Method for Ultra High Throughput Transfection by Viscoelastic Cell Stretching

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

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