Abstract
Artificial vascularized tubular liver tissue has perfusable blood vessels that allow fluid access to the tissue interior, enabling the injection of drugs and collection of metabolites, which are valuable for drug discovery. It is amenable to standard evaluation methods, such as paraffin-embedded sectioning, qPCR, and RNA sequencing, which makes it easy to implement into existing research processes. However, the application of tissues vascularized by the self-assembly of cells, (including tubular liver tissue, has not yet been tested in comprehensive proteomic analysis relevant for drug discovery. Here, we established a method to efficiently separate cells from the tubular liver tissue by adding a pipetting step during collagenase treatment. By using this method, we succeeded in obtaining a sufficient number of cells for the proteomic analysis. In addition, to validate this approach, we compared the cells separated from the tissue with those grown in 2D culture, focusing on the proteins related to drug metabolism. We found that the levels of proteins involved in metabolic phases II and III were slightly higher in the tubular liver tissue than those in the 2D cell culture. Taken together, our suggested method demonstrates the applicability of tubular liver tissue to the proteomic analysis in drug assays.
Highlights
These results indicated that the liver tissue with main blood vessels and capillary network was constructed normally, and the subsequent proteomic analysis was carried out
We have validated a method for proteomic analysis of tubular liver tissue, which is an artificial liver tissue with blood vessels, constructed using a perfusion device
By removing the tubular liver tissue from the device, and separating the cells from the collagen by collagenase treatment and mechanical dispersion, we succeeded in collecting enough cells for data-independent acquisition (DIA) proteomic analysis
Summary
Artificial liver tissue, in which hepatic parenchymal and non-parenchymal cells are combined and cultured with scaffold materials in three dimensions, has been actively developed as a tool for evaluating drug efficacy and hepatotoxicity in the field of drug discovery. This can be attributed to the fact that, unlike two-dimensional culture systems, it has a structure and function similar to that of the living liver [1,2,3,4,5,6,7]. The tubular liver tissue is highly compatible with existing molecular and cellular techniques
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