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Study on Coencapsulation of Bone Marrow Mesenchymal Stem Cells and Somatic Cells

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Tutor: WangShiBin
School: Huaqiao University
Course: Biochemical Engineering
Keywords: Coencapsulation,BMSCs,Liver cells,Isletβcells,RT-PCR
CLC: R329
Type: Master's thesis
Year:  2011
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Abstract:
Bone marrow mesenchymal stem cells (BMSCs) are widely researched as seed cells for cell transplantation, since they have rich sources, can be more easily purified and possess the potential to differentiate, therefore can be used for autologous transplantation. Microcapsules can provide a three-dimensional growth environment with a good immunoisolation and biocompatibility for stem cells. Coencapsulation provides a new technical support for stem cells since the cytokines secreted by other cells would be used to promote stem cell differentiation and increase cell signaling, consequently opening up a new alternative for cell transplantation. Alginate-chitosan-alginate (ACA) microcapsules containing BMSCs were prepared, the physico-chemical, mechanical properties and biocompatibility were investigated. Coencapsulated BMSCs/human liver cells (Chang liver) were cultured in vitro, the cell proliferation and metabolic properties were studied. Coencapsulated BMSCs/isletβcells (Beta-TC-6) for the treatment of TypeⅠdiabetic and their mechanism at the molecular level were discussed.Firstly, BMSCs were cultured in vitro and the cell growth curve was made. The surface antigens CD29, CD44 and CD117 of BMSCs were identified by immunohistochemical staining, the results showed that the CD29, CD44 and CD117 were positive, positive and negative, respectively. The in vitro experimental study on the osteogenic differentiation of BMSCs indicated that it was in the undifferentiated state when they were cultured in vitro.Secondly, the ACA microcapsules containing BMSCs were prepared by means of a high-voltage electrostatic droplet generator, the two-step method was selected as an optimized process for microencapsulating cells and the optimum parameters were determined as follows: sodium alginate 1.75%, film forming time 10~ min and cell density 1×10~6 cells/mL. The resulting microcapsules have a mean size of 300~400μm with a good spherical shape, a smooth surface, a good mechanical strength and a satisfactory cell viability, and their morphologies have no significant change in 7 days. BMSCs also grew well after they were re-cultured from broken microcapsules.Thirdly, the cytotoxicity of microencapsulated BMSCs in vitro was investigated by a CCK-8 assay. It was found that compared with the cells by plate culture and non-liquefied microencapsulated cells, the environment of microcapsule with a liquid core was more suitable for long-term growth of cells. The cell density reached a peak of 4.3×10~7 cells/mL after cultivation for 15 days. The biocompatibility in vivo of microencapsulated BMSCs was investigated through implanting the microcapsules in mice by intraperitoneal injection, the normal physiological activity was observed, and the mice remained a stable weight without toxic symptoms. After implantation for 28 days, the recovered microcapsules exhibited a good spherical shape and a smooth surface without fibrosis or macrophage aggregation.Fourthly, Chang liver cells were cultured by the ways of plate culture, microencapsulated, co-culture and coencapsulated, respectively. The metabolic properties of liver cells were examined by detecting the secretion of albumin and urea in the medium. The results revealed that the relationship of the secretion of albumin and urea among four experimental groups was coencapsulated BMSCs/Chang liver group> co-culture BMSCs/Chang liver group>microencapsulated Chang liver group>Chang liver cell group. The albumin secretion of coencapsulated BMSCs/Chang liver group reached a highest value of 3.08 mg/mL in 15 days and the secretion of urea reached a highest value of 3.75 mg/mL in 11 days.Finally, the comparative study on microcapsules containing Beta-TC-6 cells and BMSCs/Beta-TC-6 cells were carried out by culturing in vitro for 28 days, and the secretion of insulin were determined. At the 28th days, the insulin secretion of coencapsulated BMSCs/Beta-TC-6 cells was 98.60 pg/mL. The two groups of microencapsulated cells were implanted into strptozotocin-induced typeⅠdiabetic mice. The results demonstrated that insulin secretion of isletβ cells in coencapsulation group was much higher than that in the encapsulated isletβcells group. The corresponding efficiency for lowering blood glucose of diabetic mice was obtained, namely, after implantation for 28 days, the mean blood glucose value of coencapsulation group (5.08 mmol/L) was much lower than that of encapsulated isletβcells group (6.86 mmol/L), which were both obviously lower blood glucose of diabetic mice, and this also supported the results of insulin secretion measurement. The insulin gene mRNA expression was identified by RT-PCR, the results showed that BMSCs could be induced to differentiate into insulin-secreting cells to some extent while they were co-cultured with isletβcells.This study demonstrated that the microcapsules could provide a microenvironment for bone marrow mesenchymal stem cells to differentiate into somatic cells. Coencapsulating BMSCs and somatic cells has a potential application in the treatment of disease.
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