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

Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood–brain barrier coculture model

“…It is well established that changes in culture conditions and cell source can cause significant changes in protein expression of drug‐metabolizing enzymes, efflux proteins, etc., which are the focus of ongoing studies . In addition, modifications in media have been shown to have considerable effects on BMEC differentiation and tight junction formation .…”

“…To investigate the impact of direct coculture, an indirect coculture with astrocytes on the basolateral side of the Transwell was also examined. As mentioned, it is often difficult to compare models between different laboratories due to differences in culture protocol, media selection, passaging and cell source . Therefore, the indirect model was established under the same conditions and protocols as the direct contact coculture.…”

Evaluation of human peptide/histidine transporter 1 (hPHT1/SLC15A4) function: transport kinetics utilizing a HPHT1 shRNA stably transfected knockdown in the hCMEC/D3 blood-brain barrier cell line

“…These arrays were used to identify endogenous transporter expression in wild-type hCMEC/D3 cells, 1 evaluate the effect of media composition on transporter expression in HT-29 cells 30 and characterize transporter/metabolism enzymes in Caco-2 cells. 31 In the present study, these arrays were useful in identifying up or down regulation of transporters that may be regulated by the hPHT1 KD and serve as confounders for data interpretation by making a direct comparison between the hCMEC/D3 KD and hCMEC/D3-Control cells. The results presented here suggest that a variety of transporters' mRNA were either up or down regulated in response to reducing hPHT1 in the knockdown cells.…”

“…Transporter qRT-PCR arrays (SA Biosciences ® ) profiling the expression of 84 SLC and ABC transporter member sequences that are relevant to drug ADME were performed with the hCMEC/D3-hPHT1 KD and hCMEC/ D3-Control cells to assess if there were changes in drug transporter expression with respect to the knockdown of hPHT1, as described by our laboratory previously. 1,30,31 The cDNA's were prepared utilizing the SA Biosciences RT2 kit (C-03) according to the manufacturer's protocol. All qRT-PCR arrays were performed in triplicate, as described previously by our laboratory.…”

“…All qRT-PCR arrays were performed in triplicate, as described previously by our laboratory. 1,30,31 QRT-PCR was performed using the Brilliant II SYBR Green Mastermix from Stratagene. Results were normalized to the average of five housekeeping genes; β-actin, β-2 microglobulin, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1, and ribosomal protein L13a.…”