1、Designation:F299713Standard Practice forQuantification of Calcium Deposits in Osteogenic Culture ofProgenitor Cells Using Fluorescent Image Analysis1This standard is issued under the fixed designation F2997;the number immediately following the designation indicates the year oforiginal adoption or,in
2、 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 defines a method for the estimation ofcalcium content at multiple time poin
3、ts in living cell culturesthat have been cultured under conditions known to promotemineralization.The practice involves applying a fluorescentcalcium chelating dye that binds to the calcium phosphatemineral crystals present in the live cultures followed by imageanalysis of fluorescence microscopy im
4、ages of the stained cellcultures.Quantification 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
5、 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 i
6、f 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
7、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 fluorescence units or area percentage,e.g.,fra
8、ction of coverage of the area analyzed.1.4 UnitsThe 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
9、 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 Polymer
10、ic 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 Definitions:3.2.1 mineralizedmatrix,nacalciumphosphate-containing substance produced by cells typically in th
11、eosteoblast,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,nsecretory mononuclear cells that willinitiate the formation of a matrix containing characteristicproteins,s
12、uch as collagen,and non-collageneous proteins suchas bone sialoprotein and osteocalcin,that will mineralize in thepresence of a calcium and phosphate source.3.3 Definitions of Terms Specific to This Standard:3.3.1 calcium deposits,na calcium phosphate-containingsubstance synthesized in cell cultures
13、 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)fluorescently staining thecalcium deposits in a cell culture using the non-toxic calcium-chelat
14、ing dye xylenol orange,(2)collecting fluorescent micros-copy images of the stained samples,(3)collecting images of1This 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 E
15、ngineered Constructs for TEMPs.Current edition approved Dec.1,2013.Published January 2014.DOI:10.1520/F2997-13.2For referenced ASTM standards,visit the ASTM website,www.astm.org,orcontact ASTM Customer Service at serviceastm.org.For Annual Book of ASTMStandards volume information,refer to the standa
16、rds Document Summary page onthe ASTM website.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 intensity standards in bead form,and(4)conducting imageanalysis of thresholded images of the standards and thesamples to determine area percentage and mean intensity of thestained areas.4.2 The practice involves the testing and analysis of afluorescent intensity standard in order to determine standard-ized image analysis settings for imaging o
