ASTM F2739-08
(Guide)Standard Guide for Quantitating Cell Viability Within Biomaterial Scaffolds
Standard Guide for Quantitating Cell Viability Within Biomaterial Scaffolds
SIGNIFICANCE AND USE
The number and distribution of viable and non-viable cells within, or on the surface of, a biomaterial scaffold is one of several important characteristics that may determine in vivo product performance of cell/biomaterial constructs (see 5.7); therefore there is a need for standardized test methods to quantitate cell viability.
There are a variety of static and dynamic methods to seed cells on scaffolds, each with different cell seeding efficiencies. In general, static methods such as direct pipetting of cells on to scaffold surfaces have been shown to have lower cell seeding efficiencies than dynamic methods that push cells into the scaffold interior. Dynamic methods include: injection of cells into the scaffold, cell seeding on biomaterials contained in spinner flasks or perfusion chambers, or seeding that is enhanced by the application of centrifugal forces. The methods described in this guide can assist in establishing cell seeding efficiencies as a function of seeding method and for standardizing viable cell number within a given methodology.
As described in Guide F 2315, thick scaffolds or scaffolds highly loaded with cells lead to diffusion limitations during culture or implantation that can result in cell death in the center of the construct, leaving only an outer rim of viable cells. Spatial variations of viable cells such as this may be quantitated using the tests within this guide. The effectiveness of the culturing method or bioreactor conditions on the viability of the cells throughout the scaffold can also be evaluated with the methods described in this guide.
These test methods can be used to quantitate cells on hard or soft 3-D biomaterials, such as ceramics and polymer gels. The test methods also apply to cells seeded on porous coatings.
Test methods described in this guide may also be used to distinguish between proliferating and non-proliferating viable cells. Proliferating cells proceed through the DNA synthesis (S) phase and the mitosis...
SCOPE
1.1 This guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue engineered medical products (TEMPs).
1.2 In addition to providing a compendium of available techniques, this guide describes materials specific interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid, and/or account for, scaffold material/cell viability assay interactions.
1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs that are retrieved after implantation in living organisms.
1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: F2739 − 08
StandardGuide for
Quantitating Cell Viability Within Biomaterial Scaffolds
This standard is issued under the fixed designation F2739; 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 Sizing Single Cell Suspensions
F2315 Guide for Immobilization or Encapsulation of Living
1.1 This guide is a resource of cell viability test methods
Cells or Tissue in Alginate Gels
that can be used to assess the number and distribution of viable
andnon-viablecellswithinporousandnon-porous,hardorsoft
3. Terminology
biomaterial scaffolds, such as those used in tissue engineered
medical products (TEMPs).
3.1 Definitions:
3.1.1 non-viable cell, n—a cell not meeting one or more of
1.2 In addition to providing a compendium of available
the criteria for viability given in 3.1.2.
techniques, this guide describes materials specific interactions
with the cell assays that can interfere with accurate cell 3.1.2 viable cell, n—a cell capable of metabolic activity that
viability analysis, and includes guidance on how to avoid, is structurally intact with a functioning cell membrane.
and/or account for, scaffold material/cell viability assay inter-
actions.
4. Summary of Guide
1.3 These methods can be used for 3-D scaffolds containing
4.1 It is the intent of this guide to provide a compendium of
cells that have been cultured in vitro or for scaffold/cell
the commonly used methods for quantitating the number and
constructs that are retrieved after implantation in living organ-
distribution of viable and non-viable cells within, or on, a
isms.
biomaterial scaffold, because cell viability is important param-
eter of tissue engineering products used to regenerate or repair
1.4 This guide does not propose acceptance criteria based
lost or diseased tissue. The methods can be applied to cells
on the application of cell viability test methods.
residing within an intact 3-D scaffold or matrix (that is,
1.5 The values stated in SI units are to be regarded as
non-destructive methods) or to cells that have been removed
standard. No other units of measurement are included in this
from the scaffold or matrix (that is, destructive methods).
standard.
4.2 Most of the methods originate from analysis of cell
1.6 This standard does not purport to address all of the
numberon2-Dsurfaces,buthavebeenadaptedfortheanalysis
safety concerns, if any, associated with its use. It is the
of cells within 3-D constructs that are typically used in
responsibility of the user of this standard to establish appro-
regenerative medicine approaches. The mechanisms and the
priate safety and health practices and determine the applica-
sensitivity of the assays are discussed. The limitations of the
bility of regulatory limitations prior to use.
assays due to using standard curves generated from cells on
2-D surfaces are described in this document. In addition, the
2. Referenced Documents
ways in which the biomaterial scaffold itself can affect the
2.1 ASTM Standards:
viability assays are described.
F748 PracticeforSelectingGenericBiologicalTestMethods
4.3 This guide describes combinations of test methods
for Materials and Devices
which, when used together, will ensure the most accurate
F2149 Test Method for Automated Analyses of Cells—the
measure of the number and distribution of viable and non-
Electrical Sensing Zone Method of Enumerating and
viable cells.
5. Significance and Use
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
5.1 The number and distribution of viable and non-viable
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
cells within, or on the surface of, a biomaterial scaffold is one
Current edition approved Aug. 1, 2008. Published September 2008. DOI:
10.1520/F2739-08.
of several important characteristics that may determine in vivo
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
product performance of cell/biomaterial constructs (see 5.7);
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
therefore there is a need for standardized test methods to
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. quantitate cell viability.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2739 − 08
5.2 There are a variety of static and dynamic methods to 5.6 Viable cells may be under stress or undergoing apopto-
seed cells on scaffolds, each with different cell seeding sis. Assays for evaluating cell stress or apoptosis are not
efficiencies. In general, static methods such as direct pipetting addressed in this guide.
of cells on to scaffold surfaces have been shown to have lower
5.7 While cell viability is an important characteristic of a
cell seeding efficiencies than dynamic methods that push cells
TEMP, the biological performance of a TEMPis dependant on
into the scaffold interior. Dynamic methods include: injection
additional parameters.Additional tests to evaluate and confirm
ofcellsintothescaffold,cellseedingonbiomaterialscontained
the cell identity, protein expression, genetic profile, lineage
in spinner flasks or perfusion chambers, or seeding that is
progression, extent of differentiation, activation status, and
enhanced by the application of centrifugal forces.The methods
morphology are recommended.
described in this guide can assist in establishing cell seeding
5.8 Fundamental biocompatibility testing of the scaffold
efficiencies as a function of seeding method and for standard-
material itself as described in Practice F748 should be com-
izing viable cell number within a given methodology.
pleted prior to using the biomaterial with cells.
5.3 As described in Guide F2315, thick scaffolds or scaf-
folds highly loaded with cells lead to diffusion limitations
6. Selection of Test Methods
duringcultureorimplantationthatcanresultincelldeathinthe
6.1 Table 1 is a compendium of methods that can be used to
center of the construct, leaving only an outer rim of viable
quantitate cell viability on surfaces or in biomaterial scaffolds.
cells. Spatial variations of viable cells such as this may be
Importantly, a combination of the methods listed in Table 1 is
quantitated using the tests within this guide. The effectiveness
required to determine viable and non-viable (or live and dead)
of the culturing method or bioreactor conditions on the
cells quantitatively, and additional tests must be completed to
viability of the cells throughout the scaffold can also be
quantitate the subset of proliferating viable cells within the
evaluated with the methods described in this guide.
total number of viable cells. Proliferating cells are viable, but
5.4 These test methods can be used to quantitate cells on
viable cells are not necessarily proliferating. Non-viable cells
hard or soft 3-D biomaterials, such as ceramics and polymer
can be identified, even if they are not intact structurally or
gels. The test methods also apply to cells seeded on porous
metabolically,byintactnucleiordyeentryintothecellthrough
coatings.
a disrupted cytoplasmic membrane.
5.5 Testmethodsdescribedinthisguidemayalsobeusedto 6.2 The total number of cells, both alive and dead, within a
distinguish between proliferating and non-proliferating viable 3-D construct is typically determined by DNA analysis (7.2)
cells. Proliferating cells proceed through the DNA synthesis after the cells are removed destructively from the biomaterial
(S) phase and the mitosis (M) phase to produce two daughter scaffold and solubilized. Since many cell types adhere very
cells. Non-proliferating viable cells are in some phase of the well to a scaffold, significant cell lysis can occur during cell
cell cycle, but are not necessarily proceeding through the cell removal and simple manual counting of intact stained or
cycle culminating in proliferation. unstained cells (7.1) is not very reliable.
TABLE 1 Methods for Quantitating Cell Viability
Destructive Non-destructive
(Requires cell removal (Cells remain in scaffold
from scaffold or matrix) or matrix during test)
I. Total Cell Number
DNA assay X
Crystal violet X
II. Live Cell Number
Metabolic assays X X
Tetrazolium salt uptake: MTT, MTS, WST, XTT X
Alamar Blue X
Neutral Red X
Glucose Consumption X X
Cell proliferation (DNA synthesis)
[3H] Thymidine or BrDu labeling X
Dye exclusion assays
Trypan blue, erythrosin, and nigrosin X
III. Live/Dead Ratios
Live/Dead assays using dual fluorescent stains X
for plasma membrane integrity
Non-fluorescent dye exclusion assays X
IV. Imaging—density, morphology and spatial distributions of cells
Histological sectioning X
Confocal microscopy X X
Scanning electron microscopy X
F2739 − 08
6.3 If cells in a suspension are to be counted, Test Method similar). Cell cytoplasm volumes may be very different, as
F2149 may be useful. could be the number of cellular processes. In a mixed popula-
tion of cells some cells may be proliferating rapidly, whereas
6.4 To determine the quantity of live cells only, the use of a
others might be post-mitotic.
fluorescent or colorimetric metabolic indicator that fluoresces
or changes color in response to chemical reduction of growth
6.10 Some scaffolds will be translucent, others opaque.
media resulting from cell metabolic activity may be used (7.2). Some may be rigid, others very fragile. For more fragile
Metabolic assays are available in both destructive and non-
scaffolds, cells may fall off during handling, so it would be
destructive forms. The MTT or MTS assay (7.2.1)isa preferable to use a method that minimizes handling. Scaffolds
destructive, commonly used method that can be read with a
break down over time. Edges of scaffolds might be softer than
spectrophotometer. The Alamar Blue assay (7.2.2) is a non- internal portions. Scaffolds may not have uniform thickness or
destructivemethod thatrequiresafluorimeter.Cellmetabolism
density, which may affect statistical sampling.
in a 2-D environment, compared with a 3-D environment, is
significantly different, even with the same cell numbers.
7. Specific Test Methods for Determining Cell Viability
Accordingly, results for 3-D cell numbers can be erroneous
7.1 Dye Exclusion Technique to Distinguish Live from
when growth curves of cells cultured in 2-D are used for
Dead:
calibration (1). Itisimportanttonotethatmetabolicassaysare
7.1.1 One of the simplest methods to approximate cell
direct measures of intracellular enzyme activity produced by
viability is the dye exclusion technique. This method utilizes
cells.Althoughthelevelofenzymeactivityistypicallylinearly
an indicator dye to demonstrate cell membrane damage. Cells
proportional to the number of viable cells, it is possible that
which absorb the dye become stained and are considered
specific culture conditions can affect the production and
non-viable. Dyes such as trypan blue, erythrosin, and nigrosin
activity of the enzyme being assayed or that in certain
are used commonly, with trypan blue being the most common
circumstances, substrate may not be limiting. In this situation,
inpreliminarycellisolationprocedures.Cellsmustberemoved
the metabolic assay would not necessarily be linearly propor-
from the scaffold, mixed with the dye for a few minutes, and
tional to cell number. Despite these limitations, these methods
then counted manually with a hematocytometer. Cells must be
are still commonly used.
analyzed shortly (3 to 5 min) after the addition of 0.4 % trypan
6.5 The quantity of live cells within the total cell population
blue, since trypan blue is cytotoxic. There are large standard
may be determined by a proliferation or metabolic assay (7.3).
deviations with increasing cell densities; therefore samples
However, this will not provide information on the distribution
should be diluted to the densities recommended in the hemato-
of live cells within a construct; hence, an imaging technique
cytometer instructions.
(7.4) to visualize the morphology and spatial distribution of
7.2 Determination of Total Cell Number:
dye-labeled live and dead cells is typically utilized.
7.2.1 DNA Assay—DNA analysis is a commonly used
6.6 Non-destructive methods to determine cell viability of
method for determining total cell number, including both
an entire cell population within a scaffold or bioreactor are
viable and non-viable cells. There are several commercially
included in this guide and are useful for conducting kinetic
available kits for assessing DNA content, all of which can be
studies of cell number and distribution over time.
used. It is important to fully extract the cells from the scaffold
prior to analysis, using for example a solution of 0.125 mg/mL
6.7 The scaffolding material may interfere with any of the
following assays and must be included within the assay, papain and 10 mmol/L L-cysteine dihydrochloride in phos-
phate buffered EDTA in a 60°C water bath for 10 hours to
typically as a control, to determine whether it has an effect. If
the assay is affected by the presence of the scaffold, then either extract cells from a polymer matrix (2). If the cell fluorescence
will be measured, using a proteinase K digestion step ablates
theinterferenceshouldbesubtractedoutoranalternativeassay
should be selected. Notes on known interferences are included any residual endogenous fluorescence of the cells (3).
in each of the assay descriptions below. 7.2.2 Crystal Violet Staining—Another cell stain used for
determining total cell number is crystal violet which binds to
6.8 Cell density could impact accuracy of quantification.
theDNAofviableandnon-viablecells.Cellsmustberemoved
Cells grown at low density are generally harder to wash off
from the biomaterial scaffold prior to analysis. Cells are
than cells grown to confluency, where a whole sheet of cells
washed in phosphate buffered solution (PBS), stained with 0.1
may be rather easy to displace. Many of our products may be
to0.2 %crystalvioletinmethanolfor15minat37°C,andthen
seededatashighadensityaspossible.Highdensitiesmayalso
washed extensively prior to analysis. Absorbance is measured
affect dye binding.
at a wavelength of 590 nm using an ELISA plate reader.
6.9 In many instances a mixed population of cells may be
7.3 Proliferation or Metabolic Assays for Quantitating Live
present. Metabolic assays will not accurately quantify mixed
Cell Number:
cultures of cells because some cells a
...
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