A Shaking-Culture Method for Generating Bone Marrow Derived Mesenchymal Stromal/Stem Cell-Spheroids With Enhanced Multipotency in vitro

Abstract

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 chondrogenicdifferentiation 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.

Novel Mesenchymal Stem Cell Spheroids with Enhanced Stem Cell Characteristics and Bone Regeneration Ability

“…We previously established a novel 3D shaking culture method that successfully maintained LNGFR + /THY-1 + human MSC spheroids with high viability and multipotency for up to 1 month. 25 The spheroids maintained their 3D rounded shape after attaching to a plastic plate and provided undifferentiated MSCs continuously in vitro. Despite the great potential of MSC spheroids generated by this method, several MSC- or neural crest stem cell (NCSC)-related genes, such as NESTIN ( NES ) and SOX2 , showed deficient expression.…”

“…Despite the great potential of MSC spheroids generated by this method, several MSC- or neural crest stem cell (NCSC)-related genes, such as NESTIN ( NES ) and SOX2 , showed deficient expression. 25 Thus, in this study, we attempted to optimize the culture conditions of our shaking method for MSCs, taking a cue from neurosphere culture.…”

Injection Laryngoplasty of Human Adipose-derived Stem Cell Spheroids With Hyaluronic Acid-based Hydrogel Improves the Morphological and Functional Characteristics of Geriatric Larynx

“…Our previous studies showed that MSCs are potentially bene cial in tracheal regeneration and oral mucosal ulcer healing [20][21][22]. While the use of MSCs has also been studied for vocal-fold atrophy [23], spheroids of MSCs and mesenchymal stroma have not been studied despite their enhanced multipotency in vitro [24]. In the present study, we injected hASC spheroid-loaded hyaluronic-acid (HA)-based hydrogel into the geriatric larynx to evaluate its effects on therapeutic rejuvenation.…”

Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells

“… 7 In contrast, specific hydrodynamic forces in 3D shaking culture maintain and restore multipotency of mesenchymal stem cell (MSC) spheroids. 8 Low-intensity vibration attenuates adipogenic differentiation in MSCs. 9 Thus, the effects of mechanical force on stem cell responses substantially depend on many factors related to both force and cells, including force type, force magnitude, treatment duration, cell type, and cell stage.…”