Large Tissue Constructs Research Articles

Spontaneous Alignment of Myotubes Through Myogenic Progenitor Cell Migration.

In large-volume muscle injuries, widespread damage to muscle fibers and the surrounding connective tissue prevents myogenic progenitor cells (MPCs) from initiating repair. There is a clinical need to rapidly fabricate large muscle tissue constructs for integration at the site of large volume muscle injuries. Most strategies for myotube alignment require microfabricated structures or prolonged orientation times. We utilize the MPC's natural propensity to close gaps across an injury site to guide alignment on collagen I. When MPCs are exposed to an open boundary free of cells, they migrate unidirectionally into the cell-free region and align perpendicular to the original boundary direction. We study the utility of this phenomenon with biotin-streptavidin adhesion to position the cells on the substrate, and then demonstrate the robustness of this strategy with unmodified cells, creating a promising tool for MPC patterning without interrupting their natural function. We preposition MPCs in straight-line patterns separated with small gaps. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps, similar to how they migrate and align with a single open boundary. There is a directional component to the MPC migration perpendicular (90°) to the original biotin-streptavidin surface patterns. The expression of myosin heavy chain, the motor protein of muscle thick filaments, is confirmed through immunocytochemistry in myotubes generated from MPCs in our patterning process, acting as a marker of skeletal muscle differentiation. The rapid and highly specific binding of biotin-streptavidin allows for quick formation of temporary patterns, with MPC alignment based on natural regenerative behavior rather than complex fabrication techniques.

Long-Term Culture Performance of a Polyelectrolyte Complex Microcapsule Platform for Hyaline Cartilage Repair.

Articular cartilage (AC) tissue repair and regeneration remains an ongoing challenge. One component of the challenge is the limited ability to scale an engineered cartilage graft to clinically relevant sizes while maintaining uniform properties. In this paper, we report on the evaluation of our polyelectrolyte complex microcapsule (PECM) platform technology as a technique for generating cartilage-like spherical modules. Bone marrow-derived mesenchymal stem cells (bMSCs) or primary articular chondrocytes were encapsulated within PECMs composed of methacrylated hyaluronan, collagen I, and chitosan. The formation of cartilage-like tissue in the PECMs over a 90-day culture was characterized. The results showed that chondrocytes exhibited superior growth and matrix deposition compared to either chondrogenically-induced bMSCs or a mixed PECM culture containing both chondrocytes and bMSCs. The chondrocyte-generated matrix filled the PECM and produced substantial increases in capsule compressive strength. The PECM system thus appears to support intracapsular cartilage tissue formation and the capsule approach promotes efficient culture and handling of these micro tissues. Since previous studies have proven the feasibility of fusing such capsules into large tissue constructs, the results suggest that encapsulating primary chondrocytes in PECM modules may be a viable route toward achieving a functional articular cartilage graft.

Research Dynamics of Tissue Spheroids as Building Blocks: A Scientometric Analysis

Tissue spheroids represent an innovative solution for tissue engineering and regenerative medicine. They constitute an in vitro three-dimensional cell culture model capable of mimicking the complex composition of a native tissue on a micro-scale; this model can function as a building block and be assembled into larger tissue constructs. Due to the potential tissue spheroids have for the evolution of the health industry, there is a need to assess the research dynamics of this field. Thus far, there have been no studies on their use as building blocks. To fill this gap, a study was performed to characterize the evolution of research where tissue spheroids were used as building blocks to generate tissue constructs. A scientometric analysis of the literature regarding tissue spheroid technologies was developed by quantification of bibliometric performance indicators. For this purpose, articles published during the period January 1, 2015 – December 31, 2021, from the Scopus database were organized and analyzed. The main subject areas, countries, cities, journals, institutions, and top-cited articles as well as the types of techniques, cells, culture time, and principal applications were identified. This research supports the definition and growth of research and development strategies for new technologies such as tissue spheroids.

Viscous Fingering as a Rapid 3D Pattering Technique for Engineering Cell-Laden Vascular-Like Constructs.

Tissues are much larger than the diffusion limit distance, so rapidly providing blood vessels to supply embedded cells inside tissues with sufficient nutrients and oxygen is regarded as a major strategy for the success of bioengineered large and thick tissue constructs. Here, a patterning technique, viscous fingering, is developed to bioengineer vascularized-like tissues within a few minutes. By controlling viscosity, flow rate, and the volume of photo-cross-linkable prepolymer, macro- and microscale vascular network structures can be precisely engineered using the Hele-Shaw cell that is designed in this study. After cross-linking, a vascular-like gel with fingering structures is formed between the bottom and top base gels, creating a sandwich-like structure. Cells can be incorporated into the fingers, bases, or both gels. The spreading and growth direction of the embedded cells are successfully controlled and guided by manipulating the physical properties of the fingering and base gels individually. Moreover, fingering is generated, connected, and surrounded prepared cell-laden microgels in base prepolymers to form prevascularized tissue-like constructs. Taken together, the 3D cell patterning technique extends the potential for modeling and fabricating large and stackable vascularized tissue-like constructs for both ex vivo and in vivo applications.

Withdrawal: Erickson, P., Houwayek, T., Teryek, M. and Parekkadan, B. (2021), A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion. Biotechnology and Applied Biochemistry. https://doi.org/10.1002/bab.2183.

Cells absorb and secrete substances to-and-from their surroundings as part of metabolism, signaling, and other functions. These fluxes are dynamic, changing over time in response to external cues and internal programs. Static cultures are inadequate for measuring these dynamics because the environments of the cells change as substances accumulate or are depleted from medium, which affects cell behavior in unintended ways. Static cultures also offer limited time resolution due to the impracticality of frequent or prolonged manual timepoint sampling, and cannot expose cells to smooth, transient changes in stimulus concentrations. Perfusion cultures overcome these challenges by constantly replenishing medium to maintain cellular environments, while continuously sampling the effluent stream. However, many perfusion culture implementations are microfluidic devices, which cannot culture large tissue constructs and require specialized equipment and expertise to use. Previously published macrofluidic devices often use custom parts that are difficult to replicate, do not support both solute input control and measurement, and do not account for effects of dispersion on measured signals. In this study, a multiplexed macrofluidic perfusion culture platform was developed to measure secretion and absorption rates of substances by cells in a temporally controlled environment. The modular platform can handle up to 31 streams with automated sample collection using a fraction collector. This paper presents the assembly of this dynamic bioreactor and a method for quantitatively handling the effects of dispersion using residence time distributions. The system is then applied to monitor the secretion of a circadian clock gene-driven reporter from transfected cells. This article is protected by copyright. All rights reserved.

Recent Trends in the Development of Bone Regenerative Biomaterials.

The goal of a biomaterial is to support the bone tissue regeneration process at the defect site and eventually degrade in situ and get replaced with the newly generated bone tissue. Biomaterials that enhance bone regeneration have a wealth of potential clinical applications from the treatment of non-union fractures to spinal fusion. The use of bone regenerative biomaterials from bioceramics and polymeric components to support bone cell and tissue growth is a longstanding area of interest. Recently, various forms of bone repair materials such as hydrogel, nanofiber scaffolds, and 3D printing composite scaffolds are emerging. Current challenges include the engineering of biomaterials that can match both the mechanical and biological context of bone tissue matrix and support the vascularization of large tissue constructs. Biomaterials with new levels of biofunctionality that attempt to recreate nanoscale topographical, biofactor, and gene delivery cues from the extracellular environment are emerging as interesting candidate bone regenerative biomaterials. This review has been sculptured around a case-by-case basis of current research that is being undertaken in the field of bone regeneration engineering. We will highlight the current progress in the development of physicochemical properties and applications of bone defect repair materials and their perspectives in bone regeneration.

Directed self-assembly of spheroids into modular vascular beds for engineering large tissue constructs

Spheroids can be used as building-blocks for bottom-up generation of artificial vascular beds, but current biofabrication strategies are often time-consuming and complex. Also, pre-optimization of single spheroid properties is often neglected. Here, we report a simple setup for rapid biomanufacturing of spheroid-based patch-like vascular beds. Prior to patch assembly, spheroids combining mesenchymal stem/stromal cells (MSCs) and outgrowth endothelial cells (OECs) at different ratios (10:1; 5:1; 1:1; 1:5) were formed in non-adhesive microwells and monitored along 7 d. Optimal OEC retention and organization was observed at 1:1 MSC/OEC ratio. Dynamic remodelling of spheroids led to changes in both cellular and extracellular matrix components (ECMs) over time. Some OEC formed internal clusters, while others organized into a peripheral monolayer, stabilized by ECM and pericyte-like cells, with concomitant increase in surface stiffness. Along spheroid culture, OEC switched from an active to a quiescent state, and their endothelial sprouting potential was significantly abrogated, suggesting that immature spheroids may be more therapeutically relevant. Non-adhesive moulds were subsequently used for triggering rapid, one-step, spheroid formation/fusion into square-shaped patches, with spheroids uniformly interspaced via a thin cell layer. The high surface area, endothelial sprouting potential, and scalability of the developed spheroid-based patches make them stand out as artificial vascular beds for modular engineering of large tissue constructs.

Scaffold-based and Scaffold-free Strategies in Dental Pulp Regeneration

Regenerative dentistry has come a long way from pulp capping to pulp regeneration research, which aims to regenerate the pulp-dentin complex and restore its functions compromised by pulp injury and/or inflammation. Because of unique anatomic limitations of the tooth structure, engineering a suitable microenvironment that facilitates angio/vasculogenesis and innervation is a challenging task. Cell-based tissue engineering approaches have shown great potential in achieving this goal. Biomedical approaches in creating a regenerative microenvironment are mainly represented by either scaffold-based or scaffold-free strategies. The scaffold-based strategy mainly relies on the use of biomaterials to create a structural base that supports cells throughout the process of tissue formation. The scaffold could be a classic 3-dimensional construct with interconnected pores, a hydrogel with cells embedded in it, or a combination of these 2. The scaffold-free approach has been considered a bottom-up strategy that uses cell sheets, spheroids, or tissue strands as building blocks. The outcome of this strategy relies on the capacity of these building blocks to secrete a favorable extracellular matrix and to fuse into larger tissue constructs. Both the scaffold-free and scaffold-based systems are required as complementary, rather than competing, approaches for pulp regeneration. A combined synergetic strategy, through which multicellular building blocks could be integrated with robust 3-dimensional scaffolds, might represent an optimal solution to circumvent some of the major drawbacks of the current methods in pulp regeneration while concurrently fostering their advantages.

TGase-Enhanced Microtissue Assembly in 3D-Printed-Template-Scaffold (3D-MAPS) for Large Tissue Defect Reparation.

Engineering large and functional tissue constructs with complex structures (e.g., external ear or nose) for reparation and reconstruction of tissue defects remains one of the major challenges in regenerative medicine, which demands abundant cell sources, advanced biofabrication schemes, and satisfactory integration with the host for long-term efficacy post implantation. Here the 'Microtissue Assembly in 3D-Printed-template-Scaffold' (3D-MAPS), as a platform technology to rapidly fabricate centimeter-sized functional tissue constructs with complex structures, is developed. 3D-MAPS facilitates bottom-up assembly of large-scale manufactured microtissues within the 3D-printed hollow polymeric templates with pre-defined architectures. The assembly and fusion of 2×106 mesenchymal stem cell-based microtissues within the defined 3D-printed template is further enhanced by addition of a natural protein crosslinker (i.e., transglutaminase (TGase)), and thereby achieves construction of centimeter-sized tissue with high cell viability and mechanical stability in vitro within 30 min. Further in vivo implantation of the 3D-MAPS-fabricated ear-like tissue construct in rabbit models assisted by flap prefabrication technique results in increased structural vascular support and strengthened functional survival. Thus, the TGase-enhanced 3D-MAPS demonstrates its potential and feasibility as a powerful biofabrication platform for tissue engineering application.

Development of a Dual Hydrogel Model System for Vascularization

Numerous hydrogel-based culture systems are used to create in vitro model for prevascularization. Hydrogels used to induce a microenvironment conducive to microvessel formation are typically soft and fast degradable, but often suffer from maintaining a lasting perfusable channel in vitro. Here, a dual hydrogel system that consists of photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol dimethacrylate (PEGDMA) is reported. GelMA hydrogels present soft and rapidly degradable properties and show microporous structures while PEGDMA is relatively stiff, almost nondegradable in vitro, and less porous. The dual hydrogel system is sequentially photo-crosslinked to construct an endothelial cell (EC)-lined perfusable PEGDMA channel and surrounding GelMA for endothelial vascular networks. Such dual hydrogel system exhibits seamless integration of the stiff PEGDMA channel and the surrounding soft GelMA, and facilitates rapid EC sprouting and extensive microvessel formation from a stable endothelium on the PEGDMA channel into the GelMA. Furthermore, diffusivity of biomolecules in the perfusable dual hydrogel system is affected by both the structural and physicochemical properties of the hydrogel system and the microvascular networks formed in the system. The establishment of the dual hydrogel system for vascularization holds great promise as an in vitro angiogenesis model and prevascularization strategy of large tissue constructs.

Bioprinting predifferentiated adipose-derived mesenchymal stem cell spheroids with methacrylated gelatin ink for adipose tissue engineering.

The increasing number of mastectomies results in a greater demand for breast reconstruction characterized by simplicity and a low complication profile. Reconstructive surgeons are investigating tissue engineering (TE) strategies to overcome the current surgical drawbacks. 3D bioprinting is the rising technique for the fabrication of large tissue constructs which provides a potential solution for unmet clinical needs in breast reconstruction building on decades of experience in autologous fat grafting, adipose-derived mesenchymal stem cell (ASC) biology and TE. A scaffold was bioprinted using encapsulated ASC spheroids in methacrylated gelatin ink (GelMA). Uniform ASC spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. ASC spheroids in adipogenic differentiation medium (ADM) were evaluated through live/dead staining, histology (HE, Oil Red O), TEM and RT-qPCR. Viable spheroids were obtained for up to 14 days post-printing and showed multilocular microvacuoles and successful differentiation toward mature adipocytes shown by gene expression analysis. Moreover, spheroids were able to assemble at random in GelMA, creating a macrotissue. Combining the advantage of microtissues to self-assemble and the controlled organization by bioprinting technologies, these ASC spheroids can be useful as building blocks for the engineering of soft tissue implants.

A Prevascularization Strategy Using Novel Fibrous Porous Silk Scaffolds for Tissue Regeneration in Mice with Spinal Cord Injury

Spinal cord injury (SCI) represents an extremely debilitating condition for which no efficacious treatment is available. Because spinal cord does not have satisfactory capacity for revascularization after injury, it seems to be a promising way to modulate the lesion environment by reperfusion to promote a regenerative phenotype. Although engineered scaffolds provide a platform to deliver therapeutic cells and neurotrophic factors, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. In this study, we cocultured our fibrous porous silk scaffold (FPSS) with ADAMTS13-overexpressing human umbilical vein endothelial cells (HUVECs) in vitro and transplanted this prevascularized construct into an SCI mouse model. The prevascularized system exhibited a tube-like structure in vitro, promoted vascular infiltration and microvascular network formation after transplantation, and recruited more neural cells to the lesion site. Twenty-eight days later, behavioral analysis showed that locomotor recovery was significantly improved in treated animals compared with control animals. Taken together, our results suggest that the FPSS-HUVECs system promoted neovascularization, guided axon growth at the injury site, and improved the microenvironment. Therefore, this prevascularization system may provide a better therapeutic option for SCI.

Bioprinting of Multiscaled Hepatic Lobules within a Highly Vascularized Construct.

Highly vascularized complex liver tissue is generally divided into lobes, lobules, hepatocytes, and sinusoids, which can be viewed under different types of lens from the micro- to macro-scale. To engineer multiscaled heterogeneous tissues, a sophisticated and rapid tissue engineering approach is required, such as advanced 3D bioprinting. In this study, a preset extrusion bioprinting technique, which can create heterogeneous, multicellular, and multimaterial structures simultaneously, is utilized for creating a hepatic lobule (≈1 mm) array. The fabricated hepatic lobules include hepatic cells, endothelial cells, and a lumen. The endothelial cells surround the hepatic cells, the exterior of the lobules, the lumen, and finally, become interconnected with each other. Compared to hepatic cell/endothelial cell mixtures, the fabricated hepatic lobule shows higher albumin secretion, urea production, and albumin, MRP2, and CD31 protein levels, as well as, cytochrome P450 enzyme activity. It is found that each cell type with spatial cell patterning in bioink accelerates cellular organization, which could preserve structural integrity and improve cellular functions. In conclusion, preset extruded hepatic lobules within a highly vascularized construct are successfully constructed, enabling both micro- and macro-scale tissue fabrication, which can support the creation of large 3D tissue constructs for multiscale tissue engineering.

Synchronous 3D Bioprinting of Large-Scale Cell-Laden Constructs with Nutrient Networks.

Maintaining an adequate supply of nutrients/oxygen is a major challenge in the biofabrication of large tissue constructs. However, building preformed nutrient networks may be an effective strategy for engineering thick tissues. Here, a novel way for bioprinting large-scale tissue constructs with intentional nutrient networks is presented. A special nozzle is developed which can print bioink and sacrificial ink half and half synchronously in a single filament. Nutrient networks of these bioprinted constructs are formed by subsequently dissolving away gelatin, which allows for effective oxygen, nutrient, and waste diffusion, facilitating the cell activity and the generation of functional tissues. Due to the cell-laden bioink and sacrificial ink working together and promoting each other's printability to support themselves, complex soft cell-laden constructs with nutrient networks can easily be printed. Furthermore, two different cell types (osteoblast, human umbilical vein endothelial cells) encapsulated in the bioprinted large-scale constructs (≥1 cm) with nutrient networks show enhanced cell viability and spreading within a period of culture. It is envisioned that the advanced bioprinting technology may have significant potentials in facilitating the engineering of complex structures for tissue-specific needs, and bioprinting large-scale tissue constructs with nutrient networks toward applications in organ transplantation and repair.

Nanofibrillar Cellulose as an Enzymatically and Flow Driven Degradable Scaffold for Three-Dimensional Tissue Engineering

Abstract Nanofibrillar cellulose as a naturally biocompatible scaffold material is very promising for tissue engineering. It is shear thinning but has the downside of not being degradable in animals, it can only be degraded by cellulase enzymes. In this study, a newly developed bioreactor was used to culture fibroblast spheroids under flow conditions inside nanocellulose hydrogels with and without the presence of cellulase. The aim was to control the tissue size and ideally find a match between degradation and tissue formation within this promising material. Both the concentration of cellulase and the flow rate were varied and their influence on the activity and growth of fibroblast clusters was assessed. Cluster diameters, degradation, metabolic activity, and tissue production increase with higher cellulase concentration, although concentrations above 1 g/l does not have an additional benefit. Flow leads to more viable cells, more proliferation and migration, leading to overall larger tissue constructs compared to static conditions. This is most likely due to the shear thinning effect of flow on cellulose nanofibrils (CNFs) in addition to the increased nutrient supply through perfusion. At a constant cellulase concentration of 1 g/l, a flow of 2 ml/min proved to be optimal for tissue production. Therefore, degradation in combination with flow leads to more effective tissue production in CNF hydrogels, which is a very potent scaffold material for tissue engineering.

Shape-defined solid micro-objects from poly(d,l-lactic acid) as cell-supportive counterparts in bottom-up tissue engineering.

In bottom-up tissue engineering, small modular units of cells and biomaterials are assembled toward ​larger and more complex ones. In conjunction with a new implementation of this approach, a novel method to fabricate microscale objects from biopolymers by thermal imprinting on water-soluble sacrificial layers is presented. By this means, geometrically well-defined objects could be obtained without involving toxic agents in the form of photoinitiators. The micro-objects were used as cell-adhesive substrates and cell spacers in engineered tissues created by cell-guided assembly of the objects. Such constructs can be applied both for in vitro studies and clinical treatments. Clinically relevantly sized aggregates comprised of cells and micro-objects retained their viability up to 2 weeks of culture. The aggregation behavior of cells and objects showed to depend on the type and number of cells applied. To demonstrate the micro-objects’ potential for engineering vascularized tissues, small aggregates of human bone marrow stromal cells (hMSCs) and micro-objects were coated with a layer of human umbilical vein endothelial cells (HUVECs) and fused into larger tissue constructs, resulting in HUVEC-rich regions at the aggregates' interfaces. This three-dimensional network-type spatial cellular organization could foster the establishment of (premature) vascular structures as a vital prerequisite of, for example, bottom-up-engineered bone-like tissue.

Hydrogel Bioink with Multilayered Interfaces Improves Dispersibility of Encapsulated Cells in Extrusion Bioprinting.

One of the challenges for extrusion bioprinting using low-viscosity bioinks is the fast gravity-driven sedimentation of cells. Cells in a hydrogel bioink that features low viscosity tend to settle to the bottom of the bioink reservoir, and as such, their bioprintability is hindered by association with the inhomogeneous cellularized structures that are deposited. This is particularly true in cases where longer periods are required to print complex or larger tissue constructs. Increasing the bioink's viscosity efficiently retards sedimentation but gives rise to cell membranolysis or functional disruption due to increased shear stress on the cells during the extrusion process. Inspired by the rainbow cocktail, we report the development of a multilayered modification strategy for gelatin methacryloyl (GelMA) bioink to manipulate multiple liquid interfaces, providing interfacial retention to retard cell sedimentation in the bioink reservoir. Indeed, the interfacial tension in our layer-by-layer bioink system, characterized by the pendant drop method, was found to be exponentially higher than the sedimental pull (ΔGravity-Buoyancy = ∼10-9 N) of cells, indicating that the interfacial retention is crucial for preventing cell sedimentation across the adjacent layers. It was demonstrated that the encapsulated cells displayed better dispersibility in constructs bioprinted using the multilayered GelMA bioink system than that of pristine GelMA where the index of homogeneity of the cell distribution in the multilayered bioink was 4 times that of the latter.

Biofabrication of bacterial nanocellulose scaffolds with complex vascular structure

Bacterial nanocellulose (BNC) has proven to be an effective hydrogel-like material for different tissue engineering applications due to its biocompatibility and good mechanical properties. However, as for all biomaterials, in vitro biosynthesis of large tissue constructs remains challenging due to insufficient oxygen and nutrient transport in engineered scaffold-cell matrices. In this study we designed, biofabricated and evaluated bacterial nanocellulose scaffolds with a complex vascular mimetic lumen structure. As a first step a method for creating straight channeled structures within a bacterial nanocellulose scaffold was developed and evaluated by culturing of Human Umbilical Vein Endothelial Cells (HUVECs). In a second step, more complex structures within the scaffolds were produced utilizing a 3D printer. A print mimicking a vascular tree acted as a sacrificial template to produce a network within the nanoporous bacterial nanocellulose scaffolds that could be lined with endothelial cells. In a last step, a method to produce large constructs with interconnected macro porosity and vascular like lumen structure was developed. In this process patient data from x-ray computed tomography scans was used to create a mold for casting a full-sized kidney construct. By showing that the 3D printing technology can be combined with BNC biosynthesis we hope to widen the opportunities of 3D printing, while also enabling the production of BNC scaffolds constructs with tailored vascular architectures and properties.

State-of-the-Art Strategies for the Vascularization of Three-Dimensional Engineered Organs.

Engineering three-dimensional (3D) implantable tissue constructs is a promising strategy for replacing damaged or diseased tissues and organs with functional replacements. However, the efficient vascularization of new 3D organs is a major scientific and technical challenge since large tissue constructs or organs require a constant blood supply to survive in vivo. Current approaches to solving this problem generally fall into the following three major categories: (a) cell-based, (b) angiogenic factor-based, and (c) scaffold-based. In this review, we summarize state-of-the-art technologies that are used to develop complex, stable, and functional vasculature for engineered 3D tissue constructs and organs; additionally, we have suggested directions for future research.

Scaffold-free bioprinting of mesenchymal stem cells using the Regenova printer: Spheroid characterization and osteogenic differentiation

Limitations in scaffold material properties, such as sub-optimal degradation time, highlight the need for alternative approaches to engineer de novo tissues. One emerging solution for fabricating tissue constructs is scaffold-free tissue engineering. To facilitate this approach, three-dimensional (3D) bioprinting technology (Regenova Bio 3D Printer) has been developed to construct complex geometric shapes from discrete cellular spheroids without exogenous scaffolds. Optimizing spheroid fabrication and characterizing cellular behavior in the spheroid environment are important first steps prior to printing larger constructs. Here, we characterized spheroids of immortalized mouse bone marrow stromal cells (BMSCs) that were differentiated to the osteogenic lineage.Immortalized BMSCs were seeded in low attachment 96-well plates in various numbers to generate self-aggregated spheroids either under the force of gravity or centrifugation. Cells were cultured in control or osteogenic media for up to 28 days. Spheroid diameter, roundness and smoothness were measured. Cell viability, DNA content and alkaline phosphatase activity were assessed at multiple time points. Additionally, expression of osteogenic markers was determined using real time qPCR.Spheroids formed under gravity with 20 K, 30 K and 40 K cells had average diameters of 498.5 ± 8.3 μm, 580.0 ± 32.9 μm and 639.2 ± 54.0 μm, respectively, while those formed under 300G centrifugation with the same numbers of cells had average diameters of 362.3 ± 3.5 μm, 433.1 ± 6.4 μm and 491.2 ± 8.0 μm. Spheroids formed via centrifugation were superior to those formed by gravity, as evidenced by better roundness and smoothness and double the retention of DNA (cellular) content. Cells in spheroids exhibited a robust osteogenic response to the differentiation medium, including higher mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than those cultured in control medium, as well as greater alkaline phosphatase activity. The optimal spheroid fabrication technique from this study was to aggregate 40 K cells under 150–300G centrifugation. In future investigations, these spheroids will be 3D printed into larger tissue constructs.