Mechanoporation technologies achieve robust intracellular delivery while uniquely preserving cellular homeostasis.

Cellular preservation following processing on the Portal platform was evaluated via RT qPCR transcriptomic analysis of housekeeping genes and pluripotency markers in iPSCs, revealing expression profiles indistinguishable from untreated controls. These data confirm maintenance of the cellular native state and lineage fidelity which is essential for downstream therapeutic applications or maintaining proper target scaffolding in drug discovery screens.

Neuron progenitor cells, a traditionally undeliverable lineage due to high sensitivity to ionic flux and electrical stress, exhibited normal morphology and robust expression upon mRNA delivery via Portal’s technology. By maintaining native structural and functional integrity, our platform expands cell engineering opportunities in highly sensitive and underutilized cell populations.

Diffusion Preservation of Cellular Homeostasis and Native State

A single-step workflow enabled robust engineering of multiple modalities to achieve simultaneous CRISPR-mediated gene editing and high-magnitude mRNA expression. This multiplexing proficiency provides a powerful framework for complex biological modeling while completely avoiding cargo-specific biases, staggered expression timelines, metabolic burden, and reduced multi-modal yield associated with sequential transfection protocols.

We leveraged this capability to transiently engineer primary human T cells with circRNA constructs encoding a CD19-targeted chimeric antigen receptor (CAR) and membrane-bound interleukin-12 (mbIL-12). This single-step multi-modal delivery yielded rapid onset and high-magnitude co-expression of both therapeutic modalities, leading to enhanced tumor cell killing efficacy while circumventing permanent genomic manipulation and long-term cellular perturbation.

High-Fidelity Multiplexing Without Loss of Stoichiometry

Upon boosting with GFP self-amplifying RNA (saRNA), human iPSCs were used to generate cerebral organoids. Delivery of saRNA via Portal’s platform results in long-lasting GFP positive cells and does not disrupt lineage commitment or structural scaffolding during a 20-day cerebral organoid maturation protocol.

Transient Engineering for Rapid, Complex Cell Therapy

We validated three distinct implementations, serving as a foundation for a myriad of designs that capitalize on live-cell native MOA study: first, delivering the LgBiT protein directly into cells endogenously tagged with HiBiT facilitated precise, real-time kinetic monitoring of PROTAC-mediated target protein degradation; second, non-viral intracellular loading of an otherwise impermeable Src kinase tracer resulted in dose-dependent competitive displacement by Src small-molecule inhibitors; and lastly, intracellular co-delivery of the four antibody components of the Lumit immunoassay enabled live-cell, wash-free detection of intracellular kinase phosphorylation following pervanadate stimulation with signal-to-background ratios comparable to conventional lysate-based Lumit workflow. By eliminating artificial proxy models and preserving native target conformations, post-translational modifications, and cellular pathways, this direct-to-biology framework compresses validation timelines from months to hours while delivering deeper, physiologically accurate insights into candidate drug mechanisms.

Direct-to-Biology Insights for Early-Phase Drug Discovery

The porous 2D architecture of this platform enables full translational utility across operational scales with simple scaling of design footprint to accommodate throughput requirements, providing a unified framework that bridges the gap between early-stage discovery and large-scale therapeutic manufacturing. For high-throughput (HT) discovery applications, the low-pressure requirements of the silicon membrane enable seamless integration with existing automated cell dispensing instrumentation. This approach leverages a distinct attribute of mechanically-mediated poration: membrane closure can take up to a minute or more, allowing for diffusion of cargo into the cell after the poration event, and enabling a workflow in which cells can be mechanoporated during dispensing into a plate which is pre-loaded with cargo. This principle provides the foundation for automated delivery of cargo in 96- and 384-well formats, achieving uniform cargo delivery across thousands of independent samples without the need for specialized high-pressure infrastructure.

Unified Scalability from Research to Clinical Manufacturing

To evaluate the platform’s compatibility with a multi-step clinical manufacturing process, we performed a longitudinal study involving the sequential engineering of primary human T cells over a 10-day expansion period. The workflow commenced with research-scale delivery of CRISPR RNPs for B2M editing in unstimulated T cells, followed by a standard clinical activation protocol, with robust, >80-fold expansion and high viability up to clinical-scale densities. The population then underwent a second, clinical-scale boost of GFP mRNA using a porous membrane integrated in-line with the LOVO system, resulting in high-efficiency editing and near-universal mRNA expression.