Recapitulating Tumorigenesis in vitro: Opportunities and Challenges of 3D Bioprinting.

Cancer is considered one of the most predominant diseases in the world and one of the principal causes of mortality per year. The cellular and molecular mechanisms involved in the development and establishment of solid tumors can be defined as tumorigenesis. Recent technological advances in the 3D cell culture field have enabled the recapitulation of tumorigenesis in vitro, including the complexity of stromal microenvironment. The establishment of these 3D solid tumor models has a crucial role in personalized medicine and drug discovery. Recently, spheroids and organoids are being largely explored as 3D solid tumor models for recreating tumorigenesis in vitro. In spheroids, the solid tumor can be recreated from cancer cells, cancer stem cells, stromal and immune cell lineages. Organoids must be derived from tumor biopsies, including cancer and cancer stem cells. Both models are considered as a suitable model for drug assessment and high-throughput screening. The main advantages of 3D bioprinting are its ability to engineer complex and controllable 3D tissue models in a higher resolution. Although 3D bioprinting represents a promising technology, main challenges need to be addressed to improve the results in cancer research. The aim of this review is to explore (1) the principal cell components and extracellular matrix composition of solid tumor microenvironment; (2) the recapitulation of tumorigenesis in vitro using spheroids and organoids as 3D culture models; and (3) the opportunities, challenges, and applications of 3D bioprinting in this area.

Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach?

The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids.

p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.

STUDY QUESTION: Is the environmental endocrine disruptor p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters? SUMMARY ANSWER: p,p'-DDE promoted Ca(2+) flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization. WHAT IS KNOWN ALREADY: p,p'-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known. STUDY DESIGN, SIZE, DURATION: Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p'-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p'-DDE incubation periods were also performed in order to monitor sperm rapid Ca(2+) responses. All experiments were repeated on a minimum of five sperm samples from different individuals. PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: p,p'-DDE rapidly increased [Ca(2+)]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca(2+) medium demonstrated that extracellular Ca(2+) influx was responsible for this Ca(2+) increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p'-DDE-induced [Ca(2+)]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p'-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p'-DDE concentrations, suggesting that [Ca(2+)]i rise may cause premature acrosome reaction (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study, and caution must be taken when extrapolating the results. WIDER IMPLICATIONS OF THE FINDINGS: A novel non-genomic p,p'-DDE mechanism specific to sperm is shown in this study. p,p'-DDE was able to induce [Ca(2+)]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by both the Portuguese National Science Foundation (FCT; PEst-C/SAU/LA0001/2011) and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare.

Hydroalcoholic extracts of Vellozia squamata: study of its nanoemulsions for pharmaceutical or cosmetic applications

Some species of plants are notable for the wide range of biologically active constituents in their tissues. Chemical and pharmacological studies of Vellozia squamata Pohl, Velloziaceae, popularly known in Brasil as "canela-de-ema" are scarce, but showed the presence of di-and triterpenoid that may be of scientific interest. In the present study the hydroalcoholic extracts from leafs and stems of V. squamata were submitted to phytochemical prospection to identify the principal groups of constituents, and the antioxidant activity was determined by DPPH method. The hydroethanolic extracts presented higher antioxidant activity. Thus, nanoemulsion formulations were prepared using the method of phase inversion. Accelerated stability tests, such as heat stress and centrifugation were made, and physical and chemical properties of the nanoemulsions were established. Stable formulations were obtained from both extracts from leafs and stems. By the results was possible to establish the potential application of hydroalcoholic extracts from V. squamata in development of products with antioxidant properties and demonstrate a promising pharmaceutical product.

Parabens in male infertility-is there a mitochondrial connection?

Parabens are widely used as preservatives in many foods, cosmetics, toiletries, and pharmaceuticals due to their relatively low toxicity profile and to a long history of safe use. Parabens are alkyl esters of p-hydroxybenzoic acid and typically include methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, isopropylparaben and benzylparaben. These compounds are known to have a null or very weak estrogenic activity in estrogen receptor assays in vitro. In recent years, an increasing concern has emerged regarding possible adverse effects of chemicals in food and in cosmetics on human reproduction outcomes. In developed countries about 15% of human couples are affected by infertility, almost half of these cases attributed to men, through low sperm motility or/and sperm count. It is known that a significant number of cases of male infertility results from exposure to xenobiotics, and also that testis mitochondria are particularly affected by drug-induced toxicity. The present review discusses evidence that parabens may not be as safe as initially thought, and suggests that the interaction between parabens and mitochondrial function in the testis may be key in explaining the contribution of parabens for a decrease in reproductive potential.