Transcriptomic and western blot characterisation of the human CLEFF4 clone, a new rapid cell line replacement for the Caco2 model.

The CLEFF4 sub clone from stock late passage Caco2 cells has a unique property of being able to develop polarised cell monolayers with high P-gp expression and tight junctions much quicker than the original cell line. Instead of being useful for transport studies 21-24 days after initiating culture, the CLEFF4 cell line matures in 5-6 days with tight junctions surpassing that of 3 week old Caco2 cells in that time frame [1]. This has enabled the CLEFF4 cell line to provide measures of apparent permeability for potential drug candidates, so important for pre-clinical drug development, 4 times faster than the original cell line. RNA samples were collected and analysed at days 4 and 7 of culture over a 3 year period and had full RNA transcriptome analysed by the ranaseq.eu open bioinformatics platform. Protein was also collected from day 4 to day 22 of culture. Differential expression data from the FASTQ files have shown significant differences in expression in multiple genes involved with drug efflux, tight junctions, phase 2 metabolism and growth factors, which have been confirmed from protein determination that may hold the key to understanding accelerated human cell maturation. These gene expression results may be significant for other tissues beyond the gastrointestinal tract, and potentially for accelerated cell growth for the new field of laboratory grown tissues for organ replacement. The data also confirms the different genetic expression in CLEFF4 cells compared to Caco2 and the stable nature of the different expression over many years.