• Traditional 2D cell cultures are convenient but don’t accurately represent real-life conditions, especially in disease models. They struggle to mimic patient tissues for drug testing and disease modeling. In 2D cultures, drug diffusion is limited, making it hard to control dosages.
• 3D spheroids, with their 3rd dimension, better mimic in-vivo responses to external factors like physical and chemical signals. Traditional methods for introducing substances into cells, like viruses or chemicals, have issues like invasiveness and contamination risks.
• Laser-based methods, like optoporation, offer a solution for uniform and localized substance delivery. They are efficient, selective, reproducible, and don’t require physical contact. Ultrashort laser pulses are particularly effective for creating tiny pores in cell membranes, allowing substances to enter while keeping cells healthy.
• To address the above mentioned issues, the present patent is introduced.

The present patent discloses a hanging drop cell culture followed by pulse laser assisted high throughput intracellular delivery method, (Refer FIG 1, 2, 3); the present patent comprising:

• preparing hanging drop consisting of SiHa cells by trypsinizing the cultures cells wherein the supernatant are removed and cell pellets are r-suspended in cell culture medium (DMEM-F12 (Invitrogen) supplemented with 1% MEM Non-Essential Amino Acids (Gibco), 5% fetal bovine serum and 1% penicillin-streptomycin);
• incubating the spheroids with corrugated Gold (Au) nanoparticles to facilitate optoporation wherein the particular corrugated shaped Au-nanoparticles generate high surface plasmonic resonance owing to high electromagnetic field enhancement under exposure of nanosecond pulsed laser wherein cells are exposed at 680nm wavelength, 9 mJ energy for 30 sec;
• and using Propidium Iodide (PI) dye as cargo to be delivered inside spheroid in hanging drop culture wherein after photoporation cells are stained with Calcein dye to test viability of cells.