1、Designation: F 2739 08Standard Guide forQuantitating Cell Viability Within Biomaterial Scaffolds1This standard is issued under the fixed designation F 2739; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、 A 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-viabl
3、e cells 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
4、accurate 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 imp
5、lantation 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
6、address 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:2F 748 Prac
7、tice for Selecting Generic Biological Test Meth-ods for Materials and DevicesF 2149 Test Method for Automated Analyses of CellstheElectrical Sensing Zone Method of Enumerating and Siz-ing Single Cell SuspensionsF 2315 Guide for Immobilization or Encapsulation of Liv-ing Cells or Tissue in Alginate G
8、els3. Terminology3.1 Definitions:3.1.1 non-viable cell, na cell not meeting one or more ofthe criteria for viability given in 3.1.2.3.1.2 viable cell, na cell capable of metabolic activity thatis structurally intact with a functioning cell membrane.4. Summary of Guide4.1 It is the intent of this gui
9、de to provide a compendium ofthe commonly used methods for quantitating the number anddistribution of viable and non-viable cells within, or on, abiomaterial scaffold, because cell viability is important param-eter of tissue engineering products used to regenerate or repairlost or diseased tissue. T
10、he methods can be applied to cellsresiding 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).4.2 Most of the methods originate from analysis of cellnumber on 2-D surfaces, but have be
11、en adapted for the analysisof cells within 3-D constructs that are typically used 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 doc
12、ument. In addition, theways in which the biomaterial scaffold itself can affect theviability assays are described.4.3 This guide describes combinations of test methodswhich, when used together, will ensure the most accuratemeasure of the number and distribution of viable and non-viable cells.5. Sign
13、ificance 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/biomaterial constructs (see 5.7);therefore there is a need for standardi
14、zed test methods toquantitate 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 on to scaffold surfaces have been shown to have lower1This guide is
15、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 TEMPs.Current edition approved Aug. 1, 2008. Published September 2008.2For referenced ASTM standards, visit t
16、he ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,
17、 United States.cell 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 isenhanced by the application of centr
18、ifugal forces. The methodsdescribed in this guide can assist in establishing cell seedingefficiencies as a function of seeding method and for standard-izing viable cell number within a given methodology.5.3 As described in Guide F 2315, thick scaffolds or scaf-folds highly loaded with cells lead to
19、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 bequantitated using the tests within this guide. The effectivenessof the culturing method or
20、bioreactor conditions on theviability of the cells throughout the scaffold can also beevaluated with the methods described in this guide.5.4 These test methods can be used to quantitate cells onhard or soft 3-D biomaterials, such as ceramics and polymergels. The test methods also apply to cells seed
21、ed on porouscoatings.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 daughtercells. Non-proliferating viable cells
22、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.5.7 While cell viability is an import
23、ant 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, andmorphology are recommended.5.8
24、Fundamental biocompatibility testing of the scaffoldmaterial itself as described in Practice F 748 should be com-pleted prior to using the biomaterial with cells.6. Selection of Test Methods6.1 Table 1 is a compendium of methods that can be used toquantitate cell viability on surfaces or in biomater
25、ial scaffolds.Importantly, a combination of the methods listed in Table 1 isrequired to determine viable and non-viable (or live and dead)cells quantitatively, and additional tests must be completed toquantitate the subset of proliferating viable cells within thetotal number of viable cells. Prolife
26、rating cells are viable, butviable cells are not necessarily proliferating. Non-viable cellscan be identified, even if they are not intact structurally ormetabolically, by intact nuclei or dye entry into the cell througha disrupted cytoplasmic membrane.6.2 The total number of cells, both alive and d
27、ead, within a3-D construct is typically determined by DNA analysis (7.2)after the cells are removed destructively from the biomaterialscaffold and solubilized. Since many cell types adhere verywell to a scaffold, significant cell lysis can occur during cellremoval and simple manual counting of intac
28、t stained orunstained cells (7.1) is not very reliable.6.3 If cells in a suspension are to be counted, Test MethodF 2149 may be useful.6.4 To determine the quantity of live cells only, the use of afluorescent or colorimetric metabolic indicator that fluorescesor changes color in response to chemical
29、 reduction of growthmedia resulting from cell metabolic activity may be used (7.2).Metabolic assays are available in both destructive and non-destructive forms. The MTT or MTS assay (7.2.1)isadestructive, commonly used method that can be read with aTABLE 1 Methods for Quantitating Cell ViabilityDest
30、ructive(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 XXTetrazolium salt uptake: MTT, MTS, WST, XTT XAlamar Blue XNeutral Red XGlucose ConsumptionCell prol
31、iferation (DNA synthesis)3H Thymidine or BrDu labeling XDye exclusion assaysTrypan blue, erythrosin, and nigrosin XIII. Live/Dead RatiosLive/Dead assays using dual fluorescent stains for plasma membraneintegrityXNon-fluorescent dye exclusion assays XIV. Imagingdensity, morphology and spatial distrib
32、utions of cellsHistological sectioning XConfocal microscopy XXScanning electron microscopy XF2739082spectrophotometer. The Alamar Blue assay (7.2.2) is a non-destructive method that requires a fluorimeter. Cell metabolismin a 2-D environment, compared with a 3-D environment, issignificantly differen
33、t, even with the same cell numbers.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 aredirect measures of intracellular enzyme activity produced bycells.Although the level
34、of enzyme activity is typically linearlyproportional to the number of viable cells, it is possible thatspecific culture conditions can affect the production andactivity of the enzyme being assayed or that in certaincircumstances, substrate may not be limiting. In this situation,the metabolic assay w
35、ould not necessarily be linearly propor-tional to cell number. Despite these limitations, these methodsare still commonly used.6.5 The quantity of live cells within the total cell populationmay be determined by a proliferation or metabolic assay (7.3).However, this will not provide information on th
36、e distributionof live cells within a construct; hence, an imaging technique(7.4) to visualize the morphology and spatial distribution ofdye-labeled live and dead cells is typically utilized.6.6 Non-destructive methods to determine cell viability ofan entire cell population within a scaffold or biore
37、actor areincluded in this guide and are useful for conducting kineticstudies of cell number and distribution over time.6.7 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
38、 assay is affected by the presence of the scaffold, then eitherthe interference should be subtracted out or an alternative assayshould be selected. Notes on known interferences are includedin each of the assay descriptions below.6.8 Cell density could impact accuracy of quantification.Cells grown at
39、 low density are generally harder to wash offthan cells grown to confluency, where a whole sheet of cellsmay be rather easy to displace. Many of our products may beseeded at as high a density as possible. High densities may alsoaffect dye binding.6.9 In many instances a mixed population of cells may
40、 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 are probablysimilar). Cell cytoplasm volumes may be very different, ascou
41、ld be the number of cellular processes. In a mixed popula-tion of cells some cells may be proliferating rapidly, whereasothers might be post-mitotic.6.10 Some scaffolds will be translucent, others opaque.Some may be rigid, others very fragile. For more fragilescaffolds, cells may fall off during han
42、dling, 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 sampling.7. Specific Test Methods for Determining Cell Vi
43、ability7.1 Dye Exclusion Technique to Distinguish Live fromDead:7.1.1 One of the simplest methods to approximate cellviability is the dye exclusion technique. This method utilizesan indicator dye to demonstrate cell membrane damage. Cellswhich absorb the dye become stained and are considerednon-viab
44、le. Dyes such as trypan blue, erythrosin, and nigrosinare used commonly, with trypan blue being the most commonin preliminary cell isolation procedures. Cells must be removedfrom the scaffold, mixed with the dye for a few minutes, andthen counted manually with a hematocytometer. Cells must beanalyze
45、d shortly (3 to 5 min) after the addition of 0.4 % trypanblue, since trypan blue is cytotoxic. There are large standarddeviations with increasing cell densities; therefore samplesshould be diluted to the densities recommended in the hema-tocytometer instructions.7.2 Determination of Total Cell Numbe
46、r:7.2.1 DNA AssayDNA analysis is a commonly usedmethod for determining total cell number, including bothviable and non-viable cells. There are several commerciallyavailable kits for assessing DNA content, all of which can beused. It is important to fully extract the cells from the scaffoldprior to a
47、nalysis, using for example a solution of 0.125 mg/mLpapain and 10 mmol/L L-cysteine dihydrochloride in phos-phate buffered EDTA in a 60C water bath for 10 hours toextract cells from a polymer matrix (2). If the cell fluorescencewill be measured, using a proteinase K digestion step ablatesany residua
48、l endogenous fluorescence of the cells (3).7.2.2 Crystal Violet StainingAnother cell stain used fordetermining total cell number is crystal violet which binds tothe DNAof viable and non-viable cells. Cells must be removedfrom the biomaterial scaffold prior to analysis. Cells arewashed in phosphate b
49、uffered solution (PBS), stained with 0.1to 0.2 % crystal violet in methanol for 15 min at 37C, and thenwashed extensively prior to analysis. Absorbance is measuredat a wavelength of 590 nm using an ELISA plate reader.7.3 Proliferation or Metabolic Assays for Quantitating LiveCell Number:7.3.1 MTT, MTS, XTT or WST Tetrazolium Salt AssaysMetabolic activity of cells is commonly monitored colorimetri-cally by assaying the cell-based alteration of tetrazolium saltssuch as MTT (3-4,5-dimethylthiazol-2-2,5-diphenyltetrazolium bromide). The cells take up the crystalsand convert th
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