ASTM F2944-20

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 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 millimeters), or initial cell suspension volume (milliliters).

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 practice 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.

1.13 Cells and tissues commonly used in tissue engineering, regenerative medicine, and cellular therapy are routinely assayed and analyzed to define the number, prevalence, biological features, and biological potential of the original stem cell and progenitor population(s).

1.14 The number of stem cells and progenitor cells in various tissues can be assayed in vitro by liberating the cells from the tissues using methods that preserve the viability and biological potential of the underlying stem cell and/or progenitor population, and placing the tissue-derived cells in an in vitro environment that results in efficient activation and proliferation of stem and progenitor cells as clonal colonies. The true number of stem cells and progenitors (true colony forming units (tCFU)) can thereby be estimated on the basis of the number of colony-forming units observed (observed colony forming units (oCFU)) to have formed (1-3)2 (Fig. A1.1). The prevalence of stem cells and/or progenitors can be estimated on the basis of the number of observed colony-forming units (oCFU) detected, divided by the number of total cells assayed.

1.15 The automated image acquisition and analysis approach (described herein) to cell and colony enumeration has been validated and found to provide superior accuracy and precision when compared to the current “gold standard” of manual observer defined visual cel