1.1 本规程定义了在已知促进矿化的条件下培养的活细胞培养物中多个时间点的钙含量估计方法。该实践涉及应用荧光钙螯合染料，该染料与活培养物中存在的磷酸钙矿物晶体结合，然后对染色细胞培养物的荧光显微镜图像进行图像分析。阳性染色区域的量化提供了细胞培养板中钙含量的相对测量。图像分析参数与钙含量之间的精确相关性超出了本实践的范围。 1.2 分泌基质中的钙沉积是骨形成的几个特征之一( 体外 和 体内 )因此，它是一个可能指示骨形成和成骨细胞功能（即成骨细胞分化）的参数。然而，如果细胞培养中发生广泛的细胞死亡，或者如果使用大量导致人工钙基沉淀物的成骨培养基成分，则钙沉积可能与成骨细胞分化状态无关。区分与成骨细胞产生的矿化基质相关的钙沉积和病理或人工沉积，需要对矿化基质进行额外的结构和化学表征，并对细胞进行生物学表征，这超出了本实践的范围。 1.3 通过图像分析获得的参数以相对荧光单位或面积百分比（面积%）表示，例如，分析面积的覆盖率。 1.4 单位- 以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.5 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.6 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》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 体外 成骨细胞分化试验是筛选祖干细胞成为成骨细胞能力的一种方法。形成的钙沉积或矿化基质的程度 体外 可能是分化为功能性成骨细胞的指标；然而，成骨基因或蛋白质的表达是另一个重要的测量方法，可与该试验结合使用，以确定成骨细胞的存在。 5.2 本实践提供了一种染色、成像和量化与活细胞培养中矿化相关的荧光强度和面积的技术，使用非- 有毒钙螯合染料XO。细胞培养中矿化沉积物的阳性染色区域是钙含量的间接测量。测量强度以确保图像没有曝光不足或过度曝光很重要。强度和面积与钙含量没有直接关系。 5.3 XO能够在培养物的整个生命周期内重复监测钙沉积，而不会损害培养物。由于重复使用导致染料累积，因此不会干扰矿化区域的后续测量 ( 1. ) . 3. 先前已染色的钙沉积物可能看起来更亮，但这不会影响面积测量。 钙黄绿素染料也可用于此目的 ( 1. ) 但需要与XO不同的分析程序（即浓度和过滤器组），因此此处不包括。茜素红和冯·科萨不适合在活培养物上使用此程序，因为没有文件支持其在活培养物中重复使用而不会产生有害影响。 5.4 该方法可应用于任何能够产生钙沉积的细胞的培养。它也可用于记录培养物中不存在矿物质，目的是避免矿化。 5.5 在成骨细胞分化试验期间，提供成骨补充剂以诱导或协助分化过程。 如果过量使用成骨补充剂，细胞培养中可能会出现非成骨细胞介导的钙沉积，被称为营养不良、病理性或人为因素 ( 2. ) . 例如，当在培养基中使用更高浓度的β-甘油磷酸作为细胞分泌的碱性磷酸酶的底物时，游离磷酸盐显著增加，然后随Ca沉淀 ++ 离子在培养基中形成磷酸钙晶体，与祖细胞的分化状态无关。碱性磷酸酶的产生与祖细胞分化有关，并且经常被添加到培养基中的地塞米松刺激，从而增强钙沉积的形成。 因此，这些类型的钙/矿物质沉积被认为是营养不良、病理性或人为的，因为它们不是由成熟的成骨细胞启动的。因此，如果在没有基因或蛋白质表达数据的情况下单独使用，通过使用该实践获得的测量可能会导致对骨祖细胞分化状态的潜在错误解释 ( 3. , 4. ) . 5.6 由于矿化分析期间可能存在人工钙沉积 ( 2- 4. ) 建议将证明RNA信息或骨钙素和骨涎蛋白存在的基因表达分析或蛋白质分析技术与本文所述的钙沉积定量程序结合使用，以确认是否存在正在分泌矿化基质的成熟成骨细胞。 5.7 矿化物质在培养皿中的沉积不能证实所培养的细胞能够形成骨 体内 . 5.8 培养皿中矿化基质沉积的模式会有所不同，这取决于细胞传代的次数（即第一代原代细胞与多次传代的细胞，包括细胞系）。第一代原代细胞通常形成相对较大的骨祖细胞结节，可分化和矿化，而经过多次传代的细胞可在整个培养皿中形成弥漫、分散的矿物质。 这种做法与矿化模式无关，可用于分析原生细胞和细胞系中的矿化基质。 5.9 由于一些细胞的增殖速度比其他细胞慢，并且由于正在测试的一些细胞培养表面可能会影响细胞的增殖，因此可以将数据归一化为细胞总数。由于增殖减少通常会减少矿化，细胞数正常化通常不会影响结果。总DNA含量可以作为细胞数量的间接测量。有几种商用套件可用于此目的。由于DNA分析是一种破坏性的毒性分析，如果使用这种分析，必须准备额外的细胞培养物。

1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium-chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice. 1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation ( in vitro and in vivo ), and is therefore a parameter that may indicate bone formation and osteoblast function (that is, osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice. 1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage (area%), for example, fraction of coverage of the area analyzed. 1.4 Units— The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.5 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. 1.6 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 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcium deposits or mineralized matrix that form in vitro may be an indicator of differentiation to a functional osteoblast; however, expression of osteogenic genes or proteins is another important measurement to use in conjunction with this assay to determine the presence of an osteoblast. 5.2 This practice provides a technique for staining, imaging, and quantifying the fluorescence intensity and area related to the mineralization in living cell cultures using the non-toxic calcium-chelating dye, XO. The positively stained area of mineralized deposits in cell cultures is an indirect measure of calcium content. It is important to measure the intensity to ensure that the images have not been underexposed or overexposed. Intensity and area do not correlate directly to calcium content. 5.3 XO enables the monitoring of calcium deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of the mineralized area due to dye accumulation from repeated application ( 1 ) . 3 Calcium deposits that have been previously stained may appear brighter, but this does not impact the area measurement. Calcein dyes may also be used for this purpose ( 1 ) but require a different procedure for analysis than XO (that is, concentration and filter sets) and are thus not included here. Alizarin Red and Von Kossa are not suitable for use with this procedure on living cultures since there is no documentation supporting their repeated use in living cultures without deleterious effects. 5.4 The practice may be applied to cultures of any cells capable of producing calcium deposits. It may also be used to document the absence of mineral in cultures where the goal is to avoid mineralization. 5.5 During osteoblast differentiation assays, osteogenic supplements are provided to induce or assist with the differentiation process. If osteogenic supplements are used in excess, a calcium deposit that is not osteoblast-mediated and is referred to as dystrophic, pathologic, or artifactual may occur in the cell cultures ( 2 ) . For example, when higher concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca ++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell. Alkaline phosphatase production is associated with progenitor cell differentiation, and is frequently stimulated by dexamethasone addition to the medium, which enhances the formation of calcium deposits. These kinds of calcium/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. The measurement obtained by using this practice may thus result in a potentially false interpretation of the differentiation status of osteoprogenitor cells if used in isolation without gene or protein expression data ( 3 , 4 ) . 5.6 Due to the possibility of artifactual calcium deposits during mineralization assays ( 2- 4 ) , gene expression analysis or protein analysis techniques demonstrating the RNA message or the presence of osteocalcin and bone sialoprotein are recommended for use in conjunction with the calcium deposit quantification procedure described here in order to confirm the presence of mature osteoblasts that are in the process of secreting a mineralizing matrix. 5.7 The deposition of a mineralized substance in the culture dish does not confirm that the cells being cultured are capable of forming bone in vivo . 5.8 The pattern of mineralized matrix deposition in the culture dish will vary, depending on the number of times the cells have been passaged (that is, first passage primary cells versus cells that have been passaged several times, including cell lines). First passage primary cells typically form relatively large nodules of osteoprogenitor cells that differentiate and mineralize, while cells that have been passaged many times lead to the formation of diffuse, dispersed mineral throughout the culture dish. This practice is independent of the pattern of mineralization and can be used to analyze mineralized matrix in both primary cells and cell lines. 5.9 Since some cells proliferate slower than others and since some of the cell culture surfaces being tested may affect proliferation of the cells, the data can be normalized to total cell number. Since reduced proliferation typically reduces mineralization, normalization to cell number typically does not influence the outcomes. Total DNA content can be determined as an indirect measure of cell number. There are several commercially available kits for this purpose. Since DNA analysis is a destructive, toxic assay, additional cell cultures must be prepared if this assay is used.