ASTM F2944-20

4.1?The Manual Observer-Dependent AssayThe 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 4.1.1?Areas of ApplicationCell and colony enumeration (CFU assay) is becoming particularly important in the manufacture, quality assurance/control (QA/QC), and development of product safety and potency release criteria for cell-based regenerative medicine and cellular therapy. The U.S. Food and Drug Administration (FDA) has a guidance document that indicates that the CFU assay may be appropriate for testing stability of placental and umbilical cord blood-derived stem cells 4.1.1.1?Characterization of a cell source by correlating biological potential and functional potency with CFU formation.

4.1.1.2?Characterization of the effect of processing steps or biological or physical manipulation (for example, stimuli) on cells or colony formation.

4.1.1.3?Cell and colony characterization using specific fluorescent and non-fluorescent (differentiation) markers.

4.1.1.4?Extrapolation of the biological potency (for example, differentiation, proliferative, and so forth) of a larger sample from application of colony forming assay to sub-samples.

4.1.1.5?Provision of criteria for sub-colony selection of preferred colonies (specific tissue type, proliferation rate, and so forth) for use and/or further expansion.

4.2?The Technology (image acquisition, processing, and analysis)Current standards utilize user input for defining the presence and location of colonies based on visualization of an entire culture surface at low magnification through the eyepieces of a microscope. In this case, the sample may be viewed in transmission light mode (unstained or with a histochemical marker) or fluorescently with a dye or antibody. For this practice, the colony count is the only measurable output parameter. Utilizing a microscope-based imaging system to stitch together high resolution image tiles into a single mosaic image of the entire culture surface and subsequently clustering segmented cells using image processing algorithms to delineate colonies, provides a fully automated, accurate, and precise method for characterizing the biological potential and functional potency of the cultured cells. Furthermore, extracted parameters in addition to colony number provide means of further characterization and sub-classification of colony level statistics. These parameters include, but are not limited to, cell/nuclear count, cell/nuclear density, colony morphology (shape and size parameters), secondary marker coverage, effective proliferation rates, and so forth (Fig. A1.2). In addition to human connective tissue progenitors (CTPs) derived from bone, bone marrow, cartilage, adipose tissue, muscle, periosteum, and synovium, this practice and technology has been implemented in the cell and colony identification and characterization of several cell and tissue types including: umbilical cord blood hematopoietic stem cells (Fig. X1.2); adipose-derived stem cells (Fig. X1.3); and human epidermal (Fig. X1.4) and dermal (Fig. X1.5) stem cells.

4.3?Benefits of Automated Analysis of CFU AssaysAutomated analysis is expected to provide more rapid, reproducible, and precise results in comparison to the manual enumeration of cells and colonies utilizing a microscope and hemocytometer. In addition to being time consuming, labor intensive, and subjective, manual enumeration has been shown to have a significant degree of intra- and inter-observer variability, with coefficients of variation (CV) ranging from 8.1 % to 40.0 % and 22.7 % to 80 %, respectively. Standard CVs for cell viability assessment and progenitor (colony) type enumeration have been shown to range from 19.4 % to 42.9 % and 46.6 % to 100 %, respectively 4.4?Selection of Cell Culture Surface Area and Optimal Cell Seeding DensityWhen performing a CFU assay, optimizing the 4.5?Useful DocumentsA number of useful documents are available that address best practices for conducting quantitative measurements of cells using imaging approaches: Guide F2998, Guide F3294, ISO 20391-1, ISO 20391-2, and FDA Guidance on Technical Performance Assessment of Digital Pathology Whole Slide Imaging Devices, 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 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 cell and colony counting under a brightfield or fluorescent microscope with or without a hemocytometer 1.16?Advances in software and hardware now broadly enable systematic automated analytical approaches. This evolving technology creates the need for general agreement on units of measurement, nomenclature, process definitions, and analytical interpretation as presented in this practice.

1.17?Standardized methods for automated CFU analysis open opportunities to enhance the value and utility of CFU assays in several scientific and commercial domains: