Rapid, gentle, efficient and scalable cell engineering with microfluidic vortex shedding and Hydropore

Hydropore is an intracellular delivery platform based on microfluidic vortex shedding. Microfluidic vortex shedding relies on micron-scale posts and high fluid velocities to create hydrodynamic forces within a microfluidic device – these forces are capable of porating the cell membrane allowing for intracellular delivery. The Hydropore platform can be used to rapidly, gently and efficiently deliver nucleic acids and gene editing complexes to a variety of cell types such T cells and regulatory T cells. Additionally, intracellular delivery with Hydropore frequently requires significantly less construct per cell than electroporation and other nascent intracellular delivery platforms.

Genome editing T cells with Cas9 RNP and AAV typically results in >50% knockout and >20% knockin efficiencies. Editing efficiencies can be significantly improved by using homology-directed repair enhancers. Alt-R, for example, increased knockin efficiency from 24.1% to 51.5%. Hydropore-edited T cells also had a >4-fold higher initial cell viabilities when compared to Nucleofection. These very high initial cell viabilities (ie. 1-3 hrs post-processing) enhance the proliferation of Hydropore-edited T cells – a 1.6~1.9-fold increase in knockout and knock-in genome edited T cells was observed by day 7. Cumulatively, this means that Hydropore-edited cells also have improved function – a statistically significant increase in persistence or reduced activation induced cell death was observed within 6 hours for CD19 CAR-T cells. Hydropore has also shown to improve the modification of less frequent or rare cell populations. HLA-A2 CAR mRNA can be delivered to primary human regulatory T cells with >99% efficiency and 95% viability while using 3.8-fold less CAR mRNA per cell. Importantly, the same Hydropore instrument and device can be used to process between 1 and 100 million cells in less than 30 s providing a straightforward path from discovery-stage research to early stage translation.

Hydropore development is ongoing and future work is focused on scaling up and out the Hydropore platform for cell therapy while further verifying the improved function of Hydropore-edited cells in vivo. Ongoing Hydropore platform development is done in collaboration with a variety of pharmaceutical companies, biotechnology startups and academic institutes like Stanford, UC San Francisco and the Medical University of South Carolina.

• Hydropore can be used for the rapid, gentle, efficient and scalable cell engineering often with lower reagent requirements than electroporation and other nascent delivery platforms
• Hydropore improves the in vitro persistence of genome edited, CD-19 CAR-T cells