Investigating Buccal Cell Cultures To Enhance In Vitro Assessment Of Transmucosal Drug Delivery Affiong Iyire, Clifford Bailey, Afzal Mohammed School of Life & Health Sciences, Aston University, Birmingham, UK, B4 7ET, UK Email: a.u.r.mohammed@aston.ac.uk D espite advances in drug delivery via novel routes of administration, the oral route still holds high appeal because of its lower cost, ease of administration and patient acceptance/ compliance. However, setbacks of this route including drug degradation in the gastro- intestinal tract (GIT) and first-pass metabolism in the liver leading to low bioavailability, has pushed research in the direction of delivery via other non-invasive transmucosal routes such as rectal, vaginal, ocular, nasal/ pulmonary and the buccal/ oral cavity; with emphasis on oral/ buccal cavity [1]. The oral cavity is lined with stratified squamous epithelia covered by a mucus layer (figure 1). A basement membrane separates the epithelium from the supporting connective tissues called the lamina propria which is rich in blood vessels that empty into the jugular vein [2]. Below this is the submucosa. Physiologic opportunities for transbuccal drug delivery include easy accessibility, large expanse of smooth muscle and a relatively immobile mucosa when compared with that of the GIT [3], direct access to the systemic circulation (leading to high bioavailability) due to its high vascularisation, and by-pass of first-pass metabolism in the liver; substantial blood flow rate through the oral cavity vasculature which yields a somewhat constant & predictable drug concentration to the blood stream; and compared to the GIT, the oral cavity has lower enzymatic activity, a narrower pH range (5.8 – 7.6) maintained by saliva which is less mobile with less mucin/ enzymatic activities and practically no proteases [1, 3]. The major barriers to buccal drug delivery are posed by the stratified nature of the epithelium (40 - 50 cell layers), the presence of keratinisation and membrane coating granules (MCGs) present in the upper one-third of the epithelium which empty their lipid contents into the paracellular spaces there by inhibiting drug permeation; since drugs have been reported to permeate the buccal mucosa via the paracellular route [3]. One area of continuous concern is the availability of effective in vitro methods to assess buccal drug delivery which are reproducible and representative of in vivo expectations. Currently available in vitro methods involve the use of either excised animal buccal tissue mounted on diffusion (Franz cell-like) chambers, or the application of buccal cell cultures. Some setbacks with employing animal tissues include the low reproducibility due to inter-animal variations, difficulty in ensuring and maintaining viability of dissected tissue, limited availability of suitable animal model, as well as cumbersome and non-standardised methods of tissue preparation [4]. Buccal cell cultures can be utilised to overcome some of these challenges since there is high consistency and reproducibility when cells of a homogeneous population are used, and mass production can be achieved. However, inherent setbacks of cell culture including: being time consuming and requiring high sterility, the need to control lipid composition and stratification, inability to attain the same level of stratification (number of cell layers) as seen in vivo and phenotypic changes from donor organism as number of sub-cultures increase must be addressed [4]. At present, commercially available buccal cell culture models include the TR146 cell line (a continuous cell line derived from a neck node metastasis of a buccal squamous cell carcinoma), reconstituted human oral epithelium (RHOE) which are modified cells derived from the TR146 cell line showing higher differentiation than the TR146; and Epioral™ tissues which are reportedly the most developed of the available buccal cultures. However these models have not still achieved the level of stratification and differentiation expected in vivo, they are focussed on adults, and have high cost implications. Therefore, there is still a need for development of culture methods that can produce buccal cell cultures that closely resemble human buccal mucosa, including models for paediatric transbuccal delivery, using simple techniques that can be reproduced in any laboratory. The objective of this work therefore was to investigate the TR146 cell model with the larger aim of comparing the differentiation process of the cells with the anatomical make up in both paediatrics and adults. Experimental Growing TR146 cells purchased at passage 4 from Public Health England, were maintained (according to suppliers instructions) on 75cm3 T-flasks in Hams-F12 media fortified with 10% fetal bovine serum, 2mM glutamine, and antibiotics; incubated in 5% CO2 at 37⁰C. Media change occurred every 2-3 days, cells were sub-cultured at 2 Figure 1. A schematic diagram of the human buccal epithelium highlighting possible barriers to buccal drug delivery, adapted [2].