ASTM F3088-22
(Practice)Standard Practice for Use of a Centrifugation Method to Quantify/Study Cell-Material Adhesive Interactions
Standard Practice for Use of a Centrifugation Method to Quantify/Study Cell-Material Adhesive Interactions
SIGNIFICANCE AND USE
4.1 This practice describes a cell adhesion method that can be used to provide a detachment percent at a given RCF for cells that have adhered to a substrate, typically for a short time. The information generated by this practice can be used to obtain a semi-quantitative measurement of the adhesion of cells to either an uncoated or pre-coated substrate, when compared to a reference (adherent) cell type on the same substrate. As described in Reyes and Garcia (2003), it is recommended that the 50 % point be used for either ligand concentration or RCF for the most robust measurement of adhesion strength. The adhesion may vary due to changes in the phenotype of the cells or as a result of the specific properties of the surface. The substrate may include tissue culture-treated polystyrene, biomaterials, or bioactive surfaces. If the substrate is a hydrogel, care must be taken to avoid cohesive failure in the hydrogel (that is, detached cells have pulled away fragments of gel). The coating may consist of (but is not limited to) the following: natural or synthetic biomaterials, hydrogels, components of extracellular matrix (ECM), ligands, adhesion or bioactive molecules, genes, or gene products. Cell concentration is also critical, as use of too high a concentration of cells may result in cells detaching as a sheet, rather than as individual cells. This centrifugation approach, once validated, may be applicable for quality control (QC) and product development. However, until the method is correlated to other measures of cell attachment, the current method should be run in parallel with other known measures of cell adhesion.
4.2 This practice does not cover methods to quantitate changes in gene expression, or changes in biomarkers, as identified by immunostaining. This practice additionally does not cover quantitative image analysis techniques. In some cases, the change in adhesive properties may reflect on the degree of differentiation or de-differentiation of the cells...
SCOPE
1.1 This practice covers a centrifugation cell adhesion assay that can be used to detect changes in adhesive characteristics of cells with passage or treatments. This approach measures the force required to detach cells from a substrate. Adhesion, among many variables, may vary due to changes in the phenotype of the cells.
1.2 This practice does not cover methods to verify the uniformity of coating of surfaces, nor does it cover methods for characterizing surfaces.
1.3 The cells may include adult, progenitor, or stem cells from any species. The types of cells may include chondrocytes, fibroblasts, osteoblast, islet cells, or other relevant adherent cell types.
1.4 This practice does not cover methods for isolating or harvesting of cells. This practice does not cover methods to quantitate changes in gene expression, or changes in biomarker type or concentration, as identified by immunostaining. Nor does this practice cover quantitative image analysis techniques.
1.5 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.
1.6 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.
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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.
Designation: F3088 − 22
Standard Practice for
Use of a Centrifugation Method to Quantify/Study Cell-
1
Material Adhesive Interactions
This standard is issued under the fixed designation F3088; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2
1.1 Thispracticecoversacentrifugationcelladhesionassay 2.1 ASTM Standards:
thatcanbeusedtodetectchangesinadhesivecharacteristicsof F2603Guide for Interpreting Images of Polymeric Tissue
cells with passage or treatments. This approach measures the Scaffolds
force required to detach cells from a substrate. Adhesion, F2664Guide for Assessing the Attachment of Cells to
among many variables, may vary due to changes in the Biomaterial Surfaces by Physical Methods
phenotype of the cells. F2739Guide for Quantifying Cell Viability and Related
Attributes within Biomaterial Scaffolds
1.2 This practice does not cover methods to verify the
F2944Practice forAutomated Colony Forming Unit (CFU)
uniformityofcoatingofsurfaces,nordoesitcovermethodsfor
Assays—Image Acquisition and Analysis Method for
characterizing surfaces.
Enumerating and Characterizing Cells and Colonies in
1.3 The cells may include adult, progenitor, or stem cells
Culture
fromanyspecies.Thetypesofcellsmayincludechondrocytes,
F2998Guide for Using Fluorescence Microscopy to Quan-
fibroblasts,osteoblast,isletcells,orotherrelevantadherentcell
tify the Spread Area of Fixed Cells
types.
F3294Guide for Performing Quantitative Fluorescence In-
tensity Measurements in Cell-based Assays with Wide-
1.4 This practice does not cover methods for isolating or
harvesting of cells. This practice does not cover methods to field Epifluorescence Microscopy
quantitatechangesingeneexpression,orchangesinbiomarker
3. Summary of Practice
type or concentration, as identified by immunostaining. Nor
doesthispracticecoverquantitativeimageanalysistechniques. 3.1 Centrifugation Cell Assay on Cell Populations—Acon-
ventional centrifuge can be used to apply a normal or shear
1.5 This standard does not purport to address all of the
force to cells depending on the orientation of the cells with
safety concerns, if any, associated with its use. It is the
3
respect to the centrifugal force (1-3). The centrifugal force
responsibility of the user of this standard to establish appro-
that the cells are subject to can be calculated according to the
priate safety, health, and environmental practices and deter-
following formula assuming that the plates are 90° (normal) to
mine the applicability of regulatory limitations prior to use.
the spindle of the centrifuge.
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
F 5 ~p 2 p !V RCF (1)
D cell medium cell
ization established in the Decision on Principles for the
where:
Development of International Standards, Guides and Recom-
F = detachment force applied per cell,
D
mendations issued by the World Trade Organization Technical
V = cell volume,
cell
Barriers to Trade (TBT) Committee.
1 2
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
F04.43 on Cells and Tissue Engineered Constructs for TEMPs. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved April 1, 2022. Published May 2022. Originally the ASTM website.
3
approved in 2014. Last previous edition approved in 2014 as F3088–14. DOI: Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
10.1520/F3088-22. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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F3088 − 22
biomaterials, hydrogels, components of extracellular matrix
p = density of the cell,
cell
(ECM), ligands, adhesion or bioactive molecules, genes, or
p = the density of the surrounding medium, and
medium
2
gene products. Cell concentration is also critical, as use of too
RCF = Relative Centrifugal Forces = rῳ , where r =
centrifugationradiusandῳ=centrifugationspeed. high a concentration of cells may result in cells detaching as a
sheet, rather than as individual cells. This centrifugation
See X1.6 for example
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F3088 − 14 F3088 − 22
Standard Test Method Practice for
Use of a Centrifugation Method to Quantify/Study Cell-
1
Material Adhesive Interactions
This standard is issued under the fixed designation F3088; 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
1.1 This test method covers a centrifugation cell adhesion assay that can be used to detect changes in adhesive characteristics of
cells with passage or treatments. This approach measures the force required to detach cells from a substrate. Adhesion, among
many variables, may vary due to changes in the phenotype of the cells.
1.2 This test method does not cover methods to verify the uniformity of coating of surfaces, nor does it cover methods for
characterizing surfaces.
1.3 The cells may include adult, progenitor or stem cells from any species. The types of cells may include chondrocytes,
fibroblasts, osteoblast, islet cells, or other relevant adherent cell types.
1.4 This test method does not cover methods for isolating or harvesting of cells. This test method does not cover test methods to
quantitate changes in gene expression, or changes in biomarker type or concentration, as identified by immunostaining. Nor does
this test method cover quantitative image analysis techniques.
1.5 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.
2. Referenced Documents
2
2.1 ASTM Standards:
F2603 Guide for Interpreting Images of Polymeric Tissue Scaffolds
F2664 Guide for Assessing the Attachment of Cells to Biomaterial Surfaces by Physical Methods
F2739 Guide for Quantifying Cell Viability and Related Attributes within Biomaterial Scaffolds
F2944 Practice for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating
and Characterizing Cells and Colonies in Culture
3. Summary of Test Method
3.1 Centrifugation Cell Assay on Cell Populations—A conventional centrifuge can be used to apply a normal or shear force to cells
1
This test method practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of
Subcommittee F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved May 1, 2014April 1, 2022. Published June 2014May 2022. Originally approved in 2014. Last previous edition approved in 2014 as F3088 – 14.
DOI: 10.1520/F3088-14.10.1520/F3088-22.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or 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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F3088 − 22
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depending on the orientation of the cells with respect to the centrifugal force (1-3). The centrifugal force that the cells are subject
to can be calculated according to the following formula assuming that the plates are 90° (normal) to the spindle of the centrifuge.
F 5 p 2 p V RCF (1)
~ !
D cell medium cell
where:
F = detachment force applied per cell,
D
V = cell volume,
cell
p = density of the cell,
cell
p = the density of the surrounding medium, and
medium
2
RCF = Relative Centrifugal Forces = rῳ , where r = centrifugation radius and ῳ = centrifugation speed.
See X1.6 for example of a calculation of “detachment force per cell.”
3.1.1 Such tests are easy to conduct and the results represent a population average. The method can be performed in a moderately
outfitted cell biology lab with an inverted microscope or a fluorescence microplate reader and a centrifuge with a microtiter plate
holder. Three factors need to be considered when using this methodology: the potential influences of forces applied during the
period of spin up time, the maximum spin speed, and the test duration. The method is only able to correlate with cell detachment
with the maximum force applied after the centrifuge has reached its set spin speed. It should also be consi
...
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