ASTM_F_2149_-_01_2007.pdf

Designation:F214901(Reapproved 2007)Standard Test Method forAutomated Analyses of Cellsthe Electrical Sensing ZoneMethod of Enumerating and Sizing Single Cell Suspensions1This standard is issued under the fixed designation F2149;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method,provided the limitations areunderstood,covers a procedure for both the enumeration andmeasurement of size distribution of most all cell types.Theinstrumentation 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 adherentcell cultures(1).2This is a quantitative laboratory method notintended for on-line or field use.Results may be reported asnumber of cells per millilitre or total number of cells pervolume of cell suspension analyzed.Both count and sizedistribution may be expressed in cell micron diameter orvolume,femtolitres.1.2 Cells commonly used in tissue-engineered medicalproducts(2)routinely are analyzed.Examples are chondro-cytes(3),fibroblasts(4),and keratinocytes(5).Szabo et al usedthe method for both pancreatic islet number and volumemeasurements(6).In addition,instrumentation using the elec-trical sensing zone technology was used for both count and sizedistribution analyses of porcine hepatocytes placed into hollowfiber cartridge extracorporeal liver assist systems.In this study(7),and others(6,8),the automated electrical sensing zonemethod was clearly validated for superior accuracy and preci-sion when compared to the conventional manual method,visual cell counting under a microscope using a hemocytom-eter.This validation has been demonstrated over a wide varietyof cell types.In addition,the automated procedure is rapid,rugged,and cost effective;it also minimizes operator-to-operator variability inherent in manual techniques.1.3 This instrumentation is manufactured by a variety ofcompanies;however,the principle used in all is electricalimpedance.This test method,for cell counting and sizing,isbased on the detection and measurement of changes in electri-cal resistance produced by a cell,suspended in a conductiveliquid,traversing through a small aperture(see Fig.1(9).When cells are suspended in a conductive liquid,phosphate-buffered saline for instance,they function as discrete insula-tors.When the cell suspension is drawn through a smallcylindrical aperture,the passage of each cell changes theimpedance of the electrical path between two submergedelectrodes located on each side of the aperture.An electricalpulse,suitable for both counting and sizing,results from thepassage of each cell through the aperture.The path through theaperture,in which the cell is detected,is known as the“electronic sensing zone.”This test method permits the selec-tive counting of cells within very narrow size distributionranges by electronic selection of the generated pulses.Whilethe number of pulses indicates cell count,the amplitude of theelectrical pulse produced depends on the cells volume.Thebaseline resistance between the electrodes is due to theresistance of the conductive liquid within the boundaries of theaperture.The presence of cells within the“electronic sensingzone”raises the resistance of the conductive pathway thatdepends on the volume of the cell.Analyses of the behavior ofcells within the aperture demonstrates that the height of thepulse produced by the cell is the parameter that most nearlyshows proportionality to the cell volume.1.4 Limitations are discussed as follows:1.4.1 CoincidenceOccasionally,more than a single celltransverses the aperture simultaneously.Only a single largerpulse,as opposed to two individual pulses,is generated.Theresult is a lower cell count and higher cell volume measure-ment.The frequency of coincidence is a statistically predict-able function of cell concentration that is corrected by theinstrument.This is called coincidence correction(8).Thisphenomenon may be minimized,thus ensuring greater resultaccuracy,by using relatively low cell concentrations,aroundthe 5%level.1.4.2 ViabilityAutomated cell counting enumerates bothviable and nonviable cells.It does not measure percent cellviability.To measure the percent cell viability,either a vital dyeor nonvital dye,such as trypan blue,procedure must beperformed.1.4.3 Size Variation of the Cell SampleUp to 30 to 1 bycell diameter in microns;27 000 to 1 by cell volume.This issimply a function of the size range capability of the particular1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved Oct.1,2007.Pub