Optimization of an oral mucosa in vitro model based on cell line TR146

Abstract

During the last years, the popularity of saliva has been increasing for its applicability as a diagnostic fluid. Blood biomarker molecules have to cross the blood-saliva barrier (BSB) in order to appear in saliva. The BSB consists of all oral and salivary gland epithelial barriers. Within this context, the optimization of in vitro models for mechanistic studies about the transport of molecules across the oral mucosa is an important task. Here, we describe the optimization and comprehensive characterization of a Transwell model of the oral mucosa based on the epithelial cell line TR146. Through systematic media optimization investigating 12 different setups , a significant increase of barrier integrity upon airlift cultivation was achieved for TR146 cell layers. The distinct improvement of the paracellular barrier was shown by measurements of transepithelial electrical resistance (TEER) and carboxyfluorescein permeability assays. Histological characterization supported TEER data and showed a stratified, non-keratinized multilayer of the optimized TR146 model. High-Throughput qPCR using 96 selected markers for keratinization, cornification, epithelial-mesenchymal transition, aquaporins, mucins, tight junctions, receptors, and transporter proteins was applied to comprehensively characterize the systematic optimization of the cellular model and validate against human biopsy samples. Data revealed the expression of several genes in the oral mucosa epithelium for the first time and elucidated novel regulations dependent on culture conditions. Moreover, functional activity of ABC transporters ABCB1 and ABCC4 was shown indicating the applicability of the model for drug transport studies. In conclusion, a Transwell model of the oral mucosa epithelium was optimized being suitable for transport studies.

Cytokine-Mediated Inflammation in the Oral Cavity and Its Effect on Lipid Nanocarriers

“…Further work by Lin at al. showed that when culture conditions of TR146 cells were optimized by e.g., adjusting the cell culture medium, barrier properties could be improved, making the model suitable for (drug) transport and permeability studies [ 24 ]. Apart from drug permeability, TR146 cells are also used to study nanoparticle interactions and cytotoxic effects.…”

Directed Transport of CRP Across In Vitro Models of the Blood-Saliva Barrier Strengthens the Feasibility of Salivary CRP as Biomarker for Neonatal Sepsis

“…Primer sequences are shown in Table 1 and were verified with PrimerBLAST [ 25 ]. Prior to usage in qPCR, amplicon sizes were evaluated using 2% agarose gels, described in detail in Lin et al [ 21 ], followed by a two-fold dilution series for calculation of primer efficiency performed via qPCR similarly as described by Pfaffl [ 26 ]. Primer efficiency was required to be 80–120% to pass for further usage.…”

“…To further investigate the relevance of CRP as a salivary biomarker, we aimed to study the transport of CRP across in vitro models of the blood–saliva barrier (BSB), defined by the epithelial cell layers of the oral mucosa and salivary glands [ 20 ]. The human buccal mucosa carcinoma cell line TR146 was chosen as an oral mucosa model, which was recently thoroughly characterized by Lin et al [ 21 ] for its biological barrier properties including expression studies of 96 relevant barrier molecules (cytokeratins, mucins, aquaporins, epithelial-mesenchymal transition (EMT) markers, and tight junction proteins among others) and validation against human buccal biopsy samples. Additionally, the integrity of the paracellular barrier was improved by optimization of the cultivation media and conditions.…”

An In Vitro Barrier Model of the Human Submandibular Salivary Gland Epithelium Based on a Single Cell Clone of Cell Line HTB-41: Establishment and Application for Biomarker Transport Studies

“…Parental HTB-41 cells were seeded in McCoy media at passages 5-12, isolated clones from cell line HTB-41 were seeded at passage 5-12 after single cell cloning at passage 8. Seeding procedure of TR146 was described in detail previously [22].…”

“…Experiments were performed on day 29 after seeding. Cultivation and seeding procedure of TR146 cells in Transwell ® models was described in detail previously [22]. On the day of transport studies TEER was measured in both models as described above in the respective cultivation media, washed twice on the apical and basolateral side with 300 µL or 900 µL basal McCoy media (McCoy's 5A media without supplements) for clone B2 or Hank´s Balanced Salt Solution (HBSS; Sigma-Aldrich; St. Louis, MO, USA; H6648) for TR146.…”