Deterministic Mechanoporation With Single-Pore Immune Cell Delivery
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Solution Overview
Problem
Existing intracellular delivery techniques face challenges in achieving efficient and scalable delivery of large cargos to difficult-to-transfect cells, such as primary and immune cells, while maintaining cellular viability, due to the stochastic nature of poration processes that often result in uncontrolled pore formation.
Innovation Solution
A deterministic mechanoporation (DMP) platform with a substrate having capture sites and sub-micron-scale projections that penetrate cell membranes, using hydrodynamic forces to apply controlled pore formation for uniform cargo delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If stochastic poration methods are used to enable efficient uptake of large cargos, then delivery efficiency is improved, but cellular viability deteriorates due to uncontrolled pore formation
Solution Approach 1:
The invention segments the poration process by providing each cell with a dedicated capture site containing a single projection, rather than exposing all cells to uncontrolled stochastic poration. This segmentation ensures that each cell receives at most one controlled pore, preventing the formation of multiple uncontrolled pores that would compromise cell viability while still enabling efficient cargo delivery.
Solution Approach 2:
The invention applies local quality by creating a controlled pore at a specific location (the capture site) on each cell membrane, rather than allowing random pore formation across the entire cell surface. The projection penetrates the membrane at a precise location, delivering cargo through a controlled local pathway while leaving the rest of the cell membrane intact and functional.
2Productivity
If multiple large pores are formed to enable efficient cargo uptake, then delivery efficiency is improved, but cellular damage increases compromising viability
Solution Approach 1:
The invention applies partial action by forming only the minimum necessary pore (a single pore per cell) required for cargo delivery, rather than forming multiple pores. This single controlled pore is sufficient for efficient uptake of large cargos while minimizing cellular damage that would result from multiple uncontrolled pores.
3Productivity
If conventional transfection methods are used, then scalability is improved, but transfection efficiency deteriorates in difficult-to-transfect cells
Solution Approach 1:
The invention uses copying by creating identical capture sites with projections on a substrate, where each capture site is a replicated unit designed to capture and porate cells with high efficiency. The substrate contains multiple copies of the capture site pattern, enabling scalable processing of large numbers of cells while maintaining consistent high transfection efficiency across all sites.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The DMP platform achieves high transfection yields of 20-100% in immune cells, exceeding conventional methods by 2-20-fold, with minimal cellular damage and efficient delivery of large-molecule cargos, particularly relevant for CAR T cell and TCR therapies.
Implementation Method 1
capturing the cells within the capture sites by applying a first hydrodynamic force; applying a second hydrodynamic force on the captured cell and locally rupturing the membrane and/or wall of the cell with the projection
Data Source
AI summary
Intracellular delivery of a genetic construct to immune cells including: obtaining a deterministic mechanoporation (DMP) platform that includes a substrate having a surface and a plurality of capture sites, each said capture site having a boundary shape at the surface adapted and configured to support thereon a cell, and each said capture site having a bottom and including a sub-micron-scale projection extending from the bottom toward the surface of the substrate, wherein said projection is adapted and configured to penetrate a cell membrane and/or wall of the cell, and wherein the substrate has a plurality of aspiration vias situated at the bottom of the capture sites; introducing the cells to the surface in a liquid media; capturing the cells within the capture sites by applying a first hydrodynamic force; applying a second hydrodynamic force on the captured cell and locally rupturing the membrane and/or wall of the cell with the projection, introducing the genetic construct into the cells, and releasing the porated cells from the capture sites. Also disclosed are methods of chimeric antigen receptor (CAR) T cell adoptive immunotherapy and T cell receptor (TCR) therapy.