Vitrification of feline ovarian tissue: Comparison of protocols based on equilibration time and temperature.

Global contraction of biodiversity pushed most members of Felidae into threatened or endangered list except the domestic cat (Felis catus) thence preferred as the best model for conservation studies. One of the emerging conservation strategies is vitrification of ovarian tissue which is field-friendly but not yet standardized. Thus, our main goal was to establish a suitable vitrification protocol for feline ovarian tissue in field condition. Feline ovarian tissue fragments were punched with biopsy punch (1.5 mm diameter) and divided into 4 groups. Group 1 was fresh control (Fr), while the other three were exposed to 3 vitrification protocols (VIT_CT, VIT_RT1 and VIT_RT2). VIT_CT involved two step equilibrations in solutions containing dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 10 min each at 4 °C. VIT_RT1 involved three step equilibration in solutions containing DMSO, EG, polyvinylpyrrolidone and sucrose for 14 min in total at room temperature, while in VIT_RT2 all conditions remained the same as in VIT_RT1 except equilibration timing which was reduced by half. After vitrification and warming, fragments were morphologically evaluated and then cultured for six days. Subsequently, follicular morphology, cellular proliferation (expression of Ki-67, MCM-7) and apoptosis (expression of caspase-3) were evaluated, and data obtained were analysed using generalised linear mixed model and chi square tests. Proportions of intact follicles were higher in Fr (P = 0.0001) and VIT_RT2 (P = 0.0383) in comparison to the other protocols both post warming and after the six-day culture. Generally, most follicles remained at primordial state which was confirmed by the low expression of Ki-67, MCM-7 markers. In conclusion, VIT_RT2 protocol, which has lower equilibration time at room temperature has proven superior thus recommended for vitrification of feline ovarian tissue.

3D ECM-Based Scaffolds Boost Young Cell Secretome-Derived EV Rejuvenating Effects in Senescent Cells.

Aging is a complex, multifaceted degenerative process characterized by a progressive accumulation of macroscopic and microscopic modifications that cause a gradual decline of physiological functions. During the last few years, strategies to ease and counteract senescence or even rejuvenate cells and tissues were proposed. Here we investigate whether young cell secretome-derived extracellular vesicles (EVs) ameliorate the cellular and physiological hallmarks of aging in senescent cells. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli, directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after exposure to young cell secretome. The results obtained demonstrate that young cells release EVs endowed with the ability to counteract aging. In addition, comparison between young and aged cell secretomes shows a significantly higher miR-200 content in EVs produced using fibroblasts isolated from young donors. The effect exerted by young cell secretome-derived EVs is transient, but can be stabilized using a young ECM microenvironment. This finding indicates a synergistic interaction occurring among molecular effectors and ECM-derived stimuli that cooperate to control a unique program, driving the cell clock. The model described in this paper may represent a useful tool to finely dissect the complex regulations and multiple biochemical and biomechanical cues driving cellular biological age.

Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts.

PURPOSE: This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. METHODS: A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation. RESULTS: Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2- cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes. CONCLUSION: We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

Synergistic Effect of miR-200 and Young Extracellular Matrix-based Bio-scaffolds to Reduce Signs of Aging in Senescent Fibroblasts.

Aging is defined as a complex, multifaceted degenerative process that causes a gradual decline of physiological functions and a rising mortality risk with time. Stopping senescence or even rejuvenating the body represent one of the long-standing human dreams. Somatic cell nuclear transfer as well as cell reprogramming have suggested the possibility to slow or even reverse signs of aging. We exploited miR-200 family ability to induce a transient high plasticity state in human skin fibroblasts isolated from old individuals and we investigated whether this ameliorates cellular and physiological hallmarks of senescence. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after miR-200 exposure. The results show the existence of multiple factors that cooperate to control a unique program, driving the cell clock. In particular, miR-200 family directly regulates the molecular mechanisms erasing cell senescence. However, this effect is transient, reversible, and quickly lost. On the other hand, the use of an adequate young microenvironment stabilizes the miR-200-mediated rejuvenating effects, suggesting that synergistic interactions occur among molecular effectors and ECM-derived biomechanical stimuli. The model here described is a useful tool to better characterize these complex regulations and to finely dissect the multiple and concurring biochemical and biomechanical cues driving the cell biological clock.

Impact of Aging on the Ovarian Extracellular Matrix and Derived 3D Scaffolds.

Advances in medical care, improvements in sanitation, and rising living standards contribute to increased life expectancy. Although this reflects positive human development, it also poses new challenges. Among these, reproductive aging is gradually becoming a key health issue because the age of menopause has remained constant at ~50 years, leading women to live longer in suboptimal endocrine conditions. An adequate understanding of ovarian senescence mechanisms is essential to prevent age-related diseases and to promote wellbeing, health, and longevity in women. We here analyze the impact of aging on the ovarian extracellular matrix (ECM), and we demonstrate significant changes in its composition and organization with collagen, glycosaminoglycans, and laminins significantly incremented, and elastin, as well as fibronectin, decreased. This is accompanied by a dynamic response in gene expression levels of the main ECM- and protease-related genes, indicating a direct impact of aging on the transcription machinery. Furthermore, in order to study the mechanisms driving aging and identify possible strategies to counteract ovarian tissue degeneration, we here described the successful production of a 3D ECM-based biological scaffold that preserves the structural modifications taking place in vivo and that represents a powerful high predictive in vitro model for reproductive aging and its prevention.

Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro.

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an "artificial ovary" that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Current Advances in 3D Tissue and Organ Reconstruction.

Bi-dimensional culture systems have represented the most used method to study cell biology outside the body for over a century. Although they convey useful information, such systems may lose tissue-specific architecture, biomechanical effectors, and biochemical cues deriving from the native extracellular matrix, with significant alterations in several cellular functions and processes. Notably, the introduction of three-dimensional (3D) platforms that are able to re-create in vitro the structures of the native tissue, have overcome some of these issues, since they better mimic the in vivo milieu and reduce the gap between the cell culture ambient and the tissue environment. 3D culture systems are currently used in a broad range of studies, from cancer and stem cell biology, to drug testing and discovery. Here, we describe the mechanisms used by cells to perceive and respond to biomechanical cues and the main signaling pathways involved. We provide an overall perspective of the most recent 3D technologies. Given the breadth of the subject, we concentrate on the use of hydrogels, bioreactors, 3D printing and bioprinting, nanofiber-based scaffolds, and preparation of a decellularized bio-matrix. In addition, we report the possibility to combine the use of 3D cultures with functionalized nanoparticles to obtain highly predictive in vitro models for use in the nanomedicine field.