1、Designation: F2997 13Standard 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
2、, 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 defines a method for the estimation ofcalcium content at multiple
3、 time points 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 mi
4、croscopy images 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 de
5、position 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 osteoblastdiffe
6、rentiation 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-logi
7、cal 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 fluorescence units or area
8、percentage, e.g.,fraction 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
9、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 Inter
10、preting 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 Definitions:3.2.1 mineralized matrix, na calcium phosphate-containing substance pr
11、oduced 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, nsecretory mononuclear cells that willinitiate the formation of a matrix c
12、ontaining 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 Definitions of Terms Specific to This Standard:3.3.1 calcium deposits, na calcium phosphate-containing
13、substance 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) fluorescently staining thecalcium deposits in a cel
14、l culture using the non-toxic calcium-chelating 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 responsibi
15、lity of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved Dec. 1, 2013. Published January 2014. DOI: 10.1520/F2997-13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Boo
16、k of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1intensity standards in bead form, and (4) conducting imageanalysis of thresholded i
17、mages 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 of the calcified cellcultures. The u
18、se of a standard allows for the comparisonbetween different samples or different time points. Methods fordetermining area percentage and mean intensity of the standardand the samples are described.5. Significance and Use5.1 In-vitro osteoblast differentiation assays are one ap-proach to screen proge
19、nitor stem cells for their capability tobecome osteoblasts. The extent of calcified deposits or miner-alized matrix that form in-vitro may be an indicator ofdifferentiation to a functional osteoblast; however, gene ex-pression of osteogenic genes or proteins is another importantmeasurement to use in
20、 conjunction with this assay to determinethe presence of an osteoblast.5.2 This test method provides a technique for staining,imaging, and quantifying the fluorescence intensity and arearelated to the mineralization in living cell cultures using thenon-toxic calcium-chelating dye, xylenol orange. Th
21、e posi-tively stained area of mineralized deposits in cell cultures is anindirect measure of calcium content. It is important to measurethe intensity to assure that the images have not been underex-posed or overexposed. Intensity does not correlate directly tocalcium content as well as area.5.3 Xyle
22、nol orange enables the monitoring of calcifieddeposits repeatedly throughout the life of the culture withoutdetriment to the culture. There is no interference on subsequentmeasurements of mineralized area due to dye accumulationfrom repeated application (1).3Calcified deposits that havebeen previous
23、ly stained may appear brighter, but this does notimpact the area measurement. Calcein dyes may also be usedfor this purpose (1) but require a different procedure foranalysis than xylenol orange (i.e., concentration and filter sets)and are thus not included here.Alizarin Red and Von Kossa arenot suit
24、able for use with this procedure on living cultures sincethere is no documentation supporting their repeated use inliving cultures without deleterious effects.5.4 The test method may be applied to cultures of any cellscapable of producing calcified deposits. It may also be used todocument the absenc
25、e of mineral in cultures where the goal isto avoid mineralization.5.5 During osteoblast differentiation assays, osteogenicsupplements are provided to induce or assist with the differ-entiation process. If osteogenic supplements are used in excess,a calcified deposit may occur in the cell cultures th
26、at is notosteoblast-mediated and thus is referred to as dystrophic,pathologic, or artifactual (2). For example, when higher con-centrations of beta-glycerophosphate are used in the medium tofunction as a substrate for the enzyme alkaline phosphatasesecreted by the cells, there is a marked increase i
27、n freephosphate, which then precipitates with Ca+ions in the mediato form calcium phosphate crystals independently of thedifferentiation status of the progenitor cell. Alkaline phos-phatase production is associated with progenitor celldifferentiation, and is frequently stimulated by dexamethasoneadd
28、ition to the medium, which enhances the formation ofcalcified deposits. These kinds of calcified/mineral deposits arethus considered dystrophic, pathologic, or artifactual becausethey were not initiated by a mature osteoblast. The measure-ment obtained by using this practice may thus result in apote
29、ntially false interpretation of the differentiation status ofosteoprogenitor cells if used in isolation without gene orprotein expression data (3,4).5.6 Due to the potential of artifactual calcified depositsduring mineralization assays (2-4), gene expression analysis orprotein analysis techniques de
30、monstrating the RNAmessage orthe presence of osteocalcin and bone sialoprotein are recom-mended for use in conjunction with the calcified depositquantification procedure described here in order to confirm thepresence of mature osteoblasts that are in the process ofsecreting a mineralizing matrix.5.7
31、 The deposition of a mineralized substance in the culturedish does not confirm that the cells being cultured are capableof forming bone in vivo.5.8 The pattern of mineralized matrix deposition in theculture dish will vary depending on the number of times thecells have been passaged (i.e., first pass
32、age primary cellsversus cells that have been passaged several times, includingcell lines). First passage primary cells typically form relativelylarge nodules of osteoprogenitor cells that differentiate andmineralize, while cells that have been passaged many timeslead to the formation of diffuse, dis
33、persed mineral throughoutthe culture dish. This test method is independent of pattern ofmineralization and can be used to analyze mineralized matrixin both primary cells and cell lines.5.9 Since some cells proliferate slower than others and sincesome of the cell culture surfaces being tested may aff
34、ectproliferation of the cells, the data can be normalized to totalcell number. Since reduced proliferation typically reducesmineralization, normalization to cell number typically does notinfluence the outcomes. Total DNA content can be determinedas an indirect measure of cell number. There are sever
35、alcommercially available kits for this purpose. Since DNAanalysis is a destructive, toxic assay, additional cell culturesmust be prepared if this assay is used.6. Interferences6.1 Xylenol orange does not photobleach during micros-copy nor leach out of the stained mineral with time, and isstable for
36、several months; thus, stained samples can be reana-lyzed or analyzed at multiple time points without loss ofidentified areas due to previous dye application.6.2 There is no interference on stained area measurementsdue to repeated application of xylenol orange.3The boldface numbers in parentheses ref
37、er to the list of references at the end ofthis standard.F2997 1326.3 The substrate on which the cells are grown can affect thequantitation if non-specific fluorescent dye absorption to thesubstrate occurs. Tissue culture plastic commonly used forculture of cells does not interfere with this test met
38、hod, butcalcium-containing substrates and scaffolds, such as calciumphosphate or calcium carbonate, will bind the calcium-chelating dye used to identify the cell-produced mineral andcause background fluorescence that will interfere with this testmethod (5). Background values from analysis of xylenol
39、orange-stained substrates that have been exposed to osteogenicmedium for the same length of time must be determined andsubtracted from the obtained values. All substrates beyondtissue culture plastic should be tested for non-specific dyebinding prior to initiating this practice.6.4 This test method
40、is designed for use with living cells.Dead cells may become calcified and take up the calcium-chelating dyes leading to artifactual mineral deposition. Be-cause the culture medium is changed immediately beforeimaging to avoid non-specific fluorescence from unbound dye,floating dead cells that may in
41、terfere are also removed;however, the user must confirm if the cell cultures are vital toavoid possible misinterpretation of the assay.7. Apparatus7.1 Fluorescent Microscope and Digital Camera:7.1.1 A 10 objective is recommended with an additional10 in the eyepiece that results in 100 magnification
42、in total.7.1.2 Microscope filter sets specific for the dyes. Xylenolorange has an excitation wavelength of 570 nm and emissionwavelength of 610 nm and should be examined with a TRITC(tetramethyl rhodamine isothiocyanate) red filter. The filter setrange will be recorded and similar filters can be use
43、d as long asthere is no bleed through of another fluorophore in the culturewhich can be detected by imaging control cultures without thexylenol orange.7.1.3 Camera and image collection software specifications.Digital imaging system which can include either a greyscalemonochrome camera or color camer
44、a. Images obtained with amonochrome camera will be of better quality. Minimumresolution of 1000 1000 pixels, a minimum of 12 bit. Thecamera and image collection software shall be capable ofsaving the image in a lossless file format (i.e., tiff file).7.1.4 Computer with image analysis software.7.1.4.
45、1 The image analysis can be conducted using a pro-gram such as the publically available National Institutes ofHealth program called ImageJ (http:/rsbweb.nih.gov/ij/) toquantify the positively stained areas. ImageJ is image analysissoftware available through the NIH (http:/rsb.info.nih.gov/ij/)and do
46、es not require a license to use. It may be utilized onLinux, Mac OS X and Windows. It is widely used andcustomizable for specific image analysis tasks. Many imagefile types are compatible with this software including: TIFF,GIF, JPEG, BMP, PGM, FITS, ASCII and DICOM.8. Reagents and Materials8.1 Xylen
47、ol orange (C31H28N2O13SNa4) is a fluorochro-matic calcium-chelating dye suitable for staining calcifieddeposits in cell cultures. This compound binds to calcium,allowing the calcified deposits to be visualized. This dye hasproven to be reliable for assessing mineralization of osteopro-genitor cultur
48、es (2). At the concentrations used in this practiceguide the dye is safe and non-toxic to cells and can be usedwithout detriment to the cultures enabling the analysis ofmultiple time points.8.2 Xylenol orange is commercially available as a powderand should be made into a stock solution using sterile
49、 distilledwater at 20 mM and filtered through a 0.20 m filter, protectedfrom light and stored at 4C for up to three months. It isimportant to use aseptic technique and sterile reagents sincethis is an assay on live cultures. The xylenol orange stocksolution should be added directly into the cell culture mediumat a concentration of 20 M within the culture well for 1224hrs before imaging. It is important to replace the medium withfresh media that does not contain dye prior to imaging to limitbackground fluorescence. At this concentration the dye willeffectively stain an
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