New accessible multiplex imaging workflow advances spatial liver research

Spatial biology technologies are reshaping modern biomedical research by enabling the visualization of cellular organization and interactions within intact tissues. In hepatology, this is particularly important because liver diseases such as metabolic dysfunction-associated steatohepatitis (MASH), cholangiopathies, fibrosis and hepatocellular carcinoma are driven by highly heterogeneous multicellular microenvironments. However, widespread adoption of multiplex imaging technologies remains limited by high costs, specialized instrumentation and complex analytical workflows. In a recent study published in eGastroenterology, Dr. Marlene S. Kohlhepp, Dr. Adrien Guillot, and colleagues described a versatile multiplex immunofluorescence (mIF) workflow designed to overcome these barriers. The authors established an integrated platform capable of analyzing liver tissues and engineered in vitro systems while relying primarily on conventional fluorescence microscopy and open-source computational tools.

Sequential multiplex immunofluorescence using standard laboratory infrastructure

The platform is based on sequential cycles of antibody staining, imaging and antibody stripping. Following each imaging round, antibodies are chemically eluted using a β-mercaptoethanol/SDS-based stripping protocol, allowing additional markers to be sequentially applied to the same sample.

Using this approach, the researchers demonstrated successful detection of approximately 10–15 markers within a single specimen while preserving tissue architecture across multiple staining cycles. Importantly, the workflow was optimized for formalin-fixed paraffin-embedded (FFPE) liver sections, which remain the standard material used in routine pathology laboratories.

The study showed that the method could simultaneously visualize multiple hepatic cell populations, including hepatocytes, cholangiocytes, macrophages, endothelial cells and hepatic stellate cells, thereby enabling detailed spatial characterization of liver microenvironments.

Extending multiplex imaging to organoids and liver-on-a-chip systems

A major innovation of the study is the extension of multiplex imaging beyond tissue sections. The investigators adapted the protocol to intrahepatic cholangiocyte organoids, primary liver cell cultures and advanced liver-on-a-chip platforms.

In organoids, multiplex imaging enabled assessment of epithelial polarity, proliferation and cell-cell junction integrity through simultaneous analysis of markers such as CK19, β-catenin, ZO-1, Ki67 and PCNA. In primary cell cultures, the method allowed spatial characterization of hepatocytes, macrophages, endothelial cells and stellate cells cultivated within the same chamber.

The platform was also successfully implemented in a biliary niche-on-a-chip system containing multiple liver cell populations. This demonstrates the feasibility of applying high-dimensional spatial phenotyping to engineered microphysiological systems, an area of increasing importance for disease modeling and drug testing.

CytoPrixm streamlines multidimensional image processing

To address the substantial computational burden generated by sequential multiplex imaging, the authors developed CytoPrixm, an open-source image-processing software package.

CytoPrixm integrates several essential preprocessing functions, including image stitching, background correction, channel alignment and DAPI-based image registration. The software was designed to reduce manual workload and improve reproducibility while minimizing the need for advanced coding expertise.

By coupling experimental multiplex imaging with accessible computational infrastructure, the platform aims to facilitate broader implementation of spatial phenotyping approaches across research laboratories.

Implications for translational hepatology

The study highlights the growing importance of spatially resolved analyses in liver disease research. Multiplex imaging can provide mechanistic insights into immune cell recruitment, ductular reaction, fibrogenesis and tissue remodeling, while also supporting validation of findings derived from single-cell and spatial transcriptomic datasets.

Importantly, the workflow described in this study emphasizes accessibility and scalability rather than maximal marker throughput. By using commercially available reagents, standard fluorescence microscopes and open-source software, the platform may substantially lower entry barriers to spatial biology research.

As spatially resolved molecular profiling becomes increasingly integrated into translational medicine, adaptable and cost-efficient imaging platforms such as this may contribute to the development of next-generation digital pathology, disease stratification strategies and precision hepatology.