1、Designation: F2739 16Standard Guide forQuantifying Cell Viability within Biomaterial Scaffolds1This standard is issued under the fixed designation F2739; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A
2、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 guide is a resource of cell viability test methodsthat can be used to assess the number and distribution of viableand non-viable c
3、ells within porous and non-porous, hard or softbiomaterial scaffolds, such as those used in tissue-engineeredmedical products (TEMPs).1.2 In addition to providing a compendium of availabletechniques, this guide describes materials-specific interactionswith the cell assays that can interfere with acc
4、urate cellviability analysis, and includes guidance on how to avoid,and/or account for, scaffold material/cell viability assay inter-actions.1.3 These methods can be used for 3-D scaffolds containingcells that have been cultured in vitro or for scaffold/cellconstructs that are retrieved after implan
5、tation in living organ-isms.1.4 This guide does not propose acceptance criteria basedon the application of cell viability test methods.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to add
6、ress all of thesafety concerns, if any, associated with its use. It is 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:2F748 Practice
7、 for Selecting Generic Biological Test Methodsfor Materials and DevicesF2149 Test Method for Automated Analyses of CellstheElectrical Sensing Zone Method of Enumerating andSizing Single Cell SuspensionsF2315 Guide for Immobilization or Encapsulation of LivingCells or Tissue in Alginate GelsF2998 Gui
8、de for Using Fluorescence Microscopy to Quan-tify the Spread Area of Fixed Cells3. Terminology3.1 Definitions:3.1.1 non-viable cell, na cell not meeting one or more ofthe criteria for a viable cell.3.1.2 viable cell, na cell capable of metabolic activity thatis structurally intact with a functioning
9、 cell membrane.4. Summary of Guide4.1 It is the intent of this guide to provide a compendium ofthe commonly used methods for quantifying the number anddistribution of viable and non-viable cells within, or on, abiomaterial scaffold, because cell viability is an importantparameter of tissue-engineere
10、d products used to regenerate orrepair lost or diseased tissue. The methods can be applied tocells residing within an intact 3-D scaffold or matrix (that is,non-destructive methods) or to cells that have been removedfrom the scaffold or matrix (that is, destructive methods). Itshould be noted that n
11、ot all cells require a scaffold and somecell types, such as hematopoietic cells, cannot be cultured orgrown on an adherent surface.4.2 Most of the methods originate from analysis of cellnumber on 2-D surfaces, but have been adapted for the analysisof cells within 3-D constructs that are typically us
12、ed inregenerative medicine approaches. The mechanisms and thesensitivity of the assays are discussed. The limitations of theassays due to using standard curves generated from cells on2-D surfaces are described in this document. In addition, theways in which the biomaterial scaffold itself can affect
13、 theviability assays are described.4.3 This guide describes test methods which, when usedtogether, may enable accurate measure of the number anddistribution of viable and non-viable cells. Different viabilityassays have different measurands, which means that the resultsfrom different assays may not
14、correlate with one another. Forinstance, cell membrane integrity tests and cell metabolic tests1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for
15、 TEMPs.Current edition approved Oct. 1, 2016. Published November 2016. Originallyapproved in 2008. Last previous edition approved in 2008 as F2739 08. DOI:10.1520/F2739-16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For An
16、nual Book 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 States1measure fundamentally different cell properties. Although bothtests are rel
17、ated to cell viability, they may not correlate withone another.5. Significance and Use5.1 The number and distribution of viable and non-viablecells within, or on the surface of, a biomaterial scaffold is oneof several important characteristics that may determine in vivoproduct performance of cell/bi
18、omaterial constructs (see 5.7);therefore there is a need for standardized test methods toquantify cell viability.5.2 There are a variety of static and dynamic methods toseed cells on scaffolds, each with different cell seedingefficiencies. In general, static methods such as direct pipettingof cells
19、onto scaffold surfaces have been shown to have lowercell seeding efficiencies than dynamic methods that push cellsinto the scaffold interior. Dynamic methods include: injectionof cells into the scaffold, cell seeding on biomaterials containedin spinner flasks or perfusion chambers, or seeding that i
20、senhanced by the application of centrifugal forces. The methodsdescribed in this guide can assist in establishing cell seedingefficiencies as a function of seeding method and for standard-izing viable cell numbers within a given methodology.5.3 As described in Guide F2315, thick scaffolds or scaf-fo
21、lds highly loaded with cells lead to diffusion limitationsduring culture or implantation that can result in cell death in thecenter of the construct, leaving only an outer rim of viablecells. Spatial variations of viable cells such as this may bequantified using the tests within this guide. The effe
22、ctiveness ofthe culturing method or bioreactor conditions on the viabilityof the cells throughout the scaffold can also be evaluated withthe methods described in this guide.5.4 These test methods can be used to quantify cells on hardor soft 3-D biomaterials, such as ceramics and polymer gels.The tes
23、t methods also apply to cells seeded on porous coatings.5.5 Test methods described in this guide may also be used todistinguish between proliferating and non-proliferating viablecells. Proliferating cells proceed through the DNA synthesis(S) phase and the mitosis (M) phase to produce two daughtercel
24、ls. Non-proliferating viable cells are in some phase of thecell cycle, but are not necessarily proceeding through the cellcycle culminating in proliferation.5.6 Viable cells may be under stress or undergoing apopto-sis. Assays for evaluating cell stress or apoptosis are notaddressed in this guide.5.
25、7 While cell viability is an important characteristic of aTEMP, the biological performance of a TEMP is dependant onadditional parameters. Additional tests to evaluate and confirmthe cell identity, protein expression, genetic profile, lineageprogression, extent of differentiation, activation status,
26、 andmorphology are recommended.5.8 Fundamental biocompatibility testing of the scaffoldmaterial itself as described in Practice F748 should be com-pleted prior to using the biomaterial with cells.5.9 Methods that remove the cells from a 3-D scaffold mayreduce the cell number and viability due to the
27、 manipulationrequired.6. Selection of Test Methods6.1 Table 1 is a compendium of methods that can be used toquantify cell viability on surfaces or in biomaterial scaffolds.Importantly, a combination of the methods listed in Table 1 isrequired to determine viable and non-viable (or live and dead)TABL
28、E 1 Methods for Quantifying Cell ViabilityDestructive(Requires cell removalfrom scaffold or matrix)Non-destructive(Cells remain in scaffoldor matrix during test)I. Total Cell NumberDNA assay XCrystal violet XII. Live Cell NumberMetabolic assays X XTetrazolium salt uptake: MTT, MTS, WST, XTT XAlamar
29、Blue (resorufin) XNeutral Red XGlucose Consumption X XCell proliferation (DNA synthesis)3H Thymidine or BrDu (Bromodoeoxyuridine)labelingXDye exclusion assaysTrypan blue, erythrosin, and nigrosin XIII. Live/Dead RatiosLive/Dead assays using dual fluorescent stainsfor plasma membrane integrityXNon-fl
30、uorescent dye exclusion assays XIV. Imagingdensity, morphology and spatial distributions of cellsHistological sectioning XConfocal microscopy X XScanning electron microscopy XF2739 162cells quantitatively, and additional tests must be completed toquantify the subset of proliferating viable cells wit
31、hin the totalnumber of viable cells. Proliferating cells are viable, but viablecells are not necessarily proliferating. Non-viable cells can beidentified, even if they are not intact structurally ormetabolically, by intact nuclei, DNA stains or dye entry intothe cell through a disrupted cytoplasmic
32、membrane.6.2 The total number of cells, both alive and dead, within a3-D construct may be determined by DNA analysis (7.2) afterthe cells are removed destructively (lysis) from the biomaterialscaffold and solubilized (with detergents or sonication, forexample). It may not be possible to completely r
33、ecover all cellmaterial that is located deep within scaffold pores due todiffusion limitations.6.3 Counting cells harvested (by trypsinization orpassaging, for example) from scaffolds may not be reliable ifthe scaffold specimens are small (from 96-well or 48-wellplates, for example). The dilutions w
34、ith cell harvesting mediumor buffers may yield cell concentrations that are too low to beeffectively counted (by hemocytometer, for example).6.4 If cells in a suspension are to be counted, electricalsensing zone test method (F2149) or flow cytometry may beuseful.6.5 To determine the quantity of live
35、 cells only, the use of afluorescent or colorimetric metabolic indicator that fluorescesor changes color in response to cell metabolic activity may beused (7.2). Metabolic assays are available in both destructiveand non-destructive forms. The MTT (3-4,5-dimethylthiazol-2-2,5-diphenyltetrazolium brom
36、ide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays (7.2.1) are destructive,commonly used methods that can be read with a spectropho-tometer. The Alamar Blue assay (resorufin) (7.2.2) is a non-destructive method that requires a fluorimeter. C
37、ell metabolismin a 2-D environment may differ from than in a 3-Denvironment, even when the same cell numbers are the same.Accordingly, results for 3-D cell numbers can be erroneouswhen growth curves of cells cultured in 2-D are used forcalibration (1).3It is important to note that metabolic assays a
38、redirect measures of intracellular enzyme activity produced bycells. Although the level of enzyme activity may be directlyproportional to the number of viable cells, it is possible thatspecific culture conditions may affect the production andactivity of the enzyme being assayed or that the scaffold
39、mayinterfere with the measurement (matrix effects). In thissituation, the metabolic measurement may not be directlyproportional to cell number.6.6 The quantity of live cells within the total cell populationmay be determined by a proliferation or metabolic assay (7.3).It may be helpful to verify quan
40、titative results with an imagingtechnique (7.4) in order to provide visual evidence of live ordead cells. Visual evidence assures that the quantitative mea-surements can be trusted and did not arise due to experimentalartifacts (such as the scaffold reacting with assay reagents andcausing a false po
41、sitive reading). Imaging also provides infor-mation on the spatial distribution of live cells within aconstruct.6.7 Non-destructive methods to determine cell viability ofan entire cell population within a scaffold or bioreactor areincluded in this guide and are useful for conducting kineticstudies o
42、f cell number and distribution over time.6.8 The scaffolding material may interfere with any of thefollowing assays and must be included within the assay,typically as a control, to determine whether it has an effect. Ifthe assay is affected by the presence of the scaffold, then eitherthe interferenc
43、e should be subtracted out or an alternative assayshould be selected. Notes on known interferences are includedin each of the assay descriptions below.6.9 Cell density could impact accuracy of quantification.Cells grown at low density are generally harder to wash offthan cells grown to confluency, w
44、here a whole sheet of cellsmay be rather easy to displace. Many scaffolds are seeded at ashigh a cell density as possible. High densities may also affectdye binding. Also, cell density generally impacts the “health”of the whole culture, since cell-to-cell interactions are impor-tant effectors of cel
45、l state.6.10 In many instances a mixed population of cells may bepresent. Metabolic assays will not accurately quantify mixedcultures of cells because some cells are more metabolicallyactive than others. There is a similar problem with dyes:nuclear sizes may not be identical (though they may besimil
46、ar). Cell cytoplasm volumes may be very different, ascould be the number of cellular processes. In a mixed popula-tion of cells, some cells may be proliferating rapidly, whereasothers might be post-mitotic.6.11 Some scaffolds will be translucent, others opaque.Some may be rigid, others very fragile.
47、 For more fragilescaffolds, cells may fall off during handling, so it would bepreferable to use a method that minimizes handling. Scaffoldsbreak down over time. Edges of scaffolds might be softer thaninternal portions. Scaffolds may not have uniform thickness ordensity, which may affect statistical
48、sampling.7. Specific Test Methods for Determining Cell Viability7.1 Dye Exclusion Technique to Distinguish Live fromDead:7.1.1 One of the simplest methods to approximate cellviability is the dye exclusion technique. This approach is basedon the assumption that viable cells must have an intactmembran
49、e, which is required for life-associated cellular pro-cesses such as the conversion of food sources into energy,growth, and reproduction. This method utilizes an indicatordye to demonstrate cell membrane damage. Cells which absorbthe dye become stained and are considered non-viable. Dyessuch as trypan blue, erythrosin, and nigrosin are usedcommonly, with trypan blue being the most common inpreliminary cell isolation procedures. Cells must be removedfrom the scaffold, mixed with the dye, and then countedmanually with a hematocytometer. Cells must be analyzedshortly after the
