Passage number

In mouse models of skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), there is a close association between elevated XO activity in the epidermis and hyperplasia (Pence and Reiners, 1987). This association is also seen in psoriasis patients (Eisen and Seegmiller, 1961 Zimmer and Demis, 1966 Kizaki et al., 1977). In the study by Kizaki etal. (1977), the epidermis was increased about five-fold in comparison to normal. It is not known whether XO-derived ROS have any role in psoriatic epidermal hyperproliferation but low levels of hydrogen peroxide added to the culture medium are well known to induce skin fibroblast proliferation in vitro, an eflfect that is greatest at low passage numbers (Murrell et al., 1990). The generation of... 

When a fraction of the cells from an existing culture is placed in a new flask these transferred cells are said to have advanced in passage number. This action is called passaging, splitting cells, or subculture, and each cell line has... 

Procedure for the Chinese Hamster V79/Hgprt Assay. The assay usually comprises three test concentrations, each in duplicate, and four vehicle control replicates. Suitable positive controls are ethylmethane sulphonate (—S9) and dimethyl benzanthracene (+S9). V79 cells with a low nominal passage number should be used from frozen stocks to help minimize genetic drift. The procedure described includes a reseeding step for mutation expression. 

For the in vitro test, the fibroblasts are allowed to form a half-confluent monolayer within 24 h. Different concentrations of the test chemical are then incubated for 1 h with two sets of cells in parallel (typically on 96-well plates, 104 cells per well, passage number <100). After the incubation with the test substances, one set is irradiated with a nontoxic dose of UVA light (5 J/cm2), while the other set is kept in the dark. Twenty hours after irradiation, cell viability is evaluated by measuring the uptake of NR for 3 h. After the end of the absorption process, excess NR is removed and the cells are treated with an NR desorption solution (ethanol/acetic acid) to extract the dye taken up by the cells. Subsequently, the optical density of the NR solution is measured at 540 nm. As positive control, a test with chlorpromazine is performed. 

Briske-Anderson MJ, Finley JW, Newman SM (1997) The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proc Soc Exp Biol Med 214 248-257. 

Calu-3 (American type culture collection ATCC HTB-55) is a human bronchial epithelial cell line derived from an adenocarcinoma of the lung [59], This cell line has been shown to exhibit serous cell properties and form confluent monolayers of mixed cell phenotypes, including ciliated and secretory cell types [60], but the cilia are formed very irregularly and seem to disappear with increasing passage number (unpublished observations, C.E. and B.F.). Calu-3 cells have shown utility as a model to examine transport [61-63] and metabolism in human bronchial epithelial cells for many therapeutic compounds [64], Furthermore, they have been used in a number of particlecell interaction studies [65-67], The interactions between respiratory epithelial cells and particulates are discussed more in detail in Chap. 19. 

Studies were undertaken to quantify transporters and examine regional expression in the intestine. mRNA levels in the gut were studied by Englund et al. [35]. Nine transporters were examined and eight were shown to have significant regional differences in expression. In addition, up to a 20-fold difference in expression was observed for certain transporters between intestinal tissue and Caco-2 cells. Expression of transporters in cell models compared to normal tissue can be markedly different, depending on the age of the cells, passage number and culture conditions [35]. Cell models may under- or overexpress transporters. In addition cell lines may express transporters which may not be relevant in vivo. 

Significant interlaboratory differences in permeability measurements are observed with cell-based assays. It is important to standardize culture conditions and characterize a cell line within one s own laboratory. Permeability differences can be attributed to a number of factors, for example, heterogenecity of cell line, passage number, culture conditions, characteristics of the filter membrane, age of mono-layers and level of differentiation and experimental methodology used. Active... 

Add 1,990 pi complete medium containing the proper cell number (60, 70, and 80xl0 cells per dish) to the coated dishes. Optimal cell density may differ between labs, cell lines, and passage number. 

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 manufacturer must establish the maximum cultivation span for a particular cell in vitro (passage number). The characterization must include the growth rate, morphology, specific yield, and quality of the molecule of interest (Wiebe and May, 1990). The post-production cells (PPCs) must be removed from the bioreactor and tested at least once to evaluate whether new contaminants were introduced or induced by the cultivation conditions. Changes in the culture medium or production scale require new evaluation of the PPCs to determine any effects on the yield and product consistency (Levine and Castillo, 1999). 

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. 

The key to successful transfection is careful optimization of reaction conditions for each individual cell type. Cells of a lower passage number typically respond better to transfection and ensure higher efficiency than those with higher passage number. Also, some cell lines differentiate and change their features after many passages. Conversely, it is best to allow cells to settle down into... 

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