ASTM F2739-16
(Guide)Standard Guide for Quantifying Cell Viability within Biomaterial Scaffolds
Standard Guide for Quantifying Cell Viability 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 hard or soft 3-D biomaterials, such as ceramics and polymer gels. 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 proliferating and non-proliferating viable cells. Proliferating cells proceed through the DNA synthesis (S) pha...
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/or account for, 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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: F2739 − 16
Standard Guide for
1
Quantifying Cell Viability within 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 Electrical Sensing Zone Method of Enumerating and
Sizing Single Cell Suspensions
1.1 This guide is a resource of cell viability test methods
F2315 Guide for Immobilization or Encapsulation of Living
that can be used to assess the number and distribution of viable
Cells or Tissue in Alginate Gels
andnon-viablecellswithinporousandnon-porous,hardorsoft
F2998 Guide for Using Fluorescence Microscopy to Quan-
biomaterial scaffolds, such as those used in tissue-engineered
tify the Spread Area of Fixed Cells
medical products (TEMPs).
1.2 In addition to providing a compendium of available
3. Terminology
techniques, this guide describes materials-specific interactions
3.1 Definitions:
with the cell assays that can interfere with accurate cell
3.1.1 non-viable cell, n—a cell not meeting one or more of
viability analysis, and includes guidance on how to avoid,
the criteria for a viable cell.
and/or account for, scaffold material/cell viability assay inter-
actions.
3.1.2 viable cell, n—a cell capable of metabolic activity that
is structurally intact with a functioning cell membrane.
1.3 These methods can be used for 3-D scaffolds containing
cells that have been cultured in vitro or for scaffold/cell
4. Summary of Guide
constructs that are retrieved after implantation in living organ-
isms.
4.1 It is the intent of this guide to provide a compendium of
1.4 This guide does not propose acceptance criteria based the commonly used methods for quantifying the number and
on the application of cell viability test methods. distribution of viable and non-viable cells within, or on, a
biomaterial scaffold, because cell viability is an important
1.5 The values stated in SI units are to be regarded as
parameter of tissue-engineered products used to regenerate or
standard. No other units of measurement are included in this
repair lost or diseased tissue. The methods can be applied to
standard.
cells residing within an intact 3-D scaffold or matrix (that is,
1.6 This standard does not purport to address all of the
non-destructive methods) or to cells that have been removed
safety concerns, if any, associated with its use. It is the
from the scaffold or matrix (that is, destructive methods). It
responsibility of the user of this standard to establish appro-
should be noted that not all cells require a scaffold and some
priate safety and health practices and determine the applica-
cell types, such as hematopoietic cells, cannot be cultured or
bility of regulatory limitations prior to use.
grown on an adherent surface.
4.2 Most of the methods originate from analysis of cell
2. Referenced Documents
2 numberon2-Dsurfaces,buthavebeenadaptedfortheanalysis
2.1 ASTM Standards:
of cells within 3-D constructs that are typically used in
F748 PracticeforSelectingGenericBiologicalTestMethods
regenerative medicine approaches. The mechanisms and the
for Materials and Devices
sensitivity of the assays are discussed. The limitations of the
F2149 Test Method for Automated Analyses of Cells—the
assays due to using standard curves generated from cells on
2-D surfaces are described in this document. In addition, the
ways in which the biomaterial scaffold itself can affect the
1
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
viability assays are described.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
4.3 This guide describes test methods which, when used
Current edition approved Oct. 1, 2016. Published November 2016. Originally
together, may enable accurate measure of the number and
approved in 2008. Last previous edition approved in 2008 as F2739 – 08. DOI:
10.1520/F2739-16.
distribution of viable and non-viable cells. Different viability
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
assays have different measurands, which means that the results
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
from different assays may not correlate with one another. For
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. instance, cell membrane integrity tests and cell metabolic tests
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
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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 − 08 F2739 − 16
Standard Guide for
QuantitatingQuantifying Cell Viability Withinwithin
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
tissue-engineered medical products (TEMPs).
1.2 In addition to providing a compendium of available techniques, this guide describes materials specific materials-specific
interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid,
and/or account for, 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 and health practices and determine the applicability of regulatory
limitations prior to use.
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. Terminology
3.1 Definitions:
3.1.1 non-viable cell, n—a cell not meeting one or more of the criteria for viability given in a viable cell.3.1.2.
3.1.2 viable cell, n—a cell capable of metabolic activity that is structurally intact with a functioning cell membrane.
4. Summary of Guide
4.1 It is the intent of this guide to provide a compendium of the commonly used methods for quantitatingquantifying the number
and distribution of viable and non-viable cells within, or on, a biomaterial scaffold, because cell viability is an important parameter
of tissue engineering tissue-engineered products used to regenerate or repair lost or diseased tissue. The methods can be applied
to cells residing within an intact 3-D scaffold or matrix (that is, non-destructive methods) or to cells that have been removed from
the scaffold or matrix (that is, destructive methods). It should be noted that not all cells require a scaffold and some cell types, such
as hematopoietic cells, cannot be cultured or grown on an adherent surface.
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 Aug. 1, 2008Oct. 1, 2016. Published September 2008November 2016. Originally approved in 2008. Last previous edition approved in 2008 as
F2739 – 08. DOI: 10.1520/F2739-08.10.1520/F2739-16.
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’sstandard’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 ----------------------
F2739 − 16
4.2 Most of the methods originate from analysis of cell number on 2-D surfaces, but have been adapted for the analysis of cells
within 3-D constructs that are typically used in regenerative medicine approaches. The mechanisms and the sensitivity of the assays
are discussed. The limitations of the assays due to using standard curves generated from cells on 2-D surfaces are de
...
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