ASTM F2739-19
(Guide)Standard Guide for Quantifying Cell Viability and Related Attributes within Biomaterial Scaffolds
Standard Guide for Quantifying Cell Viability and Related Attributes within Biomaterial Scaffolds
SIGNIFICANCE AND USE
5.1 The number and distribution of viable and non-viable cells within, or on the surface of, a biomaterial scaffold is one of several important characteristics that may determine in vivo product performance of cell/biomaterial constructs (see 5.7); therefore, there is a need for standardized test methods to quantify cell viability.
5.2 There are a variety of static and dynamic methods to seed cells on scaffolds, each with different cell seeding efficiencies. In general, static methods such as direct pipetting of cells onto scaffold surfaces have been shown to have lower cell seeding efficiencies than dynamic methods that push cells into the scaffold interior. Dynamic methods include: injection of cells into the scaffold, cell seeding on biomaterials contained in spinner flasks or perfusion chambers, or seeding that is enhanced by the application of centrifugal forces. The methods described in this guide can assist in establishing cell seeding efficiencies as a function of seeding method and for standardizing viable cell numbers within a given methodology.
5.3 As described in Guide F2315, thick scaffolds or scaffolds highly loaded with cells lead to diffusion limitations during culture or implantation that can result in cell death in the center of the construct, leaving only an outer rim of viable cells. Spatial variations of viable cells such as this may be quantified using the tests within this guide. The effectiveness of the culturing method or bioreactor conditions on the viability of the cells throughout the scaffold can also be evaluated with the methods described in this guide.
5.4 These test methods can be used to quantify cells on non-porous or within porous hard or soft 3-D synthetic or natural-based biomaterials, such as ceramics, polymers, hydrogels, and decellularized extracellular matrices. The test methods also apply to cells seeded on porous coatings.
5.5 Test methods described in this guide may also be used to distinguish between prolifer...
SCOPE
1.1 This guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue-engineered medical products (TEMPs).
1.2 In addition to providing a compendium of available techniques, this guide describes materials-specific interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid or account for, or both, scaffold material/cell viability assay interactions.
1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs that are retrieved after implantation in living organisms.
1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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.
1.7 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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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: F2739 − 19
Standard Guide for
Quantifying Cell Viability and Related Attributes within
1
Biomaterial Scaffolds
This standard is issued under the fixed designation F2739; 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 2. Referenced Documents
2
2.1 ASTM Standards:
1.1 This guide is a resource of cell viability test methods
F748 PracticeforSelectingGenericBiologicalTestMethods
that can be used to assess the number and distribution of viable
for Materials and Devices
andnon-viablecellswithinporousandnon-porous,hardorsoft
F2149 Test Method for Automated Analyses of Cells—the
biomaterial scaffolds, such as those used in tissue-engineered
Electrical Sensing Zone Method of Enumerating and
medical products (TEMPs).
Sizing Single Cell Suspensions
1.2 In addition to providing a compendium of available
F2315 Guide for Immobilization or Encapsulation of Living
techniques, this guide describes materials-specific interactions
Cells or Tissue in Alginate Gels
with the cell assays that can interfere with accurate cell
F2998 Guide for Using Fluorescence Microscopy to Quan-
viability analysis, and includes guidance on how to avoid or
tify the Spread Area of Fixed Cells
account for, or both, scaffold material/cell viability assay
3
2.2 ICH Document:
interactions.
ICH Q2(R1) Validation of Analytical Procedures: Text and
1.3 These methods can be used for 3-D scaffolds containing Methodology
4
cells that have been cultured in vitro or for scaffold/cell
2.3 FDA Document:
constructs that are retrieved after implantation in living organ-
U.S. Food and Drug Administration (FDA) Center for
isms.
Biologics Evaluation and Research (CBER), 2015 Ana-
lytical Procedures and Methods Validation for Drugs and
1.4 This guide does not propose acceptance criteria based
Biologics—Guidance for Industry
on the application of cell viability test methods.
1.5 The values stated in SI units are to be regarded as
3. Terminology
standard. No other units of measurement are included in this
3.1 Definitions:
standard.
3.1.1 non-viable cell, n—a cell not meeting one or more of
1.6 This standard does not purport to address all of the
the criteria for a viable cell.
safety concerns, if any, associated with its use. It is the
3.1.2 viable cell, n—a cell capable of metabolic activity that
responsibility of the user of this standard to establish appro-
is structurally intact with a functioning cell membrane.
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
3.1.2.1 Discussion—The use of the term viable herein only
applies at the instant at which the measurement is conducted
1.7 This international standard was developed in accor-
and is not meant to indicate anything about the future state of
dance with internationally recognized principles on standard-
the cell.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
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.
1 3
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Available from International Conference on Harmonisation of Technical
Surgical Materials and Devices and is the direct responsibility of Subcommittee Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH
F04.43 on Cells and Tissue Engineered Constructs for TEMPs. Secretariat, 9, chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland,
Current edition approved Sept. 1, 2019. Published November 2019. Originally http://www.ich.org.
4
approved in 2008. Last previous edition approved in 2016 as F2739 – 16. DOI: Available from U.S. Food and Drug Administration (FDA), 10903 New
10.1520/F2739-19. Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2739 − 19
4. Summary of Guide the culturing method or bioreactor conditions on the viability
of the cells throughout the scaffold can also be evaluated with
4.1 It is the intent of
...
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: F2739 − 16 F2739 − 19
Standard Guide for
Quantifying Cell Viability and Related Attributes within
1
Biomaterial Scaffolds
This standard is issued under the fixed designation F2739; 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 guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and
non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue-engineered medical
products (TEMPs).
1.2 In addition to providing a compendium of available techniques, this guide describes materials-specific interactions with the
cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid, and/oravoid or account
for, or both, scaffold material/cell viability assay interactions.
1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs
that are retrieved after implantation in living organisms.
1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.7 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.
2. Referenced Documents
2
2.1 ASTM Standards:
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F2149 Test Method for Automated Analyses of Cells—the Electrical Sensing Zone Method of Enumerating and Sizing Single
Cell Suspensions
F2315 Guide for Immobilization or Encapsulation of Living Cells or Tissue in Alginate Gels
F2998 Guide for Using Fluorescence Microscopy to Quantify the Spread Area of Fixed Cells
3
2.2 ICH Document:
ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology
4
2.3 FDA Document:
U.S. Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER), 2015 Analytical Procedures
and Methods Validation for Drugs and Biologics—Guidance for Industry
3. Terminology
3.1 Definitions:
3.1.1 non-viable cell, n—a cell not meeting one or more of the criteria for a viable cell.
1
This guide 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 Oct. 1, 2016Sept. 1, 2019. Published November 2016November 2019. Originally approved in 2008. Last previous edition approved in 20082016
as F2739 – 08.16. DOI: 10.1520/F2739-16.10.1520/F2739-19.
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.
3
Available from International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH Secretariat, 9,
chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland, http://www.ich.org.
4
Available from U.S. Food and Drug Administration (FDA), 10903 New Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
F2739 − 19
3.1.2 viable cell, n—a cell capable of metabolic activity that is structurally intact with a functioning cell membrane.
3.1.2.1 Discussion—
The use of the term viable herein only applies at the instant at which the measurement is conducted and is not meant to indicate
anything
...
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