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Dolomite Bio’s Single Cell RNA-Seq System accelerating cancer research

Researchers at The Institute of Cancer Research (ICR), London, are taking advantage of the single cell encapsulation capabilities of Dolomite Bio’s Single Cell RNA-Seq System to investigate resistance mechanisms in prostate cancer. Karolina Nowakowska, a PhD student at The Institute of Cancer Research, explained: “Our team is focused on studying treatment resistance in prostate cancer patients, looking at the biochemical mechanisms responsible. My project is based on using single cell sequencing to help identify the specific genes which lead to treatment resistance, trying to identify biomarkers and, in the longer term, treatments which will allow a more personalized approach to therapy.”

Karolina-Nowakowska-ICR

“Initially I was using FACS to perform manual sorting of cells into individual wells, but this is both time consuming and laborious, limiting me to a maximum of 20 cells per experiment. As throughput is crucial in biomarker discovery, we wanted to perform our investigations on a much larger scale and, following the publication by Macosko et al1, we decided that microfluidics was the way forward. After a demonstration of the system at Dolomite Bio’s headquarters near Cambridge, we chose the Single Cell RNA-Seq System, and can now run over 1,000 cells per experiment. Although we have only just finished optimizing our protocols, the throughput is very impressive and the system offers good reproducibility. The beauty of this technique is that it allows you to view many cellular events simultaneously and, once optimized, it’s more cost effective than manually sorting and sequencing cells.” 

Disclaimer: The views stated are that of the individual, and not The Institute of Cancer Research

1 Macosko E et al. (2015). Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161(5), 1202-14.

Dolomite Bio offers flexible single cell encapsulation for autoimmune disease research

Dolomite Bio’s Single Cell RNA-Seq System is helping researchers at the University of Helsinki to investigate autoimmune diseases. Focusing on gastrointestinal conditions – such as coeliac disease and inflammatory bowel disease – the Molecular Genetics of Immunological Diseases group is using the system to study T-cell activation and response at the single cell level. Dr Päivi Saavalainen, Principal Investigator, explained: “We are primarily interested in the genetics and immunopathogenesis of these diseases, and the ability to study the molecular biology of individual lymphocytes is an important part of our work. I became interested in microfluidics for single cell encapsulation around a year ago, following the publication of the Macosko paper1. At the time, there was no bespoke system for performing the Drop-seq method but, by the time I had secured funding, Dolomite Bio was offering the purpose-made Single Cell RNA-Seq System.”

Testimonial

Dr Päivi Saavalainen, Principal Investigator at the University of Helsinki

“I’m not an expert in microfluidics, so being able to purchase a complete package – without needing to select my own chip design, pumps and tubing configuration – made the choice very easy. We have had the system since May, and I’m pleased with the throughput it offers. There was a bit of a learning curve, as we were not familiar with microfluidics technology, but we can now produce a few thousand individually encapsulated and barcoded cells in a single 15 minute run. Another nice feature of the Dolomite Bio system is that it’s quite flexible. This allows you to develop your own protocols, and we have been able to optimize the chemistry and our libraries to perfectly match our downstream processes.”

1 Macosko E et al. (2015). Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161(5), 1202-14.

Dolomite Bio lends a helping hand to synthetic biology research

Dolomite Bio's microfluidic chips are helping researchers from the Biodesign Institute at Arizona State University (ASU) to develop novel enzymesTestimonial capable of polymerising synthetic nucleotides. Using these chips, the team has created a droplet-based optical polymerase sorting (DrOPS) technique allowing rapid screening for novel polymerase activities in uniform water-in-oil microcompartments. The team’s leader, Professor John C. Chaput – formerly at ASU and currently at the University of California, Irvine – explained: “The creation of synthetic nucleic acids is of great interest to synthetic biologists but, because they are not found in nature, wild type polymerases struggle to process them. To overcome this issue, we are developing novel polymerases using directed evolution in water-in-oil microcompartments. The DrOPS methodology has significant advantages over traditional methods, which are both labour intensive and impractical to perform on a large scale due to the amount of precious artificial nucleotide reagents required for screening.”

The Biodesign Institute turned to microfluidics to allow rapid sorting and screening of novel polymerases, taking advantage of the technique’s single-cell encapsulation capabilities and picolitre reaction volumes. Dr Andrew Larsen commented: “We needed very reproducible microfluidics, and so using commercially available chips was preferable. We already had experience with Dolomite’s chips for a variety of applications within the institute, and they have always been very consistent, so the choice was obvious. These chips give us the ability to consistently generate uniform droplets – both single and double emulsion droplets – offering spatial separation between cells and allowing fluorescence-based sorting using conventional FACS technologies. Dolomite has also been very supportive of our efforts, helping to accelerate this area of research.”

Dr Adrian Briggs, Director of Molecular Biology R&D, AbVitro:

"Our aim is to develop new potential targets and therapies for a wide range of diseases, taking advantage of the specificity and sensitivity of theNews4 human immune system. To identify complete functional antibodies – pairing the correct heavy and light chains – we use a molecular barcoding technique that requires isolation of individual B or T cells. Achieving this with a flow sorter and microplate format is extremely slow and laborious, and so we looked for a reliable microfluidics solution that would enable much higher throughput."
"We investigated the various systems available, and Dolomite Bio seemed to be the most highly engineered, optimized and mature technology. Very precise control of pressure is crucial to reproducible, reliable droplet formation, and Dolomite offers high quality pumps, flow sensors and microfluidics chips, all fully automated via the Company's software."

"Since its introduction, the system has been in use almost constantly, and we have been very happy with the performance. The system's quartz chip, in particular, is very robust and long-lasting – we have been using the same chips since we first started – and the computer-controlled Mitos P-Pumps with flow sensors offer outstanding reproducibility."

"Using this set-up, we can process in excess of a million cells per experiment, allowing us to obtain over 300,000 full-length, natively-paired antibodies from a single sample. This kind of throughput would be impossible with traditional methods, as the cost and time required would be prohibitive, as well as having a poor success rate. Having a robust emulsion hardware solution has saved us significant research and development time, allowing us to proceed to product development and partnerships in studies of human disease sooner.

Associate Professor Moonsoo Jin from the Department of Radiology, Weill Cornell Medical College, New York, USA:

News2 "Some of our immunology research is focused on cloning antibody genes. In this work, it is important to ensure that individual genes are isolated from each other, and that is why we have adopted the droplet technology. It enables us to encapsulate a single human or mouse B cell, guaranteeing that antibody genes from one cell are not mixed with those from another cell."

"Biology itself is difficult enough, and so when we needed a microfluidic system for a new project we looked for a droplet generation system that was straightforward to use and not too complex."

"We chose Dolomite Bio, investing in Mitos P-Pumps and a quartz Droplet Junction Chip. We've had the system for about two years now, and our experience has been very positive. It is simple to use and reliable, and we've been able to generate stable, uniform-size droplets able to withstand subsequent molecular biology procedures in a very reproducible and robust manner."
"We plan to continue our work with B cells and, in the future, may also use the droplet generation system to look at T cells and T cell receptor diversity and specificity."

Flemming Jørgensen, Project Manager at Bioneer:

"Encapsulation of protein-producing cells inside gelatin micro droplets offers an affordable, easy to manipulate 3D cell culture technology for non-adherent cells. The small size of the droplets makes it easy for nutrients to diffuse into the droplets from the surrounding medium, and for cellular products to diffuse out, while providing a distinct 3D environment for cell growth. This technology has potential applications for the sorting and isolation of, for example, cells with very high protein yields, and we have already shown the Chinese hamster ovary (CHO) cell cultures continue to grow within this gel matrix with good viability."

"Starting with a premixed cell suspension containing 6% gelatin, the Micro Droplet System is used to create droplets approximately 200 μm in diameter. Addition of a cross linker shortly before droplet production results in the formation of soft hydrogel particles containing just a few cells – or even individual cells if desired, depending on the starting concentration – which are then resuspended in fresh cell medium."

"Dolomite's microfluidics technology makes droplet production very easy, allowing us to quickly produce large numbers of droplets for downstream analysis."