NEW YORK – Fluent BioSciences is hoping to get a slice of the single-cell transcriptomics market with its microfluidics-free, templated emulsification-based single-cell RNA sequencing technology, which the company says is flexible, scalable, and affordable.
The Watertown, Massachusetts-based firm has already commercialized its approach as scRNA-seq reagent kits, the utility of which was demonstrated in a Nature Biotechnology paper this week by a group led by Adam Abate, a professor at the University of California, San Francisco and a company cofounder.
Established in 2018, Fluent BioSciences built its core product upon so-called particle-templated instant partition (PIP) technology, invented by Abate’s lab at UCSF as a way to separate complex cell mixtures into monodispersed droplets using barcoded particle templates.
“Conceptually, what [Abate] had invented was an approach to create uniform reactions,” said Fluent BioSciences President and CEO Sepehr Kiani. “That would be very analogous to what you would see in a microfluidics device, but without the complexity.”
After raising Series A funding — led by Illumina Ventures and Samsara BioCapital — in 2020, Fluent BioSciences pivoted the PIP technology to single-cell transcriptomics applications, Kiani said. The company has since been developing particle-templated instant partition sequencing (PIP-seq) and commercializing the method into products for scRNA-seq.
“What we found was that the market had rigid solutions that were complicated to run, expensive, and didn't have the cost-scale economics that people needed,” Kiani said. “We saw there was a need for a flexible, scalable, and cost-effective solution in the [scRNA-seq] market.”
PIP-seq’s workflow starts with sample preparation where cells or nuclei are mixed with template particles (hydrogel beads with barcoded templates) to generate monodisperse emulsions by vortexing. As a result, cells and particles are co-encapsulated into droplets without the need for microfluidic instrumentation or consumables.
The cells are then heat-treated and lysed by proteinase K, releasing cellular mRNA that can be captured by the barcoded oligonucleotides that are incorporated in the template particles. After that, the captured mRNA undergoes reverse transcription to generate cDNA libraries, followed by adapter ligation, sample indexing, and cleanup steps for Illumina sequencing.
Downstream, the company has developed data analysis software called PIPseeker, which offers users primary analysis — such as summary metrics, clustering, and differential gene expression tables — for the PIP-seq sequencing libraries as well as standard feature-barcode count matrices that are compatible with tertiary analysis.
“One of the things we have really learned is that the informatics tools [for single-cell analysis] are really hard to use,” Kiani pointed out. “By producing an informatics tool that has enough tertiary analysis for a lot of the basic work, you can basically run [PIP-seq] out of the box without really any informatics experience.”
He also noted that the PIPseeker software can run locally or in a cloud environment, and that it will soon be available as an app on Illumina’s BaseSpace Sequence Hub.
In the Nature Biotechnology study, the authors concluded that PIP-seq “fills an unmet technical need by improving the speed, scalability, and ease of use of single-cell sequencing.”
Specifically, they found that PIP-seq can generate “accurate single-cell gene expression profiles” from human tissues and is compatible with multiomics measurements. The research team also applied PIP-seq to monitor the response of individuals with mixed phenotype acute leukemia (MPAL) to chemotherapy. The results showed that PIP-seq can uncover cellular heterogeneity within chemotherapy-resistant cell subsets that were missed by standard immunophenotyping.
Currently, Fluent BioSciences offers three kit configurations for PIP-seq, varying in reaction throughput. Specifically, the PIP-seq T2 kit can process up to 2,000 single cells per reaction, with each reaction priced at $300. The PIP-seq T20 kit, designed to analyze up to 20,000 single cells per reaction, sells for $900 for each reaction. Finally, the company’s PIP-seq T100 kit is capable of profiling 100,000 single cells in a single reaction and is quoted at $3,200 per reaction.
In terms of turnaround time, Fluent BioSciences said its PIP-seq kits can produce sequencing-ready libraries from single-cell suspensions “in a convenient two-day workflow.” The company also noted that after PIP-seq’s cell capture and lysis step, which takes about one and a half hours, there is a stopping point, allowing users to store the material at 0°C for up to 72 hours.
“We have a very nice stopping point,” said Kiani. “You can bring [the experiment] to the stopping point, stabilize your RNA, stabilize your sample, and be able to ship it to a core lab to be processed.”
With that leeway, he noted that while Fluent Biosciences considers the reagent kits its primary business focus, the company currently also offers PIP-seq as a service “to bridge the gap while core labs come up to speed" with the technology. Additionally, Kiani said the company has a formal program for labs to become qualified service providers, and there are several service labs currently “in the pipeline to get up and running.”
Currently, the PIP-seq RNA kits are designed for use with Illumina sequencing. However, Kiani said there were customers successfully running the product using Oxford Nanopore Technologies’ platform. As for formalin-fixed, paraffin-embedded (FFPE) samples, he said the company has not demonstrated an off-the-shelf solution yet.
Going forward, Kiani said the company plans to continue improving its products and technology. After officially launching the first PIP-seq product last July, the company released its newest chemistry at the Advances in Genome Biology and Technology (AGBT) annual meeting last month, promising a cell capture rate of up to 85 percent.
In addition to transcriptomics, Fluent BioSciences also has a PIP-seq epitope sequencing kit in early access. For use in combination with the single-cell RNA-seq kits, the epitope assay allows multiplexed protein marker detection and cell hashing using polyadenylated oligonucleotide-tagged antibodies, the company said.
While Kiani did not outline a concrete product roadmap for Fluent BioSciences, he said the company is currently “super focused” on its existing products and customers and doesn’t want to “dilute that message with other products yet.”
More than half of the firm’s customers at this point are in academia, while therapeutics companies are “really up and coming” as customers, he said. “We're somewhat market agnostic,” he added. “We're not staying out of particular markets; we are very rapidly growing our market configuration as an organization.”
As for intellectual property, Kiani said Fluent BioSciences has secured a worldwide exclusive license for the PIP-seq technology from UCSF, and the company has its own IP portfolio, as well.
With just over 40 employees, Kiani said the company is currently “a very lean and mean commercial organization” and will continue to expand based on market demands. Additionally, he said the firm is currently working to close its next financing round to support its commercial growth.
“We just came out of our cocoon seven and a half months ago,” he said. “We're just spreading our wings right now.”