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ASTM F2997-13

(Practice)

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

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

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 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 osteo...
SCOPE
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 (i.e., 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, e.g., 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 ...

General Information

Status
Historical
Publication Date
30-Nov-2013
ICS
07.100.10 - Medical microbiology
Technical Committee
F04 - Medical and Surgical Materials and Devices
Drafting Committee
F04.43 - Cells and Tissue Engineered Constructs for TEMPs
Current Stage
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ASTM F2997-13 - Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image AnalysisASTM F2997-13 - Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis
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ASTM F2997-13 - Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:F2997 −13
Standard Practice for
Quantification of Calcium Deposits in Osteogenic Culture of
1
Progenitor Cells Using Fluorescent Image Analysis
This standard is issued under the fixed designation F2997; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
1.1 This practice defines a method for the estimation of
calcium content at multiple time points in living cell cultures
2. Referenced Documents
that have been cultured under conditions known to promote
2
2.1 ASTM Standards:
mineralization. The practice involves applying a fluorescent
F2312Terminology Relating to Tissue Engineered Medical
calcium chelating dye that binds to the calcium phosphate
Products
mineral crystals present in the live cultures followed by image
F2603Guide for Interpreting Images of Polymeric Tissue
analysis of fluorescence microscopy images of the stained cell
Scaffolds
cultures.Quantificationofthepositivelystainedareasprovides
F2739Guide for Quantitating Cell Viability Within Bioma-
a relative measure of the calcium content in the cell culture
terial Scaffolds
plate.Aprecise correlation between the image analysis param-
eters and calcium content is beyond the scope of this practice.
3. Terminology
1.2 Calciumdepositioninasecretedmatrixisoneofseveral 3.1 Unless provided otherwise in 3.2, terminology shall be
features that characterize bone formation (in vitro and in vivo),
in conformance with F2312.
and is therefore a parameter that may indicate bone formation
3.2 Definitions:
and osteoblast function (i.e., osteoblastic differentiation). Cal-
3.2.1 mineralized matrix, n—a calcium phosphate-
cium deposition may, however, be unrelated to osteoblast
containing substance produced by cells typically in the
differentiation status if extensive cell death occurs in the cell
osteoblast, odontoblast, and calcifying chondrocyte lineages,
cultures or if high amounts of osteogenic medium components
which is composed of crystals of calcium phosphate and
that lead to artifactual calcium-based precipitates are used.
contains collagen Type I and other non-collagenous proteins.
Distinguishing between calcium deposition associated with
3.2.2 osteoblasts, n—secretory mononuclear cells that will
osteoblast-produced mineralized matrix and that from patho-
initiate the formation of a matrix containing characteristic
logical or artifactual deposition requires additional structural
proteins, such as collagen, and non-collageneous proteins such
and chemical characterization of the mineralized matrix and
asbonesialoproteinandosteocalcin,thatwillmineralizeinthe
biological characterization of the cell that is beyond the scope
presence of a calcium and phosphate source.
of this practice.
3.3 Definitions of Terms Specific to This Standard:
1.3 The parameters obtained by image analysis are ex-
3.3.1 calcium deposits, n—a calcium phosphate-containing
pressed in relative fluorescence units or area percentage, e.g.,
substance synthesized in cell cultures during mineralization
fraction of coverage of the area analyzed.
assays;suchas,osteoblastdifferentiationassays,thatmayhave
1.4 Units—The values stated in SI units are to be regarded
precipitated out of solution rather than being produced by the
asstandard.Nootherunitsofmeasurementareincludedinthis
cells.
standard.
4. Summary of Practice
1.5 This standard does not purport to address all of the
4.1 This practice consists of (1) fluorescently staining the
safety concerns, if any, associated with its use. It is the
calcium deposits in a cell culture using the non-toxic calcium-
responsibility of the user of this standard to establish appro-
chelatingdyexylenolorange, (2)collectingfluorescentmicros-
copy images of the stained samples, (3) collecting images of
1
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
2
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee For referenced ASTM standards, visit the ASTM website, www.astm.org, or
F04.43 on Cells and Tissue Engineered Constructs for TEMPs. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved Dec. 1, 2013. Published January 2014. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
F2997-13. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
F2997−13
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Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis

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

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 ...
SCOPE
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 responsibili...

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  • Standard
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ASTM
ASTM E1881-12(2020)(Guide)
Standard Guide for Cell Culture Analysis with SIMS

SIGNIFICANCE AND USE
5.1 The presence of cell growth medium complicates a direct analysis of cells with SIMS. Attempts to wash out the nutrient medium results in the exposure of cells to unphysiological reagents that may also alter their chemical composition. This obstacle is overcome by using a sandwich freeze-fracture method (1). This cryogenic method has provided a unique way of sampling individual cells in their native state for SIMS analysis.  
5.2 The procedure described here has been successfully used for imaging Na+ and K+ ion transport  (3), calcium alterations in stimulated cells (4,5), and localization of therapeutic drugs and isotopically labeled molecules in single cells (6). The frozen freeze-dried cells prepared according to this method have been checked for SIMS matrix effects  (7). Ion image quantification has also been achieved in this sample type (8).  
5.3 The procedure described here is amenable to a wide variety of cell cultures and provides a way for studying the response of individual cells for chemical alterations in the state of health and disease and localization of isotopically-labeled molecules and theraputic drugs in cell culture models.
SCOPE
1.1 This guide provides the Secondary Ion Mass Spectrometry (SIMS) analyst with a cryogenic method for analyzing individual tissue culture cells growing in vitro. This guide is suitable for frozen-hydrated and frozen-freeze-dried sample types. Included are procedures for correlating optical, laser scanning confocal and secondary electron microscopies to complement SIMS analysis.  
1.2 This guide is not suitable for cell cultures that do not attach to the substrate.  
1.3 This guide is not suitable for any plastic embedded cell culture specimens.  
1.4 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.  
1.5 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.

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