ASTM F3106-22

Standard Guide for <emph type="bdit"> in vitro</emph> Osteoblast Differentiation Assays

SIGNIFICANCE AND USE
4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can be used to screen for the osteogenic capability of progenitor stem cells from various human or animal sources, including mixed tissue-derived connective tissue progenitor populations, or cell populations that may be selectively isolated or manipulated through culture expansion, processing, transfection, or genetic modification.  
4.2 The osteoblast differentiation assay may be referred to as an osteogenesis assay or a mineralization assay.  
4.3 It is important to carefully select the components and conditions used for in vitro osteoblast differentiation assays since high amounts of osteogenic medium components can lead to dystrophic, pathologic, or artifactual calcium-based precipitates that do not indicate differentiation of the cells in culture to functional osteoblasts (1).4 For example, when high concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell (2, 3).  
4.4 Alkaline phosphatase production is an early event associated with osteoblast differentiation, but it can also be stimulated in other cell types by the addition of the osteogenic supplement dexamethasone to the medium. Alkaline phosphatase enhances the formation of calcified deposits prior to their natural occurrence in bone that typically coincides with bone sialoprotein and osteocalcin expression by mineralized matrix-producing osteoblasts. These kinds of calcified/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. A calcium measurement, such as that described in Practice F2997 for the Quantification of ...
SCOPE
1.1 This document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells including mesenchymal stromal cells.  
1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts.  
1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the cells.  
1.4 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.  
1.5 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.

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 11.040.40 - Implants for surgery, prosthetics and orthotics
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.43 - Cells and Tissue Engineered Constructs for TEMPs

Relations

• Refers: ASTM F2312-11(2020) - Standard Terminology Relating to Tissue Engineered Medical Products
• Effective Date: 01-Feb-2020

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: F3106 − 22
Standard Guide for
1
in vitro Osteoblast Differentiation Assays
This standard is issued under the fixed designation F3106; 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 F2997 Practice for Quantification of Calcium Deposits in
Osteogenic Culture of Progenitor Cells Using Fluorescent
1.1 This document provides guidance on how to conduct in
Image Analysis
vitro osteoblast differentiation assays with progenitor stem
3
2.2 ISO Standards:
cells including mesenchymal stromal cells.
ISO 21709 Biotechnology—Biobanking—Process and
1.2 This document describes the roles of various osteogenic
Quality Requirements for Establishment, Maintenance
supplements that are added to the cell culture medium of an
and Characterization of Mammalian Cell Lines
osteoblast differentiation assay to encourage and support the
differentiation of progenitor cells into matrix-producing osteo-
3. Terminology
blasts.
3.1 Unless provided otherwise in 3.2, terminology shall be
1.3 This document provides recommendations for the con-
in conformance with Terminology F2312.
centrations of osteogenic supplements that may prevent the
3.2 Definitions:
precipitationofartifactualmineraldepositsthatarenotdirectly
3.2.1 calcium deposits, n—a calcium phosphate-containing
produced by osteoblasts, nor correlated with osteoblastic gene
substance synthesized in cell cultures during mineralization or
expression of the cells.
osteoblast differentiation assays that may be directly produced
1.4 This standard does not purport to address all of the
by osteoblasts or precipitated out of the solution without cell
safety concerns, if any, associated with its use. It is the
participation.
responsibility of the user of this standard to establish appro-
3.2.2 mineralized matrix, n—a calcium phosphate-
priate safety, health, and environmental practices and deter-
containing substance produced by cells typically in the
mine the applicability of regulatory limitations prior to use.
osteoblast, odontoblast, and calcifying chondrocyte lineages,
1.5 This international standard was developed in accor-
which is composed of crystals of calcium phosphate and
dance with internationally recognized principles on standard-
contains collagen Type I and other non-collagenous proteins.
ization established in the Decision on Principles for the
3.2.3 osteoblasts, n—secretory mononuclear cells that will
Development of International Standards, Guides and Recom-
initiate the formation of a matrix containing characteristic
mendations issued by the World Trade Organization Technical
proteins, such as collagen, and non-collageneous proteins such
Barriers to Trade (TBT) Committee.
as bone sialoprotein and osteocalcin, that will mineralize in the
2. Referenced Documents
presence of a calcium and phosphate source.
2
2.1 ASTM Standards:
4. Significance and Use
F2312 Terminology Relating to Tissue Engineered Medical
Products
4.1 This guidance document describes the components and
F2944 Practice for Automated Colony Forming Unit (CFU)
conditions used for in vitro osteoblast differentiation assays
Assays—Image Acquisition and Analysis Method for
that can be used to screen for the osteogenic capability of
Enumerating and Characterizing Cells and Colonies in
progenitor stem cells from various human or animal sources,
Culture
including mixed tissue-derived connective tissue progenitor
populations,orcellpopulationsthatmaybeselectivelyisolated
1
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
or manipulated through culture expansion, processing,
Surgical Materials and Devices and is the direct responsibility of Subcommittee
transfection, or genetic modification.
F04.43 on Cells and Tissue Engineered Constructs for TEMPs.
Current edition approved April 1, 2022. Published April 2022. Originally
4.2 The osteoblast differentiation assay may be referred to
approved in 2014. Last previous edition approved in 2014 as F3106 – 14. DOI:
as an osteogenesis assay or a mineralization assay.
10.1520/F3106-22.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
3
Standards volume information, refer to the standard’s Document Summary page on Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
the ASTM website. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM Internati
...

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: F3106 − 14 F3106 − 22
Standard Guide for
1
in vitro Osteoblast Differentiation Assays
This standard is issued under the fixed designation F3106; 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 document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells
including mesenchymal stromal cells.
1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast
differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts.
1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation
of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the
cells.
1.4 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.
1.5 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:
F2312 Terminology Relating to Tissue Engineered Medical Products
F2944 Practice for Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis Method for Enumerating
and Characterizing Cells and Colonies in Culture
F2997 Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image
Analysis
3
2.2 ISO Standards:
ISO 21709 Biotechnology—Biobanking—Process and Quality Requirements for Establishment, Maintenance and Character-
ization of Mammalian Cell Lines
3. Terminology
3.1 Unless provided otherwise in 3.2, terminology shall be in conformance with Terminology F2312.
1
This test method 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, 2014April 1, 2022. Published February 2015April 2022. Originally approved in 2014. Last previous edition approved in 2014 as
F3106 – 14. DOI: 10.1520/F3106-14.10.1520/F3106-22.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
3
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

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F3106 − 22
3.2 Definitions:
3.2.1 calcium deposits, n—a calcium phosphate-containing substance synthesized in cell cultures during mineralization or
osteoblast differentiation assays that may be directly produced by osteoblasts or precipitated out of the solution without cell
participation.
3.2.2 mineralized matrix, n—a calcium phosphate-containing substance produced by cells typically in the osteoblast, odontoblast,
and calcifying chondrocyte lineages, which is composed of crystals of calcium phosphate and contains collagen Type I and other
non-collagenous proteins.
3.2.3 osteoblasts, n—secretory mononuclear cells that will initiate the formation of a matrix containing characteristic proteins,
such as collagen, and non-collageneous proteins such as bone sialoprotein and osteocalcin, that will mineralize in the presence of
a calcium and phosphate source.
4. Significance and Use
4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can
be used to screen for the osteogenic capability of progenitor stem cells fro
...