Efficiently delivering functional cargo to millions of cells on the time scale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent studies of intracellular delivery with thermoplasmonic structured surfaces show promising results but in most cases require time- or cost-intensive fabrication or lead to unreproducible surfaces. We designed and fabricated large-area (14 × 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.

The promise of human induced pluripotent stem cells (iPSCs) lies in their ability to serve as a starting material for autologous, or patient-specific, stem cellbased therapies. Since the first publications describing the generation of iPSCs from human tissue in 2007, a Phase I/IIa clinical trial testing an autologous iPSC-derived cell therapy has been initiated in the U.S., and several other autologous iPSC-based therapies have advanced through various stages of development. Three single-patient inhuman transplants of autologous iPSC-derived cells have taken place worldwide. None of the patients suffered serious adverse events, despite not undergoing immunosuppression. These promising outcomes support the proposed advantage of an autologous approach: a cell therapy product that can engraft without the risk of immune rejection, eliminating the need for immunosuppression and the associated side effects. Despite this advantage, there are currently more allogeneic than autologous iPSC-based cell therapy products in development due to the cost and complexity of scaling out manufacturing for each patient. In this review, we highlight recent progress toward clinical translation of autologous iPSC-based cell therapies. We also highlight technological advancements that would reduce the cost and complexity of autologous iPSC-based cell therapy production, enabling autologous iPSC-based therapies to become a more commonplace treatment modality for patients.

Intracellular delivery is crucial for cellular engineering and the development of therapeutics. Laser-activated thermoplasmonic nanostructured surfaces are a promising platform for high-efficiency, high-viability, highthroughput intracellular delivery. Their fabrication, however, typically involves complicated nanofabrication techniques, limiting the approach's applicability. Here, colloidal self-assembly and templating are used to fabricate large arrays of thermoplasmonic nanocavities simply and cost-effectively. These laseractivated substrates are used to deliver membrane-impermeable dye into cells at an efficiency of 78% and throughput of 30 000 cells min −1 while maintaining 87% cell viability. Proof-of-concept data show delivery of large cargoes ranging from 0.6 to 2000 kDa to cells without compromising viability.

Laser-exposed plasmonic substrates permeabilize the plasma membrane of cells when in close contact to deliver cell-impermeable cargo. While studies have determined the cargo delivery efficiency and viability of laser-exposed plasmonic substrates, morphological changes in a cell have not been quantified. We porated myoblast C2C12 cells on a plasmonic pyramid array using a 532-nm laser with 850-ps pulse length and time-lapse fluorescence imaging to quantify cellular changes. We obtain a poration efficiency of 80%, viability of 90%, and a pore radius of 20 nm. We quantified area changes in the plasma membrane attached to the substrate (10% decrease), nucleus (5 -10% decrease), and cytoplasm (5 -10% decrease) over 1 h after laser treatment. Cytoskeleton fibers show a change of 50% in the alignment, or coherency, of fibers, which stabilizes after 10 mins. We investigate structural and morphological changes due to the poration process to enable the safe development of this technique for therapeutic applications. Brown, "Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells," Blood 95(2),

The delivery of biomolecules into cells relies on porating the plasma membrane to allow exterior molecules to enter the cell via diffusion. Various established delivery methods, including electroporation and viral techniques, come with drawbacks such as low viability or immunotoxicity, respectively. An optics-based delivery method that uses laser pulses to excite plasmonic titanium nitride (TiN) micropyramids presents an opportunity to overcome these shortcomings. This laser excitation generates localized nano-scale heating effects and bubbles, which produce transient pores in the cell membrane for payload entry. TiN is a promising plasmonic material due to its high hardness and thermal stability. In this study, two designs of TiN micropyramid arrays are constructed and tested. These designs include inverted and upright pyramid structures, each coated with a 50-nm layer of TiN. Simulation software shows that the inverted and upright designs reach temperatures of 875 °C and 307 °C, respectively, upon laser irradiation. Collectively, experimental results show that these reusable designs achieve maximum cell poration efficiency greater than 80% and viability greater than 90% when delivering calcein dye to target cells. Overall, we demonstrate that TiN microstructures are strong candidates for future use in biomedical devices for intracellular delivery and regenerative medicine.