Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.

Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

BackgroundThere is increasing appreciation that non-cancer cells within the tumour microenvironment influence cancer progression and anti-cancer drug efficacy. For metastatic prostate cancer (PCa), the bone marrow microenvironment influences metastasis, drug response, and possibly drug resistance.MethodsUsing a novel microwell platform, the Microwell-mesh, we manufactured hundreds of 3D co-culture microtissues formed from PCa cells and bone marrow stromal cells. We used luciferase-expressing C42B PCa cells to enable quantification of the number of PCa cells in complex microtissue co-cultures. This strategy enabled us to quantify specific PCa cell growth and death in response to drug treatment, in different co-culture conditions. In parallel, we used Transwell migration assays to characterize PCa cell migration towards different 2D and 3D stromal cell populations.ResultsOur results reveal that PCa cell migration varied depending on the relative aggressiveness of the PCa cell lines, the stromal cell composition, and stromal cell 2D or 3D geometry. We found that C42B cell sensitivity to Docetaxel varied depending on culture geometry, and the presence or absence of different stromal cell populations. By contrast, the C42B cell response to Abiraterone Acetate was dependent on geometry, but not on the presence or absence of stromal cells.ConclusionIn summary, stromal cell composition and geometry influences PCa cell migration, growth and drug response. The Microwell-mesh and microtissues are powerful tools to study these complex 3D interactions.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4473-8) contains supplementary material, which is available to authorized users.

IntroductionTo characterize sperm dynamic motility patterns and chromatin integrity in infertile men with leukocytospermia.Material and methodsFifty patients with primary infertility and oligoasthenoteratozoospermia included in this prospective, controlled, blind study. All patients underwent clinical evaluation, semen peroxidase stain, computer aided semen analysis (CASA), sperm DNA integrity evaluation with acridine orange test (AOT) and fluorescence in situ hybridization (FISH) analysis of 18, X and Y chromosomes. Pregnancy outcomes were documented following antibiotic treatment of patients with leukocytospermia.ResultsInfertile men with leukocytospermia had significantly lower progressive and total sperm motility percentages compared to the control group. Sperm dynamic motility parameters by CASA including curvilinear, straight line and average pathway velocities, straightness and amplitude of lateral head displacement were significantly lower in leukocytospermia. Sperm DNA fragmentation index was significantly higher in leukocytospermia. Percentages of sperm with disomy XY and 18 were significantly higher. These changes in sperm motility parameters and DNA integrity correlated with the number of peroxidase positive leukocytes. Follow-up of 23 of the 25 patients with leukocytospermia after antibiotic treatment revealed significantly higher pregnancy rates in cured patients than in those with persistent leukocytospermia.ConclusionsLeukocytospermia has a significant impact on sperm dynamic motility patterns, DNA and chromosomal integrity in infertile men which can adversely affect the likelihood of a successful pregnancy.

The roles that eicosanoids play during pregnancy and parturition are crucial to a successful outcome. A better understanding of the regulation of eicosanoid production and the roles played by the various end products during pregnancy and parturition has led to our view that accurate measurements of a panel of those end products has exciting potential as diagnostics and prognostics of preterm labor and delivery. Exosomes and their contents represent an exciting new area for research of movement of key biological factors circulating between tissues and organs akin to a parallel endocrine system but involving key intracellular mediators. Eicosanoids and enzymes regulating their biosynthesis and metabolism as well as regulatory microRNAs have been identified within exosomes. In this review, the regulation of eicosanoid production, abundance and actions during pregnancy will be explored. Additionally, the functional significance of placental exosomes will be discussed.

Abnormal uterine function affects conception rate and embryo development, thereby leading to poor fertility and reproduction failure. Exosomes are a nanosized subclass of extracellular vesicles (EV) that have important functions as intercellular communicators. They contain and carry transferable bioactive substances including micro RNA (miRNA) for target cells. Elements of the cargo can provide epigenetic modifications of the recipient cells and may have crucial roles in mechanisms of reproduction. The dairy industry accounts for a substantial portion of the economy of many agricultural countries. Exosomes can enhance the expression of inflammatory mediators in the endometrium, which contribute to various inflammatory diseases in transition dairy cows. This results in reduced fertility which leads to reduced milk production and increased cow maintenance costs. Thus, gaining a clear knowledge of exosomal epigenetic modifiers is critical to improving the breeding success and profitability of dairy farms. This review provides a brief overview of how exosomal miRNA contributes to inflammatory diseases and hence to poor fertility, particularly in dairy cows.

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