Medium supplementation can influence the human ovarian cells in vitro

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).

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).

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.