Extracellular matrix permeability/efficacy assay tip (E-PAT) to realize three-dimensional cell-based screening

Abstract

In this work, an extracellular matrix permeability/efficacy assay tip (E-PAT), which can be used in a conventional pipette to simultaneously test drug permeability and efficacy, was developed by growing cells in the extracellular matrix (ECM) layer in a hole at the outlet (bottom) of E-PAT. In the existing permeability/efficacy assay technique conducted using the transwell assays, the Cell/ECM layers were formed on permeable polymer membranes; however, the production of small membranes to realize transwell miniaturization is expensive and difficult. To resolve this issue, in the proposed approach using E-PAT, small Cell/ECM layers were formed in the hole at the outlet (bottom) of the tip without membranes. A pipette was connected to the E-PAT to easily aspirate the Cell/ECM mixture into the hole of the tip, allowing the formation of miniature Cell/ECM layers at the bottom of the tip. Subsequently, the E-PAT was detached from the pipette and dipped into a well filled with media. The cells in the ECM grew three-dimensionally and made Cell/ECM layer in the E-PAT. The permeability/efficacy assays were performed using the Cell/ECM layer in E-PAT. The compounds or nanoparticles supplied through the inlet (hole) at the top of the E-PAT penetrated the Cell/ECM layers and were diluted by the media in the well. By measuring the concentration of the compounds or nanoparticles in the well and the cell viability in the Cell/ECM layer, the permeability and efficacy/cytotoxicity of the compounds in the proposed E-PAT could be concurrently estimated with a high throughput. In particular, E-PAT measured the IC50 (half maximal inhibitory concentration) for 3.383 μM doxorubicin (DOX) and indicated that 73 % 4 μM DOX penetrated the Cell/ECM layers, inducing the death of 57.2 % HepG2 cells. Moreover, the device demonstrated that FITC-SiO2 nanoparticles with a diameter of 25 nm permeated higher into the ECM and killed more HepG2 cells under the serum (fetal bovine serum, FBS) free media conditions than under the serum containing media conditions. The E-PAT could thus measure the compound efficacy as well as the compound permeability. The proposed tip can be a valuable tool to determine whether the efficacy/cytotoxicity of a compound is because of its low permeability.