The relationship between the food we eat and our health is clear. In the constant search for healthier foods rich in bioactive compounds that promote health and healthy ageing, a wide variety of functional foods have appeared on the market. To know the real function of these functional foods in our body, it is necessary to carry out different types of in vitro cell tests. Institut Kurz specializes in conducting in vitro cell tests for functional foods. Contact us for more information: info@institut-kurz.com https://www.institut-kurz.com/

1. INSTITUT KURZ
IN VITRO CELL TESTS
FOR FUNCTIONAL FOOD
www.institut-kurz.com
Contact: info@institut-kurz.com

2. FUNCTIONAL FOOD
(Abuajah et al., 2015; Nicoletti, 2012)
Functional foods: Dietary items that, besides providing nutrients and
energy, beneficially modulate targeted functions in the body, by enhancing
a certain physiological response and/or by reducing the risk of disease
Relationship between the food we eat and our health. → Functional
components of food can be effectively applied in the treatment and
prevention of diseases.

3. FUNCTIONAL FOOD
(Robin et al., 2018; Ferruzzi et al., 2012)
There is still much research required to better understand the role of
bioactive dietary compounds and their metabolites in human health.
In vitro methods can be used to understand and study:
✓the identity and quantity of bioactive components in food and its
metabolites.
✓mechanisms of action, absorption, bioavailability, metabolism and
biological activity.

4. In vitro is an expression of Latin which means "in the glass" and refers to
the technique of performing a certain procedure in a controlled
environment outside a living organism.
IN VITRO CELL TESTS
(OECD, 2018)
It can be performed on a wide range of cells and the biological material that
best suits the purpose of the test must be selected.
This tests can be used for:
• Drugs
• Nutraceuticals
• Food supplements
• Functional food

5. In vitro studies analyze the absorption, distribution, metabolism and
excretion of active compounds.
→Allows an assessment of their:
• performance
• toxicity
• efficacy
• side effects
IN VITRO CELL TESTS
(Ferruzzi et al., 2012)

6. In vitro tests advantages:
✓Do not require animals or humans
✓Absence of ethical restrictions
✓Avoid the need to submit animal protocols
✓Avoid/reduce the need for laboratory personnel with experience in
handling animals
✓Less security concerns
✓Lower cost
✓Faster
IN VITRO CELL TESTS
(National Academies Press, 1999)

7. The problem with the in vitro study is that it is not a complete
representation of the response of a human being to a compound.
The human body is much more complex than a simple cell culture → In
vitro studies cannot fully predict the influence that the active ingredient
will have on organs and systems, or the interaction with others.
IN VITRO CELL TESTS
(Katherine et al., 2017)
In vivo studies are necessary to clarify all this missing information.

8. There are several in vitro tests which have been applied to functional food:
• CAA (Cell Antioxidant activity)
• MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide]
• LDH (lactate dehydrogenase)
• BrdU (bromodeoxyuridine)
• AMES test
• SOD (superoxide dismutase)
• Catalase
• CAP-e (Cell-based antioxidant protection in erythrocytes)
• ROS PMN (reactive oxygen species in polymorphonuclear cells)
• CACO-2 permeability assay
EXAMPLES OF IN VITRO CELL TESTS

9. CAA
(Reşat et al., 2016)
Cell Antioxidant activity (CAA) is an assay for quantifying the
antioxidant activity of phytochemicals, food extracts, and dietary
supplements.
It is an in-vitro-measurement method that uses human or animal cell
colonies and does not require in-vivo evolvement of probands.

10. CAA
(Wolfe & Liu, 2007)
The procedure include the following steps:
1. Sample is incubated with the cells and with a precursor probe.
2. Precursor 2′,7′-dichlorofluorescin diacetate (DCFH-DA) traverses the
cell membrane and is deacetylated by cellular esterases.
3. Intracellular ROS oxidize the deacetylated precursor (DCFH) to
fluorescent dichlorofluorescein (DCF).
4. ROS generator is added to the culture media, passes through the cell
membrane and the oxidation reaction is accelerated.
5. Fluorescence is measured at 3–5min intervals for 90min or until the
curves reaches a plateau.

11. CAA

12. MTT
(van Tonder et al., 2015)
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay measures cell viability. Indirectly, it assess the cellular energy
capacity of a cell.
The yellow MTT is reduced to a purple formazan by mitochondrial
enzymes.

13. MTT
(Kuete et al., 2017)
MTT assay includes the following steps:
1. The cells and test compounds are prepared in 96-well plates,
containing 100µl/well, and incubated for the desired exposure period.
2. 10µl of MTT solution is added per well to obtain a concentration of
between 0,2-0,5mg/ml and incubated for 1 to 4 hours at 37°C.
3. 100µl of solubilization solution is added to each well to dissolve
formazan crystals into a colored solution.
4. The quantity of formazan is measured by recording changes in
absorbance at 570nm using a plate reading spectrophotometer.

14. MTT
(Sittampalam et al., 2004)
Viable cells convert MTT into a purple colored formazan. When cells die,
they lose the ability to convert MTT into formazan.→ Color formation is a
marker of the viable cells.
The signal generated depends on the following parameters:
• concentration of MTT
• length of the incubation period
• number of viable cells and their metabolic activity

15. LDH RELEASE
(Stoddart, 2011; Kumar et al., 2018)
Quantification LDH (lactate dehydrogenase) is an assay for cell viability
that provides fast, robust and reproducible insights into the potential
toxicity of compounds and products.
Well-suited correlate for the presence of damage and toxicity in tissues and
cells.
Evaluates the membrane integrity of cells by measuring the concentration
of the cytosolic LDH enzyme leaked in the extracellular medium in the
case of membrane damage.

16. LDH RELEASE
1. LDH released from cells in the culture supernatant catalyzes the
hydrolysis of lactate to pyruvate. This reaction is accompanied by the
reduction of NAD+ in NADH.
2. INT (iodonitrotetrazolium chloride), a yellow tetrazolium salt, is
reduced to a red formazan - catalyzed by diaphorase.
3. The formazan formed is measured by colorimetry at an absorbance of
490 nm.
The amount of formazan is directly proportional to the amount of LDH in
the culture → Directly proportional to the number of dead or damaged
cells.
(Kumar et al., 2018; Forest et al., 2015)

17. LDH RELEASE
(28)
LDH
LDH
Pyruvate
LDH NAD +
NADH
Lactate INT
INT formazan
Diaphorase

18. BRDU INCORPORATION
(Crane& Bhattacharya, 2013)
BrdU (5-Bromo-2’-deoxyuridine or bromodeoxyuridine) incorporation
assay is used to detect DNA synthesis → quantify cell proliferation.
Mechanisms of cell proliferation and molecular mediators that could
regulate cell proliferation can be identified through this method.

19. BRDU INCORPORATION
BrdU is incorporated into nuclear DNA during the S-phase of the cell
cycle. After that, it can be detected with immunohistochemical methods.
1. Samples are fixed and incubated with anti-BrdU monoclonal
antibodies and nucleases.
2. The sample is incubated with a secondary antibody against the
antiBrdU antibody.
3. The reaction can be observed by brightfield microscopy.
(Crane& Bhattacharya, 2013)

20. AMES TEST
(Abhishek et al., 2018)
AMES test involves microorganisms → Salmonella typhimurium is used
to assist in evaluating the potential mutagenicity and carcinogenicity of
chemicals.
The assay is based on the inability of the autotrophic histidine mutants of
Salmonella typhimurium to grow in the absence of histidine.
Through their effect on the microorganism's DNA, some compounds can
cause reversion to prototrophic forms that can grow in the absence of
histidine.

21. SOD
(Weydert & Cullen, 2010; Fukai & Ushio-Fukai, 2011)
SOD (superoxide dismutase) is the major antioxidant defense systems
against O2•− (superoxide anion) → It converts a toxic superoxide radical
into hydrogen peroxide (H2O2) and oxygen (O2).
SOD activity can be measured by biochemical method:
• Xanthine-xanthine oxidase is used to generate O2•−. Nitroblue
tetrazolium (NBT) reduction is an indicator of O2•− production.
• SOD compete with NBT for O2•− → The percent inhibition of NBT
reduction is a measure of the amount of SOD present.
• Catalase is included to remove H2O2 produced by SOD. The original
concentration of sample protein should be around 20μg/μl.

22. CATALASE
Catalase is an enzyme that dissociates H2O2 into O2 and H2O.
Catalase activity is a way to measure the protection against oxidative attack
by a given functional food or nutritional supplement.
It can be measured by a spectrophotometric procedure measuring
peroxide removal.
Direct assay with pseudo-first order kinetics. Depends on monitoring the
change of 240nm absorbance at high levels of hydrogen peroxide solution
(≥30mM).
The rate of peroxide removal by catalase is exponential. Catalase begins to
be inactivated by H2O2 (at levels greater than 0.1 M). H2O2 is consumed
and catalase is inactivated.

23. CATALASE
Catalase activity gels can also be used.
These will be in a green-blue colour with white broad bands where the
enzyme is present. After separation of native protein, the catalase enzyme
removes the peroxides from the area of the gel it occupies.
Removal of peroxide prevent the reduction of potassium ferricyanide into
potassium ferrocyanide, which reacts with ferric chloride to form a
Prussian blue precipitate.
Catalase gels will have one band that rarely saturates getting larger with
increasing catalase activity.
(Ighodaro & Akinloye, 2018)

24. CAP-E ASSAY
(Honzel, 2008)
CAP-e (cell-based antioxidant protection in erythrocytes) assay reflects
whether antioxidants from natural products can enter the cytosol of living
cells and protect them from oxidative damage.
It includes the following steps:
1. Erythrocytes are exposed to a natural product (antioxidants enter to
cells).
2. Unabsorbed antioxidants are removed, and cells are loaded with a
precursor dye.
3. After an oxidative challenge, the precursor dye emits fluorescent light
in proportion to the amount of oxidative damage. Fluorescence
reduction is proportional to antioxidant protection.

25. ROS PMN ASSAY
(Schauss et al., 2006)
ROS PMN (reactive oxygen species in polymorphonuclear cells) assay
monitors the combined effect of a test product on an inflammatory cell type
(PMN).
This cell type is an important part of our innate immune defense and can
generate ROS in response to oxidative damage and proinflammatory
stimuli.

26. ROS PMN ASSAY
(Honzel, 2008)
The PMN cell respond to compounds in 3 different mechanisms:
1. Antioxidants penetrate the cell and neutralize free radicals
2. Anti-inflammatory compounds mediate cell signaling, reprogramming
the PMN cell to a less inflammatory behavior → Reduction in
formation of ROS
3. Pro-inflammatory compounds capable of supporting innate immune
functions mediate a signal → Increase PMN cell function → Increase
ROS production.
Data reflects a combination of these mechanisms operating in parallel.

27. CACO-2 PERMEABILITY ASSAY
(Lea, 2015)
Caco-2 cells (polarized human colon carcinoma cell line) are used to
analyze the absorption process of compounds.
In the Caco-2 Permeability Assay, Caco-2 cell monolayers are cultured on a
permeable surface and placed in a cell culture plate to form donor-receiver
compartments.
It allows the measurement of compounds transport in 2 directions: apical to
basolateral and basolateral to apical.

28. CACO-2 PERMEABILITY ASSAY
(Glahn, 2009)
The bioavailable amount of the component is represented by the amount
present in the final extract.
The quantification can be done through liquid chromatography with tandem
mass spectrometry.

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