Abstract
Nanotube stamping is a technique for delivering target molecules directly into adherent cells through nanotubes by inserting an array of nanotubes into the cell membrane [1]. The ability for direct intracellular delivery has the potential to evaluate the effectivity or toxicity of drugs on cells, and even to add new functions to cells by introducing functional proteins (Fig. 1). While conventional methods such as viral vectoring and electroporation have drawbacks such as affects by viruses and voltage pulses, nanotube stamping has the advantage of achieving both a higher delivery rate (85%) and cell viability after stamping (90%). However the nanotube stamping requires subcellular control of the relative positioning (height and inclination of the nanotube membrane) because it involves inserting many nanotubes from above into cells adhering to the scaffold. Specifically, deep insertion can crush the cells, while shallow insertion introduces few molecules. When stamping on cells with weak adhesion such as neurons, more precise control of the nanotube membrane position is required.
In this study, we evaluated the feasibility of functionalization (measurement of neural activity by fluorescent calcium imaging), especially for weakly adherent neurons, by precise control of the relative positioning of the nanotube membrane to the cell/scaffold interface using a control system integrating microscope optics, image processing, and stepper motor control. The nanotube stamping system was constructed to control the relative distance between the nanotube membrane and the cell membrane in combination with real-time image processing, which enabled molecular introduction with an accuracy of approximately 1 µm without relying on the operator’s skill. In addition, the membrane tilt was corrected to make the membrane parallel to the scaffold surface, and the focus of the phase contrast microscopy observation for viewing the cells was aligned with the coaxial epi-illumination for viewing the nanotube membrane.
Fluorescent molecule Calcein (concentration, 1.6 mM; molecular weight, 622.55 Da) was introduced into fetal 18-day-old rat cerebral cortex cells. In this experiment, the membrane was held for 10 minutes after nanotube insertion to deliver Calcein to neurons by diffusion. After releasing the nanotubes from the cells, the neurons were rinsed twice to remove fluorescent molecules in the medium. Fig. 1 shows clearly that the fluorescent molecules were successfully introduced into the cytoplasm of the neurons. In the presentation, we discuss the dynamics on the nanotube insertion and the cell/scaffold on interface.
Reference
[1] Miyake, Analytical Chemistry(2024)
