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An Investigation of the Nutrient Gradients in Aggregates of Animal Cells in Culture.

Anuar, Nurina. (1998) An Investigation of the Nutrient Gradients in Aggregates of Animal Cells in Culture. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

The investigations of nutrient gradients in animal cell aggregates were conducted on three types of cell; HeLa S3, genetically engineered BHK and D36 hybridoma cells for the production of poliovirus, alkaline phosphatase enzyme and monoclonal antibody, respectively as their biological products. Agitation speed in stirred flasks controlled the size of HeLa S3 and BHK aggregate formation and had adverse an effect on hybridoma cells by reducing their viability. The three types of cells showed a preference to grow in roller bottles, where cell aggregation was promoted and cell growth was unaffected. Addition of calcium alone up to 750μM into the culture media, which already contained 423μM calcium ions, induced the formation of HeLa S3 cell aggregates without affecting cell growth. However, the cell viability was reduced when calcium was added at higher than 750μM. Relatively larger aggregates and more extensive cell growth were obtained when collagen solution was added in combination with calcium into the culture media. Because of its potential in promoting cell aggregation, a desiccated type of collagen made from calf skin, selected from the commercially available ranges via collagen fast tests, was used in these investigations. Various concentrations with or without calcium were added into the cell culture media to grow the three types of cell. Different types of cell growth were observed, through manipulation of the cell growth parameters accordingly to the aggregate sizes which enabled the indirect determination of nutrient gradients in HeLa S3 and BHK aggregates. Positive gradients in HeLa S3 and BHK aggregates showed that the cell viability and metabolic activity were increased with the enlargement of the aggregates. By contrast, efforts to achieve cell aggregation were unsuccessful with hybridoma cells where calcium and collagen additions gave adverse effects on growth and productivity. Optimum concentrations of both calcium and collagen were sought for HeLa S3 and BHK cells, for inducing the largest aggregate formation and maximum cell growth. Combinations of 750pM calcium and 0. 0025% collagen for HeLa S3 cells and 500μM calcium and 0. 0075% collagen for BHK cells were applied for the formation of the largest aggregates and maximum growth. These aggregates were used for direct determination of nutrient gradient within the aggregates by cell staining and confocal microscopy technique. No cell viability gradient was found within the aggregates due to the loosely packed viable HeLa S3 cells, while the dead cells were released from the intact aggregated cells. Meanwhile, the insertion of an O2 microelectrode into BHK aggregates depicted the pO2 level within the aggregates. The morphology of aggregates determined the pattern of pO2 profiles where spherical shaped aggregates exhibited zero and low pO2 areas towards the centre of aggregates. Addition of calcium and collagen improved cell growth where the low and zero pO2 area was reduced even though the larger aggregates were >1000um in diameter. The formation of larger aggregates from the optimum concentration of calcium and collagen addition also increased the biological production of HeLa S3 and BHK cells. HeLa S3 aggregates produced almost twice the amount of poliovirus titre over the slightly aggregated control cultures. Similarly, larger BHK aggregates produced twice to four times higher alkaline phosphatase enzyme compared with the control untreated BHK aggregate culture.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Anuar, Nurina.
Date : 1998
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1998.
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/854880

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