ASTM_F_3088_-_14.pdf

Designation:F308814Standard Test Method forUse of a Centrifugation Method to Quantify/Study Cell-Material Adhesive Interactions1This standard is issued under the fixed designation F3088;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 test method covers a centrifugation cell adhesionassay that can be used to detect changes in adhesive charac-teristics of cells with passage or treatments.This approachmeasures the force required to detach cells from a substrate.Adhesion,among many variables,may vary due to changes inthe phenotype of the cells.1.2 This test method does not cover methods to verify theuniformity of coating of surfaces,nor does it cover methods forcharacterizing surfaces.1.3 The cells may include adult,progenitor or stem cellsfrom any species.The types of cells may include chondrocytes,fibroblasts,osteoblast,islet cells,or other relevant adherent celltypes.1.4 This test method does not cover methods for isolating orharvesting of cells.This test method does not cover testmethods to quantitate changes in gene expression,or changesin biomarker type or concentration,as identified by immunos-taining.Nor does this test method cover quantitative imageanalysis techniques.1.5 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:2F2603 Guide for Interpreting Images of Polymeric TissueScaffoldsF2664 Guide for Assessing the Attachment of Cells toBiomaterial Surfaces by Physical MethodsF2739 Guide for Quantitating Cell Viability Within Bioma-terial ScaffoldsF2944 Test Method for Automated Colony Forming Unit(CFU)AssaysImage Acquisition and Analysis Methodfor Enumerating and Characterizing Cells and Colonies inCulture3.Summary of Test Method3.1 Centrifugation Cell Assay on Cell PopulationsA con-ventional centrifuge can be used to apply a normal or shearforce to cells depending on the orientation of the cells withrespect to the centrifugal force(1-3).3The centrifugal forcethat the cells are subject to can be calculated according to thefollowing formula assuming that the plates are 90(normal)tothe spindle of the centrifuge.FD5pcell2 pmedium!VcellRCF(1)where:FD=detachment force applied per cell,Vcell=cell volume,pcell=density of the cell,pmedium=the density of the surrounding medium,andRCF=Relative Centrifugal Forces=r2,where r=centrifugation radius and =centrifugation speed.See X1.6 for example of a calculation of“detachment forceper cell.”3.1.1 Such tests are easy to conduct and the results representa population average.The method can be performed in amoderately outfitted cell biology lab with an inverted micro-scope or a fluorescence microplate reader and a centrifuge witha microtiter plate holder.Three factors need to be consideredwhen using this methodology:the potential influences of forcesapplied during the period of spin up time,the maximum spinspeed,and the test duration.The method is only able tocorrelate with cell detachment with the maximum force appliedafter the centrifuge has reached its set spin speed.It should alsobe considered that the maximum force that can be applied islimited by the centrifuge,sample configuration(that is,con-figuration and type of multiwell plate)and time of centrifuga-tion.In some instances,the cell adhesion strength will be1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved May 1,2014.Published June 2014.DOI:10.1520/F3088-14.2For 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.3The boldface numbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 greater than the applied forces.Therefore,for certain cell typesthat have attached for extended periods of time,the methodmay not be able to generate forces sufficient to detach thesecells.3.1.2 Cell attachment is a complex,time-dependent,processinvolving significant morphological and structural changes ofthe cell and deposition of a bed of extracellular matrix and isa function of cellular and substrate parameters.Cell adhesionto a surface depends on a range of biological factors thatinclude culture history,species,the