The cultivation of human kidney epithelial cells in aggregate culture for the production of recombinant protein C
Abstract
Protein C is a naturally occurring serine protease which circulates in the plasma and plays a critical role in the regulation of hemostasis. It is activated at the blood vessel wall and is able to block blood clot formation by inactivating factors Va and VIIIa in the clotting cascade. Protein C could be used as an adjunct therapy with tissue plasminogen activator or as a therapeutic treatment for septic shock. The cells which secrete Protein C are anchorage dependent cells which have been adapted to grow in suspension. However, at the calcium concentrations required for proper expression of the protein, the cells attach to each other to form multicellular aggregates. As the cells in the aggregates grow, they enlarge to form tightly packed clumps commonly called multicellular spheroids. A bioreactor which allows continuous cultivation of multicellular spheroids for the production of Protein C has been designed. Although efficient protein production is achieved with this reactor through about 30 days of operation, problems associated with nutrient diffusion to cells in the interior of the spheroids appear after 10-15 days. These conditions grow more severe and ultimately lead to termination of the run. Reactor operating characteristics are described and solutions are proposed for dealing with the nutrient diffusion problems. Attempts are made to used biochemical and mechanical means to limit spheroid size. Studies of the effect of nutrient limitations on the growth of multicellular spheroids have focused on the depletion of oxygen to the center of the spheroid as the ultimate cause of necrosis. A simple mathematical model which describes oxygen uptake in human kidney cell aggregates is described. The model is able to predict spheroid enlargement at different bulk oxygen conditions satisfactorily, but it is less accurate in describing Protein C production in spheroid culture. The model takes into account the existence of three cell subpopulations, each with distinct nutrient consumption, growth, and Protein C production kinetics. The effect of external mass transfer resistance in limiting nutrient diffusion is also considered.
Degree
Ph.D.
Advisors
Tsao, Purdue University.
Subject Area
Chemical engineering|Cellular biology
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