ANIMAL TISSUE CULTURE PRESENTATION

Indirect methods of cell determination cell growth based on the measurement of an intracellular cell component such as DNA or protein or alternatively an extracellular change such as nutrient depletion or an enzyme activity released by the cells. Based on correlation between measured parameter and cell concentration. Total protein content and specific enzyme activity levels measured on a per cell basis vary substantially over the course of a culture due to changes in the growth rate and composition of the culture medium. Protein and enzyme content will be high during exponential growth but lower in lag or stationary phase.

Protein determination Total cell protein can be used as a measure of biomass. But the protein content per cell can vary during cell culture. Mammalian cell is typically 100-500pg/cell. Useful in the expression of specific enzyme activities. Two methods are used for this assay one is Lowry and other is Bradford method. Bradford assay is favoured because of speed,senstivity,and negligible interference from other cell components. Lysed cells are solubilised in NaoH and added to the reagent,coomassie blue. A blue colour develops within 10 min can be measured by spectrophotometers.

Lysed cell +NaoH+coomassie blue reagent blue coloration

DNA determination DNA content of diploid cells is usually constant, variations can occur as a result of distribution of cells through the cell cycle. Mammalian cells typically 6pg per cell. This method is best method to use as measurement of number of cells in solid tissue. Solubilised cells +diphenylamine reagent+DNA= Blue coloration. Fluorescent detection can also be used with Hoechst 33258 or DAPI which bind to DNA. Mycoplasma detection.

GLUCOSE DETERMINATION If cells are immobilized within a bioreactor then it is often difficult to obtain a cell sample. Cell growth can be monitored by changes in the concentration of key components of the culture medium i.e. the change in glucose concentration. Correlations between cell concentration and consumption of glucose. If the cell line or any of the culture conditions are altered the relationship between substrate consumption or product formation and cell number will change. Can also follow lactic acid production or oxygen consumption.

Hollow fiber bioreactor

Packed bed bioreactor