ABSTRACT
BACKGROUND:Stromal vascular fraction digested from adipose tissue is a mixture of a variety of cel s, and clone-like cel components may have the real ability to maintain the stemness of mesenchymal stem cel s OBJECTIVE:To separate, passage and culture colony-forming cel s in the stromal vascular fraction of adipose tissue, and to test its ability to repair the knee cartilage in animal models. METHODS:Stromal vascular fraction isolated from fresh adipose tissue was cultured to col ect clone-like adherent cel s that were subject to digestion and passage until the third generation. A rabbit model of osteoarthritis was established surgical y, and the third generation of colony-forming cel s were transplantated into the articular cavity of model rabbits. Model rabbits with no cel transplantation served as controls. Cartilage regeneration in the knee joint of model rabbits were observed and compared. RESULTS AND CONCLUSION:At 3 days after transplantation of stromal vascular fraction cel s, clone-like cel colonies appeared in adherent cel s;at 5 days, the amount of clone-like cel colonies and surrounding single cel s were both increased. At the passage 3, the clone-like cel colonies were also visible, which were fusiform in shape. Gross observation and MRI examination showed that these colony-forming cel s could promote cartilage repair in the joint cavity, increase secretion of synovial fluid, and help to control inflammation and narrow ulceration area. Experimental findings are beneficial for optimizing the isolation and culture of adipose-derived mesenchymal stem cel s and provide a new way for the clinical treatment of osteoarthritis.
ABSTRACT
BACKGROUND:Fluorescent magnetic nanoparticles with the properties of quantum dots and magnetic particles have good biocompatibility, and can label cels effectively through endocytosis. OBJECTIVE:To validate the feasibility of fluorescent magnetic nanoparticles in labeling human adipose-derived mesenchymal stem cels. METHODS:Healthy human adipose tissue was extracted and adipose-derived mesenchymal stem cels were isolatedin vitro by type I colagenase digestion. Passage 6 cels were incubated with the fluorescent magnetic nanoparticles overnight. Prussian blue staining and laser scanning confocal microscope were used to observe labeled adipose-derived mesenchymal stem celsin vitro after co-culturing with fluorescent magnetic nanoparticles. The tracing effect of labeled adipose-derived mesenchymal stem cels in vivo was detected by fluorescence imaging system. RESULTS AND CONCLUSION:Prussian blue staining showed that the fluorescent magnetic nanoparticles dispersed in the cytoplasm of adipose-derived mesenchymal stem cels in the form of blue particles. Under the laser scanning confocal microscope, the nuclei of adipose-derived mesenchymal stem cels were dyed blue by Hoechest33258, and the cytoplasm was dyed green. The fluorescence imaging results showed that labeled human adipose-derived mesenchymal stem cels had good imaging results. Therefore, as an efficient tracer, the fluorescent magnetic nanoparticles can label human adipose-derived stem cels in vitro and provide a new method for transplantation and transformation of adipose-derived mesenchymal stem cels.
ABSTRACT
BACKGROUND:Large-scale expansion of undifferentiated and multipotential adipose-derived stem cells using serum-free culture system is a difficult issue to be resolved. OBJECTIVE:To establish an in vitro culture system combined with the extracellular matrix in order to investigate the efficiency, effectiveness and security of extracellular matrix on expanding adipose-derived stem cells. METHODS:In vitro isolated adipose-derived stem cells were seeded in traditional two-dimensional plastic plates and extracellular matrix-coated plates supplemented with serum-free medium respectively. After in vitro expansion, total cellnumber, expression of cellsurface markers, cellsenescence degree and multipotent differentiation ability (adipogenic, osteoblastic and chondrogenic differentiation) of adipose-derived stem cells cultured under both conditions were detected and compared. Moreover, the clinical safety of adipose-derived stem cells expanded in extracellular matrix-coated plates was investigated. RESULTS AND CONCLUSION:Total cellnumber of passage 5 adipose-derived stem cells cultured in extracellular matrix-coated plates was 10 times more than that in traditional two-dimensional plastic plates. Flow-cytometric analysis showed that adipose-derived stem cells cultured with extracellular matrix expressed stem cellsurface markers. cellular senescence examination showed that almost al of passage 15 adipose-derived stem cells cultured with extracellular matrix showed no aging, while most passage 5 adipose-derived stem cells cultured by the two-dimensional system aged and lost their proliferation ability. Multidirectional induction of adipose-derived stem cells showed that passage 15 adipose-derived stem cells cultured with extracellular matrix could stil differentiate into adipocytes, osteoblasts and chondrocytes as passage 5 adipose-derived stem cells did, which performed much better than the induced differentiations of passage 5 adipose-derived stem cells cultured by the two-dimensional system. Karyotype analysis and in vivo invasion experiment insured the clinical safety of adipose-derived stem cells expanded with extracellular matrix. Al above results suggest a safe and more efficient expansion system of extracellular matrix for clinical application using the serum-free culture system combined with extracellular matrix.