Medium supplementation can influence the human ovarian cells in vitro

Abstract

Background Cells are an essential part of the triple principles of tissue engineering and a crucial component of the engineered ovary as they can induce angiogenesis, synthesize extracellular matrix and influence follicle development. Here, we hypothesize that by changing the medium supplementation, we can obtain different cell populations isolated from the human ovary to use in the engineered ovary. To this end, we have in vitro cultured cells isolated from the menopausal ovarian cortex using different additives: KnockOut serum replacement (KO), fetal bovine serum (FBS), human serum albumin (HSA), and platelet lysate (PL). Results Our results showed that most cells soon after isolation (pre-culture, control) and cells in KO and FBS groups were CD31- CD34- (D0: vs. CD31-CD34+, CD31 + CD34+, and CD31 + CD34- p < 0.0001; KO: vs. CD31-CD34+, CD31 + CD34+, and CD31 + CD34- p < 0.0001; FBS: vs. CD31-CD34+ and CD31 + CD34+ p < 0.001, and vs. CD31 + CD34- p < 0.01). Moreover, a deeper analysis of the CD31-CD34- population demonstrated a significant augmentation (more than 86%) of the CD73+ and CD90+ cells (possibly fibroblasts, mesenchymal stem cells, or pericytes) in KO- and FBS-based media compared to the control (around 16%; p < 0.001). Still, in the CD31-CD34- population, we found a higher proportion (60%) of CD90+ and PDPN+ cells (fibroblast-like cells) compared to the control (around 7%; vs PL and KO p < 0.01 and vs FBS p < 0.001). Additionally, around 70% of cells in KO- and FBS-based media were positive for CD105 and CD146, which may indicate an increase in the number of pericytes in these media compared to a low percentage (4%) in the control group (vs KO and FBS p < 0.001). On the other hand, we remarked a significant decrease of CD31- CD34+ cells after in vitro culture using all different medium additives (HSA vs D0 p < 0.001, PL, KO, and FBS vs D0 P < 0.01). We also observed a significant increase in epithelial cells (CD326+) when the medium was supplemented with KO (vs D0 p < 0.05). Interestingly, HSA and PL showed more lymphatic endothelial cells compared to other groups (CD31 + CD34+: HSA and PL vs KO and FBS p < 0.05; CD31 + CD34 + CD90 + PDPN+: HSA and PL vs D0 p < 0.01). Conclusion Our results demonstrate that medium additives can influence the cell populations, which serve as building blocks for the engineered tissue. Therefore, according to the final application, different media can be used in vitro to favor different cell types, which will be incorporated into a functional matrix.

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment

“…Although a formal assessment of cell viability was not conducted at the conclusion of the experiments, it is noteworthy that cells from all patients maintained a robust and healthy morphology throughout the entire in vitro culture period. While our study did not involve an in-depth characterization of the specific cell populations after isolation or at the end of the study, previous analyses from our group suggest that the majority of cells isolated from post-menopausal ovarian cortex are likely fibroblasts and other mesenchymal cells, with a minimal proportion of endothelial cells ( Dadashzadeh et al , 2022 ). Furthermore, supplementation with fetal bovine serum has been shown to increase the proportion of CD73+ and CD90+ cells, potentially comprising fibroblasts, mesenchymal stem cells or pericytes, while further reducing the numbers of endothelial and endothelial progenitor cells ( Dadashzadeh et al , 2022 ).…”

“…While our study did not involve an in-depth characterization of the specific cell populations after isolation or at the end of the study, previous analyses from our group suggest that the majority of cells isolated from post-menopausal ovarian cortex are likely fibroblasts and other mesenchymal cells, with a minimal proportion of endothelial cells ( Dadashzadeh et al , 2022 ). Furthermore, supplementation with fetal bovine serum has been shown to increase the proportion of CD73+ and CD90+ cells, potentially comprising fibroblasts, mesenchymal stem cells or pericytes, while further reducing the numbers of endothelial and endothelial progenitor cells ( Dadashzadeh et al , 2022 ). These cells secrete a myriad of growth factors known to exert significant influences on primordial follicle activation and/or preantral follicle growth through paracrine signaling.…”

“…Notably, these aging ovaries exhibit a characteristic inflammatory environment, which has been identified as one of the mechanisms contributing to follicle depletion ( Ansere et al , 2021 ; Lliberos et al , 2021 ; Ouni et al , 2022 ), suggesting that they are not conducive to supporting folliculogenesis. However, cells isolated from post-menopausal ovaries have demonstrated remarkable plasticity in vitro , being able to survive, proliferate, differentiate, secrete hormones, and synthesize extracellular matrix ( Asiabi et al , 2020 ; Maruhashi et al , 2020 ; Dadashzadeh et al , 2022 ). In our study, we demonstrated that a high number of these cells can be successfully isolated from frozen and thawed ovarian tissue, and their survival and proliferative capacity are evident through the formation of a confluent cell layer within a few days of in vitro culture.…”

Liposomal oxygen-generating hydrogel for enhancing cell survival under hypoxia condition

“…After procurement, ovaries were immediately frozen [30]. For ovarian cell isolation, the ovarian samples were thawed and digested using our routine protocols [31][32][33]. Briefly, the tissue fragments were mechanically minced and then digested in DPBS, 0.28 Wünsch units/mL Liberase DH, and 8…”

“…In a previous study, we conducted fluorescence-activated cell sorting on these isolated ovarian stromal cells, revealing that those supplemented with FBS exhibited the expression profile CD31-, CD34-, CD73+, and CD90+. Consequently, these cells can be classified into three distinct categories: fibroblasts, mesenchymal stem cells, and pericytes [32]. The cytotoxicity of free H2O2 and H2O2@Lip on the stromal cells was evaluated after 20 h treatment by different H2O2 concentrations (0.39, 1.56, 6.25, 25, 50 mM).…”