ASTM F2944-12
(Test Method)Standard Test Method for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture
Standard Test Method for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture
SIGNIFICANCE AND USE
The Manual Observer-Dependent Assay—The manual quantification of cell and CFU cultures based on observer-dependent criteria or judgment is an extremely tedious and time-consuming task and is significantly impacted by user bias. In order to maintain consistency in data acquisition, pharmacological and drug discovery and development studies utilizing cell- and colony-based assays often require that a single observer count cells and colonies in hundreds, and potentially thousands of cultures. Due to observer fatigue, both accuracy and reproducibility of quantification suffer severely (5). When multiple observers are employed, observer fatigue is reduced, but the accuracy and reproducibility of cell and colony enumeration is still significantly compromised due to observer bias and significant intra- and inter-observer variability (4, 13). Use of quantitative automated image analysis provides data for both the number of colonies as well as the number of cells in each colony. These data can also be used to calculate mean cells per colony. Traditional methods for quantification of colonies by hand counting coupled with an assay for cell number (for example, DNA or mitochondrial) remains a viable method that can be used to calculate the mean number of cells per colony. These traditional methods have the advantage that they are currently less labor intensive and less technically demanding (8, 9). However, the traditional assays do not, provide colony level information (for example, variation and skew), nor do they provide a means for excluding cells that are not part of a colony from the calculation of mean colony size. As a result, the measurement of the mean number of cells per colony that is obtained from these alternative methods may differ when substantial numbers of cells in a sample are not associated with colony formation. By employing state-of-the-art image acquisition, processing and analysis hardware and software, an accurate, precise, robust and automated analysi...
SCOPE
1.1 This test method, provided its limitations are understood, describes a procedure for quantitative measurement of the number and biological characteristics of colonies derived from a stem cell or progenitor population using image analysis.
1.2 This test method is applied in an in vitro laboratory setting.
1.3 This method utilizes: (a) standardized protocols for image capture of cells and colonies derived from in vitro processing of a defined population of starting cells in a defined field of view (FOV), and (b) standardized protocols for image processing and analysis.
1.4 The relevant FOV may be two-dimensional or three-dimensional, depending on the CFU assay system being interrogated.
1.5 The primary unit to be used in the outcome of analysis is the number of colonies present in the FOV. In addition, the characteristics and sub-classification of individual colonies and cells within the FOV may also be evaluated, based on extant morphological features, distributional properties, or properties elicited using secondary markers (for example, staining or labeling methods).
1.6 Imaging methods require that images of the relevant FOV be captured at sufficient resolution to enable detection and characterization of individual cells and over a FOV that is sufficient to detect, discriminate between, and characterize colonies as complete objects for assessment.
1.7 Image processing procedures applicable to two- and three-dimensional data sets are used to identify cells or colonies as discreet objects within the FOV. Imaging methods may be optimized for multiple cell types and cell features using analytical tools for segmentation and clustering to define groups of cells related to each other by proximity or morphology in a manner that is indicative of a shared lineage relationship (that is, clonal expansion of a single founding stem cell or progenitor).
1.8 The characteristics of individual colony objects (cells per...
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: F2944 − 12
Standard Test Method for
Automated Colony Forming Unit (CFU) Assays—Image
Acquisition and Analysis Method for Enumerating and
1
Characterizing Cells and Colonies in Culture
This standard is issued under the fixed designation F2944; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope groups of cells related to each other by proximity or morphol-
ogy in a manner that is indicative of a shared lineage
1.1 This test method, provided its limitations are
relationship(thatis,clonalexpansionofasinglefoundingstem
understood, describes a procedure for quantitative measure-
cell or progenitor).
ment of the number and biological characteristics of colonies
derived from a stem cell or progenitor population using image
1.8 The characteristics of individual colony objects (cells
analysis.
per colony, cell density, cell size, cell distribution, cell
heterogeneity, cell genotype or phenotype, and the pattern,
1.2 This test method is applied in an in vitro laboratory
distribution and intensity of expression of secondary markers)
setting.
are informative of differences in underlying biological proper-
1.3 This method utilizes: (a) standardized protocols for
ties of the clonal progeny.
image capture of cells and colonies derived from in vitro
1.9 Underappropriatelycontrolledexperimentalconditions,
processingofadefinedpopulationofstartingcellsinadefined
differencesbetweencoloniescanbeinformativeofthebiologi-
field of view (FOV), and (b) standardized protocols for image
calpropertiesandunderlyingheterogeneityofcolonyfounding
processing and analysis.
cells (CFUs) within a starting population.
1.4 The relevant FOV may be two-dimensional or three-
1.10 Cell and colony area/volume, number, and so forth
dimensional, depending on the CFU assay system being
may be expressed as a function of cell culture area (square
interrogated.
millimetres), or initial cell suspension volume (millilitres).
1.5 The primary unit to be used in the outcome of analysis
1.11 Sequential imaging of the FOV using two or more
is the number of colonies present in the FOV. In addition, the
optical methods may be valuable in accumulating quantitative
characteristicsandsub-classificationofindividualcoloniesand
information regarding individual cells or colony objects in the
cells within the FOV may also be evaluated, based on extant
sample. In addition, repeated imaging of the same sample will
morphological features, distributional properties, or properties
be necessary in the setting of process tracking and validation.
elicited using secondary markers (for example, staining or
Therefore, this test method requires a means of reproducible
labeling methods).
identification of the location of cells and colonies (centroids)
1.6 Imaging methods require that images of the relevant
within the FOV area/volume using a defined coordinate sys-
FOV be captured at sufficient resolution to enable detection
tem.
and characterization of individual cells and over a FOV that is
sufficient to detect, discriminate between, and characterize 1.12 To achieve a sufficiently large field-of-view (FOV),
images of sufficient resolution may be captured as multiple
colonies as complete objects for assessment.
image fields/tiles at high magnification and then combined
1.7 Image processing procedures applicable to two- and
together to form a mosaic representing the entire cell culture
three-dimensional data sets are used to identify cells or
area.
colonies as discreet objects within the FOV. Imaging methods
maybeoptimizedformultiplecelltypesandcellfeaturesusing 1.13 Cellsandtissuescommonlyusedintissueengineering,
analytical tools for segmentation and clustering to define 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 cell
1
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
and progenitor population(s).
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
1.13.1 Common applicable cell types and cell sources
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
include,butarenotlimitedto:mammalianstemandprogenitor
Current edition approved March 1, 2012. Published April 2012. DOI: 10.1520/
F2944–12. cells; adult-derived cells (for example, blood, bone marrow,
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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F2944 − 12
skin, fat, muscle, mucosa) cells, fetal-derived cells (for q
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