Air-liquid interface cultures

Typically bronchial epithelial cell layers were generated from primary epithelial cells after their propagation followed by their differentiation in air-liquid interface cultures [13, 113, 114], The cultivation in air-liquid interface conditions is absolutely essential for generating a ciliated epithelium since it resembles best the in vivo epithelium [115], Proliferative primary epithelial cells lose their capability to generate an epithelial cell layer after three to four passages. Future optimization might improve primary epithelial cell cultures in this aspect. 

Shimizu K, Endo O, Goto S, et al. (2001). Bioassay-based evaluation of toxicity of suspended particulate matter in humans integrated uses of alveolar cells (A549) in air liquid interface culture and hepatocarcinoma cells (Hep G2). Biochem. Eng. J. 22 1-9. 

Yamashita F, Mathias NR, Kim K-J, Lee VHL (1996) Dipeptide transport properties of rabbit tracheal epithelial cell monolayers cultured at an air-liquid interface. PharmRes 15 979-983. 

Of the different types of oral mucosal cell cultures that have been used [47,48], the most commonly used ones are explants of primary cultures. Small pieces of excised buccal or sublingual tissue are placed in a support system and fed with culture medium. The outgrowths obtained from these tissue explants are then transferred and grown in appropriate media. For example, outgrowths of fibroblasts [49] thus obtained have been described. Gibbs and Ponec [50] reconstructed the epithelium of mucosal tissue by placing a tissue biopsy (with the epithelial side upwards) onto a fibroblast-populated collagen gel. The explants obtained were cultured immediately at the air liquid interface until the epithelium had expanded over the gel (2-3 weeks). These explant cultures may retain many of the in vivo tissue characteristics. 

Epithelial barrier models for the skin [48,49], respiratory tract [50], BBB, and intestine [39] are constructed to study and predict the absorption, penetration, and metabolism of drugs or environmental toxins through these barriers. All the models are physically tight structures, and generally involve cells cultured at the air-liquid interface on porous membrane support, such as a porous polycarbonate filter. The use of a permeable support allows cells to be grown in a polarized state under more natural conditions promote Cell differentiation and enhance cell functions. 

Fig. 2 Workflow for air-liquid cultivation. This scheme can be adapted to most cell lines and primary cells. Cells are propagated as submerged cultures and seeded on top of porous filters. Both compartments are loaded with medium. Filter size, pore density and pore size, filter material, and coating are factors which affect the success of cultivation. Also the density at which the cells are seeded needs to be optimized. Proliferating cells from cell lines (e.g., NCI-H441, Calu3 cells) need lower densities, ranging between 20,000 and 60,000 cells/cm2, whereas primary cells need higher cell numbers ranging between 500,000 and 800,000 cells/cm2. Once cells form a stable and almost confluent cell layer, the medium has to be removed from the apical side. Usually the medium leaks into the apical compartment 2-3 days after air-liquid interface has been established. To maintain air-liquid interface conditions the medium should be removed from the apical surface at least once a day until the air-liquid interface becomes stabilized. When air-liquid interface condition has to be established, it might be necessary to replace the basolateral medium with a medium which propagates cell differentiation...

Pezzulo AA, Starner TD, Scheetz TE et al (2011) The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol 300(1 ) L25-L31... 

Lin HC, Li H, Cho HJ, et al. Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drag transport studies. J Hiarm Sci 2007 96 341-350. 

Determination of Cellulolytic Activity. The presence of cellulolytic activity of an organism was determined qualitatively by inoculating it into test-tubes containing the proper growth medium with a strip of filter paper and incubating the culture at 30 °C. If the organism is positive for cellulolytic activity, degradation of filter paper at the air-liquid interface could be noticed within 48 hrs. of incubation. Two different methods were used for quantitative determination of the activity ... 

Methods for in vitro reconstruction of three-dimensional (3D) differentiated human skin were developed during the 1980s (Bell et al., 1981). Cultured at the air-liquid interface (ALI), these in vitro reconstracted human epidermal (RhE) tissues display an in vivo-like stratified structure and functional epidermal layers including stratum comeum barrier. RhE may also be cocultured with dermal constructs containing viable fibroblasts (i.e., fuU-thickness models) as well as melanocytes (Fig. 12.1). The ALI culture format allows in vivo-like exposure and wounding scenarios including... 

Other models exist where the keratinocyte cultures grown at air-liquid interface are cultured in different substrates the SkinEthic model (Rosdy and Clauss, 1990), the Cosmital in-house model (Wella, Switzerland) (Faller et ai, 2002), and the Apligraf model (Organogenesis Inc., Novartis Pharmaceuticals Corporation, Canton, MA USA) (Medina et al, 2000). The SkinEthic is not a validated method and the other models need further development. 

The HCE-T Tissue Construct (Gillette model) uses a transfected human corneal epithelial cell line (Kahn et al., 1993) cultured on collagen-membrane cell culture inserts, which, at the air-liquid interface, stratify to form a four- to six-layer epithelium, known as the HCE-T model. Transepithelial permeabiUty (TEP) to sodium fluorescein and transepitheUal electrical resistance (TER) have been identified as physiologically relevant parameters to evaluate the barrier function of the corneal epithelium. Cell viability can be determined by the MTT assay, and histomorphology can also be used as an endpoint (Kruszewski et al., 1997). 

During cultivation, A. xylinun produces cellulose in form of pellicle with a highly swollen fiber network at the air/liquid interface of the culture medium. The cellulose pellicle is quite hydrophilic owing to the presence of pore structures and tunnels [24]. Backdahl et al. [25] reported that the nanofiber network was much denser close to the medium/air transition zone than on the opposite side, and the fine, highly entangled nanofibers with dimensions of approximately 100 nm were clearly observed in their SEM pictures. 

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