| Background：Stemness properties of mesenchymal stem cells (MSCs), which arecapable of self-renewal and multi-lineage differentiation, endow them with great potentialfor tissue engineering and regenerative medicine. Adipose-derived stem cells (ADSCs)represent an abundant source of MSCs that are easily accessible from subcutaneous adiposetissue via liposuction. MSCs comprise only a small proportion of the bone marrow oradipose tissue, and they must be extensively expanded in culture to achieve the numbersrequired for any therapeutic strategy. During in vitro expansion of common monolayercultures, however, MSCs quickly lose their primitive stemness properties, such asreplicative ability, colony-forming efficiency, and differentiation capacity. Therefore,maintaining stemness properties has become a crucial issue for future clinical applicationsof in vitro-cultured ADSCs.The stemness properties of MSCs are retained in the in vivo microenvironment, whichcomprises soluble growth factors, cell-cell interactions and cell-matrix interactions.Accumulating evidences have suggested that the cellular microenvironment plays animportant role in determining the stemness properties of cells. Compared with conventionalmonolayer cultures, three-dimensional (3D) culture methods offer a cellular niche that ismore similar to the in vivo microenvironment. Several3D cell culture methods, includingthe use of hydrogels, porous scaffolds or polymers, as well as cellular aggregates, havebeen developed to maintain the stemness properties of stem cells. Among these methods,spheroid culture is a typical scaffold-free3D cell culture system that takes advantage of thenatural self-assembly tendency of numerous cell types. Although previous studies haveindicated that spheroid formation of ADSCs on several biomaterials or using hanging dropretain their stemness properties, these methods of spheroid culture do not fully meet therequirements of large-scale expansion for clinical application. Previous studies havedemonstrated that the MB culture system exhibits unique advantages in spheroid formation and large-scale production of stem cells. However, it is unclear whether the stemnessproperties of MSCs can be maintained or improved by the spheroid culture of MBs.Methods：1. We used a non-enzymatic method of washing adipose tissue from lipoaspirate toisolate and characterize ADSCs.2. we employed a microgravity bioreactor (MB) to develop a straightforward andeffective method of biomaterial-free spheroid formation to improve the stemness propertiesof ADSCs for clinical application. The stemness properties of spheroid-derived ADSCs,including proliferative ability, colony-forming efficiency, and multipotency differentiationcapacity, were investigated.Results：1. ADSCs can be successfully isolated using a non-enzymatic method, withoutsubstantial differences in the cellular morphology, reproductive activity,immunophenotype and differentiation potential.2. Human ADSCs spontaneously formed three-dimensional spheroids in the MB.Most ADSCs within the spheroids were viable.3. Compared with conventional monolayer culture, the expression levels ofpluripotency markers including Oct4, Nanog, Sox-2and Rex-1were significantlyupregulated in ADSC spheroids.4. Spheroid-derived ADSCs exhibited higher levels of proliferative ability,colony-forming efficiency, and multipotency differentiation capacity. Our results suggestthat Spheroid formation of ADSCs in a MB enhances their stemness properties.5. Higher level of E-cadherin expression in ADSC spheroids was found comparedwith monolayer ADSCs, which may be contributed to enhancing their stemness propertiesby spheroid formation of ADSCs in the MB.Conclusions: Spheroid culture in a MB can be an efficient method to maintainstemness properties, without involvement of any biomaterials for clinical applications of invitro cultured ADSCs. |
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