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Growth Inhibitors and Promoters From High Concentration Animal Cell Cultures.

Andrews De Castro, Arcadio Garcia. (1994) Growth Inhibitors and Promoters From High Concentration Animal Cell Cultures. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

This study confirms that animal cells in culture produce factors other than metabolic by-products which affect their own growth and the growth of other cells in culture. In order to characterise these factors a number of commonly used animal cell lines namely the AFP-27 hybridoma, Chinese hamster ovary (CHO), baby hamster kidney (BHK-21), human osteosarcoma (MG-63) and African green monkey kidney (Vero) cells, were adapted to grow in serum-free conditions in HL-1 (Ventrex, USA) low protein (< 30 u/ml) medium. The adaptation all cell lines except the AFP-27 hybridomas, which normally grown in suspension, resulted in two cell subpopulations, one growing in suspension and the other remaining anchorage-dependent. The HL-1 adapted suspension BHK, Vero and MG-63 cell lines were grown to high cell concentrations in serum-free conditions using an Acusyst-Jr (Endotronics, USA) hollow fibre system. In general cell concentrations over 10 8 cell/ml were achieved, as calculated from the oxygen consumption values of the bioreactors. The hollow fibre conditioned media (HF-CM) obtained from these cultures contained a maximum protein concentrations of 2. 2 and 1. 2 mg/ml for the MG-63 and Vero cell cultures respectively. The HF-CM and their 1 kDa ultrafiltration filtrates and retentates were assessed (by adding 10% of these to the cell culture media containing 2. 25% FCS) for their effects on cell growth, metabolism and the strength of cell adhesion. All HF-CM had a growth promoting effect on BHK cells. The BHK HF-CM, Vero HF-CM and MG-63 HF-CM showed growth inhibiting effects on Vero, MG-63 and LL-24 cells in this order. No growth promoting/inhibiting effects were found in the filtrates resulting from 1 kDa cut-off membrane ultrafiltration of the HF-CM and therefore could not be due to low molecular weight metabolites but to proteinaceous materials. The metabolic activity of BHK cells was determined by an MTT conversion assay. BHK cells grown in the presence of the Vero HF-CM showed a reduced metabolic activity in comparison to BHK cells grown in culture medium. The strength of cell adhesion of Vero and MG-63 cells was not significantly affected by the presence of the various HF-CM. LL-24 and BHK cells showed a 20 to 35% reduction in the strength of cell adhesion when cells were grown in the presence of Vero HF-CM, BHK HF-CM and MG-63 HF-CM. In BHK cells this reduction was the same as that caused by HL-1 and therefore could not be attributed to cell secreted factors present in the HF-CM. The factors in the Vero HF-CM responsible for the increased growth rates and the reduction in the strength of cell adhesion of BHK cells were found to be molecules larger than 65 kDa in molecular weight. Overall, for BHK cells grown in the presence of Vero HF-CM and its ultrafiltration fractions, increased growth was matched with a decrease in the strength of cell adhesion. Thus it appeared that increased growth caused a reduction in the strength of cell adhesion.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Andrews De Castro, Arcadio Garcia.
Date : 1994
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1994.
Depositing User : EPrints Services
Date Deposited : 24 Apr 2020 15:27
Last Modified : 24 Apr 2020 15:27
URI: http://epubs.surrey.ac.uk/id/eprint/854828

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