Challenges of aortic valve tissue culture - maintenance of viability and extracellular matrix in the pulsatile dynamic microphysiological system.

BACKGROUND: Calcific aortic valve disease (CAVD) causes an increasing health burden in the 21st century due to aging population. The complex pathophysiology remains to be understood to develop novel prevention and treatment strategies. Microphysiological systems (MPSs), also known as organ-on-chip or lab-on-a-chip systems, proved promising in bridging in vitro and in vivo approaches by applying integer AV tissue and modelling biomechanical microenvironment. This study introduces a novel MPS comprising different micropumps in conjunction with a tissue-incubation-chamber (TIC) for long-term porcine and human AV incubation (pAV, hAV). RESULTS: Tissue cultures in two different MPS setups were compared and validated by a bimodal viability analysis and extracellular matrix transformation assessment. The MPS-TIC conjunction proved applicable for incubation periods of 14-26 days. An increased metabolic rate was detected for pulsatile dynamic MPS culture compared to static condition indicated by increased LDH intensity. ECM changes such as an increase of collagen fibre content in line with tissue contraction and mass reduction, also observed in early CAVD, were detected in MPS-TIC culture, as well as an increase of collagen fibre content. Glycosaminoglycans remained stable, no significant alterations of α-SMA or CD31 epitopes and no accumulation of calciumhydroxyapatite were observed after 14 days of incubation. CONCLUSIONS: The presented ex vivo MPS allows long-term AV tissue incubation and will be adopted for future investigation of CAVD pathophysiology, also implementing human tissues. The bimodal viability assessment and ECM analyses approve reliability of ex vivo CAVD investigation and comparability of parallel tissue segments with different treatment strategies regarding the AV (patho)physiology.

Mitonuclear interactions shape both direct and parental effects of diet on fitness and involve a SNP in mitoribosomal 16s rRNA.

Nutrition is a primary determinant of health, but responses to nutrition vary with genotype. Epistasis between mitochondrial and nuclear genomes may cause some of this variation, but which mitochondrial loci and nutrients participate in complex gene-by-gene-by-diet interactions? Furthermore, it remains unknown whether mitonuclear epistasis is involved only in the immediate responses to changes in diet, or whether mitonuclear genotype might modulate sensitivity to variation in parental nutrition, to shape intergenerational fitness responses. Here, in Drosophila melanogaster, we show that mitonuclear epistasis shapes fitness responses to variation in dietary lipids and amino acids. We also show that mitonuclear genotype modulates the parental effect of dietary lipid and amino acid variation on offspring fitness. Effect sizes for the interactions between diet, mitogenotype, and nucleogenotype were equal to or greater than the main effect of diet for some traits, suggesting that dietary impacts cannot be understood without first accounting for these interactions. Associating phenotype to mtDNA variation in a subset of populations implicated a C/T polymorphism in mt:lrRNA, which encodes the 16S rRNA of the mitochondrial ribosome. This association suggests that directionally different responses to dietary changes can result from variants on mtDNA that do not change protein coding sequence, dependent on epistatic interactions with variation in the nuclear genome.

Cell culture as a toolbox to generate phase I metabolites for antidoping screening.

The knowledge of the biotransformation of compounds prohibited by the World Anti Doping Agency is of high concern as doping analyses are mostly based on the detection of metabolites instead of the parent compounds abused by athletes. While the self-administration of doping-relevant compounds is from an ethical point of view a rather problematic method to investigate metabolism, the usage of cell culture systems allows for studies on biotransformation in vitro. Five cell culture models with different tissue origin (liver, ovary, skin, kidney, and testis) were comparatively incubated with testosterone and epitestosterone as well as with the synthetic testosterone derivatives 17α-methyltestosterone and 4-chlorotestosterone to investigate the impact of synthetic modifications on phase I metabolic pathways. Cell culture supernatants were analyzed by high-performance liquid chromatography-tandem mass spectrometry. All cell lines possessed the default steroid phase I biotransformation reactions. The highest conversion rate was observed in ovarian (BG-1) and liver cells (HepG2). For BG-1 and skin cells (HaCaT), the 5α-reductase products 5α-dihydrotestosterone (for both) and 5α-androstane-3α/ß,17ß-diol (for BG-1 solely) were found to be prevailing after testosterone incubation. In kidney (COS-1) and HepG2 cells, the 17ß-hydroxysteroid dehydrogenase activity was predominant as supported by the observation that the 17α-OH (epitestosterone) and the methyl group (17α-methyltestosterone) impeded the conversion rate in these cell lines. In conclusion, future work should extend the characterization of the BG-1 and HepG2 cells on phase II metabolic pathways to examine whether they are suitable models for the generation of metabolite reference collections comparable to those obtained by human excretion studies.