1、Designation: F2149 01 (Reapproved 2007)F2149 16Standard 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
2、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 are u
3、nderstood, covers a procedure for both the enumeration and measurementof size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings, hence, this test methodis not restricted to specific settings and is applicable to a wide range of cell types. The met
4、hod works best for spherical cells, andmay be less accurate if cells are not spherical, such as for discoid cells or budding yeast. The method is appropriate for suspensionas well as adherent cell cultures (1).2 This is a quantitative laboratory method not intended for on-line or field use. Results
5、maybe reported as number of cells per millilitremilliliter or total number of cells per volume of cell suspension analyzed. Both countand size Size distribution may be expressed in cell micron diameter or volume, femtolitres.volume.1.2 Cells commonly used in tissue-engineered medical products (2) ro
6、utinely are analyzed. analyzed routinely. Examples arechondrocytes (3), fibroblasts (4), and keratinocytes (5). Szabo et alal. used the method for both pancreatic islet number and volumemeasurements (6). In addition, instrumentation using the electrical sensing zone technology was used for both coun
7、t and sizedistribution analyses of porcine hepatocytes placed into hollow fiber cartridge extracorporeal liver assist systems. In this study (7),and others (6, 8), the automated electrical sensing zone method was clearly validated for superior accuracy and precision whencompared to the conventional
8、manual method, visual cell counting under a microscope using a hemocytometer. This validation hasbeen demonstrated over a wide variety of cell types. In addition, the automated procedure is rapid, rugged, and cost effective; italso minimizes operator-to-operator variability inherent in manual techni
9、ques.Currently, it is not possible to validate cell countingdevices for accuracy, since there not a way to produce a reference sample that has a known number of cells. The electrical sensingzone method shall be validated each time it is implemented in a new laboratory, it is used on a new cell type,
10、 or the cell countingprocedure is modified.1.3 This instrumentation Electrical sensing zone instrumentation (commonly referred to as a Coulter counter) is manufacturedby a variety of companies; however, the principle used in all is companies and is based upon electrical impedance. This testmethod, f
11、or cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced bya cell, suspended in a conductive liquid, traversing through a small aperture (see Fig. 1(9). When cells are suspended in aconductive liquid, phosphate-buffered saline for instance,
12、they function as discrete insulators. When the cell suspension is drawnthrough a small cylindrical aperture, the passage of each cell changes the impedance of the electrical path between two submergedelectrodes located on each side of the aperture. An electrical pulse, suitable for both counting and
13、 sizing, results from the passageof each cell through the aperture. The path through the aperture, in which the cell is detected, is known as the “electronic sensingzone.” This test method permits the selective counting of cells within very narrow size distribution ranges by electronic selectionof t
14、he generated pulses. While the number of pulses indicates cell count, the amplitude of the electrical pulse produced dependson the cells volume. The baseline resistance between the electrodes is due to the resistance of the conductive liquid within theboundaries of the aperture. The presence of cell
15、s within the “electronic sensing zone” raises the resistance of the conductivepathway that depends on the volume of the cell. Analyses of the behavior of cells within the aperture demonstrates that the heightof the pulse produced by the cell is the parameter that most nearly shows proportionality to
16、 the cell volume.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, asopposed to two individual pulses, is generated. The result is a lower cell count and higher cell volume measurement.
17、 The frequencyof coincidence is a statistically predictable function of cell concentration that is corrected by the instrument. This is called1 This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommit
18、teeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved Oct. 1, 2007Jan. 15, 2016. Published October 2007May 2016. Originally approved in 2001. Last previous edition approved in 20012007 asF2149 01.F2149 01 (2007). DOI: 10.1520/F2149-01R07.10.1520/F2149-16.2 The boldfa
19、ce numbers in parentheses refers to the list of references at the end of this standard.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to
20、 adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Cons
21、hohocken, PA 19428-2959. United States1coincidence correction (8). This phenomenon may be minimized, thus ensuring greater result accuracy, by using relatively low cellconcentrations, around the 5 % level.reduced by using lower cell concentrations.1.4.2 ViabilityAutomated Electrical sensing zone cel
22、l counting enumerates both viable and nonviable cells. It does notmeasure percent cell viability. To measure the percent cell viability, either a vital dye or nonvital dye, such as trypan blue,procedure must be performed.cells and cannot determine percent viable cells. A separate test, such as Trypa
23、n blue, is required todetermine percent viable cells.1.4.3 Size Variation of the Cell SampleCell DiameterUp to 30 to 1 by cell diameter in microns; 27 000 to 1 by cell volume.This is simply This is a function of the size range capability of the particular aperture size selected. Using this technolog
24、y,measurements Measurements may be made in the cell diameter range of about 0.6 m to 1200 m. The lower size limit is restrictedonly by thermal and electronic noise. Setting the counting size range on the instrument can affect the test results, especially if thecell size has a large distribution, and
25、 should be carefully controlled to help achieve repeatability.1.4.4 Size Range of the ApertureThe size range for a single aperture is proportional to its diameter,diameter. D.The responsehas been found to depend linearly on Ddiameter over a range from 0.022 D% to 0.8080 D%; however, the of the diame
26、ter.However, the aperture tube may become prone to blockage at levels greater than 0.6060 D.% The of diameter. Therefore, thepractical operating range, therefore, range of the aperture is considered to be 2 % to 60 % of the diameter.1.4.5 Humidity10 % to 85 %.1.4.6 Temperature10 C to 35C.35 C.1.4.7
27、Electrolyte SolutionThe diluent for cell suspension mustshall provide conductivity and have nominimal effect on cellsize. The electrolyte of choice is most often physiologic phosphate buffered commonly phosphate-buffered saline.2. Terminology2.1 Definitions:2.1.1 channelyzer, na pulse height analyze
28、r; places voltage pulses into appropriate size bins for the size distribution data.2.1.2 coincidence, nmore than one cell transversing the aperture at the same time.2.1.3 corrected count, nthe cell count corrected for coincidence.2.1.4 electrolyte, ndiluent, offering slight conductivity, in which ce
29、lls are suspended.2.1.5 femtolitre,femtoliter, na cubic micron;micrometer; a measurement of cell volume.2.1.6 micron (), n0.001 mm, also known as a micrometre; measurement of cell diameter.2.1.6 raw count, nthe enumeration of the cell population not corrected for coincidence.2.1.7 ruggedness, nthe d
30、egree of reproducibility of the same sample under a variety of normal conditions; for example,different operators.FIG. 1 Cell, Suspended in a Conductive Fluid, Traversing Through a Small ApertureF2149 1622.1.8 size thresholds, nthe instruments lower and upper size settings for the particular cell po
31、pulation; adjustable “size gate.”Cells or fragments outside the size settings are excluded from the analyses.3. Significance and Use3.1 This assay The electrical sensing zone method for cell counting is used in university tissue culture laboratories, culture,government research, and hospital, biomed
32、ical, and pharmaceutical laboratories to automate cell counting and sizing. Thisinstrumentation provides very rapid, accurate, and precise results for any tissue culture facility. In addition, as noted, since the cellsizes to be analyzed by the instrument are set by the user, the analyses may be don
33、e on virtually any species of cells and cell type;it is notfor counting and sizing cells. The method may be applicable to a wide range of cells sizes and cell types, with appropriatevalidation restricted (10to ).human cells or blood cells.3.2 The electrical sensing zone methodology was introduced in
34、 the mid 1950s mid-1950s (9). Since this time, there have beensubstantial improvements which have enhanced the operators ease of use. Among these are the elimination of the mercurymanometer, reduced size, greater automation, and availability of comprehensive statistical computer programs.3.3 This in
35、strumentation offers a rapid result as contrasted to the manual counting of cells using the hemocytometer standardcounting chamber, hemocytometer. chamber. The counting chamber is known to have an error of 10 to 30 %, as well as being verytime consuming time-consuming (1011). In addition, when count
36、ing and sizing porcine hepatocytes, Stegemann et al concludedthat the automated, electrical sensing zone method provided significantly greater accuracy, precision, and speed, for both countsand size, compared to the conventional microscopic or the cell mass-based method (7).4. Interferences4.1 Debri
37、s and Cellular FragmentsWhen these are in the cellular size ranges, they will be analyzed. Correct cell sizethreshold settings help to correct inclusion of debris or fragments in the analyses. A count of culture medium without cells can beused to assess background counts. For adherent cells, a sampl
38、e of the medium from a culture may be counted (beforetrypsinization).4.2 Cellular AggregationAggregates will be enumerated as a single cell. They will be sized larger than their individual cellcomponents. Commercially available enzyme solutions aid in the preparation of single cell suspensions. Effo
39、rts to disaggregatecells may affect cell viability. Cell sample preparations may exhibit settle, resulting in decreased counts. Gently mixing the cellsoften and immediately before counting will produce more repeatable results.4.3 SettlingCell sample preparations may exhibit settling resulting in dec
40、reased counts. Inverting the sample container severaltimes will produce more accurate and precise results.4.3 AdherenceCells may adhere to the sides of the sample vessel, thus affecting count accuracy.4.4 Line NoiseInstruments mustshall be located on a bench away from flickering lights and other equ
41、ipment such ascentrifuges.4.5 BubblesMay cause a false increase in cell counts.4.6 CurrentExcessive current across the aperture may damage cellular membranes. The optimum current is set by current canbe adjusted on the instrument.5. Hazards5.1 Warning (Chemical)Do not use nonaqueous electrolyte solu
42、tions (that in which the cells are suspended). Care shall betaken when mixing some electrolyte solutions. Violent reactions may occur. Azide shall not be used in acid solutions. Flammableelectrolyte solutions and organic solvents, as noted above, mustshall not be used.5.2 Warning (Electrical)High vo
43、ltages are present inside the instrument. Instrument shall be sited on a firm, dry work benchand mustshall be grounded correctly.5.3 Warning (Biological)Institutional, state, and OSHA approved safety action plans shall be followed.6. Procedures6.1 The procedure for this test method is similar regard
44、less of the brand of commercial instrumentation used. details in thisprocedure should be used as guidelines since modifications may be required for the cell type, instrument, or procedure beingemployed. Commercial instrumentation among manufacturers may vary as to the instruments size range, number
45、of aperturediameters available, and data acquisition capabilities using various printers or computer data acquisition. recommended cell sizerange and aperture diameters available. Certain manufacturers provide instrumentation in which both instrument function controland data analyses are computer co
46、ntrolled. In addition, some models will provide cell counts only; however, other models containa channelyzer enabling the instrument to determine cell size distribution in addition to count results.Some models provide cellcount only, while others also provide cell size distribution.F2149 1636.2 Cali
47、brate the instrument following the instrument manufacturers procedure. Instrument calibration should be performedmonthly or after the unit has been serviced. For all cell types, a commercial calibrator of known MCVvolume should be used. Lightmicroscopy can be used to verify reference particle size o
48、r cell size.6.3 Dilute the cell suspension in the electrolyte, typically physiologic phosphate-buffered saline. Gently mix the sample byinverting it seven or eight times. Perform the analyses within 20 min after sample preparation.sample. The mixing method shouldbe carefully considered and controlle
49、d since variability in this step may increase variability in the test result. The count should beperformed as soon as possible after mixing as cell settling may occur (even within a few seconds depending on cell type). It maybe necessary to assess the effect of time from mixing to counting on the test result to establish the appropriate time frame that givesa consistent result.6.4 Set the size range on the instrument for the particular cell population being analyzed. Choose the correct aperture size. Themanufacturers operator manual will
