Tissue Regeneration of Human Mesenchymal Stem Cells on Porous Gelatin Micro-Carriers by Long-Term Dynamic In Vitro Culture

BACKGROUND: Tissue engineering is a multidisciplinary field which attracted much attention in recent years. One of the most important issue in tissue engineering is how to obtain high cell numbers and tissue regeneration while maintaining appropriate cellular characteristics in vitro for restoring damaged or dysfunctional body tissues and organs. These demands can be achieved by the use of three dimensional (3D) dynamic cultures of cells combined with cell-adhesive micro-carriers. METHODS: In this study, human mesenchymal stem cells (hMSCs) were cultured in a wave-bioreactor system for up to 100 days, after seeding on Cultisphere-S porous gelatin micro-carriers. Cell counting was performed at the time points of 7, 12, 17, 31 days and compared to those of hMSCs cultured under static condition. Higher growth and proliferation rates was achieved in wave-type dynamic culture, when cell culture continued to day 31. A scanning electron microscope (SEM) photographs, both live and dead and MTT assays were taken to confirm the survival and distribution of cells on porous gelatin micro-carrier surfaces. The results of histological stains such as hematoxylin and eosin, Masson’s trichrome, Alcian blue and Alizarin red S also showed improved proliferation and tissue regeneration of hMSCs on porous gelatin micro-carriers. CONCLUSION: The experimental results demonstrated the effect and importance of both micro-carriers and bioreactor in hMSC expansion on cell proliferation and migration as well as extracellular matrix formation on the superficial and pore surfaces of the porous gelatin micro-carriers, and then their inter-connections, leading to tissue regeneration.

Stress Analysis on Elastic Substrate of in vitro Endothelial Cell Culture Device / 医用生物力学

Objective To study the stress on elastic substrate of an in vitro endothelial cell dynamic culture device, whose hemodynamic environment is designed to simulate the human body, and to test and observe the shear stress changes in elastic substrate of parallel plate flow chamber under different tensile stresses. Methods A series of static tensile tests were adopted to fit the condition of dynamic stretching. Namely, the silicone sheet with 2 different thicknesses were put into the device, and then applied with static stretch at the interval of 10% tensile rate (0%, 10%, 20%, 30%), and under the condition of maintaining its tensile rate, the chamber height after the stretch of silicon sheet was calculated. Based on the calculation of shear stress, shear stress curves at different tensile rates were obtained, to make comparative analysis on variation of the shear stress with the thickness of silicon sheet. Results The experimental result was consistent with the theoretical analysis. When the tensile rate was 30%, silicon sheet with 0.5 mm thickness would produce certain influence on shear stress of parallel plate flow chamber along with the change of tensile rate (the height of chamber), and the average and maximum shear stress were reduced by 10.1% and 10.4%, respectively. Conclusions The influence factors caused by the change of elastic substrate thickness after the introduction of tensile stress must be taken into account for the calculation of shear stress in parallel plate flow chamber. The result can provide experimental technology for the culture of endothelial cells in vitro and the design and development of novel parallel plate flow chamber.

Study for the Ureteral Reconstruction with Tissue Engineering Using Poly (glycolide/epsilon-caprolactone) Scaffold-1 / 대한비뇨기과학회지

PURPOSE: Transplantation is one modality saving human life. But not only lack of the living or cadaveric human organs but also immunologic problems or some ethical situations limit transplantation in terminal stage patients. Recent research for escaping from those problems resulted in the reconstruction of the artificial organ using patients' own cells with tissue engineering. The goal of this study is, for the better reconstruction of urinary system using tissue engineering, to perform basic researches on techniques related with seeding and viability of cells. MATERIALS AND METHODS: We used 16 adult dogs, 4 female and 12 male, for primary culture of the dog bladder mucosal cell and muscle cell. The scaffold we used was made of absorbable substance polyglycolide/epsilon-caprolactone (GL/CL) in thin sponge like shape. Fibroblast 3T3 cell was used for control and 16 primary cultured mucosal cell and smooth muscle cells were used. For dynamic culture, rocker was adapted with for 5 hours. Attached cells were evaluated by 562nm ELISA reader using BCA method and scanning electron microscope. RESULTS: Successful primary culture was achieved with cells from dog bladder, and results were much better by using male dog. The dynamic culture increased attachment of the cell in scaffold and the cell attached at deeper portion of the scaffold. Long term culture showed formation of the cellular sheets on the surface of scaffold preventing inner passage of the suggesting disadvantageous condition for cells in core of the scaffold. CONCLUSIONS: We conclude that for the better attachment of the cultured cells on scaffolds, dynamic culture would be desirable. And for the better in vivo reconstruction of the organ with primary cultured cell attached scaffold, evaluation of culture state with repeated in vitro experiments are necessary.