Maintenance Of Cell Lines Naturally Expressing nAChR

Cell lines, naturally immortalized through carcinogenesis, have been useful factories expressing some of the same kinds of nAChR found in related, non-neoplastic cells (Table 1.1).12 Limitations and attributes of cell lines in nAChR studies and examples of their uses in such work have already been described elsewhere in detail not to be duplicated here.12 However, to summarize, cell lines allow functional and ligand binding pharmacological profiles to be derived and integrated in one experimental system, taking advantage of the potentially limitless quantities of easily manipulated and comparatively homogenous material to accommodate many kinds of assays and experimental strategies. Cell lines also are suitable for somewhat indirect, but still useful and informative, assessments of nAChR pharmacology based on how receptor activation or blockade affects phenomena including gene expression, cell shape and survival, intracellular message levels, release of other chemical messengers, and enzyme activity.

If a given cell line expresses just a single nAChR subtype, then pharmacological characterization of that nAChR subtype is reasonably straightforward and usually is much simpler than pharmacological characterization of the same subtype from a tissue or brain region in which additional nAChR subtypes may be expressed. Some cell lines, such as those corresponding to autonomic neurons (e.g., PC12, SH-SY5Y, IMR-32), also may express multiple nAChR subtypes, requiring identification of

TABLE 1.1

Cell Lines Naturally Expressing Specific nAChR Subtypes nAChR Subtype

Cell Lines a1ß1y8-nAChR

TE671/RD human clonal line (rhabdomyosarcoma)

RM0 rat muscle cell line

BC3-H-1 and C2 mouse muscle cell lines

IMR-32 and SH-SY5Y human peripheral neuroblastoma lines

PC12 rat pheochromocytoma cell line

IMR-32 and SH-SY5Y human peripheral neuroblastoma lines PC12 rat pheochromocytoma cell line a3ß4*-nAChR

a7-nAChR

means for discriminating ligand binding and functional properties of those nAChR before pharmacological characterizations can be achieved confidently.

Maintenance of cell lines is both an art and a science. There are many monographs and instructional brochures pertaining to cell culture techniques that provide specific details that will not be repeated here. Use of sterile technique and manipulation of pathogen- or infectious agent-free cells in Class II Type A, laminar flow biosafety cabinets (recirculating all cabinet air through high efficiency particulate air (HEPA) filters and venting 30% of the filtered air back into the laboratory room) is central to handling the types of cell lines mentioned in this chapter that naturally express nAChR. These techniques help to ensure the safety of the investigator and also protect cells from investigator- or laboratory-introduced contamination. In cases where there are no assurances that cell lines being developed or used are pathogen-free, additional precautions, such as use of Class II Type B cabinets exhausting different proportions of cabinet air to the external (outside) environment, should be taken. Some cell lines can be safely manipulated under sterile conditions in open laboratory areas through the use of careful sterile technique and periodic ultraviolet illumination of the area, but costs of inevitable contaminations are likely to quickly surpass the investment in a suitable biosafety cabinet.

Conditions for cell incubation are quite standard, typically involving maintenance of cells at 37oC, at ~95% relative humidity to slow evaporation from media that would lead to concentration of salts and nutrients, and in 5% CO2 in air to help maintain neutral pH in bicarbonate-buffered medium. However, some studies indicate that nAChR assembly and levels of expression are higher in cultures maintained at lower temperatures.1314 In addition, it has been found that elevated CO2 levels facilitate proliferative effects of nAChR activation on pulmonary neuroendocrine carcinoma cells.15 Thus, careful consideration and pilot studies may be warranted before adopting standard cell incubation conditions for studies of cells expressing

Cell culture medium constituents can vary for maintenance of different cell lines and across laboratories. The laboratory has adapted a number of clonal cell lines to standard conditions of maintenance in Dulbecco's modified Eagle's medium (DMEM; high (4.5 mg/L) glucose, with 1 mM pyruvate, supplemented with 0.58 mg/ml (4 mM) L-glutamine), to which are added fetal calf serum (5% of DMEM

nAChR.

volume) and horse serum (10% of DMEM volume; final osmolality of serum-supplemented, "complete" medium is ~330 milliosmolar). However, studies have been performed to ensure nAChR expression by cells of the same passage is the same in this medium and in medium described in the initial report establishing the relevant cell line. In some cases, induction of nAChR expression requires change in growth medium. For example, serum deprivation and/or growth factor addition induces morphological differentiation and/or nAChR expression in some cell lines.1617 Cells are maintained on standard tissue culture plastic. Most often, 100mm diameter dishes are used for cell maintenance, but growth in 25 cm2 or 75 cm2 flasks as a precaution against microbial contamination is an option particularly recommended in climates or periods of high humidity.

Antibacterial and antifungal agents are common additives to cell culture medium to help combat microbial contamination. For example, the laboratory routinely supplements medium with penicillin (200 |/ml), streptomycin (200 |g/ml), and amphotericin B (2 |g/ml; ~2|M). Amphotericin B at ~120 |M is used for its pore forming abilities in perforated patch electrophysiological recording, but modest increases in basal 86Rb+ efflux from cells in the presence of 3 |M amphotericin B have been observed. Consequently, effects of cell culture additives on function of nAChR and rates of recovery from any such effects should be documented if those additives are to be present during or shortly before studies of nAChR function.

Each cell line has its own characteristic doubling rate. For the purpose of expedience, cells are typically passaged weekly when they approach confluence. Loosely adherent cells can be dislodged by streams of medium applied tangentially to the plate surface, whereas more adherent cells can be dislodged in this way after mild trypsinization. For the latter approach, bulk medium is aspirated and a small volume (1.5-2 ml per 100-mm dish) of 0.25% trypsin in calcium-free Hank's balanced salt solution is applied to the dish for a few seconds. Typically, the enzyme solution is aspirated and the dish is placed in the incubator for a few minutes (i.e., a period empirically determined to yield rounded-up cells dislodged from one another). For especially adherent cells or for economy, dishes can be rinsed with phosphate-buffered saline to remove serum proteins prior to digestion in 0.25% or 0.025% trypsin, respectively. Enzymatic digestion is terminated and cells are harvested by addition of about 1-10 ml of complete medium per 100-mm diameter dish; this medium is applied from a pipette as a laminar stream across the dish surface to dislodge cells. Harvested cells are suspended to a specific volume in fresh medium, serially diluted (typically no more than 1:10 in a given dilution to minimize cell clumping), and plated at initial densities that should yield cultures near confluence within one week.

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