Standard Test Method for Cumulative Population Doubling Analysis of the Proliferation of Vertebrate Tissue Cell Preparations

Importancia y uso:

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.

Subcomité:

F04.43

Volúmen:

13.02

Palabras clave:

cell counting; cell proliferation; confluency; cumulative population doubling; passage; passage basis; passage interval; serial cell culture; terminal arrest; vertebrate tissue cell;