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Method for Ultra High Throughput Transfection by Viscoelastic Cell Stretching

A microfluidic method of intracellular delivery in which cells are stretched via viscoelastic fluid at unparalleled speed and throughput


Executive summary

Drs. Mehmet Toner and Derin Sevenler have developed an ultra-high throughput microfluidic method of physically stretching the plasma membrane of cells without rotation to create temporary pores and facilitate efficient delivery of nanoscale cargoes by fluid forces alone and without touching any surface. This technology can deliver Cas9-sized biomolecules and CRISPR proteins at unparalleled speed and throughput, addressing the need for higher throughput delivery vehicles of gene and cellular therapies

While gene and cellular therapies are proving to be an emerging tool to combat genetic illnesses and cancer, manufacturing these therapies is laborious, inefficient, and expensive. For these therapies to be effective, hundreds of millions of cells are needed per dose. 

The product for this technology would be the box, disposable microfluidic chips, and the reagents, such as the transfection buffer.

The key innovation of this method is the addition of biopolymers to the solution, creating a synthetic mucus with viscoelastic fluid properties that stretch cellular membranes. Compared to electroporation, this method involves minimal cell membrane and organelle damage and triggers the natural calcium pathways that heal the cell membrane.


Unmet need

With more gene editing therapies becoming available, there is a major need for efficiently and safely transporting these therapies into cells. Existing delivery vehicles and methods have low throughput and damage the cellular membrane and organelles.

CRISPR gene therapies would require the transfection of trillions of cells, which this technology could meet. Further, CRISPR is ideally delivered in protein form, rather than as DNA/RNA. This technology can deliver CRISPR in protein form, which provides much better control over DNA-cutting activity. Other existing non-viral transfection methods cannot deliver such large cargoes.

Value proposition

This method combines best-in-class delivery efficiency of Cas9-sized biomolecules with unparalleled speed—more than 50 million cells per minute in a single microchannel. The microfluidic geometry is directly amenable to massive parallelization and scale-up, and only 10-20 channels would be needed to reach one billion cells per minute; this is between 100x and 1,000x higher throughput than all existing nonviral transfection methods.

Team

Mehmet Toner, PhD

Dr. Toner has a joint appointment as a professor of health sciences and technology at the Harvard-MIT division of health sciences and technology. He is a cofounder of the Center for Engineering in Medicine & Surgery, and founder of the NIH BioMicroElectroMechanical Systems (BioMEMS) Resource Center at Mass General.

He is internationally recognized for his multidisciplinary approach to biomedical problems in the areas of low-temperature biology and biostabilization, tissue engineering and artificial organs, and microsystems bioengineering in clinical medicine and biology.

Derin Sevenler, PhD

Dr. Sevenler is a post-doctoral fellow at the Center for Engineering in Medicine at Mass General and Harvard Medical School. His research focuses on new ways to diagnose and monitor cancer and infectious disease. He received his PhD in bioengineering and biomedical engineering from Boston University College of Engineering.

Alba Chacon Cabrera, PhD

Associate Director, Business Development & Licensing, Mass General Brigham Innovation
achaconcabrera@partners.org

Technology

Background and proof of concept

Inventors showed that viscoelastic cell stretching enables efficient delivery of effector molecules to difficult-to-transfect cell types and primary T cells (+100 million cells/minute in a single microchannel). Inventors evaluated the delivery of 70 kDa FITC-Dextran to MOLM-13, an acute myeloid leukemia cell line which is challenging to electroporate and observed > 90% delivery efficiency. The inventors had preliminary data with Jurkat cells supporting feasibility.

Advantages and progress

This technology has several advantages over existing transfection methods. This novel method of viscoelastic cell stretching results in fewer, larger pores in the cellular membrane for cargoes to be actively pumped into the cell (as opposed to diffusion). As a result, there is less cellular membrane damage and minimal damage to organelles, a major advantage over electroporation. Additionally, the microfluidic geometry is directly amenable to massive parallelization and scale-up, and only 10–20 channels would be needed to reach one billion cells per minute. This is between 100x and 1,000x higher throughput than all existing nonviral transfection methods.

Mechanism

The method is 10 uL–100 uL of delivery solution containing the cells and the cargo molecule is loaded into a length of tubing that is connected to the microfluidic chip. A valve is turned, applying regulated pneumatic pressure and driving the flow. The sample is fed through the outlet tubing to a collection tube. The throughput of a single microchannel is over 50 million cells per minute and only 10–20 channels would be needed to reach one billion cells per minute. The magnitude of the stretching force generally rises with increasing polymer concentration, larger flow rate, and higher contraction ratio of the channel.

Competitive advantages

Compared to electroporation, this method involves minimal cell membrane damage and triggers the natural calcium pathways that heal the cell membrane. This method also minimizes damage to organelles as compared to electroporation. With only 10–20 microchannels, a throughput of one billion cells per minute would be reached.

The market for DNA and RNA transfection alone is predicted to be worth $1 billion with applications across three core areas: (1) basic research, (2) biomanufacturing, and (3) cell-based therapies.

Prior strategies in the marketplace and the literature have shown efficient delivery of various difficult-to-deliver cargoes but have failed to address the field’s needs because they have major limitations compared to electroporation. Namely, they have high rates of failure (i.e., clogging), they require large amounts of expensive cargo material, and they are very slow compared to electroporation.

Publications

Leick MB, Silva H, Scarfò I, Larson R, Choi BD, Bouffard AA, Gallagher K, Schmidts A, Bailey SR, Kann MC, Jan M, Wehrli M, Grauwet K, Horick N, Frigault MJ, Maus MV. Cancer Cell. 2022 May 9;40(5):494-508.e5. doi: 10.1016/j.ccell.2022.04.001. Epub 2022 Apr 21.

Intellectual property

United States Provisional Application 63/136,669

WIPO PCT Application PCT/US2022/012098