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1.1 This test method, provided the limitations are understood, covers a procedure for both the enumeration and measurement of size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings, hence, this test method is not restricted to specific cell types. The method is appropriate for suspension as well as adherent cell cultures
1.2 Cells commonly used in tissue-engineered medical products
1.3 This instrumentation is manufactured by a variety of companies; however, the principle used in all is electrical impedance. This test method, for cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced by a cell, suspended in a conductive liquid, traversing through a small aperture (see
1.4 Limitations are discussed as follows:
1.4.1 CoincidenceOccasionally, more than a single cell transverses the aperture simultaneously. Only a single larger pulse, as opposed to two individual pulses, is generated. The result is a lower cell count and higher cell volume measurement. The frequency of coincidence is a statistically predictable function of cell concentration that is corrected by the instrument. This is called coincidence correction
1.4.2 ViabilityAutomated cell counting enumerates both viable and nonviable cells. It does not measure percent cell viability. To measure the percent cell viability, either a vital dye or nonvital dye, such as trypan blue, procedure must be performed.
1.4.3 Size Variation of the Cell Sample Up to 30 to 1 by cell diameter in microns; 27 000 to 1 by cell volume. This is simply a function of the size range capability of the particular aperture size selected. Using this technology, measurements may be made in the range of about 0.6 to 1200 m. The lower size limit is restricted only by thermal and electronic noise.
1.4.4 Size Range of the ApertureThe size range for a single aperture is proportional to its diameter, D. The response has been found to depend linearly on D over a range from 0.02 D to 0.80 D; however, the aperture tube may become prone to blockage at levels greater than 0.60 D. The practical operating range, therefore, of the aperture is considered to be 2 to 60 % of the diameter.
1.4.5 Humidity10 to 85 %.
1.4.6 Temperature10 to 35C.
1.4.7 Electrolyte SolutionThe diluent for cell suspension must provide conductivity and have no effect on cell size. The electrolyte of choice is most often physiologic phosphate buffered saline.
|Engelse titel||Standard Test Method for Automated Analyses of Cells-the Electrical Sensing Zone Method of Enumerating and Sizing Single Cell Suspensions|