ASTM_F_2739_-_08.pdf

Designation:F273908Standard Guide forQuantitating 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 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 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 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 implantation 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 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:2F748 Practice 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 Gels3.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 guide 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.The 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 been 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 document.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.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/biomaterial constructs(see 5.7);therefore there is a need for standardized test methods toquantitate cell viability.1This 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 TEMPs.Current edition approved Aug.1,2008.Published September 2008.DOI:10.1520/F2739-08.2For referenced ASTM standards,visit the 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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 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 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 seedi