Extracellular vesicles in nanomedicine and regenerative medicine: A review over the last decade.

Small extracellular vesicles (sEVs) are known to be secreted by a vast majority of cells. These sEVs, specifically exosomes, induce specific cell-to-cell interactions and can activate signaling pathways in recipient cells through fusion or interaction. These nanovesicles possess several desirable properties, making them ideal for regenerative medicine and nanomedicine applications. These properties include exceptional stability, biocompatibility, wide biodistribution, and minimal immunogenicity. However, the practical utilization of sEVs, particularly in clinical settings and at a large scale, is hindered by the expensive procedures required for their isolation, limited circulation lifetime, and suboptimal targeting capacity. Despite these challenges, sEVs have demonstrated a remarkable ability to accommodate various cargoes and have found extensive applications in the biomedical sciences. To overcome the limitations of sEVs and broaden their potential applications, researchers should strive to deepen their understanding of current isolation, loading, and characterization techniques. Additionally, acquiring fundamental knowledge about sEVs origins and employing state-of-the-art methodologies in nanomedicine and regenerative medicine can expand the sEVs research scope. This review provides a comprehensive overview of state-of-the-art exosome-based strategies in diverse nanomedicine domains, encompassing cancer therapy, immunotherapy, and biomarker applications. Furthermore, we emphasize the immense potential of exosomes in regenerative medicine.

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

STUDY QUESTION: Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? SUMMARY ANSWER: A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. WHAT IS KNOWN ALREADY: In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. STUDY DESIGN SIZE DURATION: Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. PARTICIPANTS/MATERIALS SETTING METHODS: Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 µm compared to 67.3 ± 7.2 µm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 µm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. WIDER IMPLICATIONS OF THE FINDINGS: Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

New Solutions for Old Problems: How Reproductive Tissue Engineering Has Been Revolutionizing Reproductive Medicine.

Acquired disorders and congenital defects of the male and female reproductive systems can have profound impacts on patients, causing sexual and endocrine dysfunction and infertility, as well as psychosocial consequences that affect their self-esteem, identity, sexuality, and relationships. Reproductive tissue engineering (REPROTEN) is a promising approach to restore fertility and improve the quality of life of patients with reproductive disorders by developing, replacing, or regenerating cells, tissues, and organs from the reproductive and urinary systems. In this review, we explore the latest advancements in REPROTEN techniques and their applications for addressing degenerative conditions in male and female reproductive organs. We discuss current research and clinical outcomes and highlight the potential of 3D constructs utilizing biomaterials such as scaffolds, cells, and biologically active molecules. Our review offers a comprehensive guide for researchers and clinicians, providing insights into how to reestablish reproductive tissue structure and function using innovative surgical approaches and biomaterials. We highlight the benefits of REPROTEN for patients, including preservation of fertility and hormonal production, reconstruction of uterine and cervical structures, and restoration of sexual and urinary functions. Despite significant progress, REPROTEN still faces ethical and technical challenges that need to be addressed. Our review underscores the importance of continued research in this field to advance the development of effective and safe REPROTEN approaches for patients with reproductive disorders.

The characterization and therapeutic applications of ovarian theca cells: An update.

Theca cells perform a range of roles during folliculogenesis. So far, little is known about their recruitment process and function since early research has mainly focused on the interactions between granulosa cells and the oocyte, leaving theca cells unfairly forgotten in the understanding of ovarian physiology and pathogenesis. Given that research on theca cells has greatly emerged in recent years, this review of literature aims to discuss the established theoretical concepts with the most recent findings about theca cells' characterization and origins, in vitro culture applications as models for fertility preservation and pharmacological/toxicological studies, its importance in unraveling pathogenic pathways, and stem-cell-based bioengineering for hormonal replacement therapies. Isolation and in vitro culture techniques for theca cells have led to essential advancements in their characterization as a specific cell population. Unraveling the origins of theca cells during the in vivo differentiation process in the adult ovary will assist the development of hormonal replacement therapies, reestablishment of fertility, and treatments for diseases such as premature ovarian insufficiency and polycystic ovarian syndrome, which seem to be directly influenced by theca cells.

Medium supplementation can influence the human ovarian cells in vitro.

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.