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.

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Study question Do ovarian stromal cells (OSCs) influence viability and growth of human pre-antral follicles in vitro? Summary answer Feeder layer of OSCs advanced growth and transition of primordial follicles to primary/secondary stage while keeping a high proportion of viable follicles. What is known already In the ovary, follicles require the support of ovarian cells through the secretion of essential factors for their survival and development. This was also demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of ovarian stromal cells. This co-culture significantly increased follicle survival and growth. Study design, size, duration Pre-antral follicles were isolated from human frozen-thawed ovarian tissue (OT) biopsies and encapsulated in 1% alginate scaffolds. Embedded pre-antral follicles were placed directly on the OSCs feeder layer or bottom of a culture dish for a 7-day in vitro culture (control). Follicle viability and growth and hormone production were compared between groups. Participants/materials, setting, methods Primordial and primary follicles were isolated from frozen-thawed OT of cancer patients (n = 6). OSCs were isolated from OT of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 under an inverted microscope. Viability was assessed by staining a proportion of follicles (n = 87) with calcein AM and ethidium homodimer-I. They were classified (V1/V2: healthy/minimally damaged; V3/V4: damaged/dead follicles) using confocal fluorescence microscopy. Estradiol levels were measured by ELISA. Main results and the role of chance A total of 379 human pre-antral follicles (primordial=360; primary=19) were isolated and embedded in 1% alginate hydrogels and classified according to their viability. Most follicles (96%) were viable after isolation with a diameter of 40.8±9.9 µm (mean±SD). At Day 7, pre-antral follicles have grown significantly in size in both culture conditions: on the OSCs feeder layer and without it (p &lt; 0.0001 for D0 vs. D7). However, no significant difference (p = 0.07) between culture conditions was observed. The mean diameter of follicles cultured on the OSCs layer was 80.6±11.0 μm and without it, 67.3±7.2 μm. Nonetheless, the distribution pattern of different quality grade follicles was significantly different on OSCs (V1: 62%, V2: 25%, V3: 2%, V4: 11%) and follicles without OSCs (V1: 35%, V2: 38%, V3: 0%, V4: 23%; p = 0.03). Additionally, more follicles have been activated and reached a higher developmental stage on OSCs (D0 primordial: 184, primary: 7 vs. D7 primordial: 53, primary/secondary: 93) than without stromal cells (D0 primordial: 186, primary: 4 vs. D7 primordial: 84, primary/secondary: 64; p &lt; 0.001) with 66 and 43 follicles reaching a secondary stage (75&lt;x&lt;200 μm), respectively. Estradiol level was significantly (p = 0.006) higher in media of follicles cultured on the OSCs, 54.1±14.2 pg/ml vs. 29.9±4.0 pg/ml. Limitations, reasons for caution This study was performed only in a short-term culture and no primordial/primary follicles have reached the antral stage. Further in vitro studies on follicular developmental capacity, physiology and steroidogenesis in alginate scaffold with human ovarian stromal cells are needed. Wider implications of the findings Activation and growth of human primordial follicles during in vitro short-term culture to a secondary stage has been a long-lasting challenge. Co-culture with human ovarian stromal cells can help to overcome this limitation. Trial registration number not applicable