Supplementation Of Culture Medium Research Articles

Exogenous L-Glutathione Improves Vitrification Outcomes in Murine Preimplantation Embryos.

Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6-8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status.

The dark side of foetal bovine serum in extracellular vesicle studies.

Extracellular vesicles (EVs) have been shown to be involved in cell‐cell communication and to take part in both physiological and pathological processes. Thanks to their exclusive cargo, which includes proteins, lipids, and nucleic acids from the originating cells, they are gaining interest as potential biomarkers of disease. In recent years, their appealing features have been fascinating researchers from all over the world, thus increasing the number of in vitro studies focused on EV release, content, and biological activities. Cultured cell lines are the most‐used source of EVs; however, the EVs released in cell cultures are influenced by the cell culture conditions, such as the use of foetal bovine serum (FBS). FBS is the most common supplement for cell culture media, but it is also a source of contaminants, such as exogenous bovine EVs, RNA, and protein aggregates, that can contaminate the cell‐derived EVs and influence their cargo composition. The presence of FBS contaminants in cell‐derived EV samples is a well‐known issue that limits the clinical applications of EVs, thus increasing the need for standardization. In this review, we will discuss the pros and cons of using FBS in cell cultures as a source of EVs, as well as the protocols used to remove contaminants from FBS.

A retrospective assessment of outcomes according to the concentration of protein in culture medium following vitrified oocyte warming

Background and Aim: Oocyte cryopreservation enables the preservation of fertility for patients, as well as allowing a clinic to store donated oocytes so they are readily available to recipients. Vitrification has significantly increased oocyte survival and increased pregnancy rates. Several vitrification systems are commercially available – some specifying supplementation of post warm culture media with synthetic serum substitute or dextran serum supplement at 20% (v/v), or human serum albumin at 12mg/ml.

Autologous Platelet Rich Plasma (PRGF) Preserves Genomic Stability of Gingival Fibroblasts and Alveolar Osteoblasts after Long-Term Cell Culture

Plasma rich in growth factors (PRGF) has several applications in dentistry that may require repeated applications of PRGF. Furthermore, it has been used for ex vivo expansion of human origin cells for their clinical application. One of the most relevant issues in these applications is to guarantee the genetic stability of cells. In this study, the chromosomal stability of gingival fibroblasts and alveolar osteoblasts after long-term culture was evaluated. Cells were expanded with PRGF or foetal bovine serum (FBS) as a culture medium supplement until passage 7 or 8 for gingival fibroblast or alveolar osteoblasts, respectively. A comparative genomic hybridization (CGH) array was used for the genetic stability study. This analysis was performed at passage 3 and after long-term culture with the corresponding culture medium supplements. The cell proliferative rate was superior after PRGF culture. Array CGH analysis of cells maintained with all the three supplements did not reveal the existence of alterations in copy number or genetic instability. The autologous PRGF technology preserves the genomic stability of cells and emerges as a safe substitute for FBS as a culture medium supplement for the clinical translation of cell therapy.

Novel Oocyte-Secreted Factors Improve Mouse IVM Outcomes

Background: In vitro maturation (IVM) is a technology designed to obtain mature oocytes following culture of immature cumulus–oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for patients with excessive AFC or those requiring immediate fertility preservation. However, the clinical uptake of IVM has been slow, primarily due to lower embryo yield and live birth rate relative to IVF, therefore improving IVM culture is required. Aim: To assess whether supplementation of IVM culture medium with the novel in-house engineered TGFβ proteins cumulin and super-GDF9 improves subsequent embryo development. Method: Immature mouse COCs were cultured by standard IVM or bi-phasic IVM ± cumulin or super-GDF9. Following IVM, cumulus expansion was scored and COCs were fertilized, and cultured to assess embryo development. Differential staining was performed on day 6 blastocysts following bi-phasic IVM to assess cell allocation. In a separate experiment, hyperspectral imaging of autofluorescence was carried out on oocytes and cumulus cells following standard IVM ± cumulin to assess the molecular composition of these cells. Results: Both cumulin and super-GDF9 in standard IVM significantly increased cumulus expansion (P<0001; n=104-115 COCs) and blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; P=0.006; n=382-406 oocytes). Hyperspectral imaging showed that oocytes (n=115-158) and cumulus cells (n=532-600) exposed to cumulin during IVM had a distinct spectral profile that varied dramatically (P<0.005) from untreated cells, demonstrating that cumulin has a major impact on the molecular composition of these cells, likely contributing to the improved oocyte quality. In bi-phasic IVM, cumulin did not significantly alter embryo yield (n=387-424 oocytes) or blastocyst cell number or allocation (n=84-112 blastocysts). Conclusion: Cumulin did not provide an additional beneficial effect in bi-phasic IVM, however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence suggesting that their effects in human IVM should be investigated.

Case Studies Exemplifying the Transition to Animal Component-free Cell Culture.

Cell culture techniques are strongly connected with modern scientific laboratories and production facilities. Thus, choosing the most suitable medium for the cells involved is vital, not only directly to optimise cell viability but also indirectly to maximise the reliability of the experiments performed with the cells. Fetal bovine or calf serum (FBS or FCS, respectively) is the most commonly used cell culture medium supplement, providing various nutritional factors and macromolecules essential for cell growth. Yet, the use of FBS encompasses a number of disadvantages. Scientifically, one of the most severe disadvantages is the lot-to-lot variability of animal sera that hampers reproducibility. Therefore, transitioning from the use of these ill-defined, component-variable, inconsistent, xenogenic, ethically questionable and even potentially infectious media supplements, is key to achieving better data reproducibility and thus better science. To demonstrate that the transition to animal component-free cell culture is possible and achievable, we highlight three different scenarios and provide some case studies of each, namely: i) the adaptation of single cell lines to animal component-free culture conditions by the replacement of FBS and trypsin; ii) the adaptation of multicellular models to FBS-free conditions; and (iii) the replacement of FBS with human platelet lysate (hPL) for the generation of primary stem/stromal cell cultures for clinical purposes. By highlighting these examples, we aim to foster and support the global movement towards more consistent science and provide evidence that it is indeed possible to step out of the currently smouldering scientific reproducibility crisis.

Functional properties of equine adipose-derived mesenchymal stromal cells cultured with equine platelet lysate.

Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.

Supplementation of articular cartilage-derived chondroprogenitors with bone morphogenic protein-9 enhances chondrogenesis without affecting hypertrophy.

Chondroprogenitors (CPCs) have emerged as a promising cellular therapy for cartilage-related pathologies due to their inherent primed chondrogenic potential. Studies report that the addition of growth factors such as parathyroid hormone (PTH) and Bone Morphogenic Protein (BMP) enhance the chondroinducive potential in chondrocytes and mesenchymal stem cells. This study evaluated if supplementation of the standard culture medium for cell expansion with 1-34 PTH and BMP-9 would enhance the chondrogenic potential of CPCs and reduce their hypertrophic tendency. Human chondrocytes were isolated from patients undergoing total knee replacement for osteoarthritis (n = 3). Following fibronectin adhesion assay, passage 1 CPCs were divided and further expanded under three culture conditions (a) control, i.e., cells continued under standard culture conditions, (b) 1-34 PTH group, additional intermittent 6h exposure with 1-34 PTH and (c) BMP-9 group, additional BMP-9 during culture expansion. All the groups were evaluated for population-doubling, cell cycle analysis, surface marker and gene expression for chondrogenesis, hypertrophy, multilineage differentiation and GAG (glycosaminoglycan)/DNA following chondrogenic differentiation. Concerning growth kinetics, the BMP-9 group exhibited a significantly lower S-phase and population-doubling when compared to the other two groups. Qualitative analysis for chondrogenic potential (Alcian blue, Safranin O staining and Toluidine blue for GAG) revealed that the BMP-9 group exhibited the highest uptake. The BMP-9 group also showed significantly higher COL2A1 expression than the control group, with no change in the hypertrophy marker expression. BMP-9 can potentially be used as an additive for CPCs expansion, to enhance their chondrogenic potential without affecting their low hypertrophic tendency. The mitigating effects of 1-34PTH on hypertrophy would benefit further investigation when used in combination with BMP-9 to enhance chondrogenesis whilst reducing hypertrophy.

Swiss Medical Devices for Autologous Regenerative Medicine: From Innovation to Clinical Validation

Regenerative medicine, based on the use of autologous tissues and embryonic, stem or differentiated cells, is gaining growing interest. However, their preparation, in a manner compliant with good practices and health regulations, is a technical challenge. The aim of this manuscript is to present the design of reliable CE marked medical devices for the preparation of standardized platelet-rich plasma (PRP) and other autologous biologics intended for therapeutic uses. There are numerous PRP isolation processes. Depending on the methodology used, PRP composition varies greatly in terms of platelet concentration, platelet quality, and level of contamination with red and white blood cells. This variability in PRP composition might affect the clinical outcomes. The devices presented here are based on a specific technology, patented all over the world, that allows the precise separation of blood components as a function of their density using thixotropic separator gels in closed systems. This allows the preparation, in an automated manner, of leukocyte poor PRP with a standardized composition. Production of different forms of PRP is a clinical asset to suit various therapeutic needs. Therefore, we are offering solutions to prepare PRP either in liquid or gel form, and PRP combined with hyaluronic acid. These biologics have been successfully used in many different therapeutic domains, resulting in more than 150 published clinical studies. We also developed the CuteCell technology platform for cell culture expansion for further autologous cell therapies. This technology enables the safe and rapid in vitro expansion of cells intended for therapeutic use in good manufacturing practices (GMP) and autologous conditions, using blood-derived products as culture media supplementation. We summarize in this article our 20 years’ experience of research and development for the design of PRP devices and, more recently, for PRP combined with hyaluronic acid.

Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

Role of Common Cell Culture Media Supplements on Citrate-Stabilized Gold Nanoparticle Protein Corona Formation, Aggregation State, and the Consequent Impact on Cellular Uptake

Sodium citrate-stabilized gold nanoparticles (AuNPs) are destabilized when dispersed in cell culture media (CCMs). This may promote their aggregation and subsequent sedimentation, or under the proper conditions, their interaction with dispersed proteins can lead to the formation of a NP-stabilizing protein corona. CCMs are ionic solutions that contain growth substances which are typically supplemented, in addition to serum, with different substances such as dyes, antioxidants, and antibiotics. In this study, the impact of phenol red, penicillin-streptomycin, l-glutamine, and β-mercaptoethanol on the formation of the NP-protein corona in CCMs was investigated. Similar protein coronas were obtained except in the presence of antibiotics. Under these conditions, the protein corona took more time to be formed, and its density and composition were altered, as indicated by UV-vis spectroscopy, Z potential, dynamic light scattering, and liquid chromatography-mass spectrometry analyses. As a consequence of these modifications, a significantly different AuNP cellular uptake was measured, showing that NP uptake increased as did the NP aggregate formation. AuNP uptake studies performed in the presence of clathrin- and caveolin-mediated endocytosis inhibitors showed that neither clathrin receptors nor lipid rafts were significantly involved in the internalization mechanism. These results suggest that in these conditions, NP aggregation is the main mechanism responsible for their cellular uptake.

Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3.

Human platelet lysate (hPL) contains abundant growth factors for inducing human cell proliferation and may be a suitable alternative to fetal bovine serum (FBS) as a culture medium supplement. However, the application of hPL in virological research remains blank. Parechovirus type-A3 (PeV-A3) belongs to Picornaviridae, which causes meningoencephalitis in infants and young children. To understand the suitability of hPL-cultured cells for PeV-A3 infection, the infection of PeV-A3 in both FBS- and hPL-cultured glioblastoma (GBM) cells were compared. Results showed reduced PeV-A3 infection in hPL-cultured cells compared with FBS-maintained cells. Mechanistic analysis revealed hPL stimulating type I interferon (IFN) antiviral pathway, through which phospho-signal transducer and activator of transcription 1 (STAT1), STAT2, interferon regulatory factor 3 (IRF3) were activated and antiviral genes, such as IFN-α, IFN-β, and Myxovirus resistance protein 1 (MxA), were also detected. In addition, an enhanced PeV-A3 replication was detected in the hPL-cultured GBM cells treated with STAT-1 inhibitor (fludarabine) and STAT1 shRNA. These results in vitro suggested an unexpected effect of hPL-activated type I IFN pathway response to restrict virus replication and that hPL may be a potential antiviral bioreagent.

P-248 Fallopian tube extracellular matrix hydrogels for in vitro embryo culture in rabbit models: A post-natal study

Abstract Study question Can tissue-specific extracellular matrix (ECM) hydrogels derived from decellularized (DC) fallopian tubes have phenotypic consequences in the offspring when used for in vitro embryo culture? Summary answer Tissue-specific ECM hydrogels from DC rabbit fallopian tubes influence assisted reproductive technologies (ART) outcomes, resulting in offspring with a higher body weight. What is known already The in vitro culture of embryos fails to mimic the physiological dynamism occurring in the reproductive tract. In response to this sub-optimal environment, mammalian embryo exhibits a high degree of developmental plasticity, leading to specific phenotypic deviations at birth and beyond (e.g. low weight). Recently, it has been demonstrated that using in vivo derived culture complements, these changes can be ameliorated. The ECM is one of the elements shown to be important in retaining structural and biological signals from the native source tissue. The current bioengineering techniques could provide hydrogels mimicking the native environment and offering an optimal embryo development. Study design, size, duration This study was approved by an ethics committee (2018/VSC/PEA/0116). Rabbit oviducts (n = 24) were collected, fragmented, DC and solubilized to create hydrogels. After physicochemical characterization, the tissue-specific ECM hydrogels were used for in vitro culture of pronuclear embryos during 48 hours (n = 146), which were subsequently transferred to recipient females, along with embryos cultured under control conditions (n = 138). After delivery, the rabbits were weighed during 20 weeks and blood tests were performed at weeks 8 and 20. Participants/materials, setting, methods To create hydrogels, we employed a decellularization protocol based on SDS detergent. After characterizing and validating decellularization (Francés-Herrero et al. 2021), ECM hydrogels were used to generate coatings (overnight at 37ºC) on culture plates. Pronuclear embryos were cultured until morula stage and then transferred by laparoscopy to obtain the offspring. The animals were weighed and their blood was analyzed by hemogram. T-test was used for the statistical analysis after normality verification (p &amp;lt; 0.05 statistically significant). Main results and the role of chance After 48 hours of embryo culture, no differences in development and morphology were detected between the coating and control conditions (Francés-Herrero et al. 2021). With a total of 284 embryos transferred, the implantation and live birth rates were slightly higher in the coating group than in the control group (31.26 ± 21.44% vs 26.53 ± 24.8% and 19.96 ± 12.5% vs 18.68 ± 24.68%, respectively), but the differences were not statistically significant. During the first 20 weeks of life, the animals from the coating group consistently showed higher weights than the control group. Although this difference was not significant at birth (75.15 ± 15.48 g vs 61.88 ± 17.79 g, respectively), it was significant at subsequent measurements (1117.73 ± 172.54 g vs 942 ± 105.51 g at week 5, 2513 ± 352.83 g vs 2256.25 ± 168.52 g at week 10, 3475.56 ± 297.91 g vs 3257.5 ± 189.94 g; at week 15 and 4151.11 ± 269.28 g vs 3808.75 ± 293.08 g at week 20; p &amp;lt; 0.05). The animals of both experimental groups showed normal blood parameters (white blood cells, % lymphocytes, % monocytes, % granulocytes, red blood cells and hematocrit) at 8 and 20 weeks of life. Limitations, reasons for caution This study has been carried out in an animal model and, although the offspring generated is healthy, the scope of this technique is still unknown. So, the next step would be to study the embryonic reprogramming or media metabolomic profile under ECM in vitro culture. Wider implications of the findings These findings show how culture media supplementation with fallopian tube ECM allows improving in vitro conditions and decreases postnatal phenotypic deviations after ART. This study could initiate a new embryo culture techniques era to guarantee that ART are utilized in the most efficient and safest possible practice. Trial registration number Not applicable

P-226 Efficiency of different antioxidants in reducing the supra-physiological Oxidation Reduction Potential Levels in human embryo culture media

Abstract Study question Does every antioxidant compounds perform similarly in reducing the excessive levels of Oxidation Reduction Potential (ORP) in IVF culture media? Summary answer Not all antioxidant compounds perform similarly in maintaining physiological ORP levels in IVF culture media. What is known already During assisted reproduction technologies (ART), several external factors are responsible for disturbing the ORP equilibrium in gametes and embryos. Therefore, antioxidant supplementation of culture media has been evaluated and reported. However, there is lack of consensus on the efficiency of antioxidant action in embryo culture media. Antioxidants are known to lower the levels of free radical concentration to physiological levels, preventing cellular damage caused by oxidative stress. Therefore, it is important to determine which antioxidant compound are the most effective in reducing ORP levels in embryo culture media. Study design, size, duration In this prospective study was performed at CITMER Reproductive Medicine, Mexico city. ORP levels were measured by MiOXSYS System (UAB Caerus Biotechnologies, Vilnius, Lithuania) in embryo-free culture medium (Global® Total®, LifeGlobal®, Connecticut, US). Embryo-free culture medium was supplemented with cumene hydroperoxide and seven different antioxidants were tested each day for 5 continuous days (n = 40). Follicular fluid ORP levels from dominant follicles of 40 oocyte donors were used as control and considered as a physiological target. Participants/materials, setting, methods Cumene hydroperoxide (CH) 1mM, was utilized to increase ORP values in culture media from 224 mV to 415 mV. The following compounds considered as antioxidants according to literature were supplemented at 2 mM to the CH culture media: Ascorbic acid (ASC), L-Carnitine (CAR), L-Cysteine (CYS), Glutathione (GLT), Curcumin (CU), Resveratrol (RSV) and b-Mercaptoethanol (BME). ORP levels were quickly evaluated each time. Main results and the role of chance In this study, CH was used as a culture media oxidant to measure the antioxidants capacity in lowering the excessive ORP values of CH culture media. ORP in CH solution was 415 mV ± 4.3. All antioxidants showed a statistically different capacity (p &amp;lt; 0.05) to lower the ORP values when added to CH solution: RSV decreased the ORP to 348.3 mV ± 3.6 p = 0.0449, CAR to 316.32 mV ± 4.7 (p &amp;lt; 0.0001), CU to 315.7 mV ± 4.4 (p &amp;lt; 0.0001) and GLT to 258.4 mV ± 15.1 (p = 0.00002). The compounds that showed higher antioxidant capacity were CYS 158.4 mV ± 4.7 (p &amp;lt; 0.00001), ASC 154.1 mV ± 3.1 (p &amp;lt; 0.00001) and BME 136.1 mV ± 9.6 (p &amp;lt; 0.00001). CYS, ASC and BME ORP values reached the physiological ORP values found in follicular fluid from donors, which are 89 mV±23.6 mV. Limitations, reasons for caution Further studies should focus on the comparison between physiological dynamics of antioxidant supplementation on culture media and molecular and biochemical effect of antioxidants on embryos and gametes. Wider implications of the findings According to our results, CYS, ASC and BME were significantly more potent antioxidants in lowering the ORP in culture medium to physiological levels. The measurement of antioxidant capacity in lowering excess of ROS in culture media may assist in developing the most effective antioxidant supplements, thus improving ART outcomes. Trial registration number none

Lycopene supplementation to serum-free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro.

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as β-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 μl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1μM (ITSL); and IT + DMSO-lycopene 0.1μM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.

Abstract 3020: Periodic fasting and cholesterol biosynthesis inhibitors achieve a synergistic antitumor activity in gastrointestinal cancers

Abstract Periodic fasting enhances the activity of several chemotherapeutics, molecularly-targeted drugs, endocrine agents and immune checkpoint inhibitors. Identifying drugs that are approved for non-oncological conditions, but acquire antitumor properties through fasting could pinpoint new cancer cell liabilities and define new treatment options. By screening over 800 approved drugs in PK9 pancreatic ductal adenocarcinoma (PDAC) cells, we identified several azoles (antifungal agents), including clotrimazole (CTZ), as agents whose cytotoxic activity against cancer cells is synergistically enhanced by starvation conditions. Since azoles inhibit 14alpha-demethylase, which is a key enzyme for cholesterol (CE) biosynthesis, we hypothesized that starvation and azoles would cooperate by blunting CE production in PDAC cells. Consistent with this notion, we found that simvastatin (an HMG-CoA reductase inhibitor) and terbinafine (TRB), which obstruct squalene epoxidase (SQLE), another key enzyme from the CE biosynthetic pathway, also had their antitumor effects strongly enhanced by starvation. Combined starvation and CTZ or starvation and TRB reduced intracellular CE in Capan-1 cells both in vitro and in vivo. Methyl-beta-cyclodextrin, which depletes intracellular CE, and starvation also showed a synergistic interaction in Capan-1 and in MiaPaCa2 cells (another PDAC cell line). Finally, culture media supplementation with a water-soluble CE formulation prevented the synergistic interaction between starvation and CE biosynthesis inhibitors. TRB potentiation through starvation was countered through supplementation with insulin, IGF1 and leptin, which are downregulated during fasting. Consistent with CE being an essential constituent of membrane lipid rafts, that harbor the growth- and survival-promoting PI3K/AKT signaling cascade, we found combined CTZ or TRB and starvation to markedly downregulate phosphorylated AKT in Capan-1 xenografts. Similar results were obtained in MiaPaCa2 cells. Capan-1 cell transduction with constitutively active, myristoylated AKT protected them from combined CTZ or TRB and starvation, indicating that AKT inhibition mediates the synergistic interaction between CE production inhibitors and fasting. Weekly 48h fasting enhanced CTZ and TRB antitumor effects in vivo in Capan-1 xenograft-bearing mice and, when combined with TRB, lowered circulating LDL (while increasing HDL) cholesterol as compared to TRB alone. Overall, these findings support the potential of fasting-based diets in combination with CE biosynthesis inhibitors against PDAC. Citation Format: Amr Khalifa, Ana Guijarro, Asmaa Namatalla, Moustafa Ghanem, Matteo Lambertini, Alessio Nencioni, Irene Caffa. Periodic fasting and cholesterol biosynthesis inhibitors achieve a synergistic antitumor activity in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3020.

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

Effect of sericin, a silk derived protein, on the amplification of Zika virus in insect and mammalian cell cultures

Sericin, a silk-derived non-immunogenic protein, has been used to improve cell culture performance by increasing viability, cell concentration, and promoting adherence of several cell lines. Here, we hypothesized that the properties of sericin can enhance the amplification of flaviviruses in cell cultures. The propagation of flavivirus is inefficient and limits scientific research. Zika virus (ZIKV) is an important human pathogen that has been widely studied because of its high impact on public health. There is a need to amplify Zika virus both for research and vaccine development. In this work, we show that sericin improves ZIKV amplification in insect (C6/36) and mammalian (Vero) cell cultures, and that it has a cryoprotectant capacity. Supplementation of cell culture media with sericin at 80 µg/mL resulted in a significant increase of 1 log in the concentration of ZIKV infectious particles produced from both cell lines. Furthermore, final virus yields increased between 5 and 10-fold in Vero cells and between 7 and 23-fold in C6/36 cells when sericin was supplemented, compared to control conditions. These results show that sericin is an effective supplement to increase ZIKV production by Vero and C6/36 cells. Additionally, sericin was a suitable cryoprotective agent, and hence an alternative to FBS and DMSO, for the cryopreservation of C6/36 cells but not for Vero cells.

Metabolomics Analysis and Antioxidant Potential of Endophytic Diaporthe fraxini ED2 Grown in Different Culture Media.

Endophytic fungi are a promising source of bioactive metabolites with a wide range of pharmacological activities. In the present study, MS-based metabolomics was conducted to study the metabolomes variations of endophytic Diaporthe fraxini ED2 grown in different culture media. Total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays were conducted to assess the antioxidant potential of the fungal extracts. Multivariate data analysis (MVDA) was employed in data analysis and interpretation to elucidate the complex metabolite profile. The supplemented culture medium of D. fraxini fungal extract stimulated the production of metabolites not occurring in the normal culture medium. Antioxidant activity studies revealed the potential of supplemented cultured fungal extract of D. fraxini as a source of antioxidants. The present findings highlight that fungal culture medium supplementation is an effective approach to unravelling the hidden metabolome in plant-associated fungal diversity.