ASTM F2944-20

Standard Practice for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture

SIGNIFICANCE AND USE
4.1 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 (2, 4) . 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 ana...
SCOPE
1.1 This practice, 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 practice is applied in an in vitro laboratory setting.  
1.3 This practice 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 ...

General Information

• Status: Published
• Publication Date: 31-Mar-2020
• ICS: 07.100.01 - Microbiology in general
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.43 - Cells and Tissue Engineered Constructs for TEMPs

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F2944 − 20
Standard Practice 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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope ogy in a manner that is indicative of a shared lineage
relationship (that is, clonal expansion of a single founding stem
1.1 This practice, provided its limitations are understood,
cell or progenitor).
describes a procedure for quantitative measurement of the
number and biological characteristics of colonies derived from
1.8 The characteristics of individual colony objects (cells
a stem cell or progenitor population using image analysis.
per colony, cell density, cell size, cell distribution, cell
heterogeneity, cell genotype or phenotype, and the pattern,
1.2 This practice is applied in an in vitro laboratory setting.
distribution and intensity of expression of secondary markers)
1.3 This practice utilizes: (a) standardized protocols for
are informative of differences in underlying biological proper-
image capture of cells and colonies derived from in vitro
ties of the clonal progeny.
processing of a defined population of starting cells in a defined
1.9 Under appropriately controlled experimental conditions,
field of view (FOV), and (b) standardized protocols for image
differences between colonies can be informative of the biologi-
processing and analysis.
cal properties and underlying heterogeneity of colony founding
1.4 The relevant FOV may be two-dimensional or three-
cells (CFUs) within a starting population.
dimensional, depending on the CFU assay system being
1.10 Cell and colony area/volume, number, and so forth
interrogated.
may be expressed as a function of cell culture area (square
1.5 The primary unit to be used in the outcome of analysis
millimeters), or initial cell suspension volume (milliliters).
is the number of colonies present in the FOV. In addition, the
1.11 Sequential imaging of the FOV using two or more
characteristics and sub-classification of individual colonies and
optical methods may be valuable in accumulating quantitative
cells within the FOV may also be evaluated, based on extant
information regarding individual cells or colony objects in the
morphological features, distributional properties, or properties
sample. In addition, repeated imaging of the same sample will
elicited using secondary markers (for example, staining or
be necessary in the setting of process tracking and validation.
labeling methods).
Therefore, this practice requires a means of reproducible
1.6 Imaging methods require that images of the relevant
identification of the location of cells and colonies (centroids)
FOV be captured at sufficient resolution to enable detection
within the FOV area/volume using a defined coordinate sys-
and characterization of individual cells and over a FOV that is
tem.
sufficient to detect, discriminate between, and characterize
1.12 To achieve a sufficiently large field-of-view (FOV),
colonies as complete objects for assessment.
images of sufficient resolution may be captured as multiple
1.7 Image processing procedures applicable to two- and
image fields/tiles at high magnification and then combined
three-dimensional data sets are used to identify cells or
together to form a mosaic representing the entire cell culture
colonies as discreet objects within the FOV. Imaging methods
area.
may be optimized for multiple cell types and cell features using
analytical tools for segmentation and clustering to define
1.13 Cells and tissues commonly used in tissue engineering,
groups of cells related to each other by proximity or morphol- 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 and progenitor population(s).
This practice is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
1.13.1 Common applicable cell types and cell sources
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
include, but are not limited to: mammalian stem and progenitor
Current edition approved April 1, 2020. Published June 2020. Originally
cells; adult-derived cells (for example, blood, bone marrow,
approved in 2012. Last
...

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. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F2944 − 12 F2944 − 20
Standard Test Method Practice 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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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 colony, cell density, cell size, cell distribution, cell heterogeneity,
cell genotype or phenotype, and the pattern, distribution and intensity of expression of secondary markers) are informative of
differences in underlying biological properties of the clonal progeny.
1.9 Under appropriately controlled experimental conditions, differences between colonies can be informative of the biological
properties and underlying heterogeneity of colony founding cells (CFUs) within a starting population.
1.10 Cell and colony area/volume, number, and so forth may be expressed as a function of cell culture area (square millimetres),
or initial cell suspension volume (millilitres).
1.11 Sequential imaging of the FOV using two or more optical methods may be valuable in accumulating quantitative
information regarding individual cells or colony objects in the sample. In addition, repeated imaging of the same sample will be
necessary in the setting of process tracking and validation. Therefore, this test method requires a means of reproducible
identification of the location of cells and colonies (centroids) within the FOV area/volume using a defined coordinate system.
1.12 To achieve a sufficiently large field-of-view (FOV), images of sufficient resolution may be captured as multiple image
fields/tiles at high magnification and then combined together to form a mosaic representing the entire cell culture area.
1
This test method practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of
Subcommittee F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved March 1, 2012April 1, 2020. Published April 2012June 2020. Originally approved in 2012. Last previous edition approved in 2012 as F2944–12.
DOI: 10.1520/F2944–12.10.1520/F2944–20.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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