers can be generated with high efficiency and hold great promise for furthering treatment options for patients with cancer.
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Simon, Blake E., and Mark F. Hamilton. "Acoustic radiation force and torque on a spheroid near an interface." Journal of the Acoustical Society of America 151, no. 4 (April 2022): A90. http://dx.doi.org/10.1121/10.0010754.
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Acoustic radiation force and torque on a spheroid of arbitrary size near a rigid or pressure release interface are calculated using expansions of the pressure field in terms of both spherical and spheroidal wave functions. The spheroidal expansion coefficients are obtained by satisfying the boundary conditions at the interface and at the surface of the spheroid. Conditions at the interface are satisfied using a virtual (image) spheroid, while satisfying conditions at the spheroid surface require the use of addition theorems for spheroidal wave functions. The radiation force and torque are expressed as a summation of terms involving products of the coefficients in spherical wave expansions of the incident and scattered fields [Ilinskii et al., J. Acoust. Soc. Am. 144, 568–576 (2018)]. Far-field asymptotes are used to relate the spheroidal and spherical harmonic expansion coefficients analytically. Results from the present analytical model are compared with those from a finite-element model. The effect of the interface on the radiation force and torque relative to that on a spheroid in a free field is discussed. [B.E.S. is supported by the Applied Research Laboratories Chester M. McKinney Graduate Fellowship in Acoustics.]
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Zahn, Ingrid, Tobias Braun, Clemens Gögele, and Gundula Schulze-Tanzil. "Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?" International Journal of Molecular Sciences 22, no. 20 (October 12, 2021): 11011. http://dx.doi.org/10.3390/ijms222011011.
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Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.
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Fritz, Jamie Lee, Olga Collins, Parima Saxena, Adrian Buensuceso, Yudith Ramos Valdes, Kyle E. Francis, Kevin R. Brown, et al. "A Novel Role for NUAK1 in Promoting Ovarian Cancer Metastasis through Regulation of Fibronectin Production in Spheroids." Cancers 12, no. 5 (May 15, 2020): 1250. http://dx.doi.org/10.3390/cancers12051250.
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Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.