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Updated: Jun 10, 2025
Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells
Published on: September 23, 2022
Intraovarian injection of 3D-MSC-EVs-ECM gel significantly improved rat ovarian function after chemotherapy
Yaping Zhang1,2, Dong Li3, Yi Han1,3
1Department of Pediatrics, Qilu Hospital of Shandong University, No. 107 Wenhua West Road, Jinan, Shandong Province, 250012, China.
Background:
Restoring the function of the ovary is important for chemotherapy-induced ovarian failure (COF) patients. Stem cell and extracellular vesicles (EVs) therapy show promise but need further improvement.
Methods:
Human umbilical cord mesenchymal stem cells (hUC-MSCs) were primarily cultured and further three-dimensional (3D) cultured using an ultra-low attachment surface method. The expression levels of nutritional cytokines and immunomodulatory and stemness-related genes of 3D-cultured hUC-MSCs were analyzed. EVs were isolated by ultracentrifugation and characterized. Ovaries were decellularized with sodium dodecyl sulfate to obtain extracellular matrix (ECM). Lyophilized EVs from three-dimensional (2D) or 3D hUC-MSCs were mixed with ECM to prepare the 2D/3D-MSC-EVs-ECM gels. The therapeutic effect of the MSC-EVs-ECM gel on cyclophosphamide (CTX) -treated rats was analyzed through various tests. RNA sequencing was used to analyze the expression changes of genes before and after treatment.
Results:
After culturing in ultra-low attachment dishes, hUC-MSCs aggregated into spheroids and significantly upregulated the expression levels of immunomodulatory and stemness-related genes. The total EVs yield was also upregulated (5.6-fold) after 3D culture. The cell viability of CTX-treated ovarian granulosa cells (OGCs) was significantly rescued by coculture with the 3D-MSC-EVs-ECM gel. Hormones indicative of ovarian function, AMH, E2, and FSH, were recovered in both the CTX + 2D-MSC-EVs-ECM gel group and the CTX + 3D-MSC-EVs-ECM gel group, while the apoptosis-related protein Bax was significantly downregulated. The 3D-MSC-EVs-ECM gel was more effective than the 2D-MSC-EVs-ECM gel. Significantly differentially expressed genes, such as Hbb-b1, Gpd1, and Sirpa, were detected by RNA sequencing. Hbb-b1 was increased in the ovaries of CTX-treated rats, and this increase was attenuated by injecting the 2D/3D-MSC-EVs-ECM gel. Gpd1 was increased after CTX treatment, and this increase was reversed by the 3D-MSC-EVs-ECM gel. Sirpa was decreased in the ovaries of CTX-treated rats, and this decrease was attenuated by injecting the 3D-MSC-EVs-ECM gel.
Conclusions:
Our study demonstrated that the 3D-MSC-EVs-ECM gel is an efficient strategy for the recovery of ovarian function in CTX-induced ovarian failure.
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