ASTM F2997-13
采用荧光图像分析量化祖细胞成骨培养钙沉积的标准实施规程

Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis

被代替

标准号

ASTM F2997-13

发布

2013年

发布单位

美国材料与试验协会

替代标准

ASTM F2997-21

当前最新

ASTM F2997-21

 

 

引用标准

ASTM F2312 ASTM F2603 ASTM F2739

适用范围

5.1 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcified deposits or mineralized matrix that form in-vitro may be an indicator of differentiation to a functional osteoblast; however, gene 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 test method 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, xylenol orange. 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 assure that the images have not been underexposed or overexposed. Intensity does not correlate directly to calcium content as well as area.

5.3 Xylenol orange enables the monitoring of calcified deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of mineralized area due to dye accumulation from repeated application (1).³ Calcified 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 xylenol orange (i.e., 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 test method may be applied to cultures of any cells capable of producing calcified 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 calcified deposit may occur in the cell cultures that is not osteoblast-mediated and thus is referred to as dystrophic, pathologic, or artifactual (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 calcified deposits. These kinds of calcified/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).

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