Optimization of human nasal epithelium primary culture conditions for optimal proton oligopeptide and organic cation transporters expression in vitro

• Published in: International journal of pharmaceutics 2013-01, Vol.441 (1-2), p.334-342
• Main Authors: Shao, Di, Massoud, Emad, Clarke, David, Cowley, Elizabeth, Renton, Ken, Agu, Remigius U.
• Format: Article
• Language: English
• Subjects: Animals; Cattle; cell growth; Cells, Cultured; collagen; culture media; Culture Media - chemistry; cultured cells; epithelium; gels; gene expression; Gene Expression Regulation; Humans; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; messenger RNA; Nasal epithelium; Nasal Mucosa - metabolism; nose; OCT1-3; OCTN1; OCTN2; Oligopeptides - metabolism; Organic Cation Transport Proteins - genetics; Organic Cation Transport Proteins - metabolism; Organic cation transporters; Organic Chemicals - chemistry; PEPT1; PEPT2; Peptide transporters; plastics; Polymerase Chain Reaction; polystyrenes; quantitative polymerase chain reaction; Rats; Serum - chemistry; tissue culture; Tissue Culture Techniques
• ISSN: 0378-5173; 1873-3476
• DOI: 10.1016/j.ijpharm.2012.11.023

To investigate the effect of key tissue culture conditions on cell growth, gene expression and functional uptake of peptide and organic cation transporter substrates in the human nasal epithelium (HNE). HNE were cultured on different growth surfaces (polystyrene plastic, collagen film, and hydrated collagen gel) and were maintained with three popular nasal tissue culture media supplements [DMEM/F12 supplemented with Ultroser® G (2%), FBS (10%) and NuSerum® (10%)], respectively. The expression of gene transcripts for organic cation and peptide transporters were screened using qPCR and substrate uptake studies. Cell growth surface (polystyrene plastic surface, dried collagen film and hydrated collagen gel) did not significantly alter gene expression levels. However, Ultroser® G and FBS caused significant increase in PEPT1, PEPT2, PHT1, OCT3, and OCTN1 levels (≅2–5-fold for FBS and 2–8-fold for Ultroser® G). In terms of the degree to which the supplements affected gene expression, the following observations were made: effect on OCTN1>PEPT2>OCT3>PHT1>PEPT1. Functional uptake of organic cation (4-Di-1-ASP) and peptide [β-Ala-Lys (AMCA)] transporter substrates was significantly lower in cells cultured with NuSerum® compared to Ultroser® G and FBS cultured cells (p>0.05). Tissue culture media had a major effect on SLC gene expression levels of the human nasal epithelium in primary culture. Ultroser® G was identified as the most efficient culture supplement in maintaining SLC transporter expression under most culture conditions, whereas FBS appears to be an economical choice. We do not recommend the use of NuSerum® as a supplement for growing HNE for transport studies involving SLC transporters.