Aerosol Generation for Organs-on-Chips

Introduction

In vitro cell culture has been traditionally performed with fully submerged cells, where the reactive agent to be investigated is added to the cell culture medium completely covering the cells.  For primary contact organs such as the lung, the skin, or the eye, this represents an unrealistic way of exposure, since the in vivo exposure occurs at the air-liquid interface. To better mimics physiological conditions, cells need to be cultured at the air-liquid interface and  exposed to nebulized agents of interest.

Industrial approach to aerosol generation and delivery

Current commercially available systems for studying the effects of airborne substances on cells cultured at air-liquid interface suffer from an extremely low deposition efficiency. This is  because they require generation of large air flow rates for their optimum performance (e.g., due to using pneumatic nebulizers), exhaust almost all generated aerosol through scrubbers to waste,  and deposit only a small fraction of generated aerosols onto the cells cultured at air-liquid interface. 

On-chip aerosol generation

A radically different approach is to generate only enough aerosol for the deposited dose and avoid loses by producing it in the close proximity of the cells. Generating aerosol a few hundred microns away from the cells (with unobstructed path) dramatically increases deposition efficiency, virtually eliminates generation of toxic waste, reduces safety hazards and operational cost. Additional advantage of depositing aerosol from a short distance is possibility of localized deposition; for example, different nebulized entities can be deposited a very short distance side by side.

Advantages of generating aerosol a short distance from the cells:

• Waste is practically eliminated

• Cost and hazards are reduced

• Deposited dose can be controlled by metering the liquid

• Localized deposition of various entities is possible

• The same nozzles can be used for coating with extracellular matrix protein, cell seeding, and drug delivery

Challenges:

• Controlling the exit velocity of the droplets to avoid harming the cells

Fabrication of vibrating mesh nebulizer for aerosol delivery to the cells

Aerosol for delivery to cells at air-liquid interface can be generated using an existing technology of vibrating mesh nebulizer commonly used in inhalers. The main functional parts are a nozzle plate, piezoelectric element, and medicine chamber. [pdf]

Schematic diagram

Casting the medicine chamber

Fabricated aerosol generating device

Generation of aerosol by vibrating mesh nebulizer in continuous mode

Microfabricated vibrating mesh nebulizer featuring cast dual medicine microchambers and a metal nozzle plate with 22-micron pores operated in "full blast" continuos mode at resonant frequency of ~40 kHz. In this mode, exit velocity of the droplets is too high for delivery of drugs from a short distance to cells cultured at air-liquid interface.

Controlling the exit velocity of droplets to avoid harming the cells

An effective way of drastically reducing exit droplet velocity is to operate the vibrating mesh nebulizer in pulsed mode. By using 10-microsecond long voltage pulses issued at 2.5 ms repetition rate, it is possible to slow down exit droplet velocity to 25 mm/s. This low velocity makes feasible to deliver nebulized drugs from a very short distance (e.g., hundreds of microns) to cells cultured at air-liquid interface in microfluidic devices including organs-on-chips.

Nebulization of a single droplet by vibrating mesh nebulizer in pulsed mode

High-speed imaging of droplets exiting microfluidic nebulizer operated in pulsed mode

~10 micrometer droplets separated by ~50 microns exiting microfluidic nebulizer at exit velocity of ~25mm/s. To reduce exit velocity, vibrating mesh nebulizer was operated in pulsed mode with 10-microsecond long pulses at 2.5-ms repetition rate. High-speed imaging was performed at 3600 frames/s.

Single frame from the video