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

Abstract

The blood–saliva barrier (BSB) consists of the sum of the epithelial cell layers of the oral mucosa and salivary glands. In vitro models of the BSB are inevitable to investigate and understand the transport of salivary biomarkers from blood to saliva. Up to now, standardized, cell line-based models of the epithelium of the submandibular salivary gland are still missing for this purpose. Therefore, we established epithelial barrier models of the submandibular gland derived from human cell line HTB-41 (A-253). Single clone isolation resulted in five different clones (B2, B4, B9, D3, and F11). Clones were compared to the parental cell line HTB-41 using measurements of the transepithelial electrical resistance (TEER), paracellular marker permeability assays and analysis of marker expression for acinar, ductal, and myoepithelial cells. Two clones (B9, D3) were characterized to be of acinar origin, one clone (F11) to be of myoepithelial origin and one isolation (B4) derived from two cells, to be presumably a mixture of acinar and ductal origin. Clone B2, presumably of ductal origin, showed a significantly higher paracellular barrier compared to other clones and parental HTB-41. The distinct molecular identity of clone B2 was confirmed by immunofluorescent staining, qPCR, and flow cytometry. Experiments with ferritin, a biomarker for iron storage, demonstrated the applicability of the selected model based on clone B2 for transport studies. In conclusion, five different clones originating from the submandibular gland cell line HTB-41 were successfully characterized and established as epithelial barrier models. Studies with the model based on the tightest clone B2 confirmed its suitability for transport studies in biomarker research.

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

“…The isolated clone B2 from the human submandibular cell line HTB-41 (ATCC, Virginia, MA, USA) was cultivated in T25 flasks in McCoy’s 5A (Thermo Fisher Scientific, Waltham, MA, USA, 16600082), supplemented with 10% FCS and 1% Pen/Strep, termed as McCoy media, at 37 °C, 5% CO 2 , 95% air atmosphere, and 95% humidity. The isolation was performed previously by single cell experiments using HTB-41 at passage 8 resulting in clone B2 [ 22 ]. Cells were propagated weekly at a cell density of 8 × 10 3 cells/cm 2 and seeded at a density of 8 × 10 4 /cm 2 on 24-well ThinCert in 300 µL McCoy media on the apical side, with 900 µL media provided in the basolateral side for transport studies.…”

“…As a salivary gland model, we used a barrier model based on a single clone of the human submandibular cell line HTB-41, which was previously described on expressions of α-amylase, tight junction proteins and markers specific to different cell types of the salivary gland. Paracellular integrity was verified with transepithelial electrical resistance (TEER) and permeability studies [ 22 ]. As both models of the BSB were characterized on the expression and function of transporter proteins as well as tissue specific markers, they were applied for assessment of biomarker transport with ferritin recently and confirmed their suitability for transport studies in biomarker research [ 22 ].…”

“…Paracellular integrity was verified with transepithelial electrical resistance (TEER) and permeability studies [ 22 ]. As both models of the BSB were characterized on the expression and function of transporter proteins as well as tissue specific markers, they were applied for assessment of biomarker transport with ferritin recently and confirmed their suitability for transport studies in biomarker research [ 22 ].…”

State of the Art of Chemosensors in a Biomedical Context

“…Not at least due to the COVID-19 pandemic and the fact that COVID PCR testing was successfully performed from gurgle solutions and the feasibility of detection of COVID-specific antibodies in saliva could be demonstrated [242,243], saliva has most recently attracted wide interest as promising sample matrix for point of care and home testing. Though the understanding how diagnostically relevant biomolecules from distant tissues and organs reach saliva and thus can reflect disease states is still limited and respective blood-saliva barrier models have only started to be developed, e.g., [244,245] there has been recent evidence for saliva as liquid biopsy for successful detection of EGFR mutation analysis in lung cancer patients taking advantage of a novel core technology, electric field-induced release and measurement (EFIRM), which relies on a multiple flexible electrochemical sensor [246]. In addition, there has been support from a human lung cancer mouse xenograft model for the hypothesis that systemic nucleic acid biomarkers are carried and delivered into saliva via exosomes [247].…”

Models for Oral Biology Research

“…Considering the topic of Oral Biology, the blood-saliva barrier (BSB) consists of the epithelial cell layers of the oral mucosa and salivary glands, whose study is of importance to understand the transport of salivary biomarkers from blood to saliva. Lin et al [11] established epithelial barrier models of the submandibular gland derived from the human cell line HTB-41 (A-253). Experiments with ferritin, a biomarker for iron storage, demonstrated the applicability of the selected model for transport studies, which is of great importance since cell line-based models of the epithelium of the submandibular salivary gland are still missing for this purpose.…”