Cell line maintenance

Animal cells may be anchorage-dependent. Cells that depend on a solid substratum for growth are named adherent cells. These cells normally proliferate in monolayers and show contact inhibition, with the maximum cell yield generally limited by the available surface of the culture vessel. The yield of cells in suspension is not dependent on a solid substratum. 

Since adherent cells proliferate only after cell surface adhesion, an understanding of the steps of this process is very important. The first step consists of the adsorption of adhesion factors to the substratum, such as 

Name Cell type and tissue origin Morphology 

Vero African green monkey kidney Epithelial 

Suspension cells NSO Mouse myeloma Lymphoblastoid-like 

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Cell line maintenance Relatively easy More difficult... 

Lynn DE (1999), Comparison of cell line maintenance procedures on insect cells used for producing baculoviruses, In Vitro Cell Dev. Biol. (Animal) 35 248-251. 

If available, a potency assay developed for the batch release of the biological is a good starting point because it can often be adapted for use as a Nab bioassay. For example, a neutralization assay for GM-CSF can be a modification of the proliferation assay used for potency assessment for GM-CSF. This approach is advantageous, as optimization of cell line maintenance and culture conditions had already been performed. However, this assay needs to be tailored for use as a neutralization assay, as it may not be adequately sensitive for the purpose in its original format. Therefore, the performance of the potency assay in the presence of test sample (appropriate species serum) and response to the therapeutic to yield a sufficiently sensitive assay requires validation. Ideally, the cell line should yield a functional end point upon treatment with the therapeutic protein, the assay should be simple to perform, and the biological end point should be tolerant of the test sample matrix and perform adequately over a range of concentrations. 

The biochemical-biological basis for the preparation of the catalyst (isolation, culture of pure cell lines, maintenance of strains, enzyme preparation), all the problems of raw materials (the composition of the culture media), and all the problems of analysis (chemical analysis biochemical, microbiological, and physical methods). 

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. 

Androgen AR Human cell line Cancer, andropause, menopause, Sjogren s syndrome, idiopathic hirsutism, infertility, hypertension, mood disorders, diabetes, cystic fibrosis Immune modulation, vascular hemodynamics, neuroprotection, skeletal development, reproduction, maintenance of neuronal morphology, regulation of vasopressin Vla expression, prostatic blood flow and LH production... 

The cocultivation experiments using CB CD34+ cells thus confirmed our results obtained with the cell line TFl ectopic expression of membrane-bound SCF can substitute for stroma functions like long term maintenance and expansion of multipotent progenitors. 

Haan, K.M., Aiyar, A. and Longnecker, R. (2001) Establishment of latent Epstein-Barr virus infection and stable episomal maintenance in murine B-cell lines. J. Virol., 75, 3016-3020. 

Cell bank management insures the maintenance of the cell line original characteristics, consistency, use of cells with the same passage number, and also the availability of the cells for culture when required. 

The production of heterologous proteins for therapeutic use requires selection of the producer cell line, based on yield, monoclonality (for proteins), product quality, stability, and absence of contaminants like bacteria, molds, mycoplasmas, and viruses. Progress in the production of biopharmaceuticals by cell culture is due mainly to the use of diploid cells and continuous cell lines, together with the maintenance of cells by cryo-preservation. It is important to guarantee that the expression system chosen is able to generate the product in a consistent and economically feasible way (Levine and Castillo, 1999). 

The establishment of insect cell line cultures allowed a detailed study of the infection cycle of many baculoviruses. These cell lines are easily cultured in vitro and their maintenance is relatively simple. The majority of the cell lines can be cultivated using a temperature of 25-30°C. However, the best temperature for the growth and infection of Sf9 cells is around 27-28°C (King and Possee, 1992). 

NGF (nerve growth factor) — required for maintenance of neuronal differentiation. Obtained from human melanoma cell line A37S. 

One of the problems encountered with stable cell lines has been the loss of receptor expression as a result of long term maintenance of cells in culture. This can usually be avoided by freezing down large numbers of early passage number cells which can be revived as and when required. 

Many commercial suppliers (Table 10.3) have established protocols and procedures that address many of these issues. Primary human and animal cells of a variety of types are often available, sometimes as ready-to-use frozen stock aliquots or already added to 96-or 384-well plates. Specific culturing medium and detailed protocols for the correct maintenance of the cell line in culture are usually provided as well. This should facilitate the use of primary cells for primary HTS and compound validation. 

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