ASTM F3504-21

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.
Designation: F3504 − 21
Standard Practice for
Quantifying Cell Proliferation in 3D Scaffolds by a
Nondestructive Method
This standard is issued under the fixed designation F3504; 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 2. Referenced Documents
2.1 ASTM Standards:
1.1 This practice describes how to conduct a nondestructive
F2312Terminology Relating to Tissue Engineered Medical
proliferation test for mammalian cells based on metabolic
Products
activity that can be used to assess the number of viable cells
F2664Guide for Assessing the Attachment of Cells to
withinthree-dimensional(3D)scaffoldsforregenerativemedi-
Biomaterial Surfaces by Physical Methods
cine and in tissue-engineered medical products (TEMPs).
F2739Guide for Quantifying Cell Viability and Related
1.2 This practice provides a detailed explanation of the
Attributes within Biomaterial Scaffolds
resazurin cell metabolic activity method in terms of reagent
F3163Guide for Classification of Cellular and/or Tissue-
concentrations, incubation times, cell culture media
Based Products (CTPs) for Skin Wounds
composition, calibration curve, controls, assay linearity, and
F3294Guide for Performing Quantitative Fluorescence In-
limitations of the assay.
tensity Measurements in Cell-based Assays with Wide-
field Epifluorescence Microscopy
1.3 This practice describes factors that can interfere with
2.2 ISO Standard:
accurate cell proliferation assessment.
ISO 10993Biological Evaluation of Medical Devices
1.4 Since the assay has washing steps, it is limited to
2.3 ASTM Adjuncts:
assessing cells that are immobilized, such as by adhesion to a 4
Digital Spreadsheet File
culture dish, adhesion to a scaffold, or encapsulation in a
hydrogel.
3. Terminology
1.5 The assay is limited to cell types that can metabolize 3.1 Definitions:
resazurin to provide a signal in the assay. 3.1.1 Unlessprovidedotherwisein3.2,terminologyshallbe
in conformance with Terminology F2312.
1.6 This document does not propose acceptance criteria for
3.1.2 non-viable cell, n—a cell not meeting one or more of
a cell-based product based on the application of a cell
the criteria for viability given in 3.1.6. F2739
proliferation test method.
3.1.3 proliferation competent cell, n—cell capable of
1.7 This standard does not purport to address all of the
replication. F3163
safety concerns, if any, associated with its use. It is the
3.1.4 senescence, n—invertebratecellcultures,theproperty
responsibility of the user of this standard to establish appro-
attributable to finite cell cultures, namely, their inability to
priate safety, health, and environmental practices and deter-
grow beyond a finite number of population doublings. Neither
mine the applicability of regulatory limitations prior to use.
invertebrate nor plant cell cultures exhibit this property. This
1.8 This international standard was developed in accor-
term is synonymous with in vitro senescence. F2664
dance with internationally recognized principles on standard-
3.1.5 stem cell, n—progenitor cell capable of self-
ization established in the Decision on Principles for the
replication, proliferation, and differentiation to produce cells
Development of International Standards, Guides and Recom-
that take on more specialized functions. F2312
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
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
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland the ASTM website.
Surgical Materials and Devices and is the direct responsibility of Subcommittee Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
F04.43 on Cells and Tissue Engineered Constructs for TEMPs. 4th Floor, New York, NY 10036, http://www.ansi.org.
Current edition approved Oct. 1, 2021. Published November 2021. DOI: Available from ASTM International Headquarters. Order Adjunct No.
10.1520/F3504-21. ADJF3504-EA.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3504 − 21
3.1.6 viable cell, n—acellcapableofmetabolicactivitythat methods described within this practice enable nondestructive
is structurally intact with a functioning cell membrane. F2739 testingformonitoringthecellproliferationkineticsthroughout
the culture period by repeated analysis at multiple time points
3.2 Definitions of Terms Specific to This Standard:
on the same test sample with minimal toxicity. This standard
3.2.1 cell differentiation, n—developmental process of
practice is written only for resazurin dye, a non-cytotoxic
multi-cellular organisms, through which a cell becomes spe-
reagent that should not affect cell viability and proliferation at
cialized in order to perform a specific function for further
lowconcentration.Thisisadistinctadvantageovermanyother
building different tissues and organs. (For example, bone
reagentsusedtomeasurecellnumber,suchasmeasurementsof
marrow stromal cells may differentiate into chondrocytes
the intracellular components (such as DNA, protease, orATP)
and/or osteoblasts.)
which require cell lysis and can therefore only be used for
endpoint analysis.
4. Summary of Practice
5.4 Resazurin,whichhaslowfluorescence,maybemetabo-
4.1 Thispracticeconsistsof:(1)incubatingcellsculturedon
lized by cells into resorufin, which is highly fluorescent. An
three-dimensionalscaffoldswithasolutionofametabolicdye,
resazurin,whichemitsfluorescenceafterbeingmetabolizedby increase in fluorescence caused by the conversion to resorufin
may correlate with increased dehydrogenase activity, which
living cells; (2) collecting the metabolized/reduced dye solu-
tion in a multi-well plate (for example, a 96-well plate); (3) may correlate with an increase in cell number and therefore
proliferation. Plotting the signals measured at multiple time
measuringthefluorescenceintensityusingamulti-platereader;
points enables the generation of proliferation curves. It is
and(4)conductingfluorescenceintensityanalysistodetermine
important to note that metabolic assays are intended to be
cell proliferation and viability.
measurements of intracellular dehydrogenase or reductase
4.2 The resazurin molecule can penetrate the cells by
enzyme activity produced by cells. The level of enzyme
passingthroughthecellmembraneintothecytoplasmwhereit
activity may be directly proportional to the number of viable
is reduced by cytosolic, microsomal, and mitochondrial dehy-
cellswithinarangeofcellnumberpervolume(orperscaffold)
drogenase or reductase enzymes, producing the highly fluores-
identified by a calibration curve. This is because cell metabo-
cent resorufin. Resorufin diffuses out of cells and back into the
lism rate may decrease without a loss in cell viability when
culture medium, which is collected and measured. Resazurin
cells have reached confluency or when they are differentiating.
reduction is not carried out by a single enzyme, as incubation
Some cells may be quiescent but still viable. Furthermore,
withdifferentsubcellularfractions(cytosolic,microsomal,and
certain cell types have different metabolic activity. In these
mitochondrial) leads to the formation of resorufin (1). There
situations, the relationship between cell metabolism and cell
are multiple isoforms of dehydrogenase and reductase. Dehy-
number may not be linear and other assays may be considered.
drogenase and reductase activity vary among different cell
5.5 The method may be applied to planar 2D cell cultures
lines, which may influence the assay conditions (2). Enzymes
and3Dscaffoldcellcultures.Thisassayisintendedfor96,48,
that may participate in resazurin reduction include alcohol and
and 24-well plates but could work for other size plates. Size
aldehyde oxidoreductases, nicotinamide adenine dinucleotide
and thickness of cell scaffold construct where the test can be
(NADH) dehydrogenase, NAD(P)H:quinone oxidoreductase,
flavin reductase, and cytochromes (3). applicable should be tested with control experiments. In
Reference (4), a 5mm thick scaffold in a 24-well plate was
5. Significance and Use
used.
5.1 In-vitro cell proliferation assays are used to screen the
5.6 The method may also be used to document the absence
capabilityofcellstoproliferateandself-renewwithinscaffolds
of cell proliferation in cultures.
for regenerative medicine and tissue-engineering applications.
NOTE 1—The absence or suppression of proliferation under the tested
The cell proliferation in vitro, in conjunction with other
conditionsmaybearesultoflackofreagent/nutrientdiffusionthroughthe
characteristicsofthecellssuchasgeneexpression,canbeused
scaffold. If so, the same result may not be observed if diffusion is
to determine if the cells have maintained their properties.
improved by, for example, changing from a 96-well plate to other cell
culture formats.
5.2 Cell proliferation may be an important parameter to test
5.7 The dye is not cell type specific; hence, cell identifica-
asaqualityattributeofacell-scaffoldconstruct.Thistesthelps
tion cannot be based on this method.
to assess cell colonization within a scaffold.
5.8 The assay as described herein is not designed to assess
5.3 This method provides a technique for vital assessment
cell distribution in scaffolds. It is possible that this could be
and quantification of the fluorescence intensity related to dye
achieved by sectioning the scaffolds prior to staining and
metabolism by living and proliferating cells. This method
analysis.
assumes that viable cells will have an active metabolism,
which is required to support life-associated cellular processes
6. Apparatus
such as the conversion of nutrient sources into energy and
proliferation. There may be cells that are not actively
6.1 Fluorescence reader (for example, microplate fluores-
proliferating, yet are still viable within the construct. The
cence reader, etc.) allowing excitation in the range of 500 nm
to600nmandemissionintherangeof550nmto700nm.The
platereadershouldbequalifiedandcalibrated.Thisistypically
The boldface numbers in parentheses refer to a list of references at the end of
this standard. done by automated self-checks that occur during plate reader
F3504 − 21
start-up, by a manufacturer-qualified technician or by use of a given cell density (often between 1 and 4 h). The appropriate
calibratorplatefromthemanufacturerorothervendor,orboth. incubation time and conditions should be determined by
preliminary experiments for each cell type and cell density
6.2 Black-walled, clear-bottomed, multi-well plates for
range.
fluorescence measurements (for example, 96-well plates) are
recommended to reduce fluorescent signal crosstalk and back- 8.7 The medium containing the metabolized resazurin (re-
ground. sorufin) should be collected by pipetting and 100 µL trans-
ferred into a 96-well plate for fluorescence intensity measure-
7. Reagents and Materials ment with an excitation in the range of 530 nm to 570 nm and
an emission range of 580 nm to 620 nm, with peak measure-
7.1 Resazurin dye (7-Hydroxy-3H-phenoxazin-3-one 10-
ments at 570 nm for excitation and 584 nm for emission. The
oxide). A common name for resazurin is Alamar Blue. The
fixed reading volume (100 µL in a 96-well plate) enables a
metabolized resazurin is called resorufin, which has a peak
comparison between results from scaffolds of variable size if
excitation wavelength of 579 nm and a peak emission wave-
the total quantity of resazurin available for scaffolds has been
length of 584 nm.
the same.
7.2 Cells.
8.8 Due to the variability of cells in culture, sufficient
7.3 Scaffolds for 3D testing.
replicatestomeetthestatisticalrequirementsoftheexperiment
7.4 Cell culture plates for 2D testing. are recommended. The recommended minimum would be
three biological replicates for each scaffold/condition and
conductingtheentireassaythreetimes(threeassayreplicates).
8. Procedure
Replicates are necessary to assess experimental variations in
8.1 Resazurin is commercially available as a solution or as
cell viability, seeding, and other experimental factors.
a powder and should be made into a stock solution using PBS
8.9 Tomaintainthetestsampleinculture,thesampleshould
(phosphate buffered saline) at 44 mM. If the powder is used, it
is recommended that the dye solution be filtered through a be washed twice, or more if necessary (until the culture
medium appears of its own color), with a sufficient volume of
20-µm filter to sieve any undiluted particles, protected from
light, and stored at –20°C (for up to twelve months). cell culture medium to cover the entire scaffold, to remove
resazurin and resorufin. After washing, fresh culture medium
8.2 The resazurin working solution should be prepared by
should be added to cover the entire scaffold and the plate may
diluting the stock solution into the cell culture medium to
bereturnedtotheincubatorforfurthercultureorforrecovering
obtain a 44-µM working solution (that is, a 1:1000 dilution).A
for at least 24 h before performing a new assay.
44-µM resazurin solution may be suitable for measurement of
8.10 To create of a proliferation curve over time, repeat 8.5
cells from low to high cell densities on a scaffold, such as
3 5
between1×10 cells/scaffold and8×10 cells/scaffold, as – 8.9.An interval of time of at least 24 h should be considered
between two resazurin assays on the same sample.
described for a highly porous scaffold (4), without the need to
dilute the reaction product due to high fluorescence readings
9. Hazards
outside of the linear range.
9.1 The fluorescent light source on the microscope could
8.3 After plating the cells on the 3D scaffold, the assay
cause eye damage.
cannot be performed until the constructs have been incubated
9.2 Consult the resazurin Material Safety Data Sheet
long enough to allow the cells to adhere to the scaffolds. Cells
(MSDS) for safe use.
generally take one to several hours to adhere to their substrate.
The appropriate amount of adhesion time may be determined
10. Sampling, Test Specimens, and Test Units
by preliminary experiments for each cell type and cell density
10.1 For each test sample, multiple aliquots of metabolized
range. Cells may have low activity after thawing and their
resazurin (resorufin) should be analyzed by the fluorescence
activity may increase as they recover from freezing; hence,
intensity plate reader.
cells should be cultured for at least 24 h prior to seeding on
scaffolds.
10.2 The units of analysis will be fluorescence intensity
counts.
8.4 Before performing the resazurin assay, remove the old
medium to eliminate any non-adherent living cells, rinse the
11. Controls and Calibration
scaffold twice with cell culture medium to eliminate any
11.1 Resazurin is reported to be generally nontoxic (5).
enzyme released by lysed cells, and move the scaffold into a
However, a concentration of resazurin higher than 100 µM or
new, empty well so that cells which came off the scaffold and
anincubationtimelongerthan4hmaybetoxictocellsoralter
cells adhered to the bottom of the well do not contribute to the
their natural metabolism. This kind of inhibition may be
resazurin metabolism.
reversed by washing and addition of fresh medium.
8.5 Add an appropriate volume of the resazurin solution to
11.1.1 Dye toxicity should be assessed with the cell of
entirely cover the scaffold.
choice before use. A metabolic assay may be suitable for this
8.6 Incubate in a humidified CO incubator at 37°C, at the where cells are incubated with a range of resaruzin
desired oxygen conditions or any other specific culture concentration,incubatedforappropriatetime,andthenassayed
condition, for an incubation time which is appropriate for the (6).
F3504 − 21
11.2 The working volumes need to be optimized for the have an appropriate number of replicates (three or more) as
scaffoldofinterestsothatliquidwillentirelycoverthescaffold required by the intended use of the data. The user must decide
specimen. For example, filling each well of a 24-well plate
which controls are appropriate for a given investigation.
filled with 1 mL of 44-µM resazurin in fresh medium should
11.7.1 Below are the controls that should be considered for
completely cover a 5-mm high scaffold. The appropriate
the “cell” plate (the plate that will be placed in the cell culture
volumes should be evaluated in preliminary testing.
incubator). These control wells should be treated exactly the
sameastheexperimentalwells,includingmediumchangesand
11.3 The diffusion of the dye into the 3D scaffold interior
including incubation with resazurin solution with aliquots for
maybehinderedbythescaffoldporegeometryorthepresence
of tissue generated by the cells, which may result in a low plate reader measurements. With experience, users may find
that some of the controls may only be required in preliminary
estimate of viable cell number. This may be a source of error
for this assay. testing and could be eliminated from routine runs if it is
11.3.1 The effect of the scaffold on diffusion of assay determined that they are not required for the purposes of the
componentscanbetested.Knownnumbersoflivecellscanbe assay.
seeded into scaffolds for short time periods, such as 1 h, where
11.7.1.1 Wells with cell culture medium only. This control
itcanbeassumedthatcellshavenotproliferated.Theresazurin
may determine if culture medium is providing an unwanted
assaycanthenberunandcomparedtocontrolswherethesame
source of fluorescent signal in the assay. There may be
number of cells are seeded into empty wells with culture
dehydrogenase activity in the culture medium or dehydroge-
medium without scaffolds. If the same results are obtained for
nase activity present in the culture medium may adsorb to the
both treatments, then it may be concluded that the scaffold is
cultureplates.Ifacidosisoccurs,thenlowpHmaycauseashift
not significantly affecting the results by impeding diffusion of
in the excitation/emission peaks of resorufin. There may be
assay components. If the results suggest that diffusion is
other mechanisms whereby the culture medium could provide
affecting the results, then thinner scaffolds may reduce this
a false signal in the assay.
effect.
11.7.1.2 Wellswithscaffoldonly(nocells).Thismaybethe
11.3.2 If high cell and tissue density hinder diffusion in the
most important control to run. This control may determine if
constructs, assessing earlier time points when densities are
the scaffold is providing an unwanted source of fluorescent
lower may be helpful.
signal in the assay. Dehydrogenase activity may be present in
11.4 Cell viability may be affected by the nature of the
the culture medium or dehydrogenase that is present in the
scaffoldinwhichthecellshavebeenseeded.Differenttypesof
culture medium may adsorb to the scaffolds. There may be an
scaffolds may have different effects on cell viability attributes.
interaction between the scaffold and the dye. The scaffold
Thisdocumentassumesthatthescaffoldisnontoxic.Totestfor
properties may change with incub
...