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.

1.10.1 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.

1.11.1 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.

1.13.1 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.