Majer et. al. (2024)

In this publication, Majer et. al., explore how optical microscopy techniques can be used to visualize and characterize cellular and sub-cellular features of liver cells in a liver microphysiological system (MPS). In this study, cell culture and antisense oligonucleotide (ASO) treatment were conducted using the PhysioMimix® OOC System and Liver-on-a-chip model before the 3D imaging of Liver-on-a-chip tissue.

The publication explores the groundbreaking potential of the approach to evaluate one of the biggest challenges in therapeutic ASOs: delivering oligonucleotides in sufficient concentration to the target tissue and cells of interest. This targeted delivery is crucial for reducing off-target effects and enhancing the specificity of gene silencing, both essential for safe and effective therapeutics. 

The study delves into the cellular uptake and localization of Alexa488 (A488)-labelled non-conjugated ASO and GalNAc-conjugated ASO using a PhysioMimix Liver-on-a-chip model. By employing both label and label-free imaging techniques, the authors provide a comprehensive analysis of these innovative delivery methods. 

While standard brightfield and fluorescence imaging are used to assess ASO uptake, they are limited in their ability to penetrate tissue deeply and provide 3D information. This study explored the potential of multimodal optical imaging to overcome these limitations. The team:

1. used multiphoton microscopy to track the uptake and distribution of fluorescently labeled antisense oligonucleotides (ASOs) in the liver-on-a-chip model
2. employed label-free hyperspectral stimulated Raman scattering (SRS) microscopy to analyze the chemical composition of different hepatocyte shapes and combined it with fluorescence microscopy to examine the overlap between ASOs and lipid droplets
3. demonstrated the use of light sheet fluorescence microscopy (LSFM) for 3D visualization of ASO distribution and cellular features.

One of the most intriguing observations is how the Liver-on-a-chip model narrows the gap between in vitro assays and the in vivo human liver environment. The study found that cuboidal primary human hepatocytes in the model exhibited different chemical compositions, morphologies, and ASO distributions compared to circular hepatocytes common in 2D culture. This finding queries the relevance of traditional 2D or 3D spheroid models for this purpose.

The authors cite that by replicating key aspects of liver function, such as drug metabolism, bile production, and immune responses in a controlled in vitro environment, these models offer a cost-efficient, robust, and human biology-relevant ethical alternative to traditional in vivo animal testing and the potential to accelerate drug discovery and development significantly. 

The study concludes that multimodal optical microscopy is a promising tool for visualizing and quantifying 3D cellular organization, drug distribution, and functional changes in liver-on-a-chip models, offering valuable insights into liver biology and ASO uptake.