Measurement of oxygen tension within mesenchymal stem cell spheroids

Murphy, Kaitlin C.; Hung, Ben P.; Browne-Bourne, Stephen; Zhou, Daohong; Yeung, Jenny; Genetos, Damian C.; Leach, J. Kent

doi:10.1098/rsif.2016.0851

Cited by 116 publications

(133 citation statements)

References 58 publications

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“…However, in the last phase with massive cell death there was a rebounding of oxygenation ( Figure 5B), due to both decreased total cell consumption and to spheroid shrinkage (given that the size of dying cells was set to decrease with 50%). This was observed in the notable lack of a concentration gradient at the center of the domain with further development of the necrotic core, which measured consistently with in vitro work that demonstrate the interactions of diffusive nutrients and cell viability in spheroidal aggregates [34,35].…”

Section: Coupling Oxygenation and Cell Deathsupporting

confidence: 79%

A heuristic computational model of basic cellular processes and oxygenation during spheroid-dependent biofabrication

et al. 2017

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An emerging approach in biofabrication is the creation of 3D tissue constructs through scaffold-free, cell spheroid-only methods. The basic mechanism in this technology is spheroid fusion, which is driven by the minimization of energy, the same biophysical mechanism that governs spheroid formation. However, other factors such as oxygen and metabolite accessibility within spheroids impact on spheroid properties and their ability to form larger-scale structures. The goal of our work is to develop a simulation platform eventually capable of predicting the conditions that minimize metabolism-related cell loss within spheroids. To describe the behavior and dynamic properties of the cells in response to their neighbors and to transient nutrient concentration fields, we developed a hybrid discrete-continuous heuristic model, combining a cellular Potts-type approach with field equations applied to a randomly populated spheroid cross-section of prescribed cell-type constituency. This model allows for the description of: (i) cellular adhesiveness and motility; (ii) interactions with concentration fields, including diffusivity and oxygen consumption; and (iii) concentration-dependent, stochastic cell dynamics, driven by metabolite-dependent cell death. Our model readily captured the basic steps of spheroid-based biofabrication (as specifically dedicated to scaffold-free bioprinting), including intra-spheroid cell sorting (both in 2D and 3D implementations), spheroid defect closure, and inter-spheroid fusion. Moreover, we found that when hypoxia occurring at the core of the spheroid was set to trigger cell death, this was amplified upon spheroid fusion, but could be mitigated by external oxygen supplementation. In conclusion, optimization and further development of scaffold-free bioprinting techniques could benefit from our computational model which is able to simultaneously account for both cellular dynamics and metabolism in constructs obtained by scaffold-free biofabrication.

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Section: Coupling Oxygenation and Cell Deathsupporting

confidence: 79%

A heuristic computational model of basic cellular processes and oxygenation during spheroid-dependent biofabrication

et al. 2017

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“…We previously demonstrated that MSC spheroids do not exhibit a hypoxic core due to a nutrient gradient, perhaps due to adaptive reductions in matrix deposition and packing density with increases in spheroid diameter [35]. However, the entrapment of MSC spheroids in hydrogels likely reduces diffusion-mediated transport from that observed with free spheroids, altering the oxygen microenvironment and potentially upregulating hypoxia-adaptive signals even further.…”

Section: Discussionmentioning

confidence: 99%

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

et al. 2017

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Mesenchymal stem cells (MSCs) secrete endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) that promote angiogenesis, modulate the inflammatory microenvironment, and stimulate wound repair, and MSC spheroids secrete more trophic factors than dissociated, individual MSCs. Compared to injection of cells alone, transplantation of MSCs in a biomaterial can enhance their wound healing potential by localizing cells at the defect site and upregulating trophic factor secretion. To capitalize on the therapeutic potential of spheroids, we engineered a fibrin gel delivery vehicle to simultaneously enhance the proangiogenic and anti-inflammatory potential of entrapped human MSC spheroids. We used multifactorial statistical analysis to determine the interaction between four input variables derived from fibrin gel synthesis on four output variables (gel stiffness, degradation rate, and secretion of VEGF and PGE2). Manipulation of the four input variables tuned fibrin gel biophysical properties to promote the simultaneous secretion of VEGF and PGE2 by entrapped MSC spheroids while maintaining overall gel integrity. MSC spheroids in stiffer gels secreted the most VEGF, while PGE2 secretion was highest in more compliant gels. Simultaneous VEGF and PGE2 secretion was greatest using hydrogels with intermediate mechanical properties, as small increases in stiffness increased VEGF secretion while maintaining PGE2 secretion by entrapped spheroids. The fibrin gel formulation predicted to simultaneously increase VEGF and PGE2 secretion stimulated endothelial cell proliferation, enhanced macrophage polarization, and promoted angiogenesis when used to treat a wounded three-dimensional human skin equivalent. These data demonstrate that a statistical approach is an effective strategy to formulate fibrin gel formulations that enhance the wound healing potential of human MSCs.

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“…In contrast to monolayer culture, MSCs aggregated into spheroids retain a supportive extracellular matrix (ECM) and engage in cell-cell contact that better recapitulates a native tissue environment. 10 Compared with monodispersed cells, spheroid formation can further potentiate cell survival 11 and secretion of endogenous growth factors, 12 motivating continued investigation into the mechanism and therapeutic effects of cell-cell communication within a spheroid. This complex network of communication is orchestrated via signals from the microenvironment such as paracrine or autocrine-acting soluble factors 13 and intercellular communication through gap junctions, extracellular vesicles, 14 and tunneling nanotubes (TNTs).…”

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confidence: 99%

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

et al. 2019

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The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers.However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low-and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low-and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies. K E Y W O R D Smesenchymal stem/stromal cells, p16, senescence, spheroids, tunneling nanotubes

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Measurement of oxygen tension within mesenchymal stem cell spheroids

Cited by 116 publications

References 58 publications

A heuristic computational model of basic cellular processes and oxygenation during spheroid-dependent biofabrication

A heuristic computational model of basic cellular processes and oxygenation during spheroid-dependent biofabrication

Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

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