Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates

Redondo‐Castro, Elena; Cunningham, Catriona; Miller, Jonjo; Cain, Stuart A.; Allan, Stuart M.; Pinteaux, Emmanuel

doi:10.21769/bioprotoc.2968

Cited by 22 publications

(16 citation statements)

References 19 publications

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“…The SF H4236 methylcellulose at a concentration of 0.5% was adopted because it generated only one spheroid in most of the wells with lower condensation aspect for primary hMSCs (Fig 1B). Under these culture conditions, MSCs were able to form spheroids rapidly, in as little as five hours of culture (S1 Video), which is consistent with previous studies [27,32,37].…”

Section: Establishment Of Primary Hmsc-spheroids By Cell Aggregation supporting

confidence: 90%

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

et al. 2020

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Mesenchymal stem cells (MSC)-spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSC-spheroids began to shrink after 24 hours, the size of spheroids from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, like hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.

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Section: Establishment Of Primary Hmsc-spheroids By Cell Aggregation supporting

confidence: 90%

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

et al. 2020

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“…However, by inducing cell-to-cell interactions non-adherent plates undermine the important role played by the basement membrane of the natural stem cell niche where the important role of proteins such as the laminin in controlling integrin-mediated adhesion and gene expression has clearly been demonstrated [27][28][29]. Uniquely, the different formulation of the substrates used in this work enable to evaluate the role of the integrins against that played by a substrate with the same nanotopography but different adhesion motifs thus offering a clear advantage over previous work where comparisons have been made with non-adherent substrates and mechanical drive [5,21]. Instead, the present work weighs the contribution of biospecific interactions between ligands of the extracellular matrix proteins and cell receptors against that of nanotopography play showing that, while both are important in inducing the 3D spheroid formation, the stabilisation of these structures is mainly a consequence of the receptormediated binding that can determine their behaviour including gene expression and intracellular pathway activation [6] as well as proliferation and migration [30].…”

Section: Resultsmentioning

confidence: 98%

“…It is known that, in their natural niche, stem cells divide asymmetrically leading to the formation of 3D clusters and ensuring, at the same time a reservoir of undifferentiated cells and a progeny of committed cells [19]. This has prompted a worldwide effort to develop in vitro models favouring the formation of 3D spheroids either forcing the cells towards cell-to-cell interactions in non-adherent plates or through the development of biomaterial substrates [5,[20][21][22][23][24] demonstrating that in such conditions the stem cells can uniquely maintain features otherwise lost during their culturing [4,25,26]. However, by inducing cell-to-cell interactions non-adherent plates undermine the important role played by the basement membrane of the natural stem cell niche where the important role of proteins such as the laminin in controlling integrin-mediated adhesion and gene expression has clearly been demonstrated [27][28][29].…”

Section: Resultsmentioning

confidence: 99%

“…To date, the induction of 3D spheroidal structures has been pursued either by the use of proteins found in the niche (e.g. laminin) or by non-adherent substrates and microdroplet setting facilitating cell-to-cell interactions rather than cell-substrate adhesion or by substrate topography forcing cell proliferation into the 3D volume rather than on a 2D plane [4,5].…”

Section: Introductionmentioning

confidence: 99%

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A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation

Perugini

Santin

2020

J Mater Sci: Mater Med

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The in vitro study of the properties of the human mesenchymal stem cells as well as their manipulation in culture for clinical purposes depends on the elimination of artefacts caused by the lack of their natural environment. It is now widely accepted that mesenchymal stem cells should be studied when they are organised as 3D spheroids rather than fibroblast-like colonies. Although this can be achieved with the use of some extracellular matrix proteins or by non-adherent conditions these suffer of significant limitations. The recent development of synthetic substrates resembling the physicochemical and biochemical properties of the adult stem cell niche has prompted questions about the role played by nanotopography and receptormediated adhesion. In the present paper, the influence of two types of substrates bearing the same nanostructure, but exposing either a non-specific or an integrin-specific binding motif was studied. Carboxybetaine-tethered hyperbranched poly(ɛ-lysine) dendrons showed that the hyperbranched structure was fundamental to induce spheroid formation, but these were forming more slowly, were of reduced size and less stable than those growing on substrates based on the same hyperbranched structures that had been functionalised at their uppermost branching generation by a laminin amino acid sequence, i.e. YIGSR. The study shows that both nanostructure and biorecognition need to be combined to achieve a substrate for stem cell spheroid formation as that observed in vivo in the adult stem cell niche. Graphical Abstract* Matteo Santin

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“…Due to the lack of direct contact with solid supporting interfaces, the cell-environment interaction only exists within the interior of cell spheroids. A few other similar approaches, such as pellet culture and low-attachment culture also demonstrate the spontaneous secretion of ECM proteins from the spheroid-forming cells [23][24][25], indicating that the composition of ECM proteins is the crucial environmental factor for the regulation of cell properties in these types of 3D culture systems.…”

Section: Cell-materials Interface and Interaction In Scaffold-free Stementioning

confidence: 93%

Cellular Spheroids of Mesenchymal Stem Cells and Their Perspectives in Future Healthcare

2019

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Intrinsic cellular properties of several types of cells are dramatically altered as the culture condition shifts from two-dimensional (2D) to three-dimensional (3D) environment. Currently, several lines of evidence have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) in regenerative medicine. MSCs not only replenish the lost cells, they also promote the regeneration of impaired tissues by modulating the immune responses. Following the development of 3D cell culture, the enhanced therapeutic efficacy of spheroid-forming MSCs have been identified in several animal disease models by promoting differentiation or trophic factor secretion, as compared to planar-cultured MSCs. Due to the complicated and multifunctional applications in the medical field, MSCs are recently named as medicinal signaling cells. In this review, we summarize the predominant differences of cell-environment interactions for the MSC spheroids formed by chitosan-based substrates and other scaffold-free approaches. Furthermore, several important physical and chemical factors affecting cell behaviors in the cell spheroids are discussed. Currently, the understanding of MSCs spheroid interactions is continuously expanding. Overall, this article aims to review the broad advantages and perspectives of MSC spheroids in regenerative medicine and in future healthcare. Three-Dimensional (3D) Cell Culture SystemsCell-cell and cell-environment interactions cooperatively determine the physiological phenomena in vivo. Many cell growth mechanisms and signal transduction pathways occurring in cells have been unveiled by the traditional two-dimensional (2D) culture system. Plastic-based culture materials remain the predominant cell culture platforms to date, yet some concerns have been raised with the accumulating experimental evidence.A major drawback of cell culture on 2D plastic plates is the planar cell-cell interaction that has the insufficient and inappropriate cell-environment crosstalk. In vivo, cells are grown and stacked within a three-dimensional (3D) space, and extracellular matrix (ECM) secreted by cells constitutes the supporting framework to further organize tissues and organs. Therefore, some important cell-cell and cell-environment interactions may be ignored in a planar culture platform. Primary cells, cancer cells, and stem cells have been cultured in a 3D environment, and many interesting findings have been uncovered. In general, the definition of 3D cell culture systems is that cells are cultured on/in the 3D scaffolds composed by natural or synthetic materials, or they are organized into cellular spheroids. With the continuing development of 3D cell culture, it is now generally agreed that the cell physiology and cell behavior are dramatically distinct for cells cultured within a 2D or 3D environment [1][2][3].Cancer cells are the most common cell types that have been investigated by 3D culture systems, and obvious change of gene expression as well as intracellular signaling in these 3D-cultured cancer cells have been ...

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Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates

Cited by 22 publications

References 19 publications

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation

Cellular Spheroids of Mesenchymal Stem Cells and Their Perspectives in Future Healthcare

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