Three-dimensional aggregate-suspension culture is a potential biomanufacturing method to produce a large number of human induced pluripotent stem cells (hiPSCs); however, the use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency, respectively. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device improved cell expansion up to approximately ~3.2 to 4×107 cells/mL. The high-density expansion was supported by reduction of excessive shear stress and agglomeration mediated by the addition of the functional polymer FP003. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous Nodal, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, the cost reduced in inverse proportional manner with the cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility of cost-effective mass production of hiPSCs in high-density culture.
Scalable production of avian cell lines exhibits a valuable potential on therapeutic application by producing recombinant proteins and as the substrate for virus growth due to the special glycosylation occurs in avian species. Chicken primordial germ cells (cPGCs), a germinal pluripotent avian cell type, present the ability of self-renewal, an anchorage-independent cell growth and the ability to be genetically modified. This cell type could be an interesting bioreactor system for industrial purposes. This study sought to establish an expandable culture system with defined components for three-dimensional (3D) culture of cPGCs. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium but cPGCs did not sediment in culture and efficiencies of space and nutrient utilization were thus enhanced and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1 week of culture in 3D-FAot medium, an aseric defined medium containing FP003 polymer, FGF2 and Activin A as growth factors and Ovotransferrin as protein. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. In summary, this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring, which is available for long-term culture and no loss of cellular properties was found. This system provides a platform for large-scale culture of cPGCs.
1 Technical Efficacy: Stage 3 J. MAGN. RESON. IMAGING 2018;48:389-397.
Three-dimensional aggregate-suspension culture can produce large numbers of human induced pluripotent stem cells (hiPSCs); however, use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device allowed growth-factor accumulation and improved cell expansion up to ~ 32 × 106 cells/mL, and reduction of excessive shear stress and agglomeration following addition of the functional polymer FP003 supported high-density expansion. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous NODAL, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, cost reduction increased significantly in proportional manner with cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility and cost-effective mass production of hiPSCs in high-density culture.
Background Diabetes mellitus weakens bone strength due to deterioration of bone quality; however, the histological mechanisms are still unknown. We hypothesized that histological assessment of cortical bone would enable us to determine the cause of the bone strength reduction associated with diabetes mellitus. Our aim was to evaluate the histomorphometric changes of cortical bone associated with deterioration of intrinsic bone properties and bone quality in diabetes mellitus. Methods We compared the outcomes of mechanical tests, bone mineral density measured using micro-computed tomography, and histological assessments, by applying Villanueva’s bone stain, to the tibial bones of 40-week-old diabetic and control male rats. Results With respect to mechanical testing, the maximum load and energy absorption were significantly lower in the diabetic than in the control group, although fracture displacement and stiffness were not significantly different between the two groups. Bone mineral density was significantly higher in the diabetic group than in the control group. Bone histomorphometry revealed that the diabetic rats had fewer osteocytes, greater cortical porosity, and increased mineralization in cortical bone compared with the control group. Conclusions Increased mineralization of the cortical bone with greater cortical porosity leads to a weakening of bone strength in diabetes mellitus.
Case: We describe a case of cryptococcal osteomyelitis in a 70-year-old man who presented with left elbow and shoulder pain. Subsequent imaging indicated osteomyelitis of the entire humerus, and he underwent debridement of the elbow and shoulder. Pathological findings revealed Cryptococcus neoformans infection. Surgical management was followed by a prolonged course of antibiotics. Conclusion: Although C. neoformans is rare as the etiology of infection of the entire humerus, orthopaedic surgeons should consider cryptococcosis as a potential cause of infection.
Paralabral cysts in the shoulder are a relatively rare pathology. It is sometimes difficult to detect the location of a paralabral cyst in the shoulder using arthroscopy, and it can be difficult to confirm sufficient decompression by arthroscopy. We describe the case of a 64-year-old woman who underwent arthroscopic decompression for a paralabral cyst in the shoulder. Indigo carmine was injected into the cyst under ultrasonography guidance just before the operation. The leakage point of indigo carmine was detected using arthroscopy. Arthroscopic decompression was performed until the indigo carmine was completely discharged. Her shoulder pain, limited range of motion, and muscle weakness during abduction and external rotation improved postoperatively. Magnetic resonance imaging confirmed the disappearance of the cyst. Arthroscopic decompression using an ultrasonography-guided injection of indigo carmine is a useful treatment for a paralabral cyst in the shoulder.
Background: Diabetes mellitus weakens bone strength due to deterioration of bone quality; however, the histological mechanisms are still unknown. We hypothesized that histological assessment of cortical bone would enable us to determine the cause of the bone strength reduction associated with diabetes mellitus. Our aim was to evaluate the histomorphometric changes of cortical bone associated with deterioration of intrinsic bone properties and bone quality in diabetes mellitus. Methods: We compared the outcomes of mechanical tests, bone mineral density measured using micro computed tomography, and histological assessments, by applying Villanueva’s bone stain, to the tibial bones of 40-week-old diabetic and control male rats. Results: With respect to mechanical testing, the maximum load and energy absorption were significantly lower in the diabetic than in the control group, although fracture displacement and stiffness were not significantly different between the two groups. Bone mineral density was significantly higher in the diabetic group than in the control group. Bone histomorphometry revealed that the diabetic rats had fewer osteocytes, greater cortical porosity, and increased mineralization in cortical bone compared with the control group. Conclusions: Increased mineralization of the cortical bone with greater cortical porosity leads to a weakening of bone strength in diabetes mellitus.
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