ASTM F2664-07
(Guide)Standard Guide for Assessing the Attachment of Cells to Biomaterial Surfaces by Physical Methods
Standard Guide for Assessing the Attachment of Cells to Biomaterial Surfaces by Physical Methods
SCOPE
1.1 This guide describes protocols that can be used to measure the strength of the adhesive bond that develops between a cell and a surface as well as the force required to detach cells that have adhered to a substrate. Controlling the interactions of mammalian cells with surfaces is fundamental to the development of safe and effective medical products. This guide does not cover methods for characterizing surfaces. The information generated by these methods can be used to obtain quantitative measures of the susceptibility of surfaces to cell attachment as well as measures of the adhesion of cells to a surface. This guide also highlights the importance of cell culture history and influences of cell type.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:F2664–07
Standard Guide for
Assessing the Attachment of Cells to Biomaterial Surfaces
by Physical Methods
This standard is issued under the fixed designation F2664; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope ISO 4287 Geometrical Product Specifications (GPS)—
Surface Texture: Profile Method—Terms, Definitions and
1.1 This guide describes protocols that can be used to
Surface Texture Parameters
measure the strength of the adhesive bond that develops
ISO 13565-1 Geometrical Product Specifications (GPS)—
between a cell and a surface as well as the force required to
Surface Texture: Profile Method; Surfaces Having Strati-
detach cells that have adhered to a substrate. Controlling the
fied Functional Properties—Part 1: Filtering and General
interactions of mammalian cells with surfaces is fundamental
Measurement Conditions
tothedevelopmentofsafeandeffectivemedicalproducts.This
guide does not cover methods for characterizing surfaces. The
3. Terminology
information generated by these methods can be used to obtain
3.1 Definitions:
quantitative measures of the susceptibility of surfaces to cell
3.1.1 adhesion, n—aphysiochemicalstatebywhichacellis
attachment as well as measures of the adhesion of cells to a
coupled to a non-cell surface by interfacial forces, which may
surface. This guide also highlights the importance of cell
consist of covalent or ionic forces.
culture history and influences of cell type.
3.1.2 biocompatibility, n—a material may be considered
1.2 This standard does not purport to address all of the
biocompatibleifthematerialsperformwithanappropriatehost
safety concerns, if any, associated with its use. It is the
response in a specific application. F2312
responsibility of the user of this standard to establish appro-
3.1.3 biomarker,n—biochemicalfeatureorfacetthatcanbe
priate safety and health practices and determine the applica-
used to measure the progress of disease or the effects of
bility of regulatory limitations prior to use.
treatment.
2. Referenced Documents 3.1.4 biomaterial, n—any substance (other than a drug),
synthetic or natural, that can be used as a system or part of a
2.1 ASTM Standards:
system that treats, augments, or replaces any tissue, organ, or
D4410 Terminology for Fluvial Sediment
function of the body. F2312
F22 Test Method for Hydrophobic Surface Films by the
3.1.5 detachment, n—process whereby an adhered cell or
Water-Break Test
group of cells is actively detached from a surface.
F2312 Terminology Relating to Tissue Engineered Medical
3.1.6 hydrophilic, adj—having a strong affinity for water,
Products
wettable. F22
F2603 Guide for Interpreting Images of Polymeric Tissue
3.1.7 implant, n—a substance or object that is put in the
Scaffolds
body as a prosthesis, or for treatment or diagnosis.
2.2 ISO Standards:
3.1.8 laminar flow, n—well-ordered, patterned flow of fluid
layers assumed to slide over one another. (See Ref (1).)
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
3.1.9 lay, n—direction of the predominant surface pattern.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
ISO 13565-1
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
3.1.10 passage, n—the transfer or transplantation of cells,
Current edition approved June 1, 2007. Published June 2007. DOI: 10.1520/
F2664-07.
with or without dilution, from one culture vessel to another. It
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
is understood that any time cells are transferred from one
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
3 4
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St., The boldface numbers in parentheses refer to the list of references at the end of
4th Floor, New York, NY 10036, http://www.ansi.org. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2664–07
vessel to another, a certain portion of the cells may be lost and, measure the adhesive forces that develop between cells and the
therefore, dilution of cells, whether deliberate or not, may underlying surface during attachment (Lukas and Dvorak,
occur. This term is synonymous with the term subculture. (See 2004) (5). From a practical point of view, it is much easier to
Ref (2).) measure the force required to detach or de-adhere cells from a
3.1.11 passage number, n—the number of times the cells in surface than to measure those that develop during attachment.
the culture have been subcultured or passaged. In descriptions However, in both cases, the experimental data should be
ofthisprocess,theratioordilutionofthecellsshouldbestated interpreted with a degree of caution that depends on the
so that the relative cultural age can be ascertained. (See Ref intended use of the measurements. The methods of measuring
(2).) cell adhesion described herein are measures of the force
3.1.12 Reynolds number, n—a dimensionless number ex- required to detach an adherent cell.
pressingtheratioofinertiaforcestoviscousforcesinamoving 4.5 The purpose of this guide is to provide an overview of
fluid. The number is given by VLr/m where V, is the fluid’s current generic test methods and identify the key factors that
velocity, L is a characteristic length or distance such as pipe influence the assessment of cell adhesion and detachment. It is
diameter, r is the fluid’s mass density, and m is the fluid’s anticipated that this guide will form the basis for producing a
dynamic viscosity. D4410 seriesofstandardsthatwilldescribethesetestmethodsinmore
3.1.13 scaffold, n—asupport,deliveryvehicle,ormatrixfor detail.
facilitating the migration, binding, or transport of cells or
bioactive molecules used to replace, repair, or regenerate 5. Cell Attachment Assays
tissues. F2312
5.1 Table 1 provides examples of common cell adhesion
3.1.14 senescence, n—in vertebrate cell cultures, the prop-
assays, including a brief description of the forces applied.
erty attributable to finite cell cultures; namely, their inability to
These assays are discussed in more detail in Section 6.
grow beyond a finite number of population doublings. Neither
5.2 Cell attachment assays can be performed using single
invertebrate nor plant cell cultures exhibit this property. This
cells or a population of cells. Single cell techniques can
term is synonymous with in vitro senescence. (See Ref (2).)
provide quantitative measures of the adhesive force that
3.1.15 shear stress, n—components of stress that act paral-
develops with time between a cell and a substrate or that
lel to the plane of the surface. (See Ref (3).)
required to detach an adhered cell from a substrate. Individual
3.1.16 surface profile, n—the surface profile formed by the
ligand-surface interactions can be measured directly using, for
intersection of a real surface by a specified plane. It is
example,acellmountedonanatomicforcemicroscope(AFM)
customary to select a plane that lies perpendicular to the
tip. Single cell measurements do have their disadvantages.
direction of lay unless otherwise indicated.
Variations in adhesive strength are not averaged out over a
ISO 13565-1 and ISO 4287
population and sophisticated equipment, such as an AFM, is
3.1.17 tack, n—ability of an adhesive to form a bond to a
required.
surface after brief contact under light pressure.
5.3 Cell population based assays average out variations in
cell-to-substrate adhesiveness compared with measurements
4. Significance and Use
performed on a single cell. This variation arises both because
4.1 Cell attachment or, lack of it, to biomaterials is a critical
ofvariationsinbiomaterialsurfaceproperties,andvariationsin
factor affecting the performance of a device or implant. Cell
cell phenotype used as the probe (Appendix X1 andAppendix
attachment is a complicated, time-dependent, process involv-
X2). Cell population techniques provide a usable measure of
ing significant morphological changes of the cell and deposi-
the biomaterial’s adhesiveness for a given batch of cells and
tion of a bed of extracellular matrix. Details of the adhesive
test conditions. Cell population techniques are attractive in that
bond that is formed have been reviewed by, for example,
they provide robust measurements based on a large number of
Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), and
Garcia and Gallant (2003) (6). The strength of this coupling
canbedeterminedeitherbymonitoringtheforceofattachment
TABLE 1 Assays for Measuring Cell Detachment from Surfaces
between a cell and a substrate over time or by measuring the
Cell Assay
Assay Section
force required to detach the cell once it has adhered. Requirements Description
4.2 Cell adhesion to a surface depends on a range of
Single Cell Micromanipulation Measurement of the Force 6.1.1-6.1.2
developed during attachment
biological and physical factors that include the culture history,
via an AFM
the age of the cell, the cell type, and both the chemistry and
Single Cell Micromanipulation Forces applied via a 6.1.3
morphology of the underlying surface and time. These ele-
micropipette, microprobe or
AFM
ments that need to be considered in developing a test protocol.
Cell Population Gravity Detect the number of cells 6.2.1
4.3 Devising robust methods for measuring the propensity
that remain attached after
of cells to attach to different substrates is further complicated
turning the culture vessel
upside down
sinceeithercelladhesionordetachmentcanbeassessed.These
Wash Wash off adhered cells 6.2.2
processes that are not always similar or complementary.
Centrifugation Detachment of cells using 6.2.3
4.4 Most studies of cell attachment focus on obtaining some centrifugal force
Hydrodynamic Flow Detachment of cells using 6.2.4
measure of the time-dependent force required to detach, or
shear forces generated by
de-adhere, cells that have already adhered to a surface (James
laminar flow over cells
et al, 2005) (7). More recently investigators have begun to
F2664–07
cells, which is an important consideration given the inherent 6.1.3.1 Specialized equipment, which must be calibrated to
variance of biological systems. Measurements that are based ensurethatdataarereproducibleandrepeatable,isrequiredfor
on large numbers of cells reduce the influences of local such sensitive measurements.
variations in surface chemistry and texture and in the adhe-
6.1.3.2 Careshouldbetakentoensurethatthemeasurement
siveness of the cells themselves.
relates to detachment force and is not a measure of cell
membrane strength, this can be checked by examining the
6. Measurement of Cell Detachment footprint left by the cell.
6.1.3.3 Consideration should be given as to the direction of
NOTE 1—In principle, the strength of the adhesive bond that develops
the applied force, that is, tensile, shear or some combination of
between the cell and underlying substrate will increase with time,
the two and the magnitude of the applied stress. Larger area
although in practice this will depend on the cell-surface interactions.
pipette tips will subject the cell to a lower stress than the tip of
These measurements can be performed on either populations of cells or
single cells. It should also be noted that it is not possible to conduct a an AFM for a given applied force.
series of measurements over time on the same cell, as these tests are
6.1.3.4 The period of time between exposing the cells to a
destructive. Each test described below carries its own unique sources of
surface and that at which measurements are made.
statistical error. Users should familiarize themselves with the appropriate
6.2 Cell Detachment Measurements on Cell Populations:
assay system and should consult with appropriate statistical staff to
determine the necessary statistical parameters to ensure statistical signifi- 6.2.1 Gravity—Gravity can be used to differentiate between
cance. These parameters may include, but are not limited to: sample size,
cells that are attached to a substrate and those that have not by
power of study, number of image fields counted (for microscope-based
turning the cell culture vessel upside down. Prior to using this
assays), number of cell lots tested, variability between users, what is the
approach, the user should consider the buoyancy of the cells
most appropriate statistical analysis (that is, analysis of variance, Tukeys
with respect to medium to ensure that it is negative. Consid-
test, t-test, etc.) and determination of a standard curve for analysis of
eration should be given to the test duration to improve the
detached cells.
consistency of repeat measurements.
6.1 Micromanipulation:
6.2.2 Wash Assays—A simple, convenient, widely used
6.1.1 Micromanipulation Methods (Single Cells)—Single
assay that readily provides qualitative information on adhesion
cells can be used to measure the force required to uncouple
of cells to a substrate is to wash off non-adherent cells using
cellsfromtheunderlyingsubstrate(measureofdetachment),as
culture medium. This approach may take many forms from
a result of a time-dependent adhesion. Such measurements are
mild shaking of the culture vessel to sluicing of the culture
made using micromanipulation or micropipettes. Cells can be
well. Clearly the simplicity, speed and low cost of these
seeded onto a small block of material mounted on anAFM tip,
approaches are attractive, although lack of control of the
attached to a coated AFM tip or to the tip directly. The
applied force in terms of both its magnitude and the nature of
cell-coated tip can then be used to measure the tack force that
theappliedstresslimitsthesensitivityofthemeasurement,and
develops over time.
hence reproducibility. For this reason comparisons between
6.1.2 There are some practical issues that need to be
successive tests are subject to large unquantifiable uncertain-
addressed when using this direct approach to force measure-
ties. Checks should also be made to ensure that the adherent
ment:
surface is not removed or damaged during the assay.
6.1.2.1 Care should be taken to ensure that the measure-
6.2.2.1 This assay can be used to monitor cell attachment to
ments relate to a single cell and not to contributions from a
a surface under different culture conditions, used as a measure
number of cells. This is a particular issue when a block of
of
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