1.1 本试验方法在规定的限度内，描述了进行系列试验的程序 体外 脊椎动物组织细胞的细胞培养物，其在培养时间内量化的总细胞数用于获得累积群体倍增(CPD)数据。 1.2 该测试方法描述了如何从连续组织细胞培养物的总细胞计数数据中获得CPD数据。 1.3 该测试方法描述了如何使用CPD数据对脊椎动物组织细胞群的数学形式(即线性与双曲线)、速率(即斜率)和细胞增殖程度进行科学上有效的定量比较，其中脊椎动物组织细胞群的器官或组织来源、细胞培养基(例如，补充剂和测试剂)、细胞培养条件或固有细胞特性(例如，正常与致瘤性)已知差异。1.4 该测试方法描述了来自长期连续细胞培养研究的CPD数据如何比短期细胞增殖测定提供更多关于组织细胞制剂的细胞增殖的历史、速率和程度的信息。 1.5 该测试方法应用于 体外 细胞培养实验室设置。 1.6 本试验方法不推荐使用特定的细胞计数方法。存在许多不同类型的细胞计数方法可适用于该测试方法。合适的细胞计数方法可以包括以下方法:台盼蓝血细胞计数器计数；台盼蓝自动细胞计数器；电区感应细胞计数（库尔特计数器）（试验方法 F2149 );或吖啶橙-碘化丙啶自动荧光细胞计数器。参见ISO 20391附录A-1用于细胞计数方法的总结。 1.7 尽管该测试方法可以进行活细胞和死细胞计数，但两者都不是必需的。仅需要总细胞计数。 1.8 尽管在大多数情况下，该测试方法使用单个细胞的计数，但它可以通过细胞数量变化的替代测量来进行（例如，细胞的光吸收、细胞的光散射、细胞质量）。 1.9 该测试方法可广泛应用于来自任何脊椎动物的分离细胞制剂。以下陈述是说明性的且非排他性的: 1.9.1 该测试方法可应用于从人体器官和组织中分离的哺乳动物细胞，用于再生医学应用、药物开发应用和毒理学分析应用。1.9.2 该测试方法可应用于从研究中使用的动物(例如小鼠、大鼠、狗、猴、猪、山羊、绵羊等)的器官和组织中分离的哺乳动物细胞，用于药理学和毒理学评估，并用于兽医治疗。 1.9.3 测试方法可以应用于新鲜分离的未培养的组织细胞(即原代细胞)或在不同程度的培养之后、在特定处理(例如细胞分级)之后或在其他不同操作(例如遗传修饰、肿瘤转化、亚克隆)之后的细胞群。 1.9.4 该测试方法可应用于从正常、患病或损伤的器官和组织中分离的细胞。 1.10 测试方法可以应用于任何培养形式的细胞，所述培养形式允许连续细胞培养和在每次将细胞转移到下一个培养容器时(即，在每次连续细胞培养传代时)定量培养系统中的细胞总数。1.10.1 适用的细胞培养形式包括贴壁细胞培养、悬浮细胞培养和微载体细胞培养。 1.10.2 适用的细胞培养形式包括其他细胞培养形式（例如，三维基质形式），其允许在每次连续细胞培养细胞转移时完全收获细胞以及均匀取样和定量细胞（参见例如指南 F2739 ). 1.10.3 在连续细胞培养温育期间，细胞可以作为单细胞、细胞簇或甚至以固态存在，只要它们可以被完全收获、均匀取样、计数，并且允许已知数量或分数的一部分收获的细胞转移到下一个培养容器中。 1.11 该测试方法可以用多种连续细胞培养方案进行。然而，对于不同细胞群的细胞增殖速率和程度的比较分析，对比较的细胞群使用相同的连续细胞培养时间表是至关重要的。 1.11.1 连续细胞培养可以通过在每个细胞培养传代转移从现有细胞培养物中收获的恒定数量或恒定分数的细胞来进行。 1.11.2 基于转移恒定数量的细胞或恒定级分的细胞的连续细胞培养可以在转移之间有规律的孵育时间段或在转移之间有不规则的孵育时间段的情况下进行。 1.11.3 可以进行基于转移恒定数量的细胞或恒定分数的细胞的连续细胞培养，其中当细胞培养物达到指定数量的细胞时发生转移。对于贴壁细胞培养形式，指定可以是培养物的汇合度(例如，当已经实现培养表面的100%覆盖时)。然而，可以应用细胞数量的其他独立测量(例如，吸光度水平)。 1.11.4 该测试方法可以用连续细胞培养时间表的任何多样化组合进行，只要该时间表被很好地记录用于随后的CPD测定。然而，对于不同细胞群的细胞增殖的数学形式、速率或程度的比较分析，对比较的细胞群使用相同的连续细胞培养时间表和培养形式是至关重要的。 1.12 在实验室内和实验室间评估中，已经评估了该测试方法在人原代细胞制备物增殖的数学形式、速率和程度的准确和精确比较的能力。1.13 限制描述如下: 1.13.1 该测试方法的质量取决于准确和精确的细胞计数，无论是使用细胞计数载玻片手动进行还是使用校准良好的自动化电子细胞计数器进行。 1.13.2 该测试方法的质量取决于维护良好、校准良好和操作正确的细胞培养设备、培养容器和培养基。 1.13.3 该测试方法的质量取决于在组织细胞培养维持和细胞计数程序方面受过良好培训的技术熟练的细胞培养人员。 1.13.4 该测试方法的质量取决于始终一致的技术程序，包括保持标准细胞培养传代和计数技术、方法和设备性能。 1.13.5 在贴壁细胞培养的情况下，在细胞培养间隔期间分离的死细胞将在计数中丢失。这种损失对于标准测试方法是可接受的，因为它涉及定量粘附细胞培养物中保持附着的细胞的群体倍增数量。 1.14 尽管主要基于脊椎动物组织细胞培养的经验开发，但本文所述的标准测试方法也可应用于无脊椎动物组织细胞和植物细胞的分析。 1.15 本标准并不旨在解决与其使用相关的所有安全性问题（如果有）。本标准的使用者有责任在使用前建立适当的安全、健康和环境实践并确定法规限制的适用性。1.16 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认的标准化原则制定的。 ======意义和用途====== 5.1 体外脊椎动物组织细胞增殖研究的CPD分析背景- 自从伦纳德·海弗利克早期报道 体外 组织细胞培养研究在20世纪60年代，来自连续细胞培养的CPD数据已被用于 体外 组织细胞研究作为评估细胞增殖速率和程度的基础 离体 移植的脊椎动物组织细胞 体外 细胞培养 ( 1 , 2 ) . 4 海弗利克的研究定义了现在很好的——描述了“细胞衰老”现象，其在正常原代人组织细胞群的长时间连续细胞培养后观察到。这种现象通常被称为“海弗利克极限” ( 3 ) 虽然不是所有脊椎动物来源的组织细胞都表现出海弗利克极限，但许多细胞表现出 ( 2 ) 细胞群的Hayflick极限的基本定量特征是对连续细胞培养的CPD的最大数量的限制，因为培养物中的剩余细胞经历细胞分裂的终末停滞。 5.2 CPD分析的应用- Hayflick极限的保留已成为普遍接受的正常性表型指标 体外 培养的组织细胞群。相反，海弗利克极限的丧失是 体外 细胞永生化，它可能与肿瘤细胞转化一起发生——后者可能已经开始 体内 在移植组织细胞(例如，一些肿瘤来源的组织细胞)之前或作为 体外 细胞培养效应或操作。因此，来自永生化细胞群和肿瘤转化细胞群的连续细胞培养物的CPD数据可以显示更大的斜率 ( 4 ) 具有连续细胞培养时间，并且不受最大CPD平台的限制 ( 5 ) 类似地，CPD数据分析可用于比较从不同器官、不同组织和不同物种的脊椎动物分离的组织细胞的细胞增殖特性。这种细胞类型比较分析的一个众所周知的例子是使用CPD数据来比较从不同年龄的供体分离的人皮肤成纤维细胞群的细胞增殖速率和程度 ( 1 , 6 ) . 5.2.1 以连续细胞培养CPD数据的差异已用于比较不同细胞类型群体的细胞增殖特性的相同方式，它们已用于评估补充剂对选定的感兴趣组织细胞群体的细胞增殖的数学形式、速率和程度的影响。评估试剂的实例包括候选药物、环境毒物、细胞和组织生物制造试剂以及在组织细胞研究中研究的因素。 5.3 CPD数据用于细胞增殖和细胞表型比较分析的优势- 来自连续细胞培养的CPD数据早已被认可并用于 体外 细胞研究作为评估原代脊椎动物组织细胞增殖速率和程度的细胞培养参数 ( 1- 3 , 7 , 8 ) CPD数据被认为是比较来自不同组织细胞来源、不同细胞供体或用不同细胞培养基和生长因子补充剂维持的原代细胞群增殖的更好基础（比培养传代数或培养天数） ( 9 ) 即使当比较用不同细胞培养条件繁殖的细胞群时，获得的CPD的数量也经常被用作比较细胞群未来增殖潜力差异的预测因子。培养的细胞群体的CPD的数量被认为是培养的细胞群体中发生细胞突变的风险的指标 ( 10- 13 ) 细胞群体实现的CPD的数量也可用于预测群体中细胞的表型变化（例如，干细胞特性的丧失、分化、终末分裂停滞） ( 1- 3 , 14- 19 ) . 5.4 CPD数据分析在新细胞分析技术中的作用- 最近，连续细胞培养的CPD数据的数学性质已经通过继续 体内 组织细胞动力学 体外 细胞培养。由于组织干细胞固有的不对称自我更新分裂动力学，在组织干细胞的连续稀释过程中，细胞培养物中终末停滞细胞的持续产生可以解释Hayflick极限 ( 20 , 21 ) 对Hayflick极限的细胞基础的这种新理解导致了一种新的计算方法的发展，用于定义原代组织细胞制剂中干细胞、瞬时扩增细胞和终末停滞细胞的特定级分，并定义这些级分在连续细胞培养过程中如何变化 ( 21- 23 ) 这种新的组织细胞分析创新，以及未来的其他类似创新，可能会受益于这种标准测试方法 ( 24 ) . 5.5 测定和评价连续细胞培养CPD数据的标准试验方法的意义- 在脊椎动物组织细胞科学、医学和制药工业中具有如此悠久的广泛应用历史的细胞分析程序需要标准测试方法。在连续细胞培养CPD数据的确定中存在许多因素，这些因素在实践中存在误差、技术可变性和变化。CPD数据的最有效实施需要连续细胞培养程序的一致性、细胞计数程序的一致性、细胞计数数据分析的一致性以及CPD数据分析结果的解释的一致性。这些中的每一个都可能存在高度的变化，这破坏了在持续专业发展数据分析中通过一致性可以获得的优势。目前，一个普遍的失误是从不一致的连续细胞培养程序（例如，具有不同的传代间隔或不同的传代基础）获得的CPD数据的比较中得出结论。传代时的细胞培养密度等因素的差异导致培养的组织细胞的细胞类型特异性细胞动力学的差异(例如，干细胞的对称自我更新分裂的频率的差异) ( 20 , 22 ) 这种CPD数据的标准测试方法可能有益于许多科学和医学领域 体外 脊椎动物组织细胞培养。该测试方法将为mo中使用的许多重要组织细胞群的细胞增殖的数学形式、速率和程度的科学有效比较提供所需的定量标准细胞研究、癌细胞研究、再生医学、组织工程、药物开发和毒理学评估。 5.5.1 CPD数据的数学形式(即线性或双曲线)、CPD数据斜率(即增殖速率)和CPD数据最大值(即增殖程度)的比较对于以相同培养形式按相同时间表连续培养的细胞群体是科学有效的。该测试方法提供的标准化允许在不同实验室或其他分析场所维持和使用相同类型的细胞群时，对细胞增殖的数学形式、速率和程度进行科学有效的评估。该测试方法提供的CPD数据生产的标准化为开发使用CPD数据发现和量化再生医学、细胞和组织的关键质量属性(CQA)的新技术提供了基础生物制造和药物开发。潜在CQA的一个实例是特化组织细胞亚群(诸如组织干细胞)的分数。

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. 1.15 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 practices and determine the applicability of regulatory limitations prior to use. 1.16 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 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.