ASTM F3354-19
(Guide)Standard Guide for Evaluating Extracellular Matrix Decellularization Processes
Standard Guide for Evaluating Extracellular Matrix Decellularization Processes
SIGNIFICANCE AND USE
4.1 Decellularization is used in the preparation of medical products that make use of the native structure and/or composition of the extracellular matrix derived from a specific tissue source. Upon implantation or placement, the decellullarized product is commonly intended to undergo and/or induce constructive remodeling and incorporation into the native host tissue instead of being recognized as foreign material. Typically, immune system recognition of foreign material leads to encapsulation of the material and an aggressive inflammatory response, causing the ultimate rejection or other failure of the product.
4.2 As described above, decellularization is a recognized technique which allows the use of ECM-derived products in medical treatments with a reduced risk of an adverse host immune response and immune rejection by disrupting and removing cells and/or cell contents while aiming to preserve significant features of the ECM structure and/or composition. More complete decellularization is often associated with a beneficial response (1, 2)6 but can also be associated with the loss of important ECM components and the loss of structural or biomechanical integrity from the tissue during the decellularization process (3, 4, 5, 6). Therefore, given the typical objective of producing a product that does not elicit an adverse immune response while maintaining the integrity of the tissue for its intended surgical application, this guide presents a standard approach to the evaluation of decellularization processes, including assessment of adequate decellularization to achieve this end.
4.3 An ideal decellularization process would completely remove source tissue cells and associated cellular content from a tissue or organ, while minimizing unwanted effects on the remaining ECM. However, a more widely encountered and practical representation of an optimized decellularization process exhibits partial removal and/or disruption of resident cells and cellular material to le...
SCOPE
1.1 This document provides guidance on the characterization and evaluation of the decellularization processes used to produce decellularized extracellular matrix (dECM) materials which will be used as medical products in direct or indirect contact with the body. The decellularization process may be performed on tissue from human or other mammalian sources or produced in vitro from human or other mammalian cells. The dECM may or may not be recellularized prior to use. Decellularized ECM material derived from non-mammalian tissue or cells and decellularized ECM material used for non-medical purposes may follow the framework provided but may require additional considerations outside the scope of this document.
1.2 Biological tissues are composed of a structural extracellular matrix (ECM) and embedded cells. The intent of a decellularization process is to disrupt and/or remove cells and cellular components from an ECM material while maintaining key structural and/or compositional properties of the material. Decellularization comprises process steps intended or expected to result or aid in the disruption of source tissue cells and/or removal of cellular content from the material undergoing decellularization. Actions that are intended to rinse or otherwise remove decellularization reagents or by-products should also be considered in that context as part of the decellularization process. Purifications or other isolations of specific ECM components are not considered decellularization and are outside the scope of this document.
1.3 This document describes relevant parameters of decellularization processes used to prepare extracellular matrix materials as medical products.
1.4 This document provides guidance on the measurement of specific and general properties of dECM. This includes both the analysis of cellular material as well as the assessment of the effects of decellularization on dECM properties such as composition...
General Information
- Status
- Published
- Publication Date
- 14-Sep-2019
- Technical Committee
- F04 - Medical and Surgical Materials and Devices
- Drafting Committee
- F04.42 - Biomaterials and Biomolecules for TEMPs
Relations
- Effective Date
- 01-Oct-2019
- Effective Date
- 15-Nov-2016
- Effective Date
- 01-Jul-2015
- Effective Date
- 01-Oct-2013
- Effective Date
- 01-Jun-2011
- Effective Date
- 01-Apr-2011
- Effective Date
- 01-Mar-2011
- Effective Date
- 01-Aug-2009
- Effective Date
- 01-Feb-2008
- Effective Date
- 01-Feb-2008
- Effective Date
- 01-Dec-2007
- Effective Date
- 01-Jul-2007
- Effective Date
- 01-Feb-2007
- Effective Date
- 01-Feb-2007
- Effective Date
- 10-Nov-2002
Overview
ASTM F3354-19 – Standard Guide for Evaluating Extracellular Matrix Decellularization Processes provides a comprehensive framework for characterizing and assessing the decellularization procedures used to create decellularized extracellular matrix (dECM) materials. Developed by ASTM International, this guide supports producers and researchers preparing medical products employing dECM for direct or indirect body contact. The standard specifically focuses on preserving key structural and compositional ECM attributes while maximizing cellular content removal to reduce immune responses and enhance integration with host tissues.
Decellularization is vital in regenerative medicine and tissue engineering to obtain biocompatible scaffolds from human or animal tissues. This guide outlines best practices for evaluating the effectiveness and consistency of decellularization methods to help ensure product safety and functional performance.
Key Topics
Purpose of Decellularization
The decellularization process aims to remove all cellular components from tissue while retaining the essential extracellular matrix structure and composition. This minimizes the risk of immune rejection and adverse responses during implantation.Process Evaluation Parameters
The standard recommends evaluating both the efficiency of cell-removal and the preservation of ECM by:- Measuring residual DNA, cellular debris, and membrane components
- Assessing effects on critical ECM constituents like collagen and glycosaminoglycans
- Analyzing residual reagents, and potential damage to physical or biomechanical properties of dECM
Acceptance Criteria
ASTM F3354-19 emphasizes the importance of defining project-specific acceptance criteria for decellularization, considering:- Maximum permissible levels of cellular remnants
- Limits for the retention of ECM components
- Controls on the persistence of decellularization reagents
- Consistency and capability of process metrics
Testing Recommendations
The guide outlines testing strategies for both cellularity (e.g., DNA quantification, nuclear localization) and ECM integrity (e.g., collagen content, glycosaminoglycan analysis, structural visualization).Process Documentation
Comprehensive documentation and risk assessment are advised for any changes to source materials, reagents, or key process parameters.
Applications
The guidance in ASTM F3354-19 is particularly applicable to:
Medical Device Manufacturing:
Ensuring the safety, biocompatibility, and functional integrity of tissue-engineered medical products such as scaffolds for tissue regeneration, wound healing implants, and bioprosthetics.Quality Control and Process Validation:
Supporting manufacturers in establishing robust quality assurance programs for dECM materials, including validation of analytical methods and process controls.Research and Development:
Informing protocol development for novel decellularization processes, and aiding in the refinement of existing workflows for clinical and laboratory use.Regulatory Compliance:
Providing a harmonized approach aligned with international principles, aiding compliance with regulations concerning the use of animal- or human-derived tissues in medical devices.
Related Standards
Organizations working with decellularized ECM should also reference complementary standards and guidance documents, such as:
- ASTM F2150: Guide for Characterization and Testing of Biomaterial Scaffolds Used in Tissue-Engineered Medical Products
- ASTM F2212: Guide for Characterization of Type I Collagen as Starting Material for Surgical Implants
- ISO 10993-1: Biological evaluation of medical devices – Part 1: Evaluation and testing within a risk management process
- ISO 22442 series: Medical devices utilizing animal tissues and their derivatives
- USP <1285> & <1285.1>: Procedures for preparation and histological analysis of biological specimens
Practical Value
Implementing ASTM F3354-19 in the preparation of decellularized ECM materials helps ensure:
- Rigorous evaluation of removal of immunogenic cellular remnants
- Reliable preservation of extracellular matrix essential for tissue repair and regeneration
- Improved consistency across production batches
- Better alignment with regulatory and clinical requirements for tissue-derived medical products
This standard is an essential tool for those seeking to maximize both safety and functional performance in the rapidly evolving fields of tissue engineering, regenerative medicine, and medical device manufacturing.
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Frequently Asked Questions
ASTM F3354-19 is a guide published by ASTM International. Its full title is "Standard Guide for Evaluating Extracellular Matrix Decellularization Processes". This standard covers: SIGNIFICANCE AND USE 4.1 Decellularization is used in the preparation of medical products that make use of the native structure and/or composition of the extracellular matrix derived from a specific tissue source. Upon implantation or placement, the decellullarized product is commonly intended to undergo and/or induce constructive remodeling and incorporation into the native host tissue instead of being recognized as foreign material. Typically, immune system recognition of foreign material leads to encapsulation of the material and an aggressive inflammatory response, causing the ultimate rejection or other failure of the product. 4.2 As described above, decellularization is a recognized technique which allows the use of ECM-derived products in medical treatments with a reduced risk of an adverse host immune response and immune rejection by disrupting and removing cells and/or cell contents while aiming to preserve significant features of the ECM structure and/or composition. More complete decellularization is often associated with a beneficial response (1, 2)6 but can also be associated with the loss of important ECM components and the loss of structural or biomechanical integrity from the tissue during the decellularization process (3, 4, 5, 6). Therefore, given the typical objective of producing a product that does not elicit an adverse immune response while maintaining the integrity of the tissue for its intended surgical application, this guide presents a standard approach to the evaluation of decellularization processes, including assessment of adequate decellularization to achieve this end. 4.3 An ideal decellularization process would completely remove source tissue cells and associated cellular content from a tissue or organ, while minimizing unwanted effects on the remaining ECM. However, a more widely encountered and practical representation of an optimized decellularization process exhibits partial removal and/or disruption of resident cells and cellular material to le... SCOPE 1.1 This document provides guidance on the characterization and evaluation of the decellularization processes used to produce decellularized extracellular matrix (dECM) materials which will be used as medical products in direct or indirect contact with the body. The decellularization process may be performed on tissue from human or other mammalian sources or produced in vitro from human or other mammalian cells. The dECM may or may not be recellularized prior to use. Decellularized ECM material derived from non-mammalian tissue or cells and decellularized ECM material used for non-medical purposes may follow the framework provided but may require additional considerations outside the scope of this document. 1.2 Biological tissues are composed of a structural extracellular matrix (ECM) and embedded cells. The intent of a decellularization process is to disrupt and/or remove cells and cellular components from an ECM material while maintaining key structural and/or compositional properties of the material. Decellularization comprises process steps intended or expected to result or aid in the disruption of source tissue cells and/or removal of cellular content from the material undergoing decellularization. Actions that are intended to rinse or otherwise remove decellularization reagents or by-products should also be considered in that context as part of the decellularization process. Purifications or other isolations of specific ECM components are not considered decellularization and are outside the scope of this document. 1.3 This document describes relevant parameters of decellularization processes used to prepare extracellular matrix materials as medical products. 1.4 This document provides guidance on the measurement of specific and general properties of dECM. This includes both the analysis of cellular material as well as the assessment of the effects of decellularization on dECM properties such as composition...
SIGNIFICANCE AND USE 4.1 Decellularization is used in the preparation of medical products that make use of the native structure and/or composition of the extracellular matrix derived from a specific tissue source. Upon implantation or placement, the decellullarized product is commonly intended to undergo and/or induce constructive remodeling and incorporation into the native host tissue instead of being recognized as foreign material. Typically, immune system recognition of foreign material leads to encapsulation of the material and an aggressive inflammatory response, causing the ultimate rejection or other failure of the product. 4.2 As described above, decellularization is a recognized technique which allows the use of ECM-derived products in medical treatments with a reduced risk of an adverse host immune response and immune rejection by disrupting and removing cells and/or cell contents while aiming to preserve significant features of the ECM structure and/or composition. More complete decellularization is often associated with a beneficial response (1, 2)6 but can also be associated with the loss of important ECM components and the loss of structural or biomechanical integrity from the tissue during the decellularization process (3, 4, 5, 6). Therefore, given the typical objective of producing a product that does not elicit an adverse immune response while maintaining the integrity of the tissue for its intended surgical application, this guide presents a standard approach to the evaluation of decellularization processes, including assessment of adequate decellularization to achieve this end. 4.3 An ideal decellularization process would completely remove source tissue cells and associated cellular content from a tissue or organ, while minimizing unwanted effects on the remaining ECM. However, a more widely encountered and practical representation of an optimized decellularization process exhibits partial removal and/or disruption of resident cells and cellular material to le... SCOPE 1.1 This document provides guidance on the characterization and evaluation of the decellularization processes used to produce decellularized extracellular matrix (dECM) materials which will be used as medical products in direct or indirect contact with the body. The decellularization process may be performed on tissue from human or other mammalian sources or produced in vitro from human or other mammalian cells. The dECM may or may not be recellularized prior to use. Decellularized ECM material derived from non-mammalian tissue or cells and decellularized ECM material used for non-medical purposes may follow the framework provided but may require additional considerations outside the scope of this document. 1.2 Biological tissues are composed of a structural extracellular matrix (ECM) and embedded cells. The intent of a decellularization process is to disrupt and/or remove cells and cellular components from an ECM material while maintaining key structural and/or compositional properties of the material. Decellularization comprises process steps intended or expected to result or aid in the disruption of source tissue cells and/or removal of cellular content from the material undergoing decellularization. Actions that are intended to rinse or otherwise remove decellularization reagents or by-products should also be considered in that context as part of the decellularization process. Purifications or other isolations of specific ECM components are not considered decellularization and are outside the scope of this document. 1.3 This document describes relevant parameters of decellularization processes used to prepare extracellular matrix materials as medical products. 1.4 This document provides guidance on the measurement of specific and general properties of dECM. This includes both the analysis of cellular material as well as the assessment of the effects of decellularization on dECM properties such as composition...
ASTM F3354-19 is classified under the following ICS (International Classification for Standards) categories: 11.020.20 - Medical science. The ICS classification helps identify the subject area and facilitates finding related standards.
ASTM F3354-19 has the following relationships with other standards: It is inter standard links to ASTM F2150-19, ASTM F3142-16, ASTM D6797-15, ASTM F2150-13, ASTM D6797-07(2011), ASTM F2212-11, ASTM F2903-11, ASTM F2212-09, ASTM F2212-08, ASTM F2212-08e1, ASTM F2150-07, ASTM D6797-07, ASTM F2212-02(2007)e1, ASTM F2212-02(2007), ASTM F2212-02. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.
ASTM F3354-19 is available in PDF format for immediate download after purchase. The document can be added to your cart and obtained through the secure checkout process. Digital delivery ensures instant access to the complete standard document.
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: F3354 − 19
Standard Guide for
Evaluating Extracellular Matrix Decellularization Processes
This standard is issued under the fixed designation F3354; 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 1.5 This document does not provide guidance on the assess-
ment of the host response subsequent to the implantation or
1.1 This document provides guidance on the characteriza-
other in vivo placement of dECM medical products. Such
tion and evaluation of the decellularization processes used to
assessments should instead be conducted as part of biocom-
produce decellularized extracellular matrix (dECM) materials
patibility studies or other safety and efficacy studies. At a
which will be used as medical products in direct or indirect
minimum it is recommended that the finished product com-
contact with the body. The decellularization process may be
posed of dECM material shall be assessed in a relevant model
performed on tissue from human or other mammalian sources
that represents the biological responses that the product is
or produced in vitro from human or other mammalian cells.
expected to experience to ensure that the final material is
The dECM may or may not be recellularized prior to use.
functioning in accordance with design intentions. An in vivo
Decellularized ECM material derived from non-mammalian
model will generally be used, but cellular or ex vivo models
tissue or cells and decellularized ECM material used for
may also be satisfactory when appropriate.
non-medical purposes may follow the framework provided but
may require additional considerations outside the scope of this
1.6 This document provides guidance on determining perti-
document.
nent quality attributes as well as developing and assessing
1.2 Biological tissues are composed of a structural extracel- acceptancecriteriarelatedtoensuringtheconsistentevaluation
lular matrix (ECM) and embedded cells. The intent of a and use of decellularization in manufacturing medical prod-
decellularization process is to disrupt and/or remove cells and ucts. Acceptance criteria should address the adequacy of
cellular components from an ECM material while maintaining
cellular disruption and removal of cellular remnants. Accep-
key structural and/or compositional properties of the material.
tance criteria should define acceptable levels for retention of
Decellularizationcomprisesprocessstepsintendedorexpected
extracellular matrix components. Acceptance criteria may
to result or aid in the disruption of source tissue cells and/or
place limits on damage to retained components. Acceptance
removal of cellular content from the material undergoing
criteria should place limits on the persistence of decellulariza-
decellularization. Actions that are intended to rinse or other-
tion reagents. This document also provides recommendations
wise remove decellularization reagents or by-products should
on developing process parameters and associated process
also be considered in that context as part of the decellulariza-
controls.
tion process. Purifications or other isolations of specific ECM
1.6.1 This guide recommends attributes as representative
components are not considered decellularization and are out-
measures of decellularization in the direct function of remov-
side the scope of this document.
ingcellsandcellcomponents.Theseattributescanalsobeused
1.3 This document describes relevant parameters of decel- to show process consistency, capability, or equivalency. Rec-
lularization processes used to prepare extracellular matrix
ommendation of these attributes does not confer additional
materials as medical products.
significance related to product safety and performance.
1.6.2 No consensus has been established regarding decellu-
1.4 This document provides guidance on the measurement
larization thresholds or classifications. This guide therefore
of specific and general properties of dECM.This includes both
cannot suggest acceptance criteria and instead recommends
theanalysisofcellularmaterialaswellastheassessmentofthe
commonly measured attributes to develop acceptance criteria
effects of decellularization on dECM properties such as
specific to the design of each unique material and its intended
composition, structure, and material properties.
use.
1.7 Decellularized products will require evidence of safety
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
and/or efficacy beyond that related to evaluating the decellu-
Surgical Materials and Devices and is the direct responsibility of Subcommittee
larization process. Commonly referenced standards include the
F04.42 on Biomaterials and Biomolecules for TEMPs.
ISO 10993 series (see ISO 10993-1) for biocompatibility of
Current edition approved Sept. 15, 2019. Published October 2019. DOI:
10.1520/F3354-19. medical devices and the ISO 22442 series for medical devices
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3354 − 19
utilizing animal tissues and their derivatives. These assess- 2.3 Harmonized Guidance Documents:
ments are not in the scope of this document, though they may GHTF Study Group 3 – Quality Management Systems,
help to identify relevant functional characteristics and test Process Validation Guidance– January 2004 (GHTF/SG3/
methods as discussed in 5.2.9. N99-10:2004 (Edition 2))
ICHQ2(R1) –ValidationofAnalyticalProcedures:Textand
1.8 This standard does not purport to address all of the
Methodology, Step 4
safety concerns, if any, associated with its use. It is the
2.4 USP Documents:
responsibility of the user of this standard to establish appro-
USP <1285> Preparation of Biological Specimens for
priate safety, health, and environmental practices and deter-
Histologic and Immunohistochemical Analysis
mine the applicability of regulatory limitations prior to use.
USP <1285.1> Hematoxylin and Eosin Staining of Sec-
1.9 This international standard was developed in accor-
tioned Tissue for Microscopic Examination
dance with internationally recognized principles on standard-
USP Reference Standard Bovine Acellular Dermal Matrix
ization established in the Decision on Principles for the
Reference Photomicrographs, Catalog # 1535824
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical 3. Terminology
Barriers to Trade (TBT) Committee.
3.1 Definitions of Terms Specific to This Standard:
3.1.1 decellularization, n—a process used to disrupt and/or
2. Referenced Documents
remove cells and cellular components from a biological
material, while maintaining key structural and/or composi-
2.1 ASTM Standards:
tional properties of the extracellular matrix material.
D6797 Test Method for Bursting Strength of Fabrics
Constant-Rate-of-Extension (CRE) Ball Burst Test
3.1.2 decellularized, adj—referringtoamaterial,previously
F2150 Guide for Characterization and Testing of Biomate-
containing cells, which has been subjected to decellularization.
rial Scaffolds Used in Tissue-Engineered Medical Prod-
3.1.3 detergent, n—an amphiphilic compound, containing
ucts
both a hydrophobic group and hydrophilic group, which
F2212 Guide for Characterization of Type I Collagen as
enables the solubilization of hydrophobic, hydrophilic, and
Starting Material for Surgical Implants and Substrates for
amphiphilic materials such as the lipid bilayers of cellular
Tissue Engineered Medical Products (TEMPs)
membranes. Detergents are surfactants and therefore reduce
F2903 Guide for Tissue Engineered Medical Products
surface tension in aqueous solutions.
(TEMPs) for Reinforcement of Tendon and Ligament
3.1.4 enzyme linked immunosorbent assay (ELISA), n—a
Surgical Repair
group of procedures for quantification of specific molecular
F3142 Guide for Evaluation of in vitro Release of Biomol-
antigens in a (biological) sample. ELISAmethods use antibod-
ecules from Biomaterials Scaffolds for TEMPs
ies that bind specifically to the molecule of interest. ELISA
2.2 ISO Standards:
method variations include Direct, Indirect, Sandwich, and
ISO 5840-1 Cardiovascular implants -- Cardiac valve pros-
Competitive.
theses -- Part 1: General requirements
3.1.5 immunohistochemistry (IHC), n and immunofluores-
ISO 7198 Cardiovascular implants and extracorporeal sys-
cence (IF), n—the process of detection and visualization of
tems — Vascular prostheses — Tubular vascular grafts
specific antigens/components within a tissue section (IHC) or
and vascular patches
other sample using labeled antibodies. Antibodies are labeled
ISO 10993-1 Biological evaluation of medical devices --
withchromogenicorfluorescent(IF)markersforvisualization.
Part 1: Evaluation and testing within a risk management
3.1.6 intended use, n—also, intended purpose. Use for
process
whichaproduct,processorserviceisintendedaccordingtothe
ISO/TR 10993-33 Biological evaluation of medical devices
specifications, instructions and information provided by the
— Part 33: Guidance on tests to evaluate genotoxicity —
manufacturer (ISO 14971).
Supplement to ISO 10993-3
ISO 14971 Medical devices – Application of risk manage- 3.1.7 recellularization, n—the introduction of viable cells
ment to medical devices
onto or into a decellularized material either ex vivo (in culture)
ISO 22442-1 Medical devices utilizing animal tissues and
or in vivo.
theirderivatives--Part1:Applicationofriskmanagement
4. Significance and Use
ISO 22442-2 Medical devices utilizing animal tissues and
theirderivatives--Part2:Controlsonsourcing,collection
4.1 Decellularization is used in the preparation of medical
and handling
products that make use of the native structure and/or compo-
sition of the extracellular matrix derived from a specific tissue
For referenced ASTM standards, visit the ASTM website, www.astm.org, or To obtain the referenced GHTF Study Group 3 guidance, visit the website of
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM the International Medical Device Regulators Forum, www.imdrf.org. To obtain the
Standards volume information, refer to the standard’s Document Summary page on referenced ICH guidance, visit the website of the International Council for
the ASTM website. Harmonisation (ICH), www.ich.org.
3 5
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St., Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
4th Floor, New York, NY 10036, http://www.ansi.org. Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
F3354 − 19
source. Upon implantation or placement, the decellullarized analyzed within the context of the complete process sequence
product is commonly intended to undergo and/or induce and its action upon the type of tissue. For example, a process
constructive remodeling and incorporation into the native host developedfordermiswilllikelynottranslatedirectlytoaheart
tissue instead of being recognized as foreign material. valve and the doubling of process time will affect each process
Typically,immunesystemrecognitionofforeignmaterialleads differently, so the decellularization process will have to be
to encapsulation of the material and an aggressive inflamma- adjusted to account for the difference in tissue properties and
tory response, causing the ultimate rejection or other failure of desired attributes at the conclusion of the process. Within the
the product. context of this guide, analysis of a processing step should not
suggest material testing. Analysis is meant to demonstrate an
4.2 As described above, decellularization is a recognized
understanding of the relevant mechanisms of decellularization
technique which allows the use of ECM-derived products in
and the relevant mechanisms of adverse effects on the ECM
medical treatments with a reduced risk of an adverse host
material.
immune response and immune rejection by disrupting and
removing cells and/or cell contents while aiming to preserve 4.6 Decellularization acceptance criteria and ECM integrity
acceptance criteria should be developed based on the intended
significant features of the ECM structure and/or composition.
More complete decellularization is often associated with a use of the dECM material. This guide suggests some consid-
beneficial response (1, 2) but can also be associated with the erations that should be used to develop and justify acceptance
lossofimportantECMcomponentsandthelossofstructuralor criteria.
biomechanical integrity from the tissue during the decellular- 4.6.1 Decellularization acceptance criteria already estab-
ization process (3, 4, 5, 6). Therefore, given the typical lished for a source ECM and decellularization process allow
objective of producing a product that does not elicit an adverse for controlled changes to the decellularization process. Signifi-
immune response while maintaining the integrity of the tissue cant changes include changes to the processing mechanisms,
for its intended surgical application, this guide presents a reagents/materials,reagentconcentrations,andcontrolsaswell
standard approach to the evaluation of decellularization as changes in source ECM materials. Prior to any significant
processes, including assessment of adequate decellularization change to a decellularization process, a decellularization pro-
to achieve this end. cess analysis should be conducted on the process steps which
are subject to change. In addition, testing against the estab-
4.3 An ideal decellularization process would completely
lished decellularization acceptance criteria should be con-
remove source tissue cells and associated cellular content from
ducted on dECM material produced with the proposed process
a tissue or organ, while minimizing unwanted effects on the
changes. A risk management process may then be utilized to
remaining ECM. However, a more widely encountered and
ensure that any risks associated with the proposed changes are
practical representation of an optimized decellularization pro-
acceptable.
cess exhibits partial removal and/or disruption of resident cells
and cellular material to levels within a set of product-specific 4.7 Measurements of decellularization attributes using the
ranges (acceptance criteria). This guide is intended to aid in source extracellular matrix material as a reference can provide
evaluating a decellularization process through the mechanisms a valuable frame of reference and determination of percent
andextentofdecellularizationandanypotentialimpactsonthe change for exploratory and informational purposes. However,
remaining dECM. acceptance criteria based on percent change from the source
material are more prone to variability in the final product due
4.4 This standard provides a guide to the following steps in
to variability in the source material. Acceptance criteria based
evaluating an extracellular matrix decellularization process:
on measurements of the dECM alone are more stable and
4.4.1 Selecting attributes and test methods for characteriza-
simpler to implement.
tion (Section 5)
4.7.1 The preparation of decellularized medical products
4.4.2 Developing decellularization acceptance criteria for
involvesvariabilityoriginatinginthesourcematerialaswellas
selected attributes (Section 6)
the processing; both types of variability can affect the consis-
4.4.3 Documenting and analyzing the decellularization pro-
tency of the end product (dECM) and its performance in
cess flow (Section 7)
meeting predetermined acceptance criteria. A complete char-
4.4.4 Performing a characterization of the decellularization
acterization of a decellularization process will include statisti-
process by testing decellularized ECM materials using the
cal ranges for each measured attribute. Statistical correlations
selectedattributes,methods,andacceptancecriteria(Section8)
may be explored to connect variation in source material and
4.5 Decellularization processes vary widely in practice,
processing to end product attributes. These correlations can
utilizing a variety of reagents, temperatures, pressures, and/or
help prioritize source material and process controls to address
mechanical forces in parallel and/or in sequence. While any
uncontrolled variability.
one factor may act through consistent mechanisms, its effect
will vary according to the decellularization process in its
5. Decellularized Material Attributes and Testing
entirety as well as the particular tissue structure.As such, each
Methods
part of a decellularization process should be understood and
5.1 Attributes Related to Cellularity and Cellular
Remnants—The following attributes can be used to measure
the efficacy of disruption and removal of cellular contents. to
The boldface numbers in parentheses refer to a list of references at the end of
this standard. develop a holistic understanding of the decellularization
F3354 − 19
process, its effects on ECM properties, and the consistency of 5.1.1.1 Phospholipid Quantification Methods—Several as-
thoseeffects.Theeffectofcellremnantsonhostresponseisnot say kits which quantify phospholipid content after extraction
attributabletoasinglemechanism;insteadthereisevidencefor
from tissue are available. Most methods either measure the
a variety of possible relevant mechanisms, with implications phosphorus bound to lipids or the choline groups bound to
for the end product and its eventual performance in vivo.There
lipids. Choline groups are found on a consistent proportion of
is a common practice within the tissue engineering community
human serum phospholipids (8) and should be assumed to be
in which characterization of decellularization includes, at a
consistent within any species and organ. When reporting
minimum, DNA quantification and nuclear localization (stain-
measurements using a choline-specific reaction, the results
ing). This document recommends setting acceptance criteria
should be labeled as “choline-containing phospholipids.” Ex-
for those measures as well as quantification of a representative
traction techniques for quantification of phospholipids vary,
cellular membrane component, and a representative intracellu-
but generally depend on cell lysis and membrane
lar molecule, as shown in Table 1. These attributes are
solubilization, involving the use of detergents, alcohols,
recommended to evaluate the removal of cells and cell com-
chloroform, tissue homogenization, and/or sonication.As with
ponents; this recommendation does not directly confer rel-
many tissue components, remaining phospholipids may only
evance to product safety or performance beyond measuring the
partially come into solution, especially if the extraction buffer
extent of decellularization. Producers are encouraged to use
is similar to a decellularization reagent. In these cases, an
their discretion to implement additional acceptance criteria;
orthogonal extraction technique would be preferred.
examples are provided in Table 2. The descriptions below
5.1.2 Membrane Proteins and Other Membrane
provide context on why an attribute may be relevant to a
Molecules—Similar to the detection of phospholipids, ubiqui-
product’s intended use and how to measure it. These descrip-
tous or otherwise representative membrane molecules may be
tions do not attempt to include all limitations of the referenced
considered as a stand-in for membrane-bound antigens. For
methods or all possible attributes and methods. All decellular-
example, Major Histocompatibility Complex (MHC) mol-
ization methods should be appropriately validated; for quanti-
ecules may be of interest; they mediate the T cell response in
tative measures, this should include spike recovery studies to
vertebrates against intracellular and extracellular pathogens as
measure the sensitivity of the sample preparation and testing
well as discrimination between self and non-self molecules. In
methods. In particular, inefficient extraction methods may give
humans, MHC class I proteins are found on all nucleated cells
falsely low values especially for low analyte concentrations.
and MHC class II proteins are found on antigen-presenting
Because material properties may be altered during processing,
cells.
test method validations should include source ECM to the
5.1.2.1 Membrane Molecule Quantification and Localiza-
extent that measurements of source ECM are used in evalua-
tion Methods—As with other specific molecules, immune-
tion. Further guidance on validation of test methods can be
basedtechniquesarerecommendedduetotheirhighspecificity
found in ICH Q2(R1).
and precision: ELISA for quantification and IHC or IF for
5.1.1 Phospholipids—Phospholipids comprise the cell
localization.
membrane, nuclear membrane, and the membranes of all
organelles and vacuoles. As such, the presence of phospholip- 5.1.3 αGal—Galα 1,3Galβ1,4GlcNAc (αGal) is a carbohy-
drate residue found on cell-surface and secreted glycoproteins
ids in decellularized materials is representative of the presence
of membrane molecules in general. Immunoglobulins IgG and and glycolipids in all mammals aside from humans, apes, and
Old World monkeys. Humans naturally develop antibodies
IgMhaveconsistentlybeenshowntobindtomembrane-bound
antigens identifying donor cells found in non-decellularized against αGal. The degree of relevance of αGal to the host
immune response is debated. Reduction of αGal may be
xenografts, activating the immune complement system (7) and
resulting in an adverse immune response. For standard decel- accomplishedthroughdecellularizationprocesseswithorwith-
lularization techniques the presence of these antigens as a out αGal-specific enzymatic treatments (9, 10). Testing for
group can be approximated as remaining in proportion to the αGal is not recommended if the source material does not
quantity of remaining phospholipids. contain αGal.
TABLE 1 Recommended Attributes and
Testing Methods – Cellularity and Cell Remnants
Recommended Attribute for Typical Molecule(s) of Interest Common Test Methods Section Reference
Characterization
DNA quantification DNA Hoechst, draq5, Quantifluor, Picogreen 5.1.4
Nuclear localization (staining) Cell nuclei Hematoxylin and Eosin, Feulgen (Schiff’s), 5.1.6.1
4',6-diamidino-2-phenylindole (DAPI)
Quantification of a representative cellular Phospholipids Phosphorus detection, 5.1.1
membrane component Choline detection
Membrane Proteins (for example, Major ELISA 5.1.2
Histocompatibility Molecules (MHC))
Quantification of a representative α-Smooth Muscle Actin (αSMA), β-Actin, ELISA 5.1.5
intracellular molecule Vimentin
F3354 − 19
TABLE 2 Example Discretionary Attributes and
Testing Methods – Cellularity and Cell Remnants
Discretionary Attribute for Characterization Common Characterization Modes Common Test Methods Section Reference
αGal (quantification recommended when Quantification ELISA (for example, M86, IB4 isolectin)
5.1.3
αGal reduction is intended) Localization IHC or IF (for example, M86, IB4 isolectin)
DNA Feulgen (Schiff’s), Hoechst, draq5, DAPI,
Localization
Cyanine 5.1.6
Fragment length analysis Agarose gel electrophoresis
Membrane components Localization IHC or IF (for example, MHC I)
Damage Associated Molecular Pattern Low molecular weight Hyaluronan or Fluorophore assisted carbohydrate
Molecules (DAMPs) Heparan Sulfate quantification electrophoresis (FACE)
5.1.5.1
Proteins (for example, High Mobility Group
ELISA
Box 1 (HMGB1), S100) quantification
Proteins (for example, HMGB1, S100)
IHC/IF
localization
5.1.3.1 αGal Quantification Methods—Quantification may nuclei. Regions with increased concentrations of stray DNA
be performed through standard Enzyme Linked Immunosor- indicate effective cellular disruption but reduced efficacy of
bent Assay (ELISA) methods. The IB4 isolectin, with affinity cellular remnant removal. Common DNA nucleotide stains
for αGal, may be used as a substitute for typical anti-αGal include the Feulgen stain (Schiff’s reagent) and the Hoechst,
antibodies such as the monoclonal antibody M86 (11, 12).A draq5, DAPI, and Cyanine dyes.
complete approach using the M86 antibody and appropriate 5.1.4.3 DNA Fragment Length Analysis—The lengths of
αGal positive and negative controls has been developed as an DNA fragments are commonly measured through agarose gel
industry standard in China and published (13). Alternatively, electrophoresis. This method results in bands or smears that
IHC or IF staining may be used as a semi-quantitative can be compared to DNAfragment standards to determine the
measurement. distribution of DNA fragment lengths. The DNA fragment
5.1.4 DNA—Commercially available decellularized prod- length distribution provides context for the total DNAquantity
ucts have been shown to exhibit a wide range of DNAcontent in a tissue and also indicates the action of DNAdisruption and
(14) and evidence suggests a correlation of the quantity or removal. Different decellularization mechanisms may produce
localizationofremainingDNAindecellularizedECMproducts the same measured quantity of DNA but with substantially
with the severity of adverse host inflammatory response (1, 2). different fragment distributions.
DNAcomplexes can elicit innate immunity through activation 5.1.5 Intracellular Molecules—Intracellular molecules may
of Toll Like Receptors (TLRs) or TLR-independent mecha- be measured or localized as another measure of decellulariza-
nisms (15). DNA quantification and localization have histori- tion. Some, such as αSMA (α-Smooth Muscle Actin) or
cally been used as measures of cellularity. Therefore, they, β-Actin, are ubiquitous in eukaryotic cells and may serve as
along with DNAfragment-length analysis, can be measures of representativemarkersofoverallintracellularremnants.Others
decellularization by serving as a representative of all nuclear may be indicative of remnants from cell subsets, such as the
material, even if there is a non-specific link to the immune useofVimentin,whichisgenerallyspecifictothecytoskeleton
response. In measuring DNA, the analysis may exclude single- of mesenchymal cells (16).
stranded nucleotide chains (for example, ssDNA) and very 5.1.5.1 Damage Associated Molecular Pattern Molecules
short fragments of double-stranded nucleotide chains. (DAMPs)—Evidence suggests that particular molecules asso-
5.1.4.1 DNAQuantificationMethods—Severaldyereagents, ciated with cell and/or tissue damage can initiate immune
such as the Hoechst, draq5, Quantifluor, and Picogreen dyes, responses when released from a cell or displayed on the cell
exhibit fluorescent properties upon binding with DNA mol- membrane, recreating the signals of cellular necrosis (17, 18).
ecules.Assay procedures using these dyes are commonly used Suspected DAMPs include DNA, RNA, HMGB1, ATP,
and should be qualified for the intended use. As with other adenosine, low-molecular-weight Hyaluronan, Heparan
quantification methods, DNA must be extracted or liberated Sulfate, and the S100 family of proteins.These molecules may
from ECM using qualified techniques. In most cases extraction remain in ECM after decellularization. Levels of HMGB1 in
techniques are generally applied utilizing one or more of the decellularized materials in particular have been shown to differ
following: cell lysis, tissue homogenization, enzymatic following different decellularization processes (19).
digestion, and sonication. 5.1.5.2 Intracellular Molecule Quantification and Localiza-
5.1.4.2 DNA Staining Methods—Localization of DNA tion Methods—As for other specific molecules, immune-based
through histologic staining methods can also be used to assess techniques are recommended due to their high specificity and
decellularization. Aside from providing a semi-quantitative precision: ELISA for protein quantification and IHC or IF for
measure of DNA content, localization also allows insight into protein localization.
the mechanisms of DNA persistence or removal. These meth- 5.1.6 Other Methods:
ods allow for the localization of both DNA concentrated as 5.1.6.1 Nuclear Staining—Histological nuclear staining al-
intact nuclei, nuclear remnants and stray DNA from disrupted lows for visualization and localization of intact cellular nuclei.
F3354 − 19
Regions of intact nuclei indicate reduced efficacy of cellular why they may be relevant to decellularization and how to
disruption. Common nuclear staining methods include various measure them. However, these descriptions do not attempt to
forms of Hematoxylin. USP General Chapter <1285.1> pro-
include all limitations of the referenced methods.
videsguidanceonperformingHematoxylinandEosinstaining.
5.2.1 ECM Composition Methods: General Notes—Typical
As an example, the USP Bovine Acellular Dermis Reference
methods of measuring ECM components are destructive and
Micrographsprovideexamplesofstainedsectionswithaccept-
require digestion of the ECM material through enzymatic
able and unacceptable levels of intact nuclei, according to that
and/or non-enzymatic means. Many ECM component classes,
particular tissue and use. DAPI and the Feulgen stain are
such as collagens, elastin, glycosaminoglycans, and soluble
commonly used in vitro to identify nuclei, but, as mentioned in
proteins, can be measured through partially specific biochemi-
5.1.4.2, their visualization is based on binding to and reactions
cal spectrophotometric assays. Liquid chromatography, when
with DNA molecules, respectively. Intact nuclei and localized
available, can similarly be employed for partially specific
DNA are not the same, so users of these techniques should
quantification assays. Specific quantification is generally
consider how DNA degradation, for example due to DNase
achieved through use of ELISA methods, though mass spec-
treatment, may affect results.
trometry and other proteomic techniques are increasingly
5.2 Attributes Related to ECM Integrity—Assessing ECM
common. For all techniques, sample preparation is critical in
integrity is primarily conducted through compositional mea-
maintaining the sensitivity of the measurement. Further, it is
surements that may be supported by structural analyses. The
important to test for and minimize interference from reagents
compositional portion of the assessment should include the
used in the decellularization process and/or sample preparation
relative composition of major ECM components as well as any
through method validations involving spiking studies. All
minor components or non-compositional properties with im-
ECM measurements should be appropriately validated for use
portant functional roles. Major ECM components include any
on both source ECM and decellularized ECM.
class or specific type of ECM molecules that is known or
5.2.2 ECM Structural Methods: General Notes—Histology
expected to comprise at least 5 % (50 mg of each 1 g) of the
is the most common techniq
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