Primary cell culture drug transport studies

Cultured nasal cells are reliable models for drug transport and metabolism studies, since they are known to express important biological features (e.g. tight junctions, mucin secretion, cilia, and various transporters), resembling those found in vivo systems. Moreover, easy control of experimental conditions as well as separation of the permeation step from the subsequent absorption cascade is also possible. A relatively simple primary culture condition using human nasal epithelial cells for in vitro drug transport studies has been established and applied in transport and metabolism studies of drugs. It is known that the culture condition and/or selection of culture media are critical in the recapitulation of well-differentiation features of in vivo nasal mucosal epithelium [46],... 

J. J. Yang, H. Ueda, K. Kim, and V. H. Lee. Meeting future challenges in topical ocular drug delivery Development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies. J Control Release 65 1-11 (2000). 

For in vitro toxicity studies and assessment of the barrier function, drug transport, cell physiology, and metabolism as well as the development of delivery systems, cell culture models provide powerful systems for scientific research. As the corneal epithelium is the main barrier for ocular penetration, various corneal epithelial cell cultures were established besides the corneal constructs that mimic the whole cornea and serve as reductionist models for the ocular barrier. In general, two types of cell culture models are available primary cell cultures and immortalized, continuous cell lines. 

The primary cell culture model is a more valid model for the study of absorption and transport processes of a drug via the pulmonary route. It provides a tight epithelial barrier with morphological and functional properties resembling those of the in-vivo condition. Primary alveolar epithelial cells from rats [39], rabbits [40] and humans [41] which display morphological and biochemical characteristics similar to the native epithelium have been isolated and can be used for drug transport studies. 

Over the last decades, cell and tissue cultures of renal epithelial cells have become powerful tools to study basic renal physiological functions, including transport, metabolism, and the effects of external stimuli such as oxygen tension, nutrition, hormones, and xenobiotics. Indeed, cultured renal tubular epithelial cells were one of the first cells utilized for investigating drug-induced injury. In this chapter, we describe the methods used to isolate different types of renal cells, aspects of primary cell culture, and tools to immortalize primary cells. We summarize the desired characteristics of various renal cell types and review the most widely used. Finally, we address important aspects of renal cell culture. 

In summary, primary cultures of hPT cells represent a unique and highly relevant experimental model for the study of drug metabolism, transport, mechanism of action, and nephrotoxicity. Although there exist the usual cautions with primary cell culture models, and cell culture models in general, once technique is mastered and appropriate conditions are used to minimize potential issues with the culture process, the primary hPT cells have numerous advantages over other models for obtaining information about drug action that is directly relevant to humans. 

Elbert KJ, Schafer UF, Schafers HJ, Kim KJ, Lee VHL, Lehr C-M (1999) Mono-layers of human alveolar epithelial cells in primary cultures for pulmonary drug delivery and transport studies. Pharm Res 16 601-608. 

Franke H, Gallah HJ, Beuckmann CT (2000) Primary cultures of brain microvessel endothelial cells A valid and flexible method to study drug transport through the blood-brain barrier in vitro. Brain Res Prot 5 248-256... 

The appropriateness of any cell culture model used to study drug delivery processes, whether a primary or a cell line, should be based on certain basic criteria. These include but are not limited to the presence of a restrictive paracellular pathway that allows effective characterization of transcellular permeability, the presence of physiologically realistic cell architecture reflective of the tissue barrier of interest, the expression of functional transporter mechanisms representative of the tissue barrier of interest, and the ease, convenience, and reproducibility of the culture methods.4 However, pharmaceutical scientists must realize that cell culture models in use today are not ideal with respect to these criteria, and there will be advantages and limitations with any choice. 

Recent advances in cell and tissue culture techniques provide the potential for evaluation of drug transport or metabolism processes at the placenta. Techniques are available for culturing trophoblasts of both animal and human origin.106 However, our focus here is primarily on human systems. Primary explant and isolated cell cultures of human cytotrophoblasts have been well described 106-109 however, these systems do not form confluent monolayer systems adequate for transcellular transport studies.105... 

Another barrier of interest for drug delivery studies is the blood-brain barrier (BBB). The BBB is formed by the endothelial cells of brain capillaries. The primary characteristics of the BBB are its high resistance to chemical diffusion and transport due to the presence of complex tight junctions that inhibit paracellular transport and its low endocytic activity. Several in vitro models of the BBB have been developed, and several authors have reviewed the models and their possible uses as permeability and toxicity screens (Reinhardt and Gloor, 1997 Gumbkton and Audus, 2001 Lundquist and Renftel, 2002). The most common in vitro BBB model consists of a monolayer of primary isolated brain capillary endothelial cells, primary isolated endothelial cells from elsewhere in the body, or an endothelial cell line cultured on a membrane insert. The endothelial cells are often cocultured with astrocytes or astroglial cells. In cocultures, the barrier properties of the BBB model increase. 

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