Designation: F2997 − 13Standard Practice forQuantif i cation of Calcium Deposits in Osteogenic Culture ofProgenitor Cells Using Fluorescent Image Analysis 1This standard is issued under the f i xed designation F2997; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (´) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice def i nes a method for the estimation ofcalcium content at multiple time points in living cell culturesthat have been cultured under conditions known to promotemineralization. The practice involves applying a f l uorescentcalcium chelating dye that binds to the calcium phosphatemineral crystals present in the live cultures followed by imageanalysis of f l uorescence microscopy images of the stained cellcultures. Quantif i cation of the positively stained areas providesa relative measure of the calcium content in the cell cultureplate. A precise correlation between the image analysis param-eters and calcium content is beyond the scope of this practice.1.2 Calcium deposition in a secreted matrix is one of severalfeatures that characterize bone formation (in vitro and in vivo),and is therefore a parameter that may indicate bone formationand osteoblast function (i.e., osteoblastic differentiation). Cal-cium deposition may, however, be unrelated to osteoblastdifferentiation status if extensive cell death occurs in the cellcultures or if high amounts of osteogenic medium componentsthat lead to artifactual calcium-based precipitates are used.Distinguishing between calcium deposition associated withosteoblast-produced mineralized matrix and that from patho-logical or artifactual deposition requires additional structuraland chemical characterization of the mineralized matrix andbiological characterization of the cell that is beyond the scopeof this practice.1.3 The parameters obtained by image analysis are ex-pressed in relative f l uorescence units or area percentage, e.g.,fraction of coverage of the area analyzed.1.4 Units—The values stated in SI units are to be regardedas standard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards: 2F2312 Terminology Relating to Tissue Engineered MedicalProductsF2603 Guide for Interpreting Images of Polymeric TissueScaffoldsF2739 Guide for Quantitating Cell Viability Within Bioma-terial Scaffolds3. Terminology3.1 Unless provided otherwise in 3.2, terminology shall bein conformance with F2312.3.2 Def i nitions:3.2.1 mineralized matrix, n—a calcium phosphate-containing substance produced by cells typically in theosteoblast, odontoblast, and calcifying chondrocyte lineages,which is composed of crystals of calcium phosphate andcontains collagen Type I and other non-collagenous proteins.3.2.2 osteoblasts, n—secretory mononuclear cells that willinitiate the formation of a matrix containing characteristicproteins, such as collagen, and non-collageneous proteins suchas bone sialoprotein and osteocalcin, that will mineralize in thepresence of a calcium and phosphate source.3.3 Def i nitions of Terms Specif i c to This Standard:3.3.1 calcium deposits, n—a calcium phosphate-containingsubstance synthesized in cell cultures during mineralizationassays; such as, osteoblast differentiation assays, that may haveprecipitated out of solution rather than being produced by thecells.4. Summary of Practice4.1 This practice consists of (1) f l uorescently staining thecalcium deposits in a cell culture using the non-toxic calcium-chelating dye xylenol orange, (2) collecting f l uorescent micros-copy images of the stained samples, (3) collecting images of1 This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved Dec. 1, 2013. Published January 2014. DOI: 10.1520/F2997-13.2 For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at service@astm.org. For Annual Book of ASTMStandards volume information, refer to the standard’s Document Summary page onthe ASTM website.Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1
