ASTM F2664-19e1

Standard Guide for Assessing the Attachment of Cells to Biomaterial Surfaces by Physical Methods

SIGNIFICANCE AND USE
4.1 Cell attachment or, lack of it, to biomaterials is a critical factor affecting the performance of a device or implant. Cell attachment is a complicated, time-dependent, process involving significant morphological changes of the cell and deposition of a bed of extracellular matrix. Details of the adhesive bond that is formed have been reviewed by, for example, Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), and Garcia and Gallant (2003) (6). The strength of this coupling can be determined either by monitoring the force of attachment between a cell and a substrate over time or by measuring the force required to detach the cell once it has adhered.  
4.2 Cell adhesion to a surface depends on a range of biological and physical factors that include the culture history, the age of the cell, the cell type, and both the chemistry and morphology of the underlying surface and time. These elements need to be considered in developing a test protocol.  
4.3 Devising robust methods for measuring the propensity of cells to attach to different substrates is further complicated since either cell adhesion or detachment can be assessed. These processes are not always similar or complementary.  
4.4 Most studies of cell attachment focus on obtaining some measure of the time-dependent force required to detach, or de-adhere, cells that have already adhered to a surface (James et al, 2005) (7). More recently investigators have begun to measure the adhesive forces that develop between cells and the underlying surface during attachment (Lukas and Dvorak, 2004) (5). From a practical point of view, it is much easier to measure the force required to detach or de-adhere cells from a surface than to measure those that develop during attachment. However, in both cases, the experimental data should be interpreted with a degree of caution that depends on the intended use of the measurements. The methods of measuring cell adhesion described herein are measures of the force required to ...
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.  
1.2 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 30-Sep-2019
• ICS: 07.080 - Biology. Botany. Zoology
• ICS: 07.100.01 - Microbiology in general
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.43 - Cells and Tissue Engineered Constructs for TEMPs

Relations

• Refers: ASTM F2603-06(2020) - Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
• Effective Date: 01-Aug-2020
• Refers: ASTM F2312-11(2020) - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Feb-2020
• Refers: ASTM D4410-16 - Terminology for Fluvial Sediment
• Effective Date: 01-Jan-2016
• Refers: ASTM F2603-06(2012) - Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
• Effective Date: 01-Oct-2012
• Refers: ASTM F2312-11 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Apr-2011
• Refers: ASTM F2312-10 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Jun-2010
• Refers: ASTM D4410-10 - Terminology for Fluvial Sediment
• Effective Date: 01-Feb-2010
• Refers: ASTM F22-02(2007) - Standard Test Method for Hydrophobic Surface Films by the Water-Break Test
• Effective Date: 01-Apr-2007
• Refers: ASTM F2603-06 - Standard Guide for Interpreting Images of Polymeric Tissue Scaffolds
• Effective Date: 01-Dec-2006
• Refers: ASTM F2312-04 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Nov-2004
• Refers: ASTM F2312-03 - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 10-Sep-2003
• Refers: ASTM D4410-03 - Terminology for Fluvial Sediment
• Effective Date: 10-Aug-2003
• Refers: ASTM F22-02 - Standard Test Method for Hydrophobic Surface Films by the Water-Break Test
• Effective Date: 10-Oct-2002
• Refers: ASTM F22-65(1998) - Standard Test Method for Hydrophobic Surface Films by the Water-Break Test
• Effective Date: 01-Jan-1992

Overview

ASTM F2664-19e1: Standard Guide for Assessing the Attachment of Cells to Biomaterial Surfaces by Physical Methods provides essential protocols for measuring the adhesive bond strength between mammalian cells and biomaterial surfaces. This guide, developed by ASTM International, is crucial for professionals involved in the design, testing, or manufacture of medical devices and implants, where cell attachment plays a pivotal role in biocompatibility and performance.

Cell attachment is a time-dependent, complex process involving cell morphology changes and extracellular matrix deposition. Understanding the physical interactions between cells and biomaterials enables the development of safer, more reliable medical products. This guide focuses on methods to measure either the force required to detach already-adhered cells from a biomaterial surface or the forces involved as cells attach.

Key Topics

• Cell Adhesion and Detachment: Guidance on both measuring forces during cell adhesion and the more common approach of measuring the force required to detach cells once they have adhered.
• Assay Methods:
  • Single-cell techniques: Use of atomic force microscopy (AFM), micropipettes, and microprobes for precise measurement of cell-surface interactions.
  • Population-based assays: Gravity-based, wash, centrifugation, and hydrodynamic flow methods for quantifying adhesion across cell populations.
• Influencing Factors:
  • Biological: Cell culture history, passage number, cell type, age, and cellular health.
  • Physical: Surface chemistry and morphology, assay geometry, fluid flow (laminar vs. turbulent).
• Data Interpretation: Critical review of variables affecting measurement reliability, including assay-specific limitations, statistical significance considerations, and the influence of cell/substrate heterogeneity.
• Assay Selection Guidance: Practical considerations for choosing between single-cell and population-based approaches depending on research or regulatory needs.

Applications

ASTM F2664-19e1 is widely used in industries and research fields where cell-surface interactions dictate product safety and efficacy, such as:

• Medical Devices and Implants: Assessing how cells attach to or detach from device surfaces, such as stents, orthopedic implants, dental implants, and tissue scaffolds.
• Tissue Engineering and Regenerative Medicine: Measuring how specific cell types adhere to scaffolds or matrices, supporting tissue integration and repair.
• Quality Control in Manufacturing: Ensuring consistency in cell attachment properties of biomaterial batches.
• Biomaterial Selection and Surface Engineering: Optimizing material surface properties to promote desirable cell adhesion for targeted applications.
• R&D and Regulatory Submission: Generating quantitative data required for product certification, market approval, and performance claims.

The guide is also valuable for academic research exploring the fundamentals of cell-material interactions and for laboratories conducting comparative studies across new biomaterial formulations.

Related Standards

To ensure comprehensive assessment and compatibility, consider referencing these related standards alongside ASTM F2664-19e1:

• ASTM F2312: Terminology Relating to Tissue Engineered Medical Products.
• ASTM F2603: Guide for Interpreting Images of Polymeric Tissue Scaffolds.
• ISO 4287: Geometrical Product Specifications-Surface Texture (Profile Method).
• ISO 13565-1: Surface Texture-Surfaces Having Stratified Functional Properties.
• ASTM D4410: Terminology for Fluvial Sediment (relevant for physical terminology).
• ASTM F22: Test Method for Hydrophobic Surface Films by the Water-Break Test.

Practical Value

Adopting this standard ensures standardized, reproducible results in measuring cell attachment strength-crucial for regulatory compliance, product development, and advancing biomaterials science. By leveraging recommended physical methods and considering key influences on cell adhesion, stakeholders can optimize device performance and patient outcomes, while facilitating global trade and harmonization in medical device standards.

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Keywords: cell attachment assay, biomaterial surface testing, cell adhesion measurement, medical device biocompatibility, tissue engineering, ASTM standards, physical detachment methods, quantitative adhesion assessment, implant surface analysis, quality control biomaterials.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
´1
Designation: F2664 − 19
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.
ε NOTE—Editorial corrections were made throughout in November 2019.
1. Scope F2312 Terminology Relating to Tissue Engineered Medical
Products
1.1 This guide describes protocols that can be used to
F2603 Guide for Interpreting Images of Polymeric Tissue
measure the strength of the adhesive bond that develops
Scaffolds
between a cell and a surface as well as the force required to
2.2 ISO Standards:
detach cells that have adhered to a substrate. Controlling the
ISO 4287 Geometrical Product Specifications (GPS)—
interactions of mammalian cells with surfaces is fundamental
Surface Texture: Profile Method—Terms, Definitions and
tothedevelopmentofsafeandeffectivemedicalproducts.This
Surface Texture Parameters
guide does not cover methods for characterizing surfaces. The
ISO 13565-1 Geometrical Product Specifications (GPS)—
information generated by these methods can be used to obtain
Surface Texture: Profile Method; Surfaces Having Strati-
quantitative measures of the susceptibility of surfaces to cell
fied Functional Properties—Part 1: Filtering and General
attachment as well as measures of the adhesion of cells to a
Measurement Conditions
surface. This guide also highlights the importance of cell
culture history and influences of cell type.
3. Terminology
1.2 This standard does not purport to address all of the
3.1 Definitions:
safety concerns, if any, associated with its use. It is the
3.1.1 biocompatibility, n—material may be considered bio-
responsibility of the user of this standard to establish appro-
compatible if the materials perform with an appropriate host
priate safety, health, and environmental practices and deter-
response in a specific application. F2312
mine the applicability of regulatory limitations prior to use.
1.3 This international standard was developed in accor- 3.1.2 biomarker, n—biochemical feature or facet that can be
dance with internationally recognized principles on standard- used to measure the progress of disease or the effects of
ization established in the Decision on Principles for the
treatment.
Development of International Standards, Guides and Recom-
3.1.3 biomaterial, n—any substance (other than a drug),
mendations issued by the World Trade Organization Technical
synthetic or natural, that can be used as a system or part of a
Barriers to Trade (TBT) Committee.
system that treats, augments, or replaces any tissue, organ, or
function of the body. F2312
2. Referenced Documents
3.1.4 detachment, n—process whereby an adhered cell or
2.1 ASTM Standards:
group of cells is actively detached from a surface.
D4410 Terminology for Fluvial Sediment
3.1.5 hydrophilic, adj—having a strong affinity for water,
F22 Test Method for Hydrophobic Surface Films by the
wettable. F22
Water-Break Test
3.1.6 implant, n—in medicine, object, structure, or device
intended to reside within the body for diagnostic, prosthetic, or
other therapeutic purposes.
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
3.1.7 laminar flow, n—well-ordered, patterned flow of fluid
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
layers assumed to slide over one another. See Ref (1).
Current edition approved Oct. 1, 2019. Published November 2019. Originally
approved in 2007. Last previous edition approved in 2011 as F2664 – 11. DOI:
10.1520/F2664-19E01.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM 4th Floor, New York, NY 10036, http://www.ansi.org.
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to the list of references at the end of
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
F2664 − 19
3.1.8 lay, n—direction of the predominant surface pattern. the age of the cell, the cell type, and both the chemistry and
ISO 13565-1 morphology of the underlying surface and time. These ele-
ments need to be considered in developing a test protocol.
3.1.9 passage, n—transfer or transplantation of cells, with
or without dilution, from one culture vessel to another. It is
4.3 Devising robust methods for measuring the propensity
understood that any time cells are transferred from one vessel
of cells to attach to different substrates is further complicated
to another, a certain portion of the cells may be lost and,
sinceeithercelladhesionordetachmentcanbeassessed.These
therefore, dilution of cells, whether deliberate or not, may
processes are not always similar or complementary.
occur. This term is synonymous with the term subculture. See
4.4 Most studies of cell attachment focus on obtaining some
Ref (2).
measure of the time-dependent force required to detach, or
3.1.10 passage number, n—number of times the cells in the
de-adhere, cells that have already adhered to a surface (James
culture have been subcultured or passaged. In descriptions of
et al, 2005) (7). More recently investigators have begun to
thisprocess,theratioordilutionofthecellsshouldbestatedso
measure the adhesive forces that develop between cells and the
that the relative cultural age can be ascertained. See Ref (2).
underlying surface during attachment (Lukas and Dvorak,
2004) (5). From a practical point of view, it is much easier to
3.1.11 Reynolds number, n—dimensionless number express-
measure the force required to detach or de-adhere cells from a
ing the ratio of inertia forces to viscous forces in a moving
surface than to measure those that develop during attachment.
fluid. The number is given by VLr/m where V, is the fluid’s
However, in both cases, the experimental data should be
velocity, L is a characteristic length or distance such as pipe
interpreted with a degree of caution that depends on the
diameter, r is the fluid’s mass density, and m is the fluid’s
intended use of the measurements. The methods of measuring
dynamic viscosity. D4410
cell adhesion described herein are measures of the force
3.1.12 scaffold, n—support, delivery vehicle, or matrix for
required to detach an adherent cell.
facilitating the migration, binding, or transport of cells or
bioactive molecules used to replace, repair, or regenerate 4.5 The purpose of this guide is to provide an overview of
tissues. F2312 current generic test methods and identify the key factors that
influence the assessment of cell adhesion and detachment. It is
3.1.13 surface profile, n—surface profile formed by the
anticipated that this guide will form the basis for producing a
intersection of a real surface by a specified plane. It is
seriesofstandardsthatwilldescribethesetestmethodsinmore
customary to select a plane that lies perpendicular to the
detail.
direction of lay unless otherwise indicated. ISO 13565-1 and
ISO 4287
5. Cell Attachment Assays
3.2 Definitions of Terms Specific to This Standard:
5.1 Table 1 provides examples of common cell adhesion
3.2.1 adhesion—physiochemical state by which a cell is
assays, including a brief description of the forces applied.
coupled to a non-cell surface by interfacial forces, which may
These assays are discussed in more detail in Section 6.
consist of covalent or ionic forces.
5.2 Cell attachment assays can be performed using single
3.2.2 senescence, n—in vertebrate cell cultures, property
cells or a population of cells. Single cell techniques can
attributable to finite cell cultures; namely, their inability to
provide quantitative measures of the adhesive force that
grow beyond a finite number of population doublings. Neither
invertebrate nor plant cell cultures exhibit this property. This
term is synonymous with in vitro senescence. See Ref (2). TABLE 1 Assays for Measuring Cell Detachment from Surfaces
Cell Assay
3.2.3 shear stress, n—components of stress that act parallel
Assay Section
Requirements Description
to the plane of the surface. See Ref (3).
Single Cell Micromanipulation Measurement of 6.1.1-6.1.2
3.2.4 tack, n—ability of an adhesive to form a bond to a
the Force
developed during
surface after brief contact under light pressure.
attachment via an
AFM
4. Significance and Use
Single Cell Micromanipulation Forces applied via 6.1.3
a micropipette,
4.1 Cell attachment or, lack of it, to biomaterials is a critical
microprobe or AFM
factor affecting the performance of a device or implant. Cell
Cell Population Gravity Detect the number 6.2.1
attachment is a complicated, time-dependent, process involv- of cells that remain
attached after
ing significant morphological changes of the cell and deposi-
turning the culture
tion of a bed of extracellular matrix. Details of the adhesive
vessel upside down
Wash Wash off adhered 6.2.2
bond that is formed have been reviewed by, for example,
cells
Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), and
Centrifugation Detachment of 6.2.3
Garcia and Gallant (2003) (6). The strength of this coupling
cells using
centrifugal force
canbedeterminedeitherbymonitoringtheforceofattachment
Hydrodynamic Flow Detachment of 6.2.4
between a cell and a substrate over time or by measuring the
cells using shear
force required to detach the cell once it has adhered.
forces generated
by laminar flow
4.2 Cell adhesion to a surface depends on a range of
over cells
biological and physical factors that include the culture history,
´1
F2664 − 19
develops between a cell and a substrate with time or that may be torn apart during the measurement. In this case, the
required to detach an adhered cell from a substrate. Individual mechanical properties of the cell body would be measured
ligand-surface interactions can be measured directly using, for instead of the cell adhesion force to the test material. This can
example,acellmountedonanatomicforcemicroscope(AFM) be checked by examining the cell in the microscope after the
tip. Single cell measurements do have their disadvantages. adhesion measurement to make sure the cell looks healthy and
Variations in adhesive strength are not averaged out over a is intact.
population and sophisticated equipment, such as an AFM, is
6.1.2.3 These measurements need to be made using a wet
required. cell AFM. Problems have been reported with protein adsorp-
tion on the cantilever having an adverse effect on its reflectiv-
5.3 Cell population based assays average out variations in
ity.
cell-to-substrate adhesiveness compared with measurements
6.1.3 Micropipettes, microprobes, and AFMs have been
performed on a single cell. This variation arises both because
used to measure the force required to suck or pull single cells
ofvariationsinbiomaterialsurfaceproperties,andvariationsin
away from the substrate to which they are attached (for
cell phenotype used as the probe (Appendix X1 and Appendix
example, Shao et al, 2004) (8). All these methods provide
X2). Cell population techniques provide a usable measure of
quantifiablesensitiveandreal-timedirectmeasuresoftheforce
the biomaterial’s adhesiveness for a given batch of cells and
required to detach the cell that is typically less than 10 mN (for
test conditions. Cell population techniques are attractive in that
example, Lee et al, 2004) (9). Control over the magnitude of
they provide robust measurements based on a large number of
the force and the rate at which it is applied can be used to
cells, which is an important consideration given the inherent
explore the process of cell detachment in detail. Practical
variance of biological systems. Measurements that are based
issues that need to be considered when using these methods
on large numbers of cells reduce the influences of local
include:
variations in surface chemistry and texture and in the adhe-
6.1.3.1 Specialized equipment, which must be calibrated to
siveness of the cells themselves.
ensurethatdataarerepeatableandreproducible,isrequiredfor
such sensitive measurements.
6. Measurement of Cell Detachment
NOTE 1—In principle, the strength of the adhesive bond that develops 6.1.3.2 Consideration should be given as to the direction of
between the cell and underlying substrate will increase with time,
the applied force (tensile, shear, or some combination of the
although in practice this will depend on the cell-surface interactions.
two) and the magnitude of the applied stress. Larger area
These measurements can be performed on either populations of cells or
pipette tips will subject the cell to a lower stress than the tip of
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
destructive. Each test described below carries its own unique sources of
6.1.3.3 The period of time between exposing the cells to a
statistical error. Users should familiarize themselves with the appropriate
surface and that at which measurements are made.
assay system and should consult with appropriate statistical staff to
determine the necessary statistical parameters to ensure statistical signifi-
6.2 Cell Detachment Measurements on Cell Populations:
cance. These parameters may include, but are not limited to: sample size,
6.2.1 Gravity—Gravity can be used to differentiate between
power of study, number of image fields counted (for microscope-based
cells that are attached to a substrate and those that have not by
assays), number of cell lots tested, variability between users, what is the
turning the cell culture vessel upside down. Prior to using this
most appropriate statistical analysis (that is, analysis of variance, Tukeys
approach, the user should consider the buoyancy of the cells
test, t-test, etc.) and determination of a standard curve for analysis of
detached cells.
with respect to medium to ensure that it is negative. Consid-
eration should be given to the test duration to improve the
6.1 Micromanipulation:
consistency of repeat measurements.
6.1.1 Micromanipulation Methods (Single Cells)—Single
6.2.2 Wash Assays—A simple, convenient, widely used
cells can be used to measure the force required to uncouple
assay that readily provides qualitative information on adhesion
cellsfromtheunderlyingsubstrate(measureofdetachment),as
a result of a time-dependent adhesion. Such measurements are of cells to a substrate is to wash off non-adherent cells using
culture medium. This approach may take many forms from
made using micromanipulation or micropipettes. Cells can be
seeded onto a small block of material mounted on anAFM tip, mild shaking of the culture vessel to sluicing of the culture
well. Clearly the simplicity, speed and low cost of these
attached to a coated AFM tip or to the tip directly. The
cell-coated tip can then be used to measure the tack force that approaches are attractive, although lack of control of the
applied force in terms of both its magnitude and the nature of
develops over time.
6.1.2 There are some practical issues that need to be theappliedstresslimitsthesensitivityofthemeasurement,and
hence reproducibility. For this reason comparisons between
addressed when using this direct approach to force measure-
ment: successive tests are subject to large unquantifiable uncertain-
ties. Checks should also be made to ensure that the adherent
6.1.2.1 Care should be taken to ensure that the measure-
ments relate to a single cell and not to contributions from a surface is not removed or damaged during the assay.
number of cells. This is a particular issue when a block of 6.2.2.1 This assay can be used to monitor cell attachment to
material is mounted onto the tip. a surface under different culture conditions, used as a measure
6.1.2.2 Careshouldbetakentoensurethatthemeasurement of the biocompatibility or as a route to gauging how well cells
relates to the detachment force and is not a measure of cell are attached to a substrate. This approach is also a destructive
membrane strength. If the cell adheres very tightly to both the method, so measurements should only be made using samples
culture substrate and the block of test material, then the cell that have not been previously tested.This protocol will remove
´1
F2664 − 19
any contributions from residual extra-cellular matrix of frag- subjected to a wall shear stress, τ , that is, the shear stress at
w
ments of cell membrane that may impact on the adhesiveness the wall-fluid interface according to the following equation:
of the surface.
6µQ
τ 5 (2)
6.2.3 Centrifugation—A conventional centrifuge can be
w 2
wh
used to apply a normal or shear force to cells depending on the
where:
orientation of the cells with respect to the centrifugal force (for
example, Heneweer et al, 2005) (10). The force that the cells Q = flow rate of the fluid,
w = width (channel dimension),
are subject to can be calculated according to the following
h = height (channel dimension), and
formula:
µ = fluid viscosity.
F 5 VdRω (1)
(1) This function applies to Newtonian fluids, of which
where: water is an example and assumes no influences from edge
effects. Care should be taken to ensure that these requirements
F = centrifugal force,
are met for particular test geometries and culture media.
V = cell volume,
d = differenceindensitybetweenacellandthesurrounding (2) The key element of this approach is to ensure that the
fluid flow over the cells is laminar. The wall shear stress
medium,
R = centrifugation radius, and
applied to the cells can be constant or variable, depending on
ω = centrifugation speed.
the design of the flow cell. A controlled static shear stress
gradient can easily be generated by converging either one or
6.2.3.1 Such tests are easy to conduct, do not require
both pairs of parallel sides of the flow cell.
specialized equipment or training and the results represent a
(3) The wall shear stress at a given point along the length
population average. Factors that need to be considered when
of the cell is given by Eq 2.
using this methodology include the test duration and the
potential influence of forces applied during the period of spin
6.2.4.3 Spinning Disc—The spinning disk arrangement
up. The assay only correlates cell detachment with the maxi-
shown in Fig. 2 can be used to subject the cells to a centripetal
mum force applied after the centrifuge has reached its set spin
force and complex flow field that equates to a wall shear stress,
speed.
the magnitude of which increases with increasing distance
6.2.4 Hydrodynamic Flow Assays—The basis of hydrody-
away from the pole according to:
namic test methods is to apply a known force to a population
τ 5 0.8r =ρµω (3)
w
of cells by means of controlled movement of fluid. The assays
relyonforcesgeneratedbyfluidflowoveradheredcells.There
where:
are several subtypes of hydrodynamic flow assays: (1) parallel
ω = rotational speed,
plateflowchambers, (2)spinningdiskchambers,and (3)radial
ρ = density of the culture medium,
flow chambers. The geometry of the flow cell and mode of
r = radial position, and
operationinfluenceboththemagnitudeoftheappliedforceand
µ = fluid viscosity.
its complexity, as discussed below.
6.2.4.4 Radial Flow Cell—The wall stress in the radial flow
6.2.4.1 The stresses that the cells are subjected to are
cell shown in Fig. 3 is given by:
complex and difficult to quantify. Typically cells will be
3µQ
subjected to a combination of shear stress and hydrodynamic
τ 5 (4)
w 2
πrh
dragleadingtothedevelopmentoftorque.Thegeometryofthe
cell (that is, the amount of spreading and the presence of focal
where:
adhesions) will cause the actual stress that the cell experiences
Q = flow rate,
to be different from the calculated wall stress and therefore
r = radial position,
must be considered during any quantitative analysis.
h = gap between the plates, and
6.2.4.2 Parallel and Convergent Plate Flow Chambers—
µ = viscosity of the fluid.
Fig. 1 consists of parallel plates that are a known distance
(1) This function is the same as that for the parallel plate
apart. Flow of fluid through the chamber is laminar (Reynolds
cell shown in Eq 2. The highest wall shear stress in this
number is less than 2300). In this configuration the cells are
configuration will be in the vicinity of the entrance port. A
consequence of this will be that cells detached by the highest
wallshearstressmayinfluencedetachmentofcellsinthelower
wall stress zone. Unlike the parallel plate laminar flow
FIG. 1 A Simple Parallel-Sided Flow Cell Can Be Used to Apply a
Known Shear Stress to a Bed of Adhered Cells FIG. 2 A Schematic Representation of a Spinning Disk
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F2664 − 19
7. Additional Considerations for Cell Adhesion/
Detachment Assays
7.1 Measurement Objective—The major division of detach-
ment assays is between those which measure the adhesion of a
population of cells to a surface, with the measured parameter
usually being the number of cells left adhering to the surface
after some attempt at their removal (for example, Reyes C.D.
and Garcia A. J., 2003) (11), and those which measure the
adhesion of a single cell to a surface, with the measurement
usually being of the force required to remove the cell from the
FIG. 3 A Schematic Representation of a Radial Flow Cell
surface (for example, Huang W et al 2003) (12). The approach
selected depends upon the requirements of the investigator and
methodologies seem to arise with individual laboratories. All
of these assays require a measure of the number of cells that
chamber, cells tested in the radial flow chamber will be subject
remain after the procedure has been applied. Physical,
to a complex biaxial stress field.
chemical, enzymatic or temperature-dependent mechanisms
6.3 Additional Methods of Cell Detachment—Tissue Engi-
may be applied to remove cells remaining attached following a
neered Medical Products (TEMPs), where cells may be at-
celldetachmentassay.Thesecellscanthenbesubjecttofurther
tached to, or inside of, a three-dimensional scaffold may
histomorphological, biochemical, mechanical, phenotypic,
present unique requirements for cell detachment. Detachment genetic, etc. testing to verify how closely the detached cells
resulting from physical forces, where a mechanical force or
correspond to the original seeded cell type.
shear is the driving force, may result in cell injury or death.
7.1.1 Quantification of Attached Cells—Light or phase mi-
Likewise, if the investigator’s interest is in studying the
croscopy enhanced by colorimetric means, such as fluores-
extracellular matrix proteins, mechanical forces may interfere
cence with image analysis has been used for counting remain-
with ligand-receptor studies (such as in the case where the
ing attached cells. Image analysis software can quantitate
mechanical force is applied to break or form bonds between
changes in cell shape and other relevant morphological param-
fibronectin—integrin and tissue) and subsequently impact cell eters. Other methods include radioactive labelled cells and
function. In such cases, the use of cell detachment methods bioluminescent-based ATP assays. The user is referred to
AppendixX1foradditionalinformationonsourcesofvariation
which do not involve physical forces (that is, mechanical force
is the driving force for detachment), should be applied. A resulting from the cells in cell detachment assays.
non-exhaustive list of examples of these types of cell detach-
7.2 Modes of Detachment—The following is a partial list of
ment assays include: (1) chemical chelation (that is, ethylene-
modes of detachment that can occur. Cell tethering occurs
diaminetetraacetic acid, EDTA), (2) enzyme activity (that is,
when part of the cell membrane attaches to the biomaterial
trypsin), and (3) temperature gradients (that is, use of ice to
surface and under shearing conditions becomes pulled out into
dislodge adherent cells or shift temperature to release cells
a long membranous process or tether. Peeling refers to the
from bioengineered surfaces). Users should familiarize them-
breaking of ligand-receptor bonds per unit time for the ce
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