Continuously perfusable, customisable, and matrix-free vasculature on a chip platform.

Creating vascularised cellular environments in vitro is a current challenge in tissue engineering and a bottleneck towards developing functional stem cell-derived microtissues for regenerative medicine and basic investigations. Here we have developed a new workflow to manufacture vasculature on chip (VoC) systems efficiently, quickly, and inexpensively. We have employed 3D printing for fast-prototyping of bespoke VoC and coupled them with a refined organotypic culture system (OVAA) to grow patent capillaries in vitro using tissue-specific endothelial and stromal cells. Furthermore, we have designed and implemented a pocket-size flow driver to establish physiologic perfusive flow throughout our VoC-OVAA with minimal medium use and waste. Using our platform, we have created vascularised microtissues and perfused them at physiologic flow rates for extended time (>2 weeks) observing flow-dependent vascular remodelling. Overall, we present for the first time a scalable and customisable system to grow vascularised and perfusable microtissues, a key initial step to grow mature and functional tissues in vitro. We envision that this technology will empower fast prototyping and validation of increasingly biomimetic in vitro systems, including interconnected multi-tissue systems.

The Use of Comparative Genomic Analysis for the Development of Subspecies-Specific PCR Assays for Mycobacterium abscessus.

Mycobacterium abscessus complex (MABC) is an important pathogen of immunocompromised patients. Accurate and rapid determination of MABC at the subspecies level is vital for optimal antibiotic therapy. Here we have used comparative genomics to design MABC subspecies-specific PCR assays. Analysis of single nucleotide polymorphisms and core genome multilocus sequence typing showed clustering of genomes into three distinct clusters representing the MABC subspecies M. abscessus, M. bolletii and M. massiliense. Pangenome analysis of 318 MABC genomes from the three subspecies allowed for the identification of 15 MABC subspecies-specific genes. In silico testing of primer sets against 1,663 publicly available MABC genomes and 66 other closely related Mycobacterium genomes showed that all assays had >97% sensitivity and >98% specificity. Subsequent experimental validation of two subspecies-specific genes each showed the PCR assays worked well in individual and multiplex format with no false-positivity with 5 other mycobacteria of clinical importance. In conclusion, we have developed a rapid, accurate, multiplex PCR-assay for discriminating MABC subspecies that could improve their detection, diagnosis and inform correct treatment choice.