The Effects of Media on Pharmaceutically Relevant Transporters in the Human HT-29 Adenocarcinoma Cell Line: Does Culture Media Need to be Controlled?

The effects of intralaboratory modifications to media composition and cell source on the expression of pharmaceutically relevant transporters and metabolizing genes in the Caco‐2 cell line

“…Determining whether mRNA expressional changes in other transporter or DME isoforms correlate with functional differences for other substrates is beyond the scope of this current study and would require many additional studies. The results presented here, along with results from our previously published work 33 , are intended to raise awareness and provide insights into the importance of characterizing cell lines with respect to media and culture conditions. If the data presented here are indicative of functional differences in other cell lines, then a substantial amount of variability may potentially be reduced through the use of standardized culture media.…”

“…33 Based on the results of those studies, we aimed to examine related media-dependent changes on the expression of 84 pharmaceutically relevant transporters and 84 DMEs, as well as the permeation of model substrates across two separate sources of Caco-2 cells.…”

“…33,35 Briefly, Caco-2 cells were seeded in 6-well plates in triplicate at a density of 5×10 4 cells/cm 2 . Media was changed every other day (or daily if necessary), and total RNA was harvested on day 21 post-seeding.…”

“…16,33–35 Briefly, cells were seeded onto 12 mm tissue culture treated, collagen-coated polyester membranes (0.4 μm pore size 3460 Transwells ® ) at a density of 5 × 10 4 cells/cm 2 . After seeding, the respective medium was changed every other day until the day of the study.…”

“…14 For example, recent data from our laboratory illustrated the potentially profound impact that media composition can have on genotypic, and selected phenotypic properties, of the human HT-29 adenocarcinoma cell line. 33 Based on our work with the HT-29 cell line 33 and the results presented in the current manuscript, it is our laboratory’s belief that a better understanding of induced transporter and DME expressional changes will allow pharmaceutical scientists to appreciate the limitations of permeability screening measurements and control for functional changes associated with seemingly minor differences in culture protocols across laboratories.…”