Final Report Summary - BIOCELLCHIP (Integrated Microfluidic System for Long-Term Cell Cultivation, Monitoring and Analysis) We are pleased to recognize that this project has been particularly productive and innovative, with outstanding scientific, industrial and social economic impact. The main scientific goal of this project was development of microfluidic system for isolation of single cells from heterogeneous population, and its application in biomedical and biological research. The microfluidic systems developed during the outgoing phase (Harvard University) was eventually implemented at the host institution (Vilnius University), during the returning phase and since then has been applied for various biological and biomedical applications. Among different microfluidic devices and systems developed by the fellow, two platforms worth particular emphasis. The first microfluidic system allows cell entrapment, cultivation, monitoring and analysis under well-controlled conditions; provide a spatiotemporal control over biochemical and physical environment and ability to monitor the dynamics of cellular response in the presence of external or internal stimuli. This system was used to reproduce bone marrow niche ex vivo and to investigate how physical forces and biochemical environment affect megakaryocyte cell response and adaptation. The microfluidic bioreactor represents a first in vitro model of bone marrow vasculature and provides biomedical field with unique opportunity to recreate complex physiological organs on a chip (Blood, 2014, 124(12): 1857-1867). As a biological system fellow used human-induced pluripotent stem cell derived megakaryocytes to show how share rate can increase the yields of platelet production: a biological product that has a particularly high demand in hospitals and biomedicine sector. Using developed device the fellow and co-workers showed that increased share rate on trapped cells triggers complex cytoskeleton response and improves platelet production approx. 3-fold as compared to static conditions (Blood, 2015, 125(5): 860-868). The platform constituted the technological basis for filing international patent (WO/2014/107240) and establishment of a biomedical start-up company Platelet BioGenesis Inc. and represents one of the industrial impacts of the developed system. The second microfluidic system exploits cell compartmentalization approach to create microscopic aqueous compartments (droplets) to analyze heterogeneous populations at single cell level (Cell, 2015, 161(5): p. 1187-201). Like no other technology available to-date this approach allows tens of thousands of single-cells to be barcoded and sequenced in a massively parallel fashion. The basic principle of the platform is easy to appreciate: a mixture of cells is encapsulated into microfluidic droplets together with barcoded oligonucleotide primers (attached to hydrogel beads), reverse transcription (RT) and lysis reagent mix. The mRNA released from the lysed cells remains trapped inside the same droplet and is tagged (barcoded) with oligonucleotide primers during RT reaction. After barcoding step, the material from all cells is pooled by breaking the droplets, and the copy DNA (cDNA) library is processed for next-gen sequencing. As a proof-of-concept the system has been used to sequence over 10.000 embryonic stem cells with a throughput of ~8.000 cells per hour, for a cost of only $0.04-0.06 per cell. In a comparison, with state-of-the-art Fluidigm C1 platform researchers are able to generate 70-90 single-cell profiles in few hours for the cost of over a thousand US dollars. The fellow applied inDrops technique to identify the presence of rare sub-populations expressing markers of distinct lineages that would be difficult to classify from profiling only a few hundred cells. It was found that certain transcription factors fluctuate in a correlated manner across the entire cell population, and such fluctuations might be used to associate novel gene products with distinct cell states. The technique and methods describing different aspects of developed platform have been submitted for three patent applications (US 62/072,944; US 62/065,348; US 62/066,188) in a joint effort between Vilnius University (EU) and Harvard University (USA). The technological platform became a corner stone for establishing OneCell company, which is located in Boston and is aiming at exploiting technology for single-cell applications. Socioeconomic impact: The future prosperity of the host institution critically depends on young researchers returning from abroad and brining new technological capabilities and knowledge. By implementing microfluidics platform developed at Harvard the fellow has made significant contribution not only to the Lithuanian research landscape but also to the entire Baltics region. It is unique and very powerful technology that requires highly sophisticated skills and know-how experience. The fellow has been appointed as a group leader at Vilnius University Institute of Biotechnology and is currently leading a research group composed of 1 Post-Doc, 5 PhD students, 1 master student and 1 bachelor students. Finally, the fellow has brought new international collaborations from Harvard University (Prof. Dave Weitz), Harvard Medical School (Prof. Allon Klein), Columbia University (Prof. Dana Pe’er) and Caltech (Prof. Rob Phillips). It is absolutely clear that joint projects with leading research institution will strengthen the scientific quality of our institution significantly and will likely to bring a long lasting impact.
