ASTM_F_2944_-_12.pdf

Designation:F294412Standard Test Method forAutomated Colony Forming Unit(CFU)AssaysImageAcquisition and Analysis Method for Enumerating andCharacterizing Cells and Colonies in Culture1This standard is issued under the fixed designation F2944;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 Thistestmethod,provideditslimitationsareunderstood,describes a procedure for quantitative measure-ment of the number and biological characteristics of coloniesderived from a stem cell or progenitor population using imageanalysis.1.2 This test method is applied in an in vitro laboratorysetting.1.3 This method utilizes:(a)standardized protocols forimage capture of cells and colonies derived from in vitroprocessing of a defined population of starting cells in a definedfield of view(FOV),and(b)standardized protocols for imageprocessing and analysis.1.4 The relevant FOV may be two-dimensional or three-dimensional,depending on the CFU assay system beinginterrogated.1.5 The primary unit to be used in the outcome of analysisis the number of colonies present in the FOV.In addition,thecharacteristics and sub-classification of individual colonies andcells within the FOV may also be evaluated,based on extantmorphological features,distributional properties,or propertieselicited using secondary markers(for example,staining orlabeling methods).1.6 Imaging methods require that images of the relevantFOV be captured at sufficient resolution to enable detectionand characterization of individual cells and over a FOV that issufficient to detect,discriminate between,and characterizecolonies as complete objects for assessment.1.7 Image processing procedures applicable to two-andthree-dimensional data sets are used to identify cells orcolonies as discreet objects within the FOV.Imaging methodsmay be optimized for multiple cell types and cell features usinganalytical tools for segmentation and clustering to definegroups of cells related to each other by proximity or morphol-ogy in a manner that is indicative of a shared lineagerelationship(that is,clonal expansion of a single founding stemcell or progenitor).1.8 The characteristics of individual colony objects(cellsper colony,cell density,cell size,cell distribution,cellheterogeneity,cell genotype or phenotype,and the pattern,distribution and intensity of expression of secondary markers)are informative of differences in underlying biological proper-ties of the clonal progeny.1.9 Under appropriately controlled experimental conditions,differences between colonies can be informative of the biologi-cal properties and underlying heterogeneity of colony foundingcells(CFUs)within a starting population.1.10 Cell and colony area/volume,number,and so forthmay be expressed as a function of cell culture area(squaremillimetres),or initial cell suspension volume(millilitres).1.11 Sequential imaging of the FOV using two or moreoptical methods may be valuable in accumulating quantitativeinformation regarding individual cells or colony objects in thesample.In addition,repeated imaging of the same sample willbe necessary in the setting of process tracking and validation.Therefore,this test method requires a means of reproducibleidentification of the location of cells and colonies(centroids)within the FOV area/volume using a defined coordinate sys-tem.1.12 To achieve a sufficiently large field-of-view(FOV),images of sufficient resolution may be captured as multipleimage fields/tiles at high magnification and then combinedtogether to form a mosaic representing the entire cell culturearea.1.13 Cells and tissues commonly used in tissue engineering,regenerative medicine,and cellular therapy are routinely as-sayed and analyzed to define the number,prevalence,biologi-cal features,and biological potential of the original stem celland progenitor population(s).1.13.1 Common applicable cell types and cell sourcesinclude,but are not limited to:mammalian stem and progenitorcells;adult-derived cells(for example,blood,bone marrow,1This 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 March 1,2012.Published April 2012.DOI:10.1520/F294412.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 skin,fat,muscle,mucosa)cells,fetal-derived cells(forexample,cord blood,placental/cord,amniotic fluid);embry-onic stem cells(ESC)(that is,derived from inner cell mass ofblastocysts);induced pluripotency cells(iPS)(for example,reprogrammed adult cells);culture expanded cells;and termi-nally differentiated cells of a specific type of tissue.1.13.2 Common app