Studies On Release Of Estradiol And Progesterone From Microencapsulated Ovarian Cells

Women would suffer from menopausal syndrome caused by ovarian function decline and reduced secretion of estradiol (E2) and progesterone (P4). Hormone replacement therapy (HRT) is a common clinical treatment for estrogen deficiency. However, the frequent HRT causes heavy financial burdens. Moreover, long-term HRT could result in serious side effects. This study focused on approaches of ovarian cell microencapsulation and their in vivo release of E2and P4via allotransplantation.The present study included the following four aspects:(1) The preparation of alginate-chitosan-alginate (ACA) microcapsules. An electrostatic droplet generator was employed for the preparation of Ca-alginate gel beads. The effects of needle inner diameter, electrode distance, electrostatic potential and flow rate on diameters of the Ca-alginate gel beads were investigated; The ACA microcapsules were prepared with different chitosan concentrations and crosslinking time, and their swelling behavior values (Sw) were measured as an index of mechanical strength; The ACA microcapsule permeabilities for bovine serum albumin (BSA) and y-globulin were investigated.It was showed that a small needle inner diameter, short electrode distance, high potential and low flow rate were important for preparing small-diameter Ca-alginate beads. Sw values of the ACA microcapsules decreased with the crosslinking time till10min and didn’t exhibit significant changes after that time. Among the three groups of ACA microcapsules prepared with1.2%,0.6%and0.3%(w/v) chitosan respectively, the Sw value and BSA permeation of1.2%group were the lowest while there was no significant difference between the other two groups. All the microcapsules effectively prevented the permeation of y-globulin.(2) The feasibility of Xenopus laevis as an animal model for ovarian cell microencapsulation and transplantation. The primary cultured ovarian cells of Xenopus laevis were characterized by immunohistochemistry staining with anti-FSHR and anti-LHR antibodies. The cells were cultured with different doses of pregnant mare serum gonadotrophin (PMSG) for48hours, and E2and P4concentrations in the culture media were measured by enzyme linked immunosorbent assay (ELISA). The ovarian cells were enclosed in ACA microcapsules prepared with0.3%,0.6%and1.2%(w/v) chitosan solutions respectively and corresponding cell survival percents were measured by CFDA-SE/PI treatment for28days. The ovarian cell microcapsules prepared with0.3%(w/v) chitosan solution were cultured in vitro and intraperitoneally transplanted to ovariectomized female Xenopus laevis; E2and P4levels in culture media and rat serum were measured by ELISA for60days.It was showed that the primary cultured ovarian cells were positively stained with anti-FSHR and anti-LHR antibodies; The optimum stimulating dose of PMSG for E2and P4secretion was20IU/mL; The ACA microcapsules prepared with0.3%(w/v) chitosan solution were most suitable for cell survival. The ACA microencapsulated cells kept secreting E2and P4in vitro for60days; After transplantation, serum E2and P4levels in ovariectomized Xenopus laevis remained elevated for60days;90%of the retrieved ACA microcapsules maintained intact and smooth.(3) Co-microencapsulation of ovarian granulosa cells (GCs) and theca cells (TCs). Primary cultured rat GCs and TCs were characterized by immunohistochemistry staining with anti-FSHR, anti-LHR and anti-CYP17A1antibodies respectively; Different cell number ratios of GCs and TCs were mixed, co-microencapsulated in ACA microcapsules and cultured for48h in vitro, followed by E2and P4levels measuring; Serum levels of E2and P4in ovariectomized rats were measured for60days after the rats were transplanted with the GCs/TCs microencapsules (GCs:TCs=1:2).It was showed that E2secretion in vitro was enhanced by co-encapsulation of GCs/TCs, and the ratio for the maximum secretion was1:2; Serum E2and P4levels could be maintained normal for60days by the co-microencapsulated GCs and TCs;90%of the retrieved microcapsules maintained intact and smooth.(4) A microencapsulation mode of ovarian cell simulating native follicle structure. Primary rat GCs were cultured on microcarriers (GCsMs) and four groups of microcapsules enclosing GCs. GCs+TCs. GCsMs and GCsMs+TCs (simulated follicle) were prepared, respectively; The cell viabilities of microencapsulated GCs and GCsMs groups were measured by WST-1assay for the first24h, and E2and P4levels in culture media of all groups of microcapsules were measured after48h culture; The GCsMs+TCs microcapsules were transplanted to ovariectomized rats and the serum levels of E2and P4were measured for60days.It was showed that the microencapsulated GCsMs exhibited enhanced viability and promoted secreting ability of E2and P4compared with the microencapsulated GCs; The microencapsulated GCsMs+TCs had the highest E2secretion in vitro; Transplantation of co-microencapsulated GCs on microcarriers and TCs maintained serum E2and P4at normal levels for60days;90%of the retrieved microcapsules maintained intact and smooth.In summary, ovarian cells were enclosed in ACA microcapsules with the electrostatic droplet method. All the Xenopus laevis ovarian cell microcapsules, rat GCs/TCs microcapsules and simulated follicle could secrete E2and P4in vitro, and they maintained E2and P4release after allotransplantation for60days, providing a new idea for the treatment of menopausal syndrome.