Materials Measurement Laboratory • Biosystems & Biomaterials Division • Biomaterials 

National Institute of Standards and Technology

United States Department of Commerce

Tissue Engineering Measurands

Carl G. Simon, Jr., Ph.D.
Tissue Engineering Measurands

• During the development lifecycle of a TEMP (tissue engineered med

product), many product properties are measured over many years a

way to a product release. 

• Measurands for TEMPs are often complex, hard to measure and dif

define. 

• The selection of each measurand is motivated by the ultimate goal t

establish that the product is safe and efficacious (product success)

Arora D, Babakhanova G, Simon Jr C

measurands. ACS Biomaterials Scien

https://doi.org/10.1021/acsbiomate
Complexity of Tissue Engineered Constructs Makes

Them Difficult to Assess and Describe

• Complexity can come from cells, which are

• Responsive to their environment

• Inhomogenous• Composed of thousands of different molecules

• Composition can’t be defined as with a small molecule drug

Challenging to identify a measurand to meaningfully describe such a complex 
Tissue Engineering Measurands Are Difficult to Select

• Desirable measurands have a bearing on product success, clinical efficacy

• A good place to start is the mechanism of action (MOA)

• Challenging to confirm the MOA

• The only way to do this is a human clinical trial

• Often not possible to test a hypothesis in human since the proper con

be ethical

• Empty scaffold (no cells) could cause harm

• Scaffold seeded with dead cells could cause harm

• Immunosuppressive drugs may be required, which have harsh sid

• Sham surgery could be highly invasive

Challenging to identify a measurand which has a bearing on clinical efficacy 

success.
Miscommunication During Discussions of

How to Characterize a Tissue Engineered Medical Product (

What are we really measuring?

We o

prope

with 

How do you validate that 

these markers are related 

to patient outcomes?

Is it biologically 

relevant? If not, 

then why are we 

measuring it?



	




Yes, but the platereader

is detecting photons, 

not cell viability.

This may not have 

anything to do with 

curing the patient...

Sca

Cell Therapy Workshop, NIST, 2015

Cell Therapy Workshop, NIST, 2015

Cell Therapy Workshop, NIST, 2015

Phacilitate, Miami, 2020

Scaffold Workshop, ARMI|BiofabUSA, 2018

You can’t c

isn’t wha
“the process that is undertaken to generate an estimate of the true value of a property o

along with an evaluation of the uncertainty associated with that estimate, which is inten

support of decision‐making”

Possolo, A. NIST Technical Note 1900: Simple Guide for Evaluating and Expressing the Uncertainty of 

Results. NIST, U.S. Department of Commerce, 2015.

“measurement is typically done in the context of an experiment designed to make the p

interest accessible to a sensor in a measuring instrument”

Possolo, A.; Iyer, H.K.; Concepts and Tools for the Evaluation of Measurement Uncertainty. Review of 

2017, 88, 011301.

“process of experimentally obtaining one or more quantity values that can reasonably b

quantity”

Note: “presupposes a description of the quantity commensurate with the inten

measurement result”

VIM 3rd Edition: International Vocabulary of Metrology ‐ Basic and General Concepts and Associated 

Edition. Joint Committee for Guides in Metrology (JCGM) 200:2012.

Measurement
Measurand

“the quantity or property intended to be measured”• International Vocabulary of Metrology ‐ Basic and General Concepts and Associated Ter

Edition. Joint Committee for Guides in Metrology (JCGM) 200:2012.

• Use of the word “intended” in this definition is intentional

• It may be impossible to measure what you intend to measure

• What is actually measured may not be what you intended to measure (due to expe

artifacts)

Nevertheless, the measurand is the property that you intend to mea

whether or not this is possible or achieved.

• Measurand is personal• No one can define it for you• It is what you say it is

Can comchaeach of wis & 
Measurand Chart:

Cell Viability in a Scaffold
Cell Viability in a Scaffold

• A TEMP is often composed of a three‐dimensional template, called a scaffo

has been seeded with cells

• The mechanism of action (MOA) for a scaffold‐cell construct may be:

• The cells build a new tissue in the patient through the processes of ce

proliferation and deposition of extracellular matrix

• The cells secreting factors that induce tissue regeneration or repair

• Since these MOAs may require live cells, a common and important measur

scaffold‐cell construct is “cell viability in a scaffold”
I assume this may be true for all measurement systems

[that different measurements of an attribute may not agree]

High Dehydrogenase Activity => MTT => Live

Poor Membrane Integrity => Trypan blue => Dead

Low Dehydrogenase Activity 

Good Membrane Integrity =

Measuring Cell Viability: There are a ~100 different methods for assessing cell viability

• Each method has a unique measurand (they measure different things)

• Results from the various methods may not agree with one another, even when analyzing th

Case 1: Cell under a stress Case 2: Cell under a differen

My Definition of a Viable Cell: “a cell that has 1) an intact membrane and the 2) capacity for m

motility, 4) proliferation and 5) reaction to stimuli” (Merriam‐Webster’s Dictionary, Encyclope

ASTM F2739)

Cell Viability
The route from live to dead may contain multiple

steps and could be a continuum.

• The order of the steps may change

• Not all of the steps may be observed

Cell Viability May Be a Continuum

Davey HM (2011) Ap
• Must make the allowance that ther

measurands per measurement

• Similar to

a process flow diagram

• Note that all possible measurands f

present

• The relationships depicted by the c

meant to facilitate discussion

Measurand Chart: Cell Viability in

a Scaffold (Every Box is

a Me

• Graphic to visualize the various measurands that may be

present in

a measurement

• A way to think about complex measurements to

• Improve strategic thinking

• Reveal the hidden assumptions

Amount of

ATP in

a cell

Number of

emitted

photons

detected

during ATP

catalysis

ATP con‐

centration

calculated

from lumi‐

nescence

∝ ∝

Number of

fluors able to

bind to DNA

Number of

emitted

photons

detected

DNA con‐

centration

calculated

from fluor‐

escence

∝ ∝

ATP/DNA

(∝ ATP/Cell)

∝ 

Amount of

DNA in

a cell

∝

Amount of

DNA

released

from lysed

cells

Amount of

ATP released

from lysed

cells

∝

Amount of

ATP able to

reach the

luciferase

enzyme

∝

∝ Picogreen DNA Assay

LuciferaseATP Assay

1/

∝

∝

Arora D, Babakhanova G, Simon Jr CG (2020) Tissue engineering measurands. ACS Biomaterials

Science

& Engineering 6, 5368‐5376. https://doi.org/10.1021/acsbiomaterials.0c00475
Measurement Model 

& 

Multiple Measurands
GUM: JCGM, 

2008.

GUM Is a Model for Expressing [Output Quantities] in Terms of 

Possolo, A. NIST Technical Note 1900, 2012.

Input quantities: 

accessible to a 

measurement

Output 

Not acc

mea
Multiple Measurands:

Volume of the Cylinder

V = 

V = VA ‐ 

• Two different systems for measuring cylinder volume

are shown

1) Measure radius & height with a ruler

2) Measure volume of water in a graduated

cylinder before & after immersion of the

cylinder

• In both cases, you have two different input values

that are used to determine the measurand

• However, the primary measurand is the same:

“Volume of the cylinder”
Multiple Measurands: NIST SRM 972a “Vitamin 

Metabolites in Frozen Human Serum”

• Frozen serum with certified values of 25-

hydroxyvitamin D

2 & 25-hydroxyvitamin D

3

• The sum of D

2 and D

3 is generally considered a 

reliable indicator of “vitamin D status” (primary measurand the physician desires to assess health)
Measurand Function 

Y General model for measurands: Y ൌ fሺX1, X2,..., XNሻ 

Volume of

a Cylinder

V

=

π

r2h 

Vitamin

D Metabolites in

Frozen Human Serum

[25‐hydroxyvitamin

D2] +

[25‐hydroxyvitamin

D3]

Cell Viability in

a Scaffold 
Definitional Uncertainty
Definitional Uncertainty

True value:

• The “true value (of a quantity)” is the “value consistent with the definition of a given pa

(GUM, 2008)

• True value “is a value that would be obtained by a perfect measurement” (GUM, 2008)

• “true values are by nature indeterminate” (GUM, 2008)

Definitional uncertainty:• The “component of measurement uncertainty resulting from the finite amount of detai

measurand” (VIM, 2012)

• “a measurand cannot be completely described without an infinite amount of informatio

Consequences• The true value of a measurand is what you say it is (true value is dependent on how you 

value)

• Accuracy does not exist (not in an absolute sense)

• Discussing product characterization will be challenging if you don’t agree on a common 

measurand
Definitional Uncertainty: Mass of the Sun

Example from Possolo is the mass of the sun:

• Possolo, A. NIST Technical Note 1900, 2015.

• The mass of the sun rapidly changes from moment to

moment

• Even at a given instant, the mass of the sun is

dependent upon how the volume of the sun is defined
Definitional Uncertainty: Velocity of Sound

• The air composition, the temperature and the pressure cannot be

defined with infinite precision, which introduces uncertainty into t

definition of the measurand
0

0000000001

0 1 2 3 4 5 6 7 8 9 

Frequency

ATP Concentrat

Measured 

Values

Error

00000000001

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Frequency

ATP Concentration

Measured 

Values

True

Value

Error

Definitional Uncertainty

Charles Ehrlich. Terminological aspects of the Guide to the Expression of UncertaiMeasurement (GUM). Metrologia 51 (2014) S145–S154.
Defining what constitutes a cell:

• Cell numbers in a culture rapidly change as the cells divide• Cell cultures contain many cell fragments that contain DNA. Are these consider• How dead would a cell have to be to no longer be considered a cell?

• If the membrane degrades and the cytoplasm has dissipated but there is st

structure that contains DNA, would this count as a cell?

Defining ATP/cell:• Cell preparations are typically inhomogenous & ATP/cell may vary between subp

• There may be differences in ATP/cell within the human population

• For an autologous indication that uses patient‐derived cells, then each pati

will have a different true value for the ATP/cell in the scaffold

Cell Viability May Have Large Definitional Uncertainty: AT
Defining ATP concentration:

• As with the mass of the sun, the ATP levels in cells may rapidly fluctuate

• ATP is continuously dephosphorylated to release energy to support cell

functions

• ATP is continuously synthesized as the energy from food sources is used to

drive the rephosphorylation of AMP & ADP

• There may be a challenge with defining what constitutes a molecule of ATP

• Some fraction of ATP molecules will be complexed with enzymes, some by

covalent bonds, as they are metabolized

• Mitochondria, where most of the ATP is synthesized, comprise up to a

quarter of the cell volume, which means that a significant fraction of ATP

molecules may be complexed

Cell Viability May Have Large Definitional Uncertainty: AT

“Li

invo

enzym

the a
Defining the Measurand
Measurand Should Be Defined Both Philosophically & Practi

• Philosophical measurand

• Establishes a goal for the measurement

• Can be abstract, impractical or impossible to measure

• Should not mention a specific measurement system

• Practical measurand

• Mentions the measurement system

• Required for a coherent discussion of the value of a particular measu

• Need to have both for a complex measurand such as “cell viability in a sc

M

in
Measurand Should Be Defined Both Philosophically & Practi

Without reference 

to a measurement

In t

me

Philosophical Measurand

(does not mention

measurement system)

Practical Measurand(includes measurement system)

Mass of a penny Position of weights on balance

when pointer is horizontal on a

triple beam balance

Cell viability [Number of non‐blue cells]

divided by [total number of

cells] as observed on a

brightfield microscope following

trypan blue staining
Amount of

ATP in a cell

Number of

emitted

photons

detected

during ATP

catalysis

ATP con‐

centration

calculated

from lumi‐

nescence

∝ ∝

Number of

fluors able to

bind to DNA

Number of

emitted

photons

detected

DNA con‐

centration

calculated

from fluor‐

escence

∝ ∝

ATP/DNA

(∝ ATP/Cell)

∝ 

Amount of

DNA in a cell

∝

Amount of

DNA

released

from lysed

cells

Amount of

ATP released

from lysed

cells

∝

Amount of

ATP able to

reach the

luciferase

enzyme

∝

∝ Picogreen DNA Assay

LuciferaseATP Assay

Cell Viability in a Scaffold Measurand Chart

Practical

Measurands

We only want to measure 

properties that correlate 

with clinical efficacy.

Yes, but the platereader

is detecting photons, 

not cell viability.

Scaffold Workshop, ARMI|BiofabUSA, 2018 Cell Therapy Workshop, NIST, 2015

Y

s
• An example where we mixed the philosophical & practical

measurands

• “viability” is the philosophical measurand (agnostic to the

measurement system)

• “related attributes” was meant to address the practical

measurands (trypan blue, MTT, ATP, etc.)

Yo

si

Philosophical 

Measurand

Practical

Measurand
Closing
Measurand Chart

• Graphical rendering of the relationship between measurands to make the 

more accessible & easily consumed

• Formally map the chain of measurands & their relationships in a measure

• Reveal unrecognized assumptions

• Improve communication & strategic thinking

• Enhance interpretation of measurement results (what are we really meas

• Help assess the value of measuring a given attribute

• Should not serve as an absolute; should be used to facilitate discussion
Summary

• Tissue engineering measurements can have many measurands

• Different test methods do not measure the same thing (different measurands)

• Tissue engineering measurands have large definitional uncertainty (inability to precisely defi

• Coherent discussion requires that you define the measurand both philosophically & practica

• Philosophically: without reference to a measurement system

• Practically: in terms of the measurement system

Awareness of these issues enables strategic thinking when deciding 1) which product attribut

characterized and 2) how they should be measured
VIM, JCGM

GUM, JCGM

NIST Technical Note 1900 (G

Malcolm Moos (FD

Richard McFarland (FDA / B

Becky Robinson-Zeigler (FDA 

Acknowledgements

NIST

Deepika Arora

Greta Babakhanova

Dave Duewer 

John Elliott

Charles Ehrlich 

Sheng Lin-Gibson

Will Guthrie

Anne Plant

Antonio Possolo 

Marc Salit

Sumona Sarkar

Robert Waters 

Thank yo

Arora D, Babakhanova G, Simon Jr CG (2020

measurands. ACS Biomaterials Science & En

https://doi.org/10.1021/acsbiomaterials.0c