Cell Lines and Primary Cells

FIGURE 16.1 Morphology of commonly used gastrointestinal cell lines in monolayer culture. Representative gastrointestinal cell lines were cultured on 75 cm cell culture flasks with appropriate growth media. 

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Several opiate receptors have been identified on cells of the nervous systems of animals and humans, with mu (p), kappa (k), and gamma (y) subtypes being predominant. These classical opiate receptors are G- protein coupled 7-transmembrane molecules.27 Opiates predominantly affect immune responses directly by ligation of p, k, and y opiate receptors, as well as non-classical opiate-like receptors, on immune cells and indirectly by binding to receptors on CNS cells. Studies conducted in vitro with opiate-treated immune cells demonstrated receptor-mediated reduced phagocytosis, chemotaxis and cytokine and chemokine production. These effects are linked to modulation of host resistance to bacterial, protozoan, viral and fungal infections using animal models, cell lines and primary cells. 

The Bcl-2 family has been extensively investigated and has become the target of many approaches to modulate its expression and function. Bcl-2 antisense oligonucleotides are able to induce apoptosis in leukemia cell line and primary cells, and enhance chemotherapy-induced apoptosis (K4). This approach has already been implemented successfully in a phase I study in treating lymphoma patients (W7). 

Over the last four decades, there has been substantial progress in the development of dedicated, highly specialized delivery systems for nucleic acids in vitro as well as in vivo. Numerous different approaches addressing multiple established cell lines and primary cell cultures have been developed and refined. For most of the... 

Brand, A., Engelmann, J., Leibfritz, D. (1992). A 13C NMR study on fluxes into the TCA cycle of neuronal and glial tumour cell lines and primary cells. Biochemie 74,941-948. 

Krebs, F. C., Miller, S. R., Catalone, B. J., Fichorova, R., Anderson, D., Malamud, D., Howett, M. K., and Wigdahl, B. (2002), Comparative in vitro sensitivities of human immune cell lines, vaginal and cervical epithelial cell lines, and primary cells to candidate microbicides nonoxynol 9, C31G, and sodium dodecyl sulfate, Antimicrob. Agents Che-mother., 46,2292-2298. 

The use of nonneuronal cell types may provide initial information on whether a chemical may have differential effects, or display different potencies, in neuronal versus nonneuronal cells. For example, a battery of seventeen different cell types, including cell lines and primary cells (both neuronal and glial), human and rat cells, and nervous system and nonnervous system cells, was utilized to assess the toxicity of known developmental neurotoxicants, such as methyl-mercury and polychlorinated biphenyls (PCBs).24 Endpoints were cell viability and cell proliferation, and a summary of results for methylmercury and PCB-153 is shown in Table 8.5. This simple approach would flag methylmercury as a potential neurotoxicant, as toxicity was greater in neuronal cells than in other cell types. PCB-153 would also be flagged as a potential neurotoxicant, though... 

There are many other endothelial, epithelial, fibroblast cell lines, and primary cells that have been used for on-chip cultures. Some examples are as follows ... 

Cell culture models to evaluate the pulmonary fate of inhaled drugs have not been used extensively for characterizing inhalation drugs. However, this technique is promising and will be briefly discussed. This section focuses on airway and alveolar epithelial models using cell lines and primary cell culture methods. 

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...

Three-dimensional brain cell cultures can been obtained from different sources such as cell lines [38], primary cells [39], and stem cells [40]. Depending of the cells used the 3D culturing techniques can vary, e.g., stem cells are commonly forming 3D structures spontaneously, while cell lines and primary cells often need support by for example extracellular matrix, hanging drop, or rotation-mediated culture techniques. 

Morris, K.V., J. Gilbert, et al. 2004. Transduction of cell lines and primary cells by FIV-packaged HIV vectors. Mol Ther 10, 181-190. 

Emphasizing the importance of stealth, PMPC-PDPA polymersomes were able to encapsulate DNA physically with high transfection efficiencies in both cell line and primary cells [51,113], modestly affecting cell viability. Furthermore, the presence of the biocompatible PMPC chains on the external corona, coupled with the 200 00 nm vesicles dimensions, are likely to promote relatively long in vivo circulation times. 

Kamp, H.G., Eisenbrand, G., Schlatter, J., WOrth, K. Janzowski, C. (2005a) Ochratoxin A induction of (oxidative) DMA damage, cytotoxicity and apoptosis in mammalian cell lines and primary cells. Toxicology 206, 413-425. 

Mammalian cell cultures have been used as the basis of several systemsin detect ng the potential carcinogenic activity of chemicals. Basically, two general approaches have been utilized continuous cell lines and primary cell cultures. Cell lines have the advantage of ease of use, in that cultures do not have to be obtained fresh from animals prior to each test, but may be maintained for months to years by proper subculturing techniques. They have the disadvantage of possessing one or more "transformed" characteristics (e.g., immortality). In some cases cell lines may also lack certain enzyme systems required for metabolic activation of chemicals. Some of the cell lines used for transformation assays include the murine (BALB/3T3) A31 system ( ), and the baby kidney-21 (BHK-21) systems (43). 

As non-viral delivery vectors of nucleic acids, dendrimers have shown to be able to form stable dendriplexes by electrostatic interaction with nucleic acids these dendriplexes in general are biocompatible over a wide concentrations range, protect the nucleic material from attack of serum proteins or nucleases and are able to internalize these nucleic acids in a large number of cell-lines and primary cell cultures with great efficiency. 

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