Direct Methods, Acoustic Resonance Densitometry, Linear Relationship, Spectroscopy, Methods based on electrical properties, Exclusively Dependent

Back to Home

Home >>Applied Biotechnology >> Cell and Tissue Culture Technology >>Direct Methods

Back to Home

	Direct Methods I. Acoustic resonance densitometry (ARD) This is making use of the linear relationship between density of the fluid and the resonance frequency of the cell culture sample. Cell culture sample is enclosed in a test chamber and is excited electromagnetically to vibrate at its natural frequency. Fluid density is measured from the square of the oscillation period of the sample. A hollow fibre device filters out the supernatant and its density is also determined. This is then subtracted from the density of the cell culture and gives the density due to cells.
This method has been successfully used for measuring the concentration of hybridoma and human lymphoma cell culture. 2. NMR spectroscopyIt is based. on the measurement of total Na + per unit volume of cell culture. The culture is placed in a magnetic field and exposed to electromagnetic radiation and the reaction of the Na + nuclei is measured. Since intracellular Na + concentration is low relative to extracellular Na + concentration, with increase in cell concentration, total Na + will decrease and will give an estimate of the cell concentration. NMR continuously monitors the high density of viable cells in hollow- fibre bioreactors. Other methods fail at these high concentrations.
3. Methods based on electrical propertiesElectrical properties of a culture solution are exclusively dependent on the cell concentration, and so can be made use of for measuring cell concentrations. The electrochemical method (fuel cell) measures the intensity of electric current between two electrodes immersed in a cell culture. Since the current results from direct oxidation of cells on the surface of the anode, it gives a direct measure of cell concentration. A conductivity meter can measure electrical conductivity. This is directly proportional to the cell concentration and can be easily measured in a hollow fibre reactor.
Another electrical property made use of for measuring cell concentration is capacitance (C). Each viable cell works as a capacitor. 4. Optical techniques These are the most popular techniques, but cannot distinguish between viable and non-viable cells. FluorescenceCells are irradiated with UV (340 nm), and fluorescence due to NADPH at 460 nm is measured. Fluorescence is proportional to cell concentration, but at high concentration it is less sensitive. Improvements are being made to overcome this problem.
	Light absorbance Scattering is also used as a measurement of cell concentration. Most of the commercial instruments are designed to measure high cell density and are less sensitive at low concentration. An image analysis system with a CCD camera may lead to a breakthrough in bioprocess monitoring. The cells can be viewed on the screen and cell concentration can be monitored. Laser-beam-based commercial equipment These are being used in microbial bioprocess monitoring and may be used for cell culture bioprocess monitoring in future. They measure real-time particle size or cell concentration