1、Designation: F2664 11Standard Guide forAssessing the Attachment of Cells to Biomaterial Surfacesby Physical Methods1This standard is issued under the fixed designation F2664; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、 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 describes protocols that can be used tomeasure the strength of the adhesive bond that developsbetween a
3、 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. Thein
4、formation 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 standard
5、 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
6、Standards:2D4410 Terminology for Fluvial SedimentF22 Test Method for Hydrophobic Surface Films by theWater-Break TestF2312 Terminology Relating to Tissue Engineered MedicalProductsF2603 Guide for Interpreting Images of Polymeric TissueScaffolds2.2 ISO Standards:3ISO 4287 Geometrical Product Specific
7、ations (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 Defini
8、tions: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 specific
9、 application. F23123.1.3 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 replac
10、es any tissue, organ, orfunction of the body. F23123.1.5 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, nin medicine, an object, structure, or de-vice intended to r
11、eside within the body for diagnostic, pros-thetic, or other therapeutic purposes.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 tra
12、nsplantation of cells,with or without dilution, 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
13、 with the term subculture. (SeeRef (2).)3.1.11 passage number, nthe number of times the cells inthe culture have been subcultured or passaged. In descriptions1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of S
14、ubcommitteeF04.43 on Cells and Tissue Engineered Constructs for TEMPs.Current edition approved April 1, 2011. Published May 2011. Originallyapproved in 2007. Last previous edition approved in 2007 as F2664 07. DOI:10.1520/F2664-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org,
15、 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, New York, NY 10036, http:/www.ansi.org.4Th
16、e 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.of this process, the ratio or dilution of the cells should be statedso that the relative cul
17、tural age can be ascertained. (See Ref(2).)3.1.12 Reynolds number, na dimensionless number ex-pressing the ratio of inertia forces to viscous 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
18、fluids mass density, and m is the fluidsdynamic viscosity. D44103.1.13 scaffold, na support, delivery vehicle, or matrix forfacilitating the migration, binding, or transport of cells orbioactive molecules used to replace, repair, or regeneratetissues. F23123.1.14 senescence, nin vertebrate cell cult
19、ures, the prop-erty attributable to finite cell cultures; namely, their inability togrow beyond a finite number of population doublings. Neitherinvertebrate nor plant cell cultures exhibit this property. Thisterm is synonymous with in vitro senescence. (See Ref (2).)3.1.15 shear stress, ncomponents
20、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 theintersection 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-
21、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 Use4.1 Cell attachment or, lack of it, to biomaterials is a criticalfactor affecting the performance of a device or implant. Cellattachment is a complicated, tim
22、e-dependent, process involv-ing significant morphological changes of the cell and deposi-tion of a bed of extracellular matrix. Details of the 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 s
23、trength of this couplingcan be determined either by monitoring the force of attachmentbetween a cell and a substrate over time or by measuring 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
24、 culture history,the age of the cell, the cell type, and both the chemistry andmorphology of the underlying surface and time. These ele-ments 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
25、 further complicatedsince either cell adhesion or detachment can be assessed. Theseprocesses that are not always similar or complementary.4.4 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
26、 a surface (Jameset al, 2005) (7). More recently investigators have begun tomeasure the adhesive forces that develop between cells and theunderlying 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-a
27、dhere cells from asurface than to measure those that develop during attachment.However, in both cases, the experimental data should beinterpreted with a degree of caution that depends on theintended use of the measurements. The methods of measuringcell adhesion described herein are measures of the f
28、orcerequired to detach an adherent cell.4.5 The purpose of this guide is to provide an overview ofcurrent generic test methods and identify the key factors thatinfluence the assessment of cell adhesion and detachment. It isanticipated that this guide will form the basis for producing aseries of stan
29、dards that will describe these test methods in moredetail.5. Cell Attachment Assays5.1 Table 1 provides examples of common cell adhesionassays, 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
30、 singlecells or a population of cells. Single cell techniques canprovide quantitative measures of the adhesive force thatdevelops with time between a cell and a substrate or thatrequired to detach an adhered cell from a substrate. Individualligand-surface interactions can be measured directly using,
31、 forexample, a cell mounted on an atomic force microscope (AFM)tip. Single cell measurements do have their disadvantages.Variations in adhesive strength are not averaged out over apopulation and sophisticated equipment, such as an AFM, isrequired.5.3 Cell population based assays average out variatio
32、ns incell-to-substrate adhesiveness compared with measurementsperformed on a single cell. This variation arises both becauseof variations in biomaterial surface properties, and variations incell phenotype used as the probe (Appendix X1 and AppendixX2). Cell population techniques provide a usable mea
33、sure ofthe biomaterials adhesiveness for a given batch of cells andtest conditions. Cell population techniques are attractive in thatthey provide robust measurements based on a large number ofcells, which is an important consideration given the inherentvariance of biological systems. Measurements th
34、at are basedon large numbers of cells reduce the influences of localvariations in surface chemistry and texture and in the adhe-siveness of the cells themselves.TABLE 1 Assays for Measuring Cell Detachment from SurfacesCellRequirementsAssayAssayDescriptionSectionSingle Cell Micromanipulation Measure
35、ment of the Forcedeveloped during attachmentvia an AFM6.1.1-6.1.2Single Cell Micromanipulation Forces applied via amicropipette, microprobe 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.
36、2Centrifugation Detachment of cells usingcentrifugal force6.2.3Hydrodynamic Flow Detachment of cells usingshear forces generated bylaminar flow over cells6.2.4F2664 1126. Measurement of Cell DetachmentNOTE 1In principle, the strength of the adhesive bond that developsbetween the cell and underlying
37、substrate will increase with time,although in practice this will depend on the cell-surface interactions.These measurements can be performed 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, a
38、s these tests aredestructive. Each test described below carries its own unique sources ofstatistical error. Users should familiarize themselves with the appropriateassay system and should consult with appropriate statistical staff todetermine the necessary statistical parameters to ensure statistica
39、l signifi-cance. These parameters may include, but are not limited to: sample size,power of study, number of image fields counted (for microscope-basedassays), number of cell lots tested, variability between users, what is themost appropriate statistical analysis (that is, analysis of variance, Tuke
40、ystest, t-test, etc.) and determination of a standard curve for analysis ofdetached cells.6.1 Micromanipulation:6.1.1 Micromanipulation Methods (Single Cells)Singlecells can be used to measure the force required to uncouplecells from the underlying substrate (measure of detachment), asa result of a
41、time-dependent adhesion. Such measurements aremade using micromanipulation or micropipettes. Cells can beseeded onto a small block of material 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 ti
42、me.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 be 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 ofmate
43、rial 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 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. Proble
44、ms have been reported with protein adsorp-tion on the cantilever having an adverse effect on its reflectiv-ity.6.1.3 Micropipettes, microprobes, 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
45、, 2004) (8). All these methods providequantifiable sensitive and real time direct measures of the forcerequired to detach the cell that is typically 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
46、 process of cell detachment in detail. Practicalissues that need to be considered when using these methodsinclude:6.1.3.1 Specialized equipment, 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 ens
47、ure that the measurementrelates to detachment force and is not a measure of cellmembrane strength, this can be checked by examining thefootprint 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 t
48、he 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 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 Popul
49、ations:6.2.1 GravityGravity can be used to differentiate betweencells that are attached to a substrate and those that have not byturning the 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 of
