Cell culture immortalization

Occasionally, some of the animal cells in short-term cultures do not die, but instead survive indefinitely. These types of animal cell cultures, which can divide indefinitely, are called established cell lines. Established animal cell lines have been obtained from both normal and tumorigenic cells. Immortalized animal cell lines have also been successfully obtained from short-term cultures following their transformation with appropriate oncogenes. 

Monoclonal antibody technology entails isolation of such B-lymphocytes, with subsequent fusion of these cells with transformed (myeloma) cells. Many of the resultant hybrid cells retain immortal characteristics, while producing large quantities of the monospecific antibody. These hybridoma cells can be cultured long term to effectively produce an inexhaustible supply of the monoclonal antibody of choice. 

For in vitro toxicity studies and assessment of the barrier function, drug transport, cell physiology, and metabolism as well as the development of delivery systems, cell culture models provide powerful systems for scientific research. As the corneal epithelium is the main barrier for ocular penetration, various corneal epithelial cell cultures were established besides the corneal constructs that mimic the whole cornea and serve as reductionist models for the ocular barrier. In general, two types of cell culture models are available primary cell cultures and immortalized, continuous cell lines. 

Table 12.1 Immortalized human corneal epithelial cell culture models.

Reconstituted HCE (order no. RHC/S/5) is available from Skinethic, Nice, France. It consists of transformed human corneal epithelial cells immortalized by Roger Beuerman from the Louisiana State University-Eye Center, New Orleans, USA. This reconstituted human corneal epithelium forms a multilayered cell culture model that does not exhibit tight junctions and is therefore unsuitable for in vitro drug transport studies. Main focus of the model is eye irritation and toxicity testing and it is commonly used in this area with good prediction qualities. Major advantage of this model is the high reproducibility and uniform appearance. The commercial availability provides a ready-to-use model that is easy to handle. 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

The body s cells are normally subject to strict social control. They only divide until they come into contact with neighboring cells cell division then ceases due to contact inhibition. Exceptions to this rule include embryonic cells, cells of the intestinal epithelium (where the cells are constantly being replaced), cells in the bone marrow (where formation of blood cells takes place), and tumor cells. Uncontrolled cell proliferation is an important indicator of the presence of a tumor. While normal cells in cell culture only divide 20-60 times, tumor cells are potentially immortal and are not subject to contact inhibition. 

Caralt, S. D., Uriz, M. J., and Wijffels, R. FI. (2007). Cell culture from sponges Pluripotency and immortality. Trends Biotechnol. 25, 467-471. 

Although efforts are underway to identify markers in serum and prostate tissue, the question arose as to whether metastatic prostate cancer cell lines accurately represent in vivo disease. It was found that in vitro cell cultures (LnCaP and PC3) shared less than 20% of proteins when compared to in vivo LCM procured malignant prostate cancer. 2D-PAGE protein profiles were used to compare normal and malignant cells to immortalized cells from the same patient. Protein expression patterns were dramatically altered when cells were grown in culture and immortalized most notably, a loss of prostate specific antigen expression was observed [18]. Thus, caution must be used when working with immortalized cell lines to discover potential disease markers. 

Transcript 1 mRNAs represent 11 percent of the desat transcripts. They are principally found in Schneider cell culture (Schneider cells originated from embryonic cells which became immortalized) but are less represented in embryos and larvae (10 to 12 percent of the desatl transcripts). In the adult they constitute only 3 percent of the desat transcripts in head but seem more abundant in testis (two out of two) and ovary (two out of five). In the proximal 5 -non-transcribed region are found two TATA and two CAAT boxes. 

Permeability coefficients for paracellular diffusion of 3H-sucrose and 14C-inulin across monolayers of HCEC and immortalized SV-HCEC and transendothe-lial resistances have been compared to human lung microvascular endothelial cells to evaluate the BBB phenotype of the cerebromicrovascular endothelial cell cultures (Muruganandam et al. 1997). 

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