Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops.

Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.

Highly Photostable Carbon Dots from Citric Acid for Bioimaging.

Bioimaging supported by nanoparticles requires low cost, highly emissive and photostable systems with low cytotoxicity. Carbon dots (C-dots) offer a possible solution, even if controlling their properties is not always straightforward, not to mention their potentially simple synthesis and the fact that they do not exhibit long-term photostability in general. In the present work, we synthesized two C-dots starting from citric acid and tris (hydroxymethyl)-aminomethane (tris) or arginine methyl ester dihydrochloride. Cellular uptake and bioimaging were tested in vitro using murine neuroblastoma and ovine fibroblast cells. The C-dots are highly biocompatible, and after 24 h of incubation with the cells, 100% viability was still observed. Furthermore, the C-dots synthesized using tris have an average dimension of 2 nm, a quantum yield of 37%, high photostability and a zeta potential (ζ) around -12 mV. These properties favor cellular uptake without damaging cells and allow for very effective bioimaging.

Intrafollicular oocyte transfer (IFOT): Potential feasibility in the ovine species.

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 µL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

Use of Virus-Mimicking Nanoparticles to Investigate Early Infection Events in Upper Airway 3D Models.

The current coronavirus disease-19 (COVID-19) pandemic, caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), underscores the threat posed by newly emerging viruses. The understanding of the mechanisms driving early infection events, that are crucial for the exponential spread of the disease, is mandatory and can be significantly implemented generating 3D in vitro models as experimental platforms to investigate the infection substrates and how the virus invades and ravages the tissues.We here describe a protocol for the creation of a synthetic hydrogel-based 3D culture system that mimics in vitro the complex architectures and mechanical cues distinctive of the upper airway epithelia. We then expose the in vitro generated 3D nasal and tracheal epithelia to gold nanoparticles (AuNPs) that display the typical shape and size distinctive of SARS-CoV-2 and of the majority of Coronaviridae presently known.The infection platform here described provides an efficient and highly physiological in vitro model that reproduces the host-pathogen early interactions, using virus-mimicking nanoparticles, and offers a flexible tool to study virus entry into the cell. At the same time, it reduces the risk of accidental infection/spillovers for researchers, which represents a crucial aspect when dealing with a virus that is highly contagious, virulent, and even deadly.

DuoStim - a reproducible strategy to obtain more oocytes and competent embryos in a short time-frame aimed at fertility preservation and IVF purposes. A systematic review.

Recent evidence suggests that follicular development occurs in a wave-like model during the ovarian cycle, where up to three cohorts of follicles are recruited to complete folliculogenesis. This understanding overtakes the previous dogma stating that follicles grow only during the follicular phase of the menstrual cycle. Therefore, in in vitro fertilization (IVF), novel protocols regarding ovarian stimulation have been theorized based on the use of gonadotrophins to prompt the growth of antral follicles at any stage of the menstrual cycle. These unconventional protocols for ovarian stimulation aim at a more efficient management of poor-prognosis patients, otherwise exposed to conflicting outcomes after conventional approaches. DuoStim appears among these unconventional stimulation protocols as one of the most promising. It combines two consecutive stimulations in the follicular and luteal phases of the same ovarian cycle, aimed at increasing the number of oocytes retrieved and embryos produced in the short time-frame. This protocol has been suggested for the treatment of all conditions requiring a maximal and urgent exploitation of the ovarian reserve, such as oncological patients and poor responders at an advanced maternal age. At present, data from independent studies have outlined the consistency and reproducibility of this approach, which might also reduce the drop-out between consecutive failed IVF cycles in poor-prognosis patients. However, the protocol must be standardized, and more robust studies and cost-benefit analyses are needed to highlight the true clinical pros and cons deriving from DuoStim implementation in IVF.

Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours.

BACKGROUND: Cerium oxide nanoparticles (CeO2 NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO2 NPs for 96 h on the main structural and kinematic parameters of spermatozoa. METHODS: The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO2 NPs (0, 44 and 220 µg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H2DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni's as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. RESULTS: Exposure of ram spermatozoa to increasing doses of CeO2 NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO2 NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. CONCLUSIONS: We reported beneficial effects of CeO2 NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO2 NPs. These effects could not be related to an antioxidant activity of CeO2 NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

Microvesicles secreted from equine amniotic-derived cells and their potential role in reducing inflammation in endometrial cells in an in-vitro model.

BACKGROUND: It is known that a paracrine mechanism exists between mesenchymal stem cells and target cells. This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication. METHODS: In this context, this study aims to understand the efficacy of MVs in in-vitro endometrial stressed cells in view of potential healing in in-vivo studies. For this purpose, the presence and type of MVs secreted by amniotic mesenchymal stem cells (AMCs) were investigated and the response of endometrial cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract the in vitro stress in endometrial cells induced by lipopolysaccharide was studied by measuring the rate of apoptosis and cell proliferation, the expression of some pro-inflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin 1ß (IL-1ß), and metalloproteinases (MMP) 1 and 13, and the release of some pro- or anti-inflammatory cytokines. RESULTS: MVs secreted by the AMCs ranged in size from 100 to 200 nm. The incorporation of MVs was gradual over time and peaked at 72 h. MVs reduced the apoptosis rate, increased cell proliferation values, downregulated pro-inflammatory gene expression, and decreased the secretion of pro-inflammatory cytokines. CONCLUSION: Our data suggest that some microRNAs could contribute to counteracting in-vivo inflammation of endometrial tissue.

In vitro production and cryotolerance of prepubertal and adult goat blastocysts obtained from oocytes collected by laparoscopic oocyte-pick-up (LOPU) after FSH treatment.

This study compares the developmental capacity and cryotolerance of embryos produced from oocytes of stimulated prepubertal and adult Sarda goats. Twelve prepubertal and 13 adult goats were each given 110 and 175 IU FSH, respectively, and cumulus-oocyte complexes (COCs) were collected by laparoscopic oocyte-pick-up (LOPU). After in vitro maturation, fertilisation and culture (IVMFC), blastocysts were vitrified, warmed and blastocoel re-expansion and gene expression were evaluated. Prepubertal goats produced a higher COCs number than adults (mean +/- s.e.m., 89.67 +/- 5.74 and 26.69 +/- 3.66, respectively; P < 0.01). Lower developmental competence was demonstrated in the prepubertal oocytes as shown by a higher number of COCs discarded before IVM (21.1% and 14.7% for prepubertals and adults, respectively; P < 0.01) and IVF (23.4% v. 9.1%; P < 0.01) and by the lower cleavage (55.6% and 70.3%, respectively; P < 0.01) and blastocyst rates (24.2% and 33.9%, respectively; P < 0.05). Compared with the adult, prepubertal vitrified/warmed blastocysts showed significantly (P < 0.05) lower in vitro viability, as determined by the re-expansion rate (62.5% and 40.3%). No differences were observed in the time required for blastocoel re-expansion or in cyclin B1, E-cadherin, Na/K ATPase, HSP90beta and aquaporin 3 messenger RNA quantity. These results show that in vitro-produced embryos produced from prepubertal goat oocytes have a lower developmental rate and cryotolerance compared with their adult counterparts. However, we can assume that the quality of re-expanded embryos does not differ between the two groups.

Maternal-fetal transplacental leakage of mitochondrial DNA in bovine nuclear transfer pregnancies: potential implications for offspring and recipients.

The synepitheliochorial placenta of ruminants is constructed of multiple tissue layers that separate maternal and fetal blood. In nuclear transfer cloned ruminants, however, placental anomalies such as abnormal vascular development and hemorrhagic cotyledons have been reported. We have investigated the possible exchange of genetic material between somatic cell nuclear transfer cloned (SCNT) bovine fetuses and recipients at day 80 of gestation using mitochondrial DNA (mtDNA) as a marker. Twenty-three recovered SCNT-fetuses and their recipients were screened for divergent and thus informative mtDNA combinations. Twenty-one fetuses generated by in vitro fertilization (IVF) or multiple ovulation embryo transfer (MOET) and the corresponding recipients served as controls. A search for recipient mtDNA haplotype in DNA extracts from fetal blood by PCR-RFLP analysis revealed three cases of chimerism (two SCNT, one IVF) among a total of 19 informative fetus-recipient pairs (eight SCNT, seven IVF, four MOET). Placental anomalies have also been observed in some IVF fetuses and the present data therefore suggests transplacental leakage of cell components or cells from the recipient into some fetuses generated by in vitro techniques. Further studies are necessary to determine (i) the nature of leaked material, (ii) whether there is bi-directional leakage, and (iii) whether leaked material is present in recipients and calves after parturition, i.e. whether leakage takes place in vivo. If recipients were chimeric for DNA or cells derived from genetically modified SCNT (or IVF) embryos, their subsequent utilization might be affected.

Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes.

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.

Influence of cadmium exposure on in vitro ovine gamete dysfunction.

This study was conducted to determine the in vitro effects of three different cadmium concentrations (0, 2, and 20 microM CdCl(2)) on oocyte maturation, fertilisation, and acrosome integrity and sperm viability in sheep. Cumulus-oocyte complexes were recovered from ovaries of slaughtered sheep and sperm were collected by artificial vagina from adult rams. The oocyte maturation rate was significantly affected (P < 0.001) by Cd at both concentrations, with a metaphase II (MII) rate of 96.8, 63.8, and 32.0% for 0, 2, and 20 microM cadmium, respectively. In the second experiment, the presence of Cd significantly decreased (P < 0.01) the rate of oocytes resting in MII after 24-h postmaturation culture, compared with the control group (93.8 versus 29.0 and 19.8%, respectively, for 0, 2, and 20 microM Cd). Oocytes cultured with Cd 2 microM showed a higher activation rate (59.5%, P < 0.001) with one or two pronucleus than with 0 and 20 microM Cd (6.2 and 22.9%, respectively). During fertilisation the presence of fertilised oocytes was decreased in both culture systems with Cd compared with the control (76.1, 25.9, and 4.7% for 0, 2, and 20 microM Cd, respectively; P < 0.001) while polyspermy was increased in the 2 microM Cd group (23.5 for 2 microM versus 6.7 and 0%, respectively, for 0 and 20 microM groups). In both experiments Cd significantly increased (P < 0.001) the rates of oocyte degeneration. In the third experiment, Cd 20 microM significantly decreased (P < 0.01) the viability rate (35.6%) of spermatozoa compared with 2 microM (57.6%) and 0 microM (54.4%) while Cd 2 microM increased (P < 0.01) acrosome-reacted spermatozoa (45.2%) compared with 20 microM (32.5%) and control (31.9%). The results suggest that in vitro cadmium at the lowest dose tested affects the physiological function of both ovine gametes but at higher dose tested can compromise cell viability.