ASTM F2997-21

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Designation: F2997 − 13 F2997 − 21
Standard Practice for
Quantification of Calcium Deposits in Osteogenic Culture of
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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
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., (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, e.g., 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 responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.6 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.
This test method practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of
Subcommittee F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved Dec. 1, 2013June 15, 2021. Published January 2014June 2021. Originally approved in 2013. Last previous edition approved in 2013 as
F2997 – 13. DOI: 10.1520/F2997-13.10.1520/F2997-21.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2997 − 21
2. Referenced Documents
2.1 ASTM Standards:
F2312 Terminology Relating to Tissue Engineered Medical Products
F2603F3294 Guide for Interpreting Images of Polymeric Tissue ScaffoldsPerforming Quantitative Fluorescence Intensity
Measurements in Cell-based Assays with Widefield Epifluorescence Microscopy
F2739 Guide for Quantifying Cell Viability and Related Attributes within Biomaterial Scaffolds
3. Terminology
3.1 Unless provided otherwise in 3.2, terminology shall be in conformance with Terminology F2312.
3.2 Definitions:
3.2.1 calcium deposit, n—a calcium phosphate-containing substance synthesized in cell cultures during mineralization or
osteoblast differentiation assays that may be directly produced by osteoblasts or precipitated out of the solution without cell
participation.
3.2.2 mineralized matrix, n—a calcium phosphate-containing substance produced by cells typically in the osteoblast, odontoblast,
and calcifying chondrocyte lineages, which is composed of crystals of calcium phosphate and contains Type I collagen and other
non-collagenous proteins.
3.3 Definitions:Definitions of Terms Specific to This Standard:
3.2.1 mineralized matrix, n—a calcium phosphate-containing substance produced by cells typically in the osteoblast, odontoblast,
and calcifying chondrocyte lineages, which is composed of crystals of calcium phosphate and contains collagen Type I and other
non-collagenous proteins.
3.3.1 osteoblasts,osteoblast, n—secretory mononuclear cellscell that will initiate the formation of a matrix containing character-
istic proteins, such as collagen, and non-collageneous proteins such as bone sialoprotein and osteocalcin, that will mineralize in
the presence of a calcium and phosphate source.
3.3 Definitions of Terms Specific to This Standard:
3.3.1 calcium deposits, n—a calcium phosphate-containing substance synthesized in cell cultures during mineralization assays;
such as, osteoblast differentiation assays, that may have precipitated out of solution rather than being produced by the cells.
4. Summary of Practice
4.1 This practice consists of (1) fluorescently staining the calcium deposits in a cell culture using the non-toxic calcium-chelating
dye xylenol orange, orange (XO), (2) collecting fluorescent microscopy images of the stained samples, (3) collecting images of
intensity standards in bead form, and (4) conducting image analysis of thresholded images of the standards and the samples to
determine area percentage area% and mean intensity of the stained areas.
4.2 The practice involves the testing and analysis of a fluorescent intensity standard in order to determine standardized image
analysis settings for imaging of the calcified cell cultures. The use of a standard allows for the comparison between different
samples or different time points. Methods for determining area percentage area% and mean intensity of the standard and the
samples are described.
5. 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 calcifiedcalcium deposits or mineralized matrix that form in-vitroin 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.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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5.2 This test method 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, xylenol orange. 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 assureensure that the images have not been underexposed or overexposed. Intensity does and area do not correlate
directly to calcium content as well as area.content.
5.3 Xylenol orange XO enables the monitoring of calcifiedcalcium 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). CalcifiedCalcium 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.,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 test method practice may be applied to cultures of any cells capable of producing calcifiedcalcium 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 calcium deposit that is not
osteoblast-mediated and thus is referred to as dystrophic, pathologic, or artifactual may occur in the cell cultures (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 calcifiedcalcium deposits. These kinds of calcified/mineralcalcium/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).
5.6 Due to the potentialpossibility of artifactual calcifiedcalcium deposits during mineralization assays (2-4), gene expression
analysis or protein analysis techniques demonstrating the RNA message or the presence of osteocalcin and bone sialoprotein are
recommended for use in conjunction with the calcifiedcalcium deposit quantification procedure described here in order to confirm
the presence of mature osteoblasts that are in the process of secreting a mineralizing matrix.
5.7 The deposition of a mineralized substance in the culture dish does not confirm that the cells being cultured are capable of
forming bone in vivo.
5.8 The pattern of mineralized matrix deposition in the culture dish will vary, depending on the number of times the cells have
been passaged (i.e., (that is, first passage primary cells versus cells that have been passaged several times, including cell lines).
First passage primary cells typically form relatively large nodules of osteoprogenitor cells that differentiate and mineralize, while
cells that have been passaged many times lead to the formation of diffuse, dispersed mineral throughout the culture dish. This test
method practice is independent of the pattern of mineralization and can be used to analyze mineralized matrix in both primary cells
and cell lines.
5.9 Since some cells proliferate slower than others and since some of the cell culture surfaces being tested may affect proliferation
of the cells, the data can be normalized to total cell number. Since reduced proliferation typically reduces mineralization,
normalization to cell number typically does not influence the outcomes. Total DNA content can be determined as an indirect
measure of cell number. There are several commercially available kits for this purpose. Since DNA analysis is a destructive, toxic
assay, additional cell cultures must be prepared if this assay is used.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
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6. Interferences
6.1 Xylenol orange XO does not photobleach during microscopy nor leach out of the stained mineral with time, and is stable for
several months; thus, stained samples can be reanalyzed or analyzed at multiple time points without loss of identified areas due
to previous dye application.
6.2 There is no interference on stained area measurements due to repeated application of xylenol orange.XO.
6.3 The substrate on which the cells are grown can affect the quantitation if non-specific fluorescent dye absorption tointo the
substrate occurs. Tissue culture plastic commonly used for culture of cells does not interfere with this test method, practice, but
calcium-containing substrates and scaffolds, suchscaffolds (such as calcium phosphate or calcium carbonate,carbonate) will bind
the calcium-chelating dye used to identify the cell-produced mineral and cause background fluorescence that will interfere with
this test method practice (5). Background values from analysis of xylenol orange-stained XO-stained substrates that have been
exposed to osteogenic medium for the same length of time must be determined and subtracted from the obtained values. All
substrates beyond tissue culture plastic should be tested for non-specific dye binding prior to initiating this practice.
6.4 This test method practice is designed for use with living cells. Dead cells may become calcified and take up the
calcium-chelating dyes, leading to artifactual mineral deposition. Because the culture medium is changed immediately before
imaging to avoid non-specific fluorescence from unbound dye, floating dead cells that may interfere are also removed; however,
the user must confirm if the cell cultures are vitalviable to avoid possible misinterpretation of the assay.
7. Apparatus
7.1 Fluorescent Microscope and Digital Camera:
7.1.1 A 10× objective is recommended with an additional 10× in the eyepiece that results in 100× magnification in total.a total
of 100× magnification.
7.1.2 Microscope filter sets specific for the dyes. Xylenol orange XO has an excitation wavelength of 570 nm and emission
wavelength of 610 nm and should be examined with a TRITC (tetramethyl rhodamine isothiocyanate) red filter. The filter set range
willshall be recorded and similar filters can be used as long as there is no bleed through bleed-through of another fluorophore in
the culture, which can be detected by imaging control cultures without the xylenol orange.XO.
7.1.3 Camera and image collection software specifications. Digital imaging system which can include either a greyscale
monochrome camera or color camera. Images obtained with a monochrome camera will be of better quality. Minimum resolution
of 1000 × 1000 pixels, a minimum of 12 bit. The camera and image collection software shall be capable of saving the image in
a lossless file format (i.e., (for example, tiff file).
7.1.4 Computer with image analysis software.
7.1.4.1 The image analysis can be conducted using a program such as the publicallypublicly available National Institutes of Health
(NIH) program called ImageJ (http://rsbweb.nih.gov/ij/)(https://imagej.net) to quantify the positively stained areas. ImageJ is
image analysis software available through the NIH (http://rsb.info.nih.gov/ij/) and does not require a license to use. It may be
utilized on Linux, Mac OS X, and Windows. It is widely used and customizable for specific image analysis tasks. Many image
file types are compatible with this software including: TIFF, GIF, JPEG, BMP, PGM, FITS, ASCII, and DICOM.
8. Reagents and Materials
8.1 Xylenol orange (XO) (C H N O SNa ) (3,3'-Bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein tetraso-
31 28 2 13 4
dium salt) is a fluorochromatic calcium-chelating dye suitable for staining calcifiedcalcium deposits in cell cultures. This
compound binds to calcium, allowing the calcifiedcalcium deposits to be visualized. This dye has proven to be reliable for
assessing mineralization of osteoprogenitor cultures (2). At the concentrations used in this practice guide the dye is safe and
non-toxic to cells and can be used without detriment to the cultures, enabling the analysis of multiple time points.
8.2 Xylenol orange XO is commercially available as a powder and should be made into a stock solution using sterile distilled water
at 20 mM and filtered through a 0.20 μm filter, protected from light, and stored at 4°C4 °C for up to three months. It is important
to use aseptic technique and sterile reagents since this is an assay on live cultures. The xylenol orange XO stock solution should
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be added directly into the cell culture medium at a concentration of 20 μM within the culture well for 12–24 hrs 12 h to 24 h before
imaging. It is important to replace the medium with fresh mediamedium that does not contain dye prior to imaging to limit
background fluorescence. At this concentration the dye will effectively stain an area of mineralized matrix in a similar manner to
von Kossa staining which is commonly used in osteogenic cultures (2). It is thus important to use xylenol orange XO at this
concentration and not other untested dyes which may reduce or enlarge the mineralized areas from their actual size.
8.3 A fluorescent intensity standard in the form of a suspension of fluorescence microspheres is used in this standard practice. For
xylenol orange, a commercially available fluorescent intensity standard is InSpeck Red (580/605 nm) Kit (6 μm, Cat.# I14787)
available from Invitrogen Corp. Standards from other suppliers can be used. to calibrate image intensity (InSpeck Red, excitation
580 nm, emission 605 nm, 2.5 μm diameter beads, ThermoFisher, Cat. # I7224). Beads can be purchased from many vendors. For
consistency between groups, and between tests conducted on different days or in different labs, the same standards should be used.
Each kit includes may include six separate suspensions of InSpeck fluorescent microspheres with relative fluorescence intensities
of 100%, 30%, 10%, 3%, 1% and 0.3%.100 %, 30 %, 10 %, 3 %, 1 %, and 0.3 %. Beads of each one of the six types have the same
diameter (6 μm). diameter. Guidance from the manufacturer should be followed with respect to storage and shelf-life.shelf life
should be followed.
8.4 Cell Culture:
8.4.1 Living cell cultures that have been exposed to mineralizing conditions, such as osteogenic supplements described in
references (3-5) to induce calcifiedcalcium deposits are needed for this test method.practice.
8.4.2 Cell cultures that have not been exposed to mineralizing conditions, but cultured for the same time period, as well as tissue
culture dishes containing medium only, should also be included as negative controls.
8.4.3 If biomaterial substrates are used, tissue culture wells containing the biomaterial substrates with osteogenic medium should
also be prepared as controls and kept in the incubator for the same time as the samples with cells.
8.4.4 Cultures prior to xylenol orange applicationPrior to XO application, cultures should be examined prior to commencing this
procedure to to determine if there is any background from the cells or biomaterials.
8.4.5 Cell culture plates of various sizes can be used. The examples in the appendix describe analysis and imaging of 6 well
six-well cell culture plates.
8.4.6 Sample size will depend on the test groups that are specific to a given experiment. Appropriate statistical methods should
be used to determine the number of replicates or cell culture wells used per group.
8.4.7 Additional cell cultures may be required if the imaging data is going to be normalized to cell number, which can be quantified
by measuring total DNA content. Total DNA content is a destructive assay and thus requires additional control wells or plates for
this purpose.
9. Hazards
9.1 The fluorescent light source on the microscope can cause eye damage. Avoid direct contact with eyes.
9.2 Consult the xylenol orange XO materials safety data sheet (MSDS) for safe use.
10. Sampling, Test Specimens, and Test Units
10.1 Within each sample to be analyzed, multiple images that form a representative field of view shall be acquired. The cell culture
dish should be examined throughout to determine the pattern of mineralized matrix deposition and the presence of cells before
beginning to acquire images. A uniform pattern of calcifiedcalcium deposits throughout the cell culture is required in order for the
recommended image acquisition procedure to be representative and accurate. Samples for analysis should be restricted to those
with intact culturecell monolayers that are not retracted or otherwise structurally perturbed. The center area of a cell culture plate
has the best focus for image collection and ideally should be used. An array of 1 × 10 adjacent images at 100× magnification (10×
from the eye piece and 10× from the objective) with the center point of the array in the middle of the cell culture is adequate for
a 6-wellsix-well plate. Linear array image collection should be conducted in relation to culture well dimensions and should not
extend beyond the edge of the culture plate. Linear sample array collection is based on previous measurements in human osteoblast
progenitor cultures where up to 400 images (20 × 20) images at this magnification were obtained without any difference in the
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value of area% of calcifiedcalcium deposits. Additional images may be obtained and used; however, it is important to randomly
select the images and not deliberately select fields with fluorescence. The 10ten adjacent images may be stitched together into one
image for the image analysis using an automated or manual stitching program. Stitching the images together saves analysis time,
but is not required.
10.2 A linear array of images is preferred because it represents a slice of the cell culture dish and thus more broadly samples the
potential heterogeneities within the cell culture such as the areas of higher cell density in the center of the well.
10.3 The units of analysis will be area percentage area% and mean intensity in relative fluorescence units.
11. Calibration and Standardization
11.1 Fluorescence Reference Standards:
11.1.1 Fluorescent microspheres that match the excitation and emission wavelengths of xylenol orange XO and are certified for
emission fluorescence intensity and for diameter are available. These shall be used as reference standards to ensure that
comparisons can be made between different microscope acquisition systems and different users of the same systems.
11.1.2 It should be determined if there is a linear response to exposure time by varying exposure time and determining intensity
and plotting the exposure time vs intensity. versus intensity (Guide F3294). This is necessary to ensure that the lamp and imaging
settings are being used within the correct range. The data to be used for this standard practice should be obtained in the linear range
of exposure time and intensity.
11.1.3 The calcium content is related to the fluorescent area, not the intensity; however, an intensity measurement it is important
to make an intensity measurement in order to confirm that the images are not overexposed.
11.1.4 This practice includes a procedure to measure the area of the standard beads to verify that the area of the standard beads
has not been falsely enlarged due to excessive exposure times, incorrect focus or use of the procedure that can artifactually enlarge
the bead diameter. The intensity standards and area standards (e.g., (for example, recommended concentration of beads to use as
a standard) have been selected to match that of typical calcified osteogenic cultures.
11.2 Image Acquisition Sett
...