Download ANIMAL TISSUE CULTURE PRESENTATION

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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.
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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
coloration
blue
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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.
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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