Indicator cell lines

The particular cell line used in our laboratory, AHH-1 TK-(-/- cells (Crespi and Thilly, 1984), is a versatile indicator cell line for a variety of in vitro toxicological end points (Crespi et al., 1993 Crofton-Sleigh et al.,... 

The RMK cell line, which exhibits cytopathic effect (CPE) in the presence of Influenza A(H1N1) or Avian Influenza A (H3N2) virus, was used as the indicator cell line in the infectivity assays. Cells in multiwell culture dishes were inoculated in quadruplicate with 0.1 ml of the dilutions prepared from test and control groups. Uninfected indicator cell cultures (cell controls) were inoculated with test medium alone. The cultures were incubated at 36-38° C. in a humidified atmosphere of 5-7% C02 in sterile disposable cell culture labware. The cultures were scored periodically for approximately seven days for the absence or presence of CPE, cytotoxicity and for viability. 

Lleonart, R., Naf, D., Browning, H., and Weissman, C. (1990). A novel, quantitative bioassay for type 1 interferon using a recombinant indicator cell line. Bio/Technology 8,1263 1267. 

A recommended indicator cell line is the Vero African green monkey kidney cell line, which has a high cytoplasm/nucleus ratio. The indicator cells are added at a concentration of approximately 1 x 10" cells ml to sterile coverslips in tissue culture dishes 10-24 h before inoculation. The test sample should be added at a concentration of approximately 5 x 10 cells ml" to give a semi-confluent mono-layer at the time of observation (at 1 and 3 days post-inoculation of the sample). Slides are prepared in the same way as those prepared by the direct method. 

In an alternative system, cells of the test culture can be inoculated onto coverslips preinoculated with an indicator cell line, such as the Vero African Green Monkey cell line. In this case the Vero cell should be inoculated at a cell concentration of 1 x 104 cells/mL and left for 4—24 h prior to addition of the test sample. The major advantage of this system, which overcomes the additional time required to set up, is the increased sensitivity achieved by the increased surface area of cytoplasm in Vero cells, which aids in revealing the mycoplasma. This system also enables the mycoplasma screening of serum and other reagents that can be inoculated directly onto the indicator cell line. 

HIV indicator cell line, Rev-CEM (NIH AIDS Research Reference Reagent Program, Cat 11467). 

Virus titer (TCID ) can be measured by infection of a Rev-dependent indicator cell line, Rev-CEM (23) see Note 4). An example of the result using Rev-CEM is shown in Fig. 2. 

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

Most recently, a phase-I-study defined a dose of 13-ris-retinoic acid that was tolerable in patients after myeloablative therapy, and a phase-III-trial showed that postconsolidation therapy with 13-cis-retinoic acid improved EFS for patients with high-risk neuroblastoma [7]. Preclinical studies in neuroblastoma indicate that ATRA or 13-cw-RA can antagonize cytotoxic chemotherapy and radiation, such that use of 13-cis-RA in neuroblastoma is limited to maintenance after completion of cytotoxic chemotherapy and radiation. It is likely that recurrent disease seen during or after 13-cis-RA therapy in neuroblastoma is due to tumor cell resistance to retinoid-mediated differentiation induction. Studies in neuroblastoma cell lines resistant to 13-cw-RA and ATRA have shown that they can be sensitive, and in some cases collaterally hypersensitive, to the cytotoxic retinoid fenretinide. Here, fenretinide induces tumor cell cytotoxicity rather than differentiation, acts independently from RA receptors, and in initial phase-I-trials has been well tolerated. Clinical trials of fenretinide, alone and in combination with ceramide modulators, are in development. 

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