Size-controlled Spheroids Research Articles

Fabrication and Cell Culture Applications of Core-Shell Hydrogel Fibers Composed of Chitosan/DNA Interfacial Polyelectrolyte Complexation and Calcium Alginate: Straight and Beaded Core Variations.

Core-shell hydrogel fibers are widely used in cell culture applications. A simple and rapid method is presented for fabricating core-shell hydrogel fibers, consisting of straight or beaded core fibers, for cell culture applications. The core fibers are prepared using interfacial polyelectrolyte complexation (IPC) with chitosan and DNA. Briefly, two droplets of chitosan and DNA are brought in contact to form an IPC film, which is dragged to prepare an IPC fiber. The incubation time and DNA concentration are adjusted to prepare straight and beaded IPC fibers. The fibers with Ca2+ are immersed in an alginate solution to form calcium alginate shell hydrogels around the core IPC fibers. To the best of the knowledge, this is the first report of core-shell hydrogel fibers with IPC fiber cores. To demonstrate cell culture, straight hydrogel fibers are applied to fabricate hepatic models consisting of HepG2 and 3T3 fibroblasts, and vascular models consisting of human umbilical vein endothelial cells and 3T3 fibroblasts. To evaluate the effect of co-culture, albumin secretion, and angiogenesis are evaluated. Beaded hydrogel fibers are used to fabricate many size-controlled spheroids for fiber and cloning applications. This method can be widely applied in tissue engineering and cell analysis.

Development of a Simple Spheroid Production Method Using Fluoropolymers with Reduced Chemical and Physical Damage

Establishing an in vitro–based cell culture system that can realistically simulate in vivo cell dynamics is desirable. It is thus necessary to develop a method for producing a large amount of cell aggregates (i.e., spheroids) that are uniform in size and quality. Various methods have been proposed for the preparation of spheroids; however, none of them satisfy all requirements, such as cost, size uniformity, and throughput. Herein, we successfully developed a new cell culture method by combining fluoropolymers and dot patterned extracellular matrix substrates to achieve size-controlled spheroids. First, the spheroids were spontaneously formed by culturing them two-dimensionally, after which the cells were detached with a weak liquid flow and cultured in suspension without enzyme treatment. Stable quality spheroids were easily produced, and it is expected that the introduction and running costs of the technique will be low; therefore, this method shows potential for application in the field of regenerative medicine.

Engineering Multi-Cellular Spheroids for Tissue Engineering and Regenerative Medicine.

Multi-cellular spheroids are formed as a 3D structure with dense cell-cell/cell-extracellular matrix interactions, and thus, have been widely utilized as implantable therapeutics and various ex vivo tissue models in tissue engineering. In principle, spheroid culture methods maximize cell-cell cohesion and induce spontaneous cellular assembly while minimizing cellular interactions with substrates by using physical forces such as gravitational or centrifugal forces, protein-repellant biomaterials, and micro-structured surfaces. In addition, biofunctional materials including magnetic nanoparticles, polymer microspheres, and nanofiber particles are combined with cells to harvest composite spheroids, to accelerate spheroid formation, to increase the mechanical properties and viability of spheroids, and to direct differentiation of stem cells into desirable cell types. Biocompatible hydrogels are developed to produce microgels for the fabrication of size-controlled spheroids with high efficiency. Recently, spheroids have been further engineered to fabricate structurally and functionally reliable in vitro artificial 3D tissues of the desired shape with enhanced specific biological functions. This paper reviews the overall characteristics of spheroids and general/advanced spheroid culture techniques. Significant roles of functional biomaterials in advanced spheroid engineering with emphasis on the use of spheroids in the reconstruction of artificial 3D tissue for tissue engineering are also thoroughly discussed.

Engineering of Primary Pancreatic Islet Cell Spheroids for Three-dimensional Culture or Transplantation: A Methodological Comparative Study

Three-dimensional (3D) cell culture by engineering spheroids has gained increasing attention in recent years because of the potential advantages of such systems over conventional two-dimensional (2D) tissue culture. Benefits include the ability of 3D to provide a more physiologically relevant environment, for the generation of uniform, size-controlled spheroids with organ-like microarchitecture and morphology. In recent years, different techniques have been described for the generation of cellular spheroids. Here, we have compared the efficiency of four different methods of islet cell aggregation. Rat pancreatic islets were dissociated into single cells before reaggregation. Spheroids were generated either by (i) self-aggregation in nonadherent petri dishes, (ii) in 3D hanging drop culture, (iii) in agarose microwell plates or (iv) using the Sphericalplate 5D™. Generated spheroids consisted of 250 cells, except for the self-aggregation method, where the number of cells per spheroid cannot be controlled. Cell function and morphology were assessed by glucose stimulated insulin secretion (GSIS) test and histology, respectively. The quantity of material, labor intensity, and time necessary for spheroid production were compared between the different techniques. Results were also compared with native islets. Native islets and self-aggregated spheroids showed an important heterogeneity in terms of size and shape and were larger than spheroids generated with the other methods. Spheroids generated in hanging drops, in the Sphericalplate 5D™, and in agarose microwell plates were homogeneous, with well-defined round shape and a mean diameter of 90 µm. GSIS results showed improved insulin secretion in response to glucose in comparison with native islets and self-aggregated spheroids. Spheroids can be generated using different techniques and each of them present advantages and inconveniences. For islet cell aggregation, we recommend, based on our results, to use the hanging drop technique, the agarose microwell plates, or the Sphericalplate 5D™ depending on the experiments, the latter being the only option available for large-scale spheroids production.

Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes.

We previously established a method for a directed differentiation of human pluripotent stem cells into classical brown adipocytes (BA) by forming aggregates via massive floating culture in the presence of a specific cytokine cocktail. However, use of recombinant cytokines requires significant cost. Moreover, an enforced differentiation by exogenously added cytokines may amend skewed differentiation propensity of patient’s pluripotent stem cells, providing unsatisfactory disease models. Therefore, an exogenous cytokine-free method, where cytokines required for differentiation are provided in an auto/paracrine manner mimicking natural developmental process, is beneficial. Here we show that, if human pluripotent stem cells are cultured as size-controlled spheroids (100–120 µm radius, 2000–2500 cells/spheroid) in a mutually segregated manner with half-change of the medium every other day, they differentiate into classical BA via an authentic MYF5-positive myoblast route in the absence of exogenous cytokines. Differentiated BA exerted thermogenic activity in transplanted mice in response to beta-adrenergic receptor agonist stimuli. The cytokine-free differentiation method has further advantages in exploring BATokines, BA-derived physiologically active substances. Indeed, we have found that BA produces an unknown small (<1000 Da), highly hydrophilic molecule that augments insulin secretion from pancreatic beta cells. Our upgraded technique will contribute to an advancement of stem cell study for diverse purposes.

Study of oxygen tension variation within live tumor spheroids using microfluidic devices and multi-photon laser scanning microscopy.

Three-dimensional cell spheroid culture using microfluidic devices provides a convenient in vitro model for studying tumour spheroid structures and internal microenvironments. Recent studies suggest that oxygen deprived zones inside solid tumors are responsible for stimulating local cytokines and endothelial vasculature proliferation during angiogenesis. In this work, we develop an integrated approach combining microfluidic devices and multi-photon laser scanning microscopy (MPLSM) to study variations in oxygen tension within live spheroids of human osteosarcoma cells. Uniform shaped, size-controlled spheroids are grown and then harvested using a polydimethylsiloxane (PDMS) based microfluidic device. Fluorescence live imaging of the harvested spheroids is performed using MPLSM and a commercially available oxygen sensitive dye, Image-iT Red, to observe the oxygen tension variation within the spheroids and those co-cultured with monolayers of human umbilical vein endothelial cells (HUVECs). Oxygen tension variations are observed within the spheroids with diameters ranging from 90 ± 10 μm to 140 ± 10 μm. The fluorescence images show that the low-oxygenated cores diminish when spheroids are co-cultured with HUVEC monolayers for 6 hours to 8 hours. In the experiments, spheroids subjected to HUVEC conditioned medium treatment and with a cell adherent substrate are also measured and analyzed to study their significance on oxygen tension within the spheroids. The results show that the oxygenation within the spheroids is improved when the spheroids are cultured under those conditions. Our work presents an efficient method to study oxygen tension variation within live tumor spheroids under the influence of endothelial cells and conditioned medium. The method can be exploited for further investigation of tumor oxygen microenvironments during angiogenesis.

3D hydrogel-based microwell arrays as a tumor microenvironment model to study breast cancer growth

The tumor microenvironment (TME) is distinctly heterogeneous and is involved in tumor growth, metastasis, and drug resistance. Mimicking this diverse microenvironment is essential for understanding tumor growth and metastasis. Despite the substantial scientific progress made with traditional cell culture methods, microfabricated three-dimensional (3D) cell cultures that can be precisely controlled to mimic the changes occur in the TME over tumor progression are necessary for simulating organ-specific TME in vitro. In this research, to simulate the breast cancer TME, microwell arrays of defined geometry and dimensions were fabricated using photo-reactive hydrogels for a cancer cell line and primary explant tissue culture. Microwell arrays fabricated from 4-arm polyethylene glycol acrylate and methacrylated gelatin with different degrees of methacrylation for controlled cell–matrix interactions and tunable stiffness were used to create a platform for studying the effects of distinct hydrogel compositions and stiffness on tumor formation. Using these microwell arrays, size-controlled spheroids of human breast cancer cell line HCC1806 were formed and the cell attachment properties, viability, metabolic activity, and migration levels of these spheroids were examined. In addition, primary mammary organoid tissues explanted from mice were successfully cultured in these hydrogel-based microwell arrays and the organoid morphology and viability, as well as organoid branching were studied. The microwell array platform developed and characterized in this study could be useful for generating a tissue-specific TME for in vitro high throughput studies of breast cancer development and progression as well as in drug screening studies for breast cancer treatment.

Preparation and characterization of directed, one-day-self-assembled millimeter-size spheroids of adipose-derived mesenchymal stem cells.

Three-dimensional cell spheroids prepared without using any artificial scaffold materials are desirable for cell-based transplants. However, conventional cell culture systems are inefficient for rapid, large-scale and non-cytotoxic generation of size-controlled spheroids (>1 mm diameter) that are required for tissue regenerative therapy application. In this study, we prepared millimeter-order spheroids of adipose-derived mesenchymal stem cells (ADSCs) by controlling the spheroid size (diameter range: 0.4-2.5 mm). Notably, spheroid generation required only one day of culture on charged culture dishes. Almost all spheroid-derived ADSCs were viable and produced adhesion molecules and growth factors, which play an important role in tissue regeneration. Moreover, spheroid-derived ADSCs could infiltrate and recellularize collagenous tissue membranes in vitro. The ADSC spheroids developed in this study could be directly (without additional processing) used for cell-based tissue regeneration therapy. Furthermore, the rapid scale-up process and noncytotoxic generation of spheroids would also enable other applications such as use as screening models for drug discovery.

Robotic production of cancer cell spheroids with an aqueous two-phase system for drug testing.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

Microfluidic experimental platform for producing size-controlled three-dimensional spheroids

We propose a microfluidic experimental platform for producing size-controlled spheroids. Cells were aggregated into chambers arranged in an array by microrotational flow within 120 s to form spheroids. The cell density of the initial medium and hydrodynamic flow in the developed array could be adjusted while keeping the device geometry the same to control spheroid size with a standard deviation of less than 19% of the mean. Using this device, spheroids of HepG2 cells of various size categories could be maintained for three days in the chamber with medium exchange and could be continuously evaluated for topology and hepatic functions. Furthermore, CYP1A1 activities were found to increase with time to a constant level at three days. These results demonstrate that this device is readily applicable to producing and maintaining spheroids for in vitro drug screening and biological research.

Parallel Formation of Three-Dimensional Spheroid Using Microrotational Flow

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.

Calcium alginate microcapsules with spherical liquid cores templated by gelatin microparticles for mass production of multicellular spheroids

Multicellular spheroids are important in biomedical applications, such as drug research and regenerative medicine. We developed microcapsules from sodium alginate and gelatin for mass production of size-controlled spheroids with diameters <200μm. The microcapsules were composed of calcium alginate gels with spherical liquid cores (diameter≈150μm) for formation of spheroids. The spherical liquid cores were prepared by incubating calcium alginate microcapsules containing thermally gelled, cell-enclosing gelatin microparticles about 150μm in diameter, at 37°C. The gelatin microparticles were encapsulated within the microcapsules by dropping a sodium alginate solution containing suspended gelatin microparticles into 100mM CaCl2. The enclosed feline renal fibroblast cell line, CRFK, cells showed 93.8% viability immediately after encapsulation, then grew and completely filled the spherical cores. Multicellular spheroids were collected within 1min by soaking microcapsules in a medium containing alginate lyase.