Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation.
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ABSTRACT:
SUBMITTER: Jeger-Madiot N
PROVIDER: S-EPMC8052426 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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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Project description:To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 10(6) cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3?days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10?days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10?day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.
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Project description:The simplest and most commonly used acoustic levitator is comprised of a transmitter and an opposing reflecting surface. This type of device, however, is only able to levitate objects along one direction, at distances multiple of half of a wavelength. In this work, we show how a customised reflective acoustic metamaterial enables the levitation of multiple particles, not necessarily on a line and with arbitrary mutual distances, starting with a generic input wave. We establish a heuristic optimisation technique for the design of the metamaterial, where the local height of the surface is used to introduce delay patterns to the reflected signals. Our method stands for any type and number of sources, spatial resolution of the metamaterial and system's variables (i.e. source position, phase and amplitude, metamaterial's geometry, relative position of the levitation points, etc.). Finally, we explore how the strength of multiple levitation points changes with their relative distance, demonstrating sub-wavelength field control over levitating polystyrene beads into various configurations.
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Project description:Mesenchymal stromal/stem cells (MSCs), which generally expand into adherent monolayers, readily lose their proliferative and multilineage potential following repeated passages. Floating culture systems can be used to generate MSC spheroids, which are expected to overcome limitations associated with conventional adherent cultures while facilitating scaffold-free cell transplantation. However, the phenotypic characteristics of spheroids after long-term culture are unknown. In addition, regenerative therapies require new culture systems to maintain their undifferentiated state. In this study, we established a novel culture method employing three-dimensional (3D) "shaking" to generate MSC spheroids using bone marrow derived MSCs. Floating 3D cultures of mouse or human MSCs formed spheroids after shaking (85-95 rpm), within 1 month. These spheroids maintained their osteogenic-, adipogenic-, and chondrogenic-differentiation capacity. The adipogenic-differentiation capacity of adherent cultured mouse and human MSCs, which is lost following several passages, was remarkedly restored by shaking-culture. Notably, human MSC spheroids exhibited a renewable "undifferentiated MSC-pool" property, wherein undifferentiated MSCs grew from spheroids seeded repeatedly on a plastic culture dish. These data suggest that the shaking-culture method maintains and restores multipotency that is lost following monolayer expansion and thereby shows potential as a promising strategy for regenerative therapies with mesenchymal tissues.
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Project description:Substrate stiffness is known to alter cell behavior and drive stem cell differentiation, though most research in this area has been restricted to traditional, two-dimensional culture systems rather than more physiologically relevant, three-dimensional (3D) platforms. In this study, we utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to human adipose derived stem cells in 3D spheroid culture to examine changes in adipogenic differentiation response and mechanophenotype. After 21 days of adipogenic induction, spheroids containing CMMPs (composite spheroids) stiffened in accordance with CMMP elasticity such that spheroids containing the stiffest, ~?10 kPa, CMMPs were over 27% stiffer than those incorporating the most compliant, ~?0.25 kPa CMMPs. Adipogenically induced, cell-only spheroids were over 180% larger and 50% more compliant than matched controls. Interestingly, composite spheroids cultured without chemical induction factors dissociated when presented with CMMPs stiffer than ~?1 kPa, while adipogenic induction factors mitigated this behavior. Gene expression for PPARG and FABP4 were upregulated more than 45-fold in adipogenically induced samples compared to controls but were unaffected by CMMP elasticity, attributed to insufficient cell-CMMP contacts throughout the composite spheroid. In summary, mechanically tuned CMMPs influenced whole-spheroid mechanophenotype and stability but minimally affected differentiation response.
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Project description:To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 ?m, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.
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Project description:Mesenchymal stem cells (MSCs) exhibit self-renewal, multi-lineage differentiation potential and immunomodulatory properties, and are promising candidates for cellular therapy of various tissues. Despite the effective function of MSCs, the gradual loss of stem cell characteristics that occurs with repeated passages may significantly limit their therapeutic potential. A novel 3D shaking method was previously established to generate MSC spheroids in growth medium (GM-spheroids) and successfully maintain the multipotency of expanded MSCs, yet the expression of MSC-related genes was still low. In this study, we used a neurosphere culture technique to optimize the shaking culture method using human bone marrow-derived MSCs (BM-MSCs). MSC spheroids generated in neurosphere medium (NM-spheroids) maintained high expression of MSC-related genes during 3 weeks of prolonged shaking culture. Moreover, NM-spheroids generated from expanded MSCs showed high viability, upregulation of MSC-related and immune-related genes, and recovery of differentiation potential in vitro. Expanded adherent MSCs, GM-spheroids, and NM-spheroids were transplanted into a rat femur bone defect model to investigate their therapeutic potential in bone repair. Adherent MSCs and GM-spheroids showed delayed bone healing. In contrast, NM-spheroids showed high transplantation efficiency and enhanced bone regeneration. These data suggest that NM-spheroids generated using modified neurosphere culture conditions under continuous shaking recovered their stem cell characteristics in vitro and enhanced bone regeneration in vivo. Therefore, NM-spheroids should have great clinical potential for bone and tissue regenerative therapies as a stem cell-based biomaterial therapy.
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Project description:Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.
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Project description:BackgroundRecently, various studies have revealed that 3D cell spheroids have several advantages over 2D cells in stem cell culture. However, conventional 3D spheroid culture methods have some disadvantages and limitations such as time required for spheroid formation and complexity of the experimental process. Here, we used acoustic levitation as cell culture platform to overcome the limitation of conventional 3D culture methods.MethodsIn our anti-gravity bioreactor, continuous standing sonic waves created pressure field for 3D culture of human mesenchymal stem cells (hMSCs). hMSCs were trapped and aggerated in pressure field and consequently formed spheroids. The structure, viability, gene and protein expression of spheroids formed in the anti-gravity bioreactor were analyzed by electron microscope, immunostaining, polymerase chain reaction, and western blot. We injected hMSC spheroids fabricated by anti-gravity bioreactor into the mouse hindlimb ischemia model. Limb salvage was quantified to evaluate therapeutic efficacy of hMSC spheroids.ResultsThe acoustic levitation in anti-gravity bioreactor made spheroids faster and more compact compared to the conventional hanging drop method, which resulted in the upregulation of angiogenic paracrine factors of hMSCs, such as vascular endothelial growth factor and angiopoietin 2. Injected hMSCs spheroids cultured in the anti-gravity bioreactor exhibited improved therapeutic efficacy, including the degree of limb salvage, capillary formation, and attenuation of fibrosis and inflammation, for mouse hindlimb ischemia model compared to spheroids formed by the conventional hanging drop method.ConclusionOur stem cell culture system using acoustic levitation will be proposed as a new platform for the future 3D cell culture system.
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