ASTM F3225-17(2022)

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: F3225 − 17 (Reapproved 2022)
Standard Guide for
Characterization and Assessment of Vascular Graft Tissue
Engineered Medical Products (TEMPs)
This standard is issued under the fixed designation F3225; 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 F2210Guide for Processing Cells, Tissues, and Organs for
Use in Tissue Engineered Medical Products (Withdrawn
1.1 This guide is intended as a resource for individuals and
2015)
organizations involved in the development, production,
F2211Classification for Tissue-Engineered Medical Prod-
delivery, and regulation of tissue engineered medical products
ucts (TEMPs)
(TEMPs) intended for use in the surgical repair, replacement,
F2212Guide for Characterization of Type I Collagen as
shunting, and/or bypass of blood vessels. This guide is in-
Starting Material for Surgical Implants and Substrates for
tended for use related to the in vitro assessment of TEMP
Tissue Engineered Medical Products (TEMPs)
vascular grafts. In vitro cellular characterization and in vivo
F2312Terminology Relating to Tissue Engineered Medical
testing are not within scope for this standard guide.
Products
1.2 This standard does not purport to address all of the
F2382Test Method for Assessment of Circulating Blood-
safety concerns, if any, associated with its use. It is the
Contacting Medical Device Materials on Partial Throm-
responsibility of the user of this standard to establish appro-
boplastin Time (PTT)
priate safety, health, and environmental practices and deter-
F2739Guide for Quantifying Cell Viability and Related
mine the applicability of regulatory limitations prior to use.
Attributes within Biomaterial Scaffolds
1.3 This international standard was developed in accor-
STP 997-EBCompositional Analysis by Thermogravimetry
dance with internationally recognized principles on standard- 4
2.2 US FDA Regulations and Guidance Documents:
ization established in the Decision on Principles for the
21 CFR 610.12General Biological Products Standards—
Development of International Standards, Guides and Recom-
Sterility
mendations issued by the World Trade Organization Technical
21 CFR 1270Human Tissue Intended for Transplantation
Barriers to Trade (TBT) Committee.
21 CFR 1271Human Cells, Tissues, and Cellular and
Tissue-Based Products
2. Referenced Documents
FDA Guidance for IndustryPyrogen and Endotoxins Test-
2.1 ASTM Standards:
ing: Questions and Answers
F1635Test Method for in vitro Degradation Testing of
Guidance for IndustryEligibility Determination for Donors
HydrolyticallyDegradablePolymerResinsandFabricated
of Human Cells, Tissues, and Cellular and Tissue-Based
Forms for Surgical Implants
Products (HCT/Ps)
F2150Guide for Characterization and Testing of Biomate-
FDA Guidance for IndustryContainer and Closure System
rial Scaffolds Used in Regenerative Medicine and Tissue-
Integrity Testing in Lieu of Sterility Testing as a Compo-
Engineered Medical Products
nent of the Stability Protocol for Sterile Products
Guidance for Industry and Food and Drug Administration
StaffUseofInternationalStandardISO-10993-1,Biologi-
This guide is under the jurisdiction ofASTM Committee F04 on Medical and
cal Evaluation of Medical Devices—Part 1: Evaluation
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.44 on Assessment for TEMPs. and testing within a risk management process
Current edition approved Sept. 1, 2022. Published September 2022. Originally
approved in 2017. Last previous edition approved in 2017 as F3225–17. DOI:
10.1520/F3225-17R22. The last approved version of this historical standard is referenced on
For referenced ASTM standards, visit the ASTM website, www.astm.org, or www.astm.org.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Available from U.S. Government Printing Office, Superintendent of
Standardsvolumeinformation,refertothestandard’sDocumentSummarypageon Documents, 732 N. Capitol St., NW, Washington, DC 20401-0001, http://
the ASTM website. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3225 − 17 (2022)
2.3 ISO Standards: 3.3 Throughout this guide, the reader is referred to other
ISO 7198 Cardiovascular implants and extracorporeal documents that may provide specific information that can be
systems—Vascular prostheses—Tubular vascular grafts applied in the manufacture and testing of TEMPs. Although
and vascular patches many of these documents were not written with TEMPs in
ISO 10993Biological evaluation of medical devices mind, parts are often applicable. Most of the potentially
ISO 11135Sterilization of health-care products—Ethylene applicablepositionpapersandguidancedocumentsfrommany
oxide—Requirementsforthedevelopment,validationand regions of the world can be accessed via the internet. New
routine control of a sterilization process for medical documents are continually produced, and existing documents
devices are continually updated.
ISO 11137 (Parts 1, 2 and 3)Sterilization of health care
3.4 Theapplicationofthisguidedoesnotguaranteeclinical
products – Radiation
success of a finished product, but will help develop and
ISO 11737-1 Sterilization of medical devices—
characterize a given vascular graft TEMP developed for the
Microbiological methods—Part 1: Determination of a
purpose of surgically replacing, bypassing, or forming shunts
population of microorganisms on products
between sections of the vascular system.
ISO 11737-2 Sterilization of medical devices—
3.5 This guide does not suggest that all listed tests be
Microbiological methods—Part 2: Tests of sterility per-
conducted. The decision regarding applicability or suitability
formed in the definition, validation and maintenance of a
of any particular test method remains the responsibility of the
sterilization process
supplier, user, or regulator of the material based on risk,
ISO 22442-1Medical devices utilizing animal tissues and
applicable regulations, characterizations, and preclinical/
theirderivatives—Part1:Applicationofriskmanagement
clinical testing.
ISO 22442-3Medical devices utilizing animal tissues and
their derivatives—Part 3: Validation of the elimination
4. Significance and Use
and/or inactivation of viruses and transmissible spongi-
form encephalopathy (TSE) agents
4.1 A common therapy to mitigate the pathological effects
2.4 Other Documents:
of blood vessel occlusion or aneurysm-related vascular wall
United States Pharmacopeia XXVII <71>Sterility Tests
weakening is to reroute blood flow around the diseased
ICH Harmonized Tripartite GuidelineViral Safety Evalua- vascular regions. Autologous and non-autologous grafts are
tion of Biotechnology Products Derived from Cell Lines
often used as vascular substitutes surgically to achieve this
of Human or Animal Origin, Q5A(R1) therapeutic intervention. Vascular graft TEMPs may also be
AmericanAssociationofTissueBanks(AATB)AATBStan-
used for these purposes. They may also be used to create or
dards for Tissue Banking revise arteriovenous shunts.
ANSI/AAMI ST72Bacterial Endotoxins—Test Methods,
4.2 Coronary, carotid, renal, common iliac, external iliac,
Routine Monitoring, and Alternative to Batch Testing
superficial femoral, and popliteal arteries are examples of
vascular sites commonly requiring bypass surgery.
3. Summary of Guide
4.3 TEMPs may be composed of biological products (for
3.1 Itistheintentofthisguidetoprovideacompendiumof
example,cells,organs,andtissues),biomaterials(forexample,
informationthatmayberelatedtothefunctionalcharacteristics
substrates and scaffolds composed of polymers or collagen),
ofvasculargraftTEMPsintendedtosurgicallyreplace,bypass,
biomolecules (for example, recombinant proteins, native/
or form shunts between sections of the vascular system.
biological proteins, amino acids, peptides, fatty acids, sugars,
Examplesoffunctionalcharacteristicsincludevasoactivityand
and other macromolecules), and various combinations thereof
mechanical properties (e.g., burst pressure, tensile strength,
(see Terminology F2312). Examples of TEMPs are listed in
creep) suitable for implantation. TEMPs may be composed of
Classification F2211.
biological products (e.g., cells, organs, tissues, and processed
biologics), biomaterials (e.g., substrates and scaffolds com-
4.4 TEMPs may be used with the intent of facilitating the
posed of polymers or extracellular matrix (ECM) components
surgical outcome by improving the biological repair and/or
such as collagen), and/or biomolecules (e.g., recombinant
reconstruction, by accommodating the mechanical loads at the
proteins) (see Terminology F2312). Examples of TEMPs are
repair site, or by a combination of these mechanisms.
listed in Classification F2211.
4.5 Clinical evidence of improved surgical outcomes may
3.2 ISO 7198 provides basic requirements for sterile vascu-
includepatency,reducedincidenceofrevisionsurgery,reduced
lar prostheses and the methods of testing which will enable
rate of implant infection, and improved functionality after
evaluation of vascular prostheses. The degree of sterility
surgery.
–3 –6
(sterility assurance level of1×10 versus1×10 ) will be
determined by the materials of construction and their ability to
5. Synthetic Biomaterials
be sterilized without compromising their function once im-
5.1 Polymer Types—The biomaterial used may be formed
planted.
fromsyntheticpolymers,shouldelicitanacceptablebiological
response with minimal toxicity, and may be degradable or
Available from International Organization for Standardization (ISO), ISO
non-degradable. Examples of degradable polymers are
Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
Geneva, Switzerland, http://www.iso.org. glycolide, lactide, trimethylene carbonate, dioxanone,
F3225 − 17 (2022)
caprolactone, ortho esters, and polymers and copolymers of a specific shape (for example, tube), and may be cross-linked
some of these. Other examples of degradable polymers, in to maintain its shape and to reduce degradation rates.
order of fast to slow degradation time, include polyglycolic
6.2 Plant-derived biomaterials such as starch and cellulose
acid, poly-lactic acid, and polycaprolactone. Non-absorbable
may also be used.
materials include polypropylene, polyethylene, polyamide,
6.3 Cell Culture-Derived Components—Cellsmaybemain-
polyalkylene terephthalate, polyvinylidene fluoride,
tained in culture to synthesize ECM components that are
polytetrafluoroethylene, and blends and copolymers of these.
secreted into the tissue culture media. These components may
Non-synthetic polymers, such as silk, may also be used.
include collagen, elastin, proteoglycans, hyaluronan and other
5.2 Structure—The biomaterial is typically manufactured
proteins, and glycoproteins. These components may then be
intoastructuralmaterialappropriateforavascularTEMPsuch
isolated, purified if necessary, and used to develop a biomate-
as a sheet or strip or tube, by weaving or knitting using a fiber
rial with a specific shape (for example, tube), and may be
as a base material, or a felt with random fiber orientation, as a
cross-linkedtomaintainitsshapeandtomodulatedegradation
membrane, or by using a different technique such as electro-
rates. Alternatively, cells may be seeded onto a biomaterial,
spinning. The structure of the material is selected based on its
cultured in vitro to synthesize an ECM, and then be decellu-
required function, for example to support cells and matrix
larized.
deposition, to provide mechanical support for certain loading
6.4 Biomaterials fabricated from combinations of native
conditions,and/ortointegrateintothesurroundingtissues.For
matrices, plant-derived matrices, and cell culture-derived pro-
example, multi-layered biomaterials may be used to confer
teins may be considered. An adverse immunologic response
desired properties by using layers with distinct capabilities.
may occur due to the presence of cells or cellular debris.
Two-dimensional structures, such as sheets, may be rendered
tubularbysuturing,whilethree-dimensionalstructuresmaybe
7. Biomaterial Characterization
manufactured in tubular form by casting. Geometries other
7.1 Biomaterials may be synthetic, non-synthetic, or ECM.
than tubes may be appropriate depending on the implantation
While biomaterial characterization is beyond the scope of this
anatomic site. Additive manufacturing can be used to create
document, there are several applicable reference documents,
more complex geometries.
including the following: Guide F2210, Guide F2150, and
5.3 Degradation—The biomaterial may be selected to be
Guide F2212 (also see 21 CFR 1270).
non-degradable, or to exhibit specific degradation characteris-
tics (e.g., rapidly or slowly degrading) based on the time
8. Cells
necessary for the TEMPto perform its desired function. If the
8.1 Cell Types—The cell population used may be of one or
function of the material is primarily to support cell attachment
multiple cell types such as (a) arterial, venous, or dermal
and matrix deposition, the biomaterial may be relatively
fibroblasts; (b) arterial or venous smooth muscle cells; (c)
rapidly degrading. If the function is to provide mechanical
arterial or venous endothelial cells; (d) stromal cells; or (e)
support in vivo, the biomaterial should degrade at a rate that
other progenitor cells and may be derived from various tissue
allows natural repair to occur to a level that enables the new
sources. These cells are likely to have undergone expansion
tissue ingrowth to accommodate the mechanical loading at the
priortobeingseededintotheTEMP,andthecellkaryotypeand
repair site.
phenotype should be characterized and compared to a popula-
tionoffreshlyisolatedorearlypassagecellspriortouseinthe
6. Extracellular Matrices
TEMP. Characteristics of the final product should match its
6.1 Native Matrices—Human- and animal-derived tissues
intended use.
may be used to provide an ECM with an appropriate form.
8.2 Cell Performance Requirements—In formation of the
Tissuesthatmaybeappropriateinclude,butarenotlimitedto,
TEMPs in vitro or in situ, the cells may be combined with the
skin, submucosa, arteries, and veins. The tissue should be
biomaterial and/or ECM, and must be able to attach to the
processed to ensure that cell debris and other immunogenic
biomaterial and/or ECM of theTEMPs. For someTEMPs, the
processingreagentsareminimized.Forhuman-derivedtissues,
cellsshouldbeabletoproliferateandsecreteafunctionalECM
asepticprocessingtechniquesshouldbeemployedasdescribed
in vitro. When implanted, the cells may be required to
byFreshney (1) topreventtheintroductionortransmissionof
synthesizeanECMinvivoorfunctioninbiologicrepairbutthe
communicable diseases (also see 21 CFR 1271). For animal-
cells should not induce an inflammatory or immune response
derived tissues or ECM, herd information should be provided
that may have a negative effect on repair. Distribution of
and testing should be conducted to confirm that there are no
seeded cells in biomaterials should be evaluated for intended
potential transmissible diseases. Human- or animal-derived
use.
proteins and glycoproteins such as collagen, elastin,
8.3 Cellular characterization guidelines are beyond the
proteoglycans, and hyaluronan may be used. Collagen may be
scopeofthisdocument.RefertoFDAGuidancedocumentsfor
purified from native tissues (for example, skin, tendon, or
moreinformationunderTissueGuidancesandCellular&Gene
ligament).Thecollagenshouldbemadeintoabiomaterialwith
Therapy Guidances as well as to references Guide F2210;
GuidanceforIndustry:EligibilityDeterminationforDonorsof
HumanCells,Tissues,andCellularandTissue-BasedProducts
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. (HCT/Ps); and ICH Harmonized Tripartite Guideline—Viral
F3225 − 17 (2022)
Safety Evaluation of Biotechnology Products Derived from glycosaminoglycansthroughthedimethylmethylenebluespec-
Cell Lines of Human or Animal Origin, Q5A(R1). trophotometric assay (4). There are many other commercial
assay kits available for ECM quantification that may be used.
9. Attachment of the TEMPs in vivo
12.2 DNA—For grafts that contain live cells, DNA is
9.1 Attachment in vivo—The TEMPs should be able to be
typically used to measure cell number by converting DNA
attached firmly to the adjacent vessels without leaks such that
content per given cell type into the number of cells. The
they can function as patent fluid-tight vessels capable of
Hoechst assay, which stains cell nuclei with Hoechst dye
withstanding the intravascular pressures and hemodynamic
33258,isoftenused (5, 6).Briefly,vasculargraftsarefrozenat
shear stresses in vivo. If the TEMP is to be secured in vivo
–20°C, lyophilized, and the construct dry weight is measured.
using sutures, then it should be able to retain them in the
ECM is digested by overnight incubation with proteinase K
mannerthatisappropriateforthesurgicalimplantationsiteand
solution at 55°C. Digested samples are stored at –20°C until
forces. Once implanted, the TEMPshould be retained in place
used for biochemical assays. A small aliquot sample of the
for the time required for it to complete its functional require-
digested sample is then mixed with the Hoechst dye solution,
ments and should remain patent and free from thrombosis.
placedina96-wellplate,anditsopticaldensityismeasuredin
10. Sterilization amicroplatespectrophotometeratexcitation/emission365/460
wavelengths. DNA content is calibrated using standards such
10.1 TEMPs must be provided sterile to the clinical field.
as calf thymus DNA. Cell number is calculated from DNA
Acellular products may be sterilized after manufacture by
content by assuming a given DNA content per cell type of
ethylene oxide, liquid chemical sterilization, gamma
interest (e.g., 7.6 pg of DNA per smooth muscle cell) (5).
irradiation, or other sterilization techniques if there is no
Alternatively, a DNA standard curve may be created using
negative impact on functionality. If the TEMP is cellular, it
DNAextractedfromaknownnumberofcellsusedinthegraft
must be manufactured using standard aseptic techniques (1)
fabrication. Other assays may also be used such as rapid UV
and/oraclosedculturesystem.SomeTEMPproperties,includ-
detection of DNA at 260/280nm (7).
ingbutnotlimitedtodegradationprofiles,couldbeaffectedby
sterilization. Consequently, it is recommended that potentially
12.2.1 To obtain the DNA content of cellular tissue the
affected properties be evaluated for design compliance after
above method can be utilized; however, a standard addition
sterilization processing. Sterilization is beyond the scope of
approach should be taken to account for matrix interference
this document; consult reference documents
...