ASTM F3716-25

1.1 This test method, within the limitations defined, describes procedures for performing serial in vitro cell cultures of vertebrate tissue cells whose quantified total cell numbers over culture time are used to derive cumulative population doubling (CPD) data.

1.2 This test method describes how to derive CPD data from total cell count data from serial tissue cell cultures.

1.3 This test method describes how CPD data can be used to perform scientifically valid quantitative comparisons of the mathematical form (that is, linear versus hyperbolic), rate (that is, slope), and extent of cell proliferation of vertebrate tissue cell populations with known differences in their organ or tissue source, their cell culture media (for example, supplements and test agents), their cell culture conditions, or their intrinsic cellular properties (for example, normal versus tumorigenic).

1.4 This test method describes how CPD data from long-term serial cell culture studies provide more information about the history, rate, and extent of cell proliferation by tissue cell preparations than short-term cell proliferation assays.

1.5 This test method is applied in an in vitro cell culture laboratory setting.

1.6 This test method does not recommend use of a specific cell counting method. Many different types of cell counting methods exist that may be suitable for this test method. Suitable cell counting methods may include the following:

• trypan blue hemocytometer counting;
• trypan blue automated cell counter;
• electrical zone sensing cell counting (Coulter counter) (Test Method F2149);
• or acridine orange-propidium iodide automated fluorescence cell counter.

See Annex A of ISO 20391-1 for a summary of cell counting methods.

1.7 Although live cell and dead cell counting may be performed for this test method, neither is required. Only total cell counts are necessary.

1.8 Although in most cases counts of individual cells are used for this test method, it can be performed with surrogate measures of changes in cell number (for example, light absorbance by cells, light scatter by cells, cell mass).

1.9 This test method can be broadly applied to isolated cell preparations from any vertebrate animal. The following statements are illustrative and non-exclusive:

• 1.9.1 The test method can be applied to mammalian cells isolated from human organs and tissues for regenerative medicine applications, pharmaceutical drug development applications, and toxicological analysis applications.
• 1.9.2 The test method can be applied to mammalian cells isolated from the organs and tissues of animals used in research (for example, mice, rats, dogs, monkeys, pigs, goats, sheep, etc.), used for pharmaceutical and toxicological evaluations, and treated in veterinary medicine.
• 1.9.3 The test method can be applied to freshly isolated uncultured tissue cells (that is, primary cells) or to cell populations after varying degrees of culture, after specific processing (for example, cell fractionation) or after other varied manipulations (for example, genetic modification, neoplastic transformation, subcloning).
• 1.9.4 The test method can be applied to cells isolated from normal, diseased, or injured organs and tissues.

1.10 The test method can be applied to cells in any culture format that permits serial cell culture and quantification of the total number of cells in the culture system at each transfer of cells to a next culture vessel (that is, at each serial cell culture passage).

• 1.10.1 Applicable cell culture formats include adherent cell culture, suspension cell culture, and microcarrier cell culture.
• 1.10.2 Applicable cell culture formats include other cell culture formats (for example, three-dimensional matrix formats) that permit complete cell harvest and uniform sampling and quantification of cells at each serial cell culture cell transfer (see, for example, Guide F2739).
• 1.10.3 During serial cell culture incubation periods, cells may exist as single cells, cell clusters, or even in solid states, as long as they can be completely harvested, uniformly sampled, counted, and allow a portion of the harvested cells, of known number or fraction, to be transferred into the next culture vessel.

1.11 This test method may be performed with a variety of serial cell culture schedules. However, for comparative analyses of the rate and extent of the cell proliferation of different cell populations, it is crucial that the same serial cell culture schedule is used for the compared cell populations.

• 1.11.1 The serial cell culture can be performed by transferring either a constant number or a constant fraction of the cells harvested from the existing cell culture at each cell culture passage.
• 1.11.2 Serial cell culture based on transferring either a constant number of cells or a constant fraction of cells can be performed with either regular time periods of incubation between transfers or irregular time periods of incubation between transfers.
• 1.11.3 Serial cell culture based on transferring either a constant number of cells or a constant fraction of cells can be performed with transfers occurring when cell cultures have reached a designated quantity of cells. With adherent cell culture formats, the designation can be the cultures’ degree of confluency (for example, when 100 % coverage of the culture surface has been achieved). However, other independent measures of the quantity of cells may be applied (for example, absorbance level).
• 1.11.4 This test method can be performed with any variegating combination of serial cell culture schedules, as long as the schedules are well documented for subsequent CPD determinations. However, for comparative analyses of the mathematical form, rate, or extent of the cell proliferation of different cell populations, it is crucial that the same serial cell culture schedule and culture format are used for the compared cell populations.

1.12 This test method has been evaluated (herein) for its ability to provide accurate and precise comparisons of the mathematical form, rate, and extent of proliferation by human primary cell preparations in both intralaboratory and interlaboratory evaluations.

1.13 Limitations are described as follows:

• 1.13.1 The quality of this test method depends on accurate and precise cell counting, whether performed manually with cell counting slides or with well-calibrated automated electronic cell counters.
• 1.13.2 The quality of this test method depends on well-maintained, well-calibrated, and properly operated cell culture equipment, culture vessels, and culture media.
• 1.13.3 The quality of this test method depends on technically proficient cell culture personnel who are well trained in tissue cell culture maintenance and cell counting procedures.
• 1.13.4 The quality of this test method depends on consistent technical procedures throughout, including maintaining standard cell culture passaging and counting techniques, methods, and equipment performance.
• 1.13.5 In the case of adherent cell cultures, dead cells that detach during the cell culture interval will be lost to the accounting. This loss is acceptable for the standard test method, as it is concerned with quantifying the number of population doublings by cells that remain attached in adherent cell cultures.

1.14 Though developed primarily based on experience with vertebrate tissue cell culture, the standard test method described herein may also be applied to analyses of invertebrate tissue cells and plant cells.

1.1 This test method, within the limitations defined, describes procedures for performing serial in vitro cell cultures of vertebrate tissue cells whose quantified total cell numbers over culture time are used to derive cumulative population doubling (CPD) data.

1.2 This test method describes how to derive CPD data from total cell count data from serial tissue cell cultures.

1.3 This test method describes how CPD data can be used to perform scientifically valid quantitative comparisons of the mathematical form (that is, linear versus hyperbolic), rate (that is, slope), and extent of cell proliferation of vertebrate tissue cell populations with known differences in their organ or tissue source, their cell culture media (for example, supplements and test agents), their cell culture conditions, or their intrinsic cellular properties (for example, normal versus tumorigenic).

1.4 This test method describes how CPD data from long-term serial cell culture studies provide more information about the history, rate, and extent of cell proliferation by tissue cell preparations than short-term cell proliferation assays.

1.5 This test method is applied in an in vitro cell culture laboratory setting.

1.6 This test method does not recommend use of a specific cell counting method. Many different types of cell counting methods exist that may be suitable for this test method. Suitable cell counting methods may include the following:

• trypan blue hemocytometer counting;
• trypan blue automated cell counter;
• electrical zone sensing cell counting (Coulter counter) (Test Method F2149);
• or acridine orange-propidium iodide automated fluorescence cell counter.

1.7 Although live cell and dead cell counting may be performed for this test method, neither is required. Only total cell counts are necessary.

1.8 Although in most cases counts of individual cells are used for this test method, it can be performed with surrogate measures of changes in cell number (for example, light absorbance by cells, light scatter by cells, cell mass).

1.9 This test method can be broadly applied to isolated cell preparations from any vertebrate animal. The following statements are illustrative and non-exclusive:

• 1.9.1 The test method can be applied to mammalian cells isolated from human organs and tissues for regenerative medicine applications, pharmaceutical drug development applications, and toxicological analysis applications.
• 1.9.2 The test method can be applied to mammalian cells isolated from the organs and tissues of animals used in research (for example, mice, rats, dogs, monkeys, pigs, goats, sheep, etc.), used for pharmaceutical and toxicological evaluations, and treated in veterinary medicine.
• 1.9.3 The test method can be applied to freshly isolated uncultured tissue cells (that is, primary cells) or to cell populations after varying degrees of culture, after specific processing (for example, cell fractionation) or after other varied manipulations (for example, genetic modification, neoplastic transformation, subcloning).
• 1.9.4 The test method can be applied to cells isolated from normal, diseased, or injured organs and tissues.

1.10 The test method can be applied to cells in any culture format that permits serial cell culture and quantification of the total number of cells in the culture system at each transfer of cells to a next culture vessel (that is, at each serial cell culture passage).

• 1.10.1 Applicable cell culture formats include adherent cell culture, suspension cell culture, and microcarrier cell culture.
• 1.10.2 Applicable cell culture formats include other cell culture formats (for example, three-dimensional matrix formats) that permit complete cell harvest and uniform sampling and quantification of cells at each serial cell culture cell transfer (see, for example, Guide F2739).
• 1.10.3 During serial cell culture incubation periods, cells may exist as single cells, cell clusters, or even in solid states, as long as they can be completely harvested, uniformly sampled, counted, and allow a portion of the harvested cells, of known number or fraction, to be transferred into the next culture vessel.

1.11 This test method may be performed with a variety of serial cell culture schedules. However, for comparative analyses of the rate and extent of the cell proliferation of different cell populations, it is crucial that the same serial cell culture schedule is used for the compared cell populations.

• 1.11.1 The serial cell culture can be performed by transferring either a constant number or a constant fraction of the cells harvested from the existing cell culture at each cell culture passage.
• 1.11.2 Serial cell culture based on transferring either a constant number of cells or a constant fraction of cells can be performed with either regular time periods of incubation between transfers or irregular time periods of incubation between transfers.
• 1.11.3 Serial cell culture based on transferring either a constant number of cells or a constant fraction of cells can be performed with transfers occurring when cell cultures have reached a designated quantity of cells. With adherent cell culture formats, the designation can be the cultures’ degree of confluency (for example, when 100 % coverage of the culture surface has been achieved). However, other independent measures of the quantity of cells may be applied (for example, absorbance level).
• 1.11.4 This test method can be performed with any variegating combination of serial cell culture schedules, as long as the schedules are well documented for subsequent CPD determinations. However, for comparative analyses of the mathematical form, rate, or extent of the cell proliferation of different cell populations, it is crucial that the same serial cell culture schedule and culture format are used for the compared cell populations.

1.12 This test method has been evaluated (herein) for its ability to provide accurate and precise comparisons of the mathematical form, rate, and extent of proliferation by human primary cell preparations in both intralaboratory and interlaboratory evaluations.

1.13 Limitations are described as follows:

• 1.13.1 The quality of this test method depends on accurate and precise cell counting, whether performed manually with cell counting slides or with well-calibrated automated electronic cell counters.
• 1.13.2 The quality of this test method depends on well-maintained, well-calibrated, and properly operated cell culture equipment, culture vessels, and culture media.
• 1.13.3 The quality of this test method depends on technically proficient cell culture personnel who are well trained in tissue cell culture maintenance and cell counting procedures.
• 1.13.4 The quality of this test method depends on consistent technical procedures throughout, including maintaining standard cell culture passaging and counting techniques, methods, and equipment performance.
• 1.13.5 In the case of adherent cell cultures, dead cells that detach during the cell culture interval will be lost to the accounting. This loss is acceptable for the standard test method, as it is concerned with quantifying the number of population doublings by cells that remain attached in adherent cell cultures.

1.14 Though developed primarily based on experience with vertebrate tissue cell culture, the standard test method described herein may also be applied to analyses of invertebrate tissue cells and plant cells.

1.15 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental

5.1 Background for CPD Analyses for in vitro Vertebrate Tissue Cell Proliferation Studies— Since Leonard Hayflick’s early reports on in vitro tissue cell culture studies in the 1960s, CPD data derived from serial cell culture have been used in in vitro tissue cell research as a basis for evaluating the rate and extent of cell proliferation by ex vivo explanted vertebrate tissue cells in in vitro cell culture (1, 2).⁴ Hayflick’s studies defined the now well-described phenomenon of “cellular senescence,” which is observed after extended periods of serial cell culture for normal primary human tissue cell populations. This phenomenon is commonly referred to as the “Hayflick limit” (3). Though not all vertebrate-derived tissue cells exhibit the Hayflick limit, many do (2). The essential quantitative feature of the Hayflick limit for a cell population is limitation to a maximum number of CPDs with serial cell culture because the remaining cells in the culture undergo a terminal arrest of cell division.

5.2 Applications of CPD Analyses— Retention of the Hayflick limit has become a universally accepted phenotypic indicator of the normalcy of in vitro cultured tissue cell populations. Conversely, loss of the Hayflick limit is a phenotypic indicator of in vitro cellular immortalization, and it may occur with neoplastic cellular transformation—the latter of which may have initiated in vivo before tissue cells were explanted (for example, some tumor-derived tissue cells) or as a result of in vitro cell culture effects or manipulations. Accordingly, CPD data from the serial cell culture of immortalized cell populations and neoplastic-transformed cell populations can show greater slopes (4) with serial cell culture time and are not limited by a maximum CPD plateau (5). Similarly, CPD data analyses can be used to compare the cell proliferation properties of tissue cells isolated from different organs, different tissues, and different species of vertebrate animals. One well-known example of such cell type comparison analyses is the use of CPD data to compare the rate and extent of cell proliferation for populations of human skin fibroblast cells isolated from donors of different ages (1, 6).

• 5.2.1 In the same manner that differences in serial cell culture CPD data have been used to compare the cell proliferation properties of populations of different cell types, they have been used to evaluate effects of supplemented agents on the mathematical form, rate, and extent of cell proliferation of selected tissue cell populations of interest. Examples of evaluated agents include drug candidates, environmental toxicants, cell and tissue biomanufacturing agents, and factors investigated in tissue cell research.

5.3 Advantages of CPD Data for Comparative Analyses of Cell Proliferation and Cell Phenotype— CPD data from serial cell cultures have long been recognized and used in in vitro cell research as a cell culture parameter for evaluation of the rate and extent of the proliferation of primary vertebrate tissue cells (1-3, 7, 8). CPD data are recognized as a better basis (than culture passage number or number of days of culture) for comparing the proliferation of primary cell populations from different tissue cell sources, different cell donors, or maintained with different cell culture media and growth factor supplements (9). Even when comparing cell populations propagated with different cell culture conditions, the number of CPDs achieved is often used as a predictor of differences in the future proliferation potential of compared cell populations. The number of CPDs of a cultured cell population is considered an indicator of the risk of cell mutations occurring in cultured cell populations (10-13). The number of CPDs achieved by a cell population can also be used to predict phenotypic changes in cells in the population (for example, loss of stem cell properties, differentiation, terminal division arrest) (1-3, 14-19).

5.4 Roles for CPD Data Analyses in New Cell Analysis Technologies— Recently, the mathematical properties of CPD data with serial cell culture have been accounted for by the continuation of in vivo tissue cell kinetics in in vitro cell culture. The Hayflick limit can be explained by the continued production of terminally arrested cells in cell culture during serial dilution of tissue stem cells because of the intrinsic asymmetric self-renewal division kinetics of tissue stem cells (20, 21). This new understanding of the cellular basis for the Hayflick limit led to the development of a new computational approach for defining the specific fractions of stem cells, transiently amplifying cells, and terminally arrested cells in primary tissue cell preparations and to defining how those fractions change during serial cell culture (21-23). This new tissue cell analysis innovation, and others like it in the future, may benefit from this standard test method (24).

5.5 Significance of a Standard Test Method for Determination and Evaluation of Serial Cell Culture CPD Data— A standard test method is needed for a cell analysis procedure that has such a long history of widespread applications in vertebrate tissue cell science, medicine, and the pharmaceutical industry. There are many elements in the determination of serial cell culture CPD data that are subject to error, technical variability, and variation in practice. The most effective implementation of CPD data requires consistency in serial cell culture procedures, consistency in cell counting procedures, consistency in cell count data analysis, and consistency in the interpretation of CPD data analysis results. There can be a high degree of variation in each of these, which undermines the advantages that could be obtained with consistency in CPD data analysis. Currently, a pervasive misstep is making conclusions from comparisons of CPD data that were derived with incongruent serial cell culture procedures (for example, with different passage intervals or a different passage basis). Differences in factors such as cell culture density at the time of passage cause differences in the cell-type-specific cell kinetics of cultured tissue cells (for example, differences in the frequency of symmetric self-renewal divisions by stem cells) (20, 22). This standard test method for CPD data may benefit many areas of science and medicine that utilize in vitro vertebrate tissue cell culture. This test method will provide a needed quantitative standard for scientifically valid comparison of the mathematical form, rate, and extent of cell proliferation by many important tissue cell populations used in molecular cell research, cancer cell research, regenerative medicine, tissue engineering, pharmaceutical drug development, and toxicological assessments.

• 5.5.1 Comparison of the mathematical form of CPD data (that is, linear or hyperbolic), the CPD data slope (that is, the rate of proliferation), and the CPD data maximum (that is, the extent of proliferation) is scientifically valid for cell populations serially cultured on the same schedule with the same culture format. The standardization provided by this test method allows scientifically valid evaluations of the mathematical form, rate, and extent of cell proliferation by the same types of cell populations when maintained and used in different laboratories or other sites of analysis. The standardization of CPD data production provided by this test method provides a foundation for the development of new technologies that use CPD data to discover and quantify critical quality attributes (CQAs) for regenerative medicine, cell and tissue biomanufacturing, and drug development. An example of a potential CQA is the fraction of specialized tissue cell subpopulations, such as tissue stem cells.