1、Designation: F 2664 07Standard Guide forAssessing the Attachment of Cells to Biomaterial Surfacesby Physical Methods1This standard is issued under the fixed designation F 2664; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the ye
2、ar of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide describes protocols that can be used tomeasure the strength of the adhesive bond that developsbetwee
3、n a cell and a surface as well as the force required todetach cells that have adhered to a substrate. Controlling theinteractions of mammalian cells with surfaces is fundamentalto the development of safe and effective medical products. Thisguide does not cover methods for characterizing surfaces. Th
4、einformation generated by these methods can be used to obtainquantitative measures of the susceptibility of surfaces to cellattachment as well as measures of the adhesion of cells to asurface. This guide also highlights the importance of cellculture history and influences of cell type.1.2 This stand
5、ard 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 AS
6、TM Standards:2D 4410 Terminology for Fluvial SedimentF22 Test Method for Hydrophobic Surface Films by theWater-Break TestF 2312 Terminology Relating to Tissue Engineered MedicalProductsF 2603 Guide for Interpreting Images of Polymeric TissueScaffolds2.2 ISO Standards:3ISO 4287 Geometrical Product Sp
7、ecifications (GPS)Surface Texture: Profile MethodTerms, Definitions andSurface Texture ParametersISO 13565-1 Geometrical Product Specifications (GPS)Surface Texture: Profile Method; Surfaces Having Strati-fied Functional PropertiesPart 1: Filtering and GeneralMeasurement Conditions3. Terminology3.1
8、Definitions:3.1.1 adhesion, na physiochemical state by which a cell iscoupled to a non-cell surface by interfacial forces, which mayconsist of covalent or ionic forces.3.1.2 biocompatibility, na material may be consideredbiocompatible if the materials perform with an appropriate hostresponse in a sp
9、ecific application. F 23123.1.3 biomarker, nbiochemical feature or facet that can beused to measure the progress of disease or the effects oftreatment.3.1.4 biomaterial, nany substance (other than a drug),synthetic or natural, that can be used as a system or part of asystem that treats, augments, or
10、 replaces any tissue, organ, orfunction of the body. F 23123.1.5 detachment, nprocess whereby an adhered cell orgroup of cells is actively detached from a surface.3.1.6 hydrophilic, adjhaving a strong affinity for water,wettable. F223.1.7 implant, na substance or object that is put in thebody as a p
11、rosthesis, or for treatment or diagnosis.3.1.8 laminar flow, nwell-ordered, patterned flow of fluidlayers assumed to slide over one another. (See Ref (1).)43.1.9 lay, ndirection of the predominant surface pattern.ISO 13565-13.1.10 passage, nthe transfer or transplantation of cells,with or without di
12、lution, from one culture vessel to another. Itis understood that any time cells are transferred from onevessel to another, a certain portion of the cells may be lost and,therefore, dilution of cells, whether deliberate or not, mayoccur. This term is synonymous with the term subculture. (SeeRef (2).)
13、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 June 1, 2007. Published June 2007.2For referenced ASTM standard
14、s, 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.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, Ne
15、w York, NY 10036, http:/www.ansi.org.4The boldface numbers in parentheses refer to the list of references at the end ofthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.11 passage number, nthe number of times the cell
16、s inthe culture have been subcultured or passaged. In descriptionsof this process, the ratio or dilution of the cells should be statedso that the relative cultural age can be ascertained. (See Ref(2).)3.1.12 Reynolds number, na dimensionless number ex-pressing the ratio of inertia forces to viscous
17、forces in a movingfluid. The number is given by VLr/m where V, is the fluidsvelocity, L is a characteristic length or distance such as pipediameter, r is the fluids mass density, and m is the fluidsdynamic viscosity. D 44103.1.13 scaffold, na support, delivery vehicle, or matrix forfacilitating the
18、migration, binding, or transport of cells orbioactive molecules used to replace, repair, or regeneratetissues. F 23123.1.14 senescence, nin vertebrate cell cultures, the prop-erty attributable to finite cell cultures; namely, their inability togrow beyond a finite number of population doublings. Nei
19、therinvertebrate nor plant cell cultures exhibit this property. Thisterm is synonymous with in vitro senescence. (See Ref (2).)3.1.15 shear stress, ncomponents of stress that act paral-lel to the plane of the surface. (See Ref (3).)3.1.16 surface profile, nthe surface profile formed by theintersecti
20、on of a real surface by a specified plane. It iscustomary to select a plane that lies perpendicular to thedirection of lay unless otherwise indicated.ISO 13565-1 and ISO 42873.1.17 tack, nability of an adhesive to form a bond to asurface after brief contact under light pressure.4. Significance and U
21、se4.1 Cell attachment or, lack of it, to biomaterials is a criticalfactor affecting the performance of a device or implant. Cellattachment is a complicated, time-dependent, process involv-ing significant morphological changes of the cell and deposi-tion of a bed of extracellular matrix. Details of t
22、he adhesivebond that is formed have been reviewed by, for example,Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), andGarcia and Gallant (2003) (6). The strength of this couplingcan be determined either by monitoring the force of attachmentbetween a cell and a substrate over time or by measuri
23、ng theforce required to detach the cell once it has adhered.4.2 Cell adhesion to a surface depends on a range ofbiological and physical factors that include the culture history,the age of the cell, the cell type, and both the chemistry andmorphology of the underlying surface and time. These ele-ment
24、s that need to be considered in developing a test protocol.4.3 Devising robust methods for measuring the propensityof cells to attach to different substrates is further complicatedsince either cell adhesion or detachment can be assessed. Theseprocesses that are not always similar or complementary.4.
25、4 Most studies of cell attachment focus on obtaining somemeasure of the time-dependent force required to detach, orde-adhere, cells that have already adhered to a surface (Jameset al, 2005) (7). More recently investigators have begun tomeasure the adhesive forces that develop between cells and theun
26、derlying surface during attachment (Lukas and Dvorak,2004) (5). From a practical point of view, it is much easier tomeasure the force required to detach or de-adhere cells from asurface than to measure those that develop during attachment.However, in both cases, the experimental data should beinterp
27、reted with a degree of caution that depends on theintended use of the measurements. The methods of measuringcell adhesion described herein are measures of the forcerequired to detach an adherent cell.4.5 The purpose of this guide is to provide an overview ofcurrent generic test methods and identify
28、the key factors thatinfluence the assessment of cell adhesion and detachment. It isanticipated that this guide will form the basis for producing aseries of standards that will describe these test methods in moredetail.5. Cell Attachment Assays5.1 Table 1 provides examples of common cell adhesionassa
29、ys, including a brief description of the forces applied.These assays are discussed in more detail in Section 6.5.2 Cell attachment assays can be performed using singlecells or a population of cells. Single cell techniques canprovide quantitative measures of the adhesive force thatdevelops with time
30、between a cell and a substrate or thatrequired to detach an adhered cell from a substrate. Individualligand-surface interactions can be measured directly using, forexample, a cell mounted on an atomic force microscope (AFM)tip. Single cell measurements do have their disadvantages.Variations in adhes
31、ive strength are not averaged out over apopulation and sophisticated equipment, such as an AFM, isrequired.5.3 Cell population based assays average out variations incell-to-substrate adhesiveness compared with measurementsperformed on a single cell. This variation arises both becauseof variations in
32、 biomaterial surface properties, and variations incell phenotype used as the probe (Appendix X1 and AppendixX2). Cell population techniques provide a usable measure ofthe biomaterials adhesiveness for a given batch of cells andtest conditions. Cell population techniques are attractive in thatthey pr
33、ovide robust measurements based on a large number ofcells, which is an important consideration given the inherentvariance of biological systems. Measurements that are basedon large numbers of cells reduce the influences of localvariations in surface chemistry and texture and in the adhe-siveness of
34、the cells themselves.TABLE 1 Assays for Measuring Cell Detachment from SurfacesCellRequirementsAssayAssayDescriptionSectionSingle Cell Micromanipulation Measurement of the Forcedeveloped during attachmentvia an AFM6.1.1-6.1.2Single Cell Micromanipulation Forces applied via amicropipette, microprobe
35、orAFM6.1.3Cell Population Gravity Detect the number of cellsthat remain attached afterturning the culture vesselupside down6.2.1Wash Wash off adhered cells 6.2.2Centrifugation Detachment of cells usingcentrifugal force6.2.3Hydrodynamic Flow Detachment of cells usingshear forces generated bylaminar f
36、low over cells6.2.4F26640726. Measurement of Cell DetachmentNOTE 1In principle, the strength of the adhesive bond that developsbetween the cell and underlying substrate will increase with time,although in practice this will depend on the cell-surface interactions.These measurements can be performed
37、on either populations of cells orsingle cells. It should also be noted that it is not possible to conduct aseries of measurements over time on the same cell, as these tests aredestructive. Each test described below carries its own unique sources ofstatistical error. Users should familiarize themselv
38、es with the appropriateassay system and should consult with appropriate statistical staff todetermine the necessary statistical parameters to ensure statistical signifi-cance. These parameters may include, but are not limited to: sample size,power of study, number of image fields counted (for micros
39、cope-basedassays), number of cell lots tested, variability between users, what is themost appropriate statistical analysis (that is, analysis of variance, Tukeystest, t-test, etc.) and determination of a standard curve for analysis ofdetached cells.6.1 Micromanipulation:6.1.1 Micromanipulation Metho
40、ds (Single Cells)Singlecells can be used to measure the force required to uncouplecells from the underlying substrate (measure of detachment), asa result of a time-dependent adhesion. Such measurements aremade using micromanipulation or micropipettes. Cells can beseeded onto a small block of materia
41、l mounted on an AFM tip,attached to a coated AFM tip or to the tip directly. Thecell-coated tip can then be used to measure the tack force thatdevelops over time.6.1.2 There are some practical issues that need to beaddressed when using this direct approach to force measure-ment:6.1.2.1 Care should b
42、e taken to ensure that the measure-ments relate to a single cell and not to contributions from anumber of cells. This is a particular issue when a block ofmaterial is mounted onto the tip.6.1.2.2 Care should be taken to ensure that the measurementrelates to the detachment force and is not a measure
43、of cellmembrane strength; this can be checked by examining thefootprint left by the cell.6.1.2.3 These measurements need to be made using a wetcell AFM. Problems have been reported with protein adsorp-tion on the cantilever having an adverse effect on its reflectiv-ity.6.1.3 Micropipettes, microprob
44、es, and AFMs have beenused to measure the force required to suck or pull single cellsaway from the substrate to which they are attached (forexample, Shao et al, 2004) (8). All these methods providequantifiable sensitive and real time direct measures of the forcerequired to detach the cell that is ty
45、pically less than 10 mN (forexample, Lee et al, 2004) (9). Control over the magnitude ofthe force and the rate at which it is applied can be used toexplore the process of cell detachment in detail. Practicalissues that need to be considered when using these methodsinclude:6.1.3.1 Specialized equipme
46、nt, which must be calibrated toensure that data are reproducible and repeatable, is required forsuch sensitive measurements.6.1.3.2 Care should be taken to ensure that the measurementrelates to detachment force and is not a measure of cellmembrane strength, this can be checked by examining thefootpr
47、int left by the cell.6.1.3.3 Consideration should be given as to the direction ofthe applied force, that is, tensile, shear or some combination ofthe two and the magnitude of the applied stress. Larger areapipette tips will subject the cell to a lower stress than the tip ofan AFM for a given applied
48、 force.6.1.3.4 The period of time between exposing the cells to asurface and that at which measurements are made.6.2 Cell Detachment Measurements on Cell Populations:6.2.1 GravityGravity can be used to differentiate betweencells that are attached to a substrate and those that have not byturning the
49、cell culture vessel upside down. Prior to using thisapproach, the user should consider the buoyancy of the cellswith respect to medium to ensure that it is negative. Consid-eration should be given to the test duration to improve theconsistency of repeat measurements.6.2.2 Wash AssaysA simple, convenient, widely usedassay that readily provides qualitative information on adhesionof cells to a substrate is to wash off non-adherent cells usingculture medium. This approach may take many forms frommild shaking of the culture vessel to sluicing of the culturewell
