FR AUNHOFER INSTITUTE FOR INTERFACIAL ENGINEERING AND BIOTECHNOLOGY IGB
HUMAN ORGAN-LIKE THREE-DIMENSIONAL
TEST SYSTEMS
ALTERNATIVES TO ANIMAL TESTING
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3D TEST SYSTEMS
NEW OPPORTUNITIES FOR
DRUG DEVELOPMENT AND
SUBSTANCE TESTING IN VITRO
N e w d r u g s a n d su b s t a n ce s a re re q uire d to b e te s te d fo r qua li t y, s afe t y an d e f f i c a c y b e fo re mar ke t au t h o r iz at i o n. Du e to t h e la ck of e qui val e nt alte r nat i ve m et h o d s , animal e x p e r im e nt s are an imp o r t ant s t andard
i n s t r u m e nt i n d r u g r e s e a rc h . D u e to s p e c i e s - s p e c i f i c d i f fe r e n c e s , h o w e v e r, a n i m a l e x p e r i m e nt s a r e n o t
a l way s sui t a b l e fo r t h e au t h o r iz at i o n of n e w su b s t an ce s o r t h e a da pt at i o n of n e w t h e ra p i e s to humans .
Therefore, the Fraunhofer IGB has been increasingly engaged
3D test systems for various applications for substance testing
in the development of alternative human test systems that
or stem cell differentiation tests have already been devel-
mimic the complex characteristics of the body and permit the
oped:
investigation of materials according to the ADMET criteria
(absorption, distribution, metabolism, excretion and toxicity).
These test systems are based on in vitro cultured human pri-
3D test systems
mary cells, cell lines, induced-pluripotent stem cells or adult
stem cells. In order to ensure cell functionality in vitro, culture
Skin equivalent
conditions are created that are similar to the natural microen-
Skin cancer model
vironment of the cell in the body. For specific applications,
Caco-2 intestine model
co-cultures with other cell types and custom-designed carrier
Trachea model
substrates must be employed.
Cardiac muscle model
Blood vessel model
The Department of Cell and Tissue Engineering is specialized
in constructing human three-dimensional (3D) tissues. The 3D
nature of the scaffolds considerably affect parameters such
as metabolic activity, viability, division, morphology and differentiation status and thus, ultimately, the function of the
tissue as a test model.
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Why ADMET?
Basic drug discovery research can identify chemical compounds as effective drug candidates. However, questions
regarding compound safety must be answered. ADMET is
an acronym in pharmacokinetics and pharmacology for absorption, distribution, metabolism, excretion and toxicology. These criteria are important for describing the properties of a drug candidate.
With the investigation of ADMET parameters, it is possible
to describe the disposition of pharmaceutical compounds
within an organism, e.g. their absorption in the digestive
tract or their distribution over the bloodstream. In the
body, substances are subject to biochemical conversions
that can lead to ineffective or toxic degradation products.
Therefore, if and how a substance is metabolized and excreted is also subject to investigation.
With our 3D test systems, we can examine various ADMET
criteria in vitro at an earlier stage of active substance development to make relevant projections on the in vivo effects that are to be expected. Such early and cost-effective
indications can play a major role in a company’s decision to
pursue a drug candidate, saving millions in R&D costs.
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SKIN EQUIVALENT
After many years of development, the Fraunhofer IGB has a
In vitro model for human squamous cell carcinoma
patented three-dimensional two-layer human skin equivalent
(patent ID: EP 1 290 145B1 and US8 222 031B2), which very
With 400,000–600,000 new cases per year worldwide,
closely matches natural skin. The dermis layer of the model is
human squamous cell carcinoma (SCC) is one of the most
composed of dermal fibroblasts. They are embedded into a
common types of skin cancer. SCC has its origin in the
biomatrix consisting of tissue-typical matrix proteins and serve
development of atypical epidermal keratinocytes and may
as a scaffold for the epidermal keratinocytes seeded on them.
result from so-called precancerous tissue changes associated
with an increased risk of cancer, such as actinic keratosis or
During a three-week culture in special culture conditions, the
Bowen’s disease. Caused by chronic photo damage, white
keratinocytes differentiate into a multilevel epidermis with
skin cancer occurs mainly in fair-skinned people with light-
Stratum basale, Stratum spinosum, Stratum granulosum and
sensitive skin after years of exposure to UV. Despite high cure
Stratum corneum. The horny layer plays an important barrier
rates, the early treatment of superficial skin cancer is recom-
function for substance penetration. Due to the interaction of
mended because the formation of cancer metastases is still
fibroblasts and keratinocytes between dermal and epidermal
a potential threat. A promising new therapeutic approach is
sections of the skin model, a functional basal membrane con-
photodynamic therapy (PDT), in which a chemical compound
sisting of matrix proteins develops in the model.
accumulates selectively in the tumor cells and makes them
more sensitive to light. Subsequently, the tumor and the
The defined two-layered structure of the skin equivalent
healthy tissue surrounding it are irradiated with light of a suit-
permits the analysis of a wide variety of interactions between
able wavelength. This photochemical process generates toxic
epidermal and cutaneous cells. The skin model is certified for
substances, leading to cell death.
the examination of the biocompatibility of medical devices
(DIN ISO 10993-5) and can be extended – as required – by
At the Fraunhofer IGB, we have developed a white skin cancer
other cells such as melanocytes, skin tumor cells or micro-
model for the testing of new photosensitizers that allow for
vascular endothelial cells. Like the skin equivalent, these ex-
the optimization of novel therapy approaches [1]. We intro-
tended models can be applied as a preliminary stage to animal
duced white skin cancer cells (cell line SCC-25) into our well-
experiments for investigations of functional parameters such
established three-dimensional skin model creating the first
as the penetration, distribution and metabolization of test
in vitro model for squamous cell carcinoma. When the SCC
substances in various tissue layers. Additionally, effects con-
cells are simultaneously introduced with healthy keratinocytes
cerning the proliferation, differentiation, cell death (necrosis,
in defined ratios, both cell types can be co-cultured in the
apoptosis) and the initiation and graduation of tumors of the
skin model producing a test system that is morphologically
applied cell types can be examined.
comparable to the early stage of squamous cell carcinoma
in humans. For late stages of the disease, full-thickness skin
models were developed where the epidermis is composed
1 3D skin model.
solely of SCC cells. In addition, SCC cells were integrated into
2 Skin model with tumor nests.
the dermal part of the model to allow the development of
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tumor nests, which is similar to nests found in the very late
In this project, human melanocytes and keratinocytes were
stage of squamous cell carcinoma. Using Raman spectroscopy,
successfully combined to establish the test system. The
we were able to non-destructively distinguish healthy kera-
melanocyte cell markers melan-a and hmb-45 were positively
tinocytes from tumor cells in non-fixed models without the
identified in the skin model and Fontana-Masson staining
need to use of cell-specific markers.
showed that the tissue morphology of the epidermis models
was comparable with that of native human skin. Further-
Our model allows the investigation of new photosensitizing
more, a spectrometric melanin quantification method and a
substances and their effects on healthy and sick cells. We can
L-dihydroxyphenylalanine (DOPA) test procedure for topically
further use the model to test different irradiation protocols
applied melanogenic substances was successfully established
for photodynamic therapy and develop comparative studies
and validated. The melanin content of pigmented epidermis
of various radiation sources to apply to the tumor cells more
models was significantly increased in a dose-dependent
effectively. Furthermore, the model can be used for the devel-
manner by treatment with DOPA. The effect of melanogenic
opment of new photosensitizer formulations that reach tumor
sun protection agents was analyzed with a newly developed
cells located in the deep layers of the skin. In collaboration
parameter used to describe the UV radiation sensitivity of in
with the University of Stuttgart, we have also developed a
vitro epidermis models.
skin model for skin melanoma.
ArtiVasc – Vascularized and standardized artificial skin
In vitro pigmented skin model
equivalent
Skin color is determined by a pigment known as melanin
As part of the circulatory system, vascularization is one of the
which is made by melanocytes. A person’s skin color is
most important and highly challenging issues in the develop-
determined by the amount and type of melanin. Melanin
ment of soft tissue [2]. The role of the circulatory system is to
levels depend on race and amount of sunlight exposure, as
transport nutrients, oxygen, carbon dioxide and blood cells
well as hormonal changes. Exposure to sunlight increases
to and from cells throughout the body and within multilayer
melanin production to protect the skin against ultraviolet
tissues, such as the skin. Without this transport, tissues would
rays. Skin pigmentation disorders such as postinflamma-
become ischemic and die. As part of the twenty-partner
tory hyperpigmentation, melasma and vitiligo affect a great
collaboration within the EU FP7 funded project “ArtiVasc”,
number of people. Pharmaceuticals to treat skin disorders
engineers, physicists, chemists, biologists and physicians are
are typically tested on animal models. At the Fraunhofer IGB,
working on developing standardized processes for producing
we have developed a pigmented in vitro skin model for the
vascularized scaffolds as well as processes to culture cells
characterization of cosmetic and pharmaceutical substances
within the scaffolds in order to rapidly and inexpensively pro-
that modulate the pigmentation of the skin, as well as mela-
duce standardized and vascularized artificial skin.
nogenic self-tanning agents used to increase the natural sun
protection of the skin.
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Contact
Dipl.-Biol. (t.o.) Sibylle Thude
The two main applications for the research into vascularized
Phone +49 711 970-4152
artificial skin are in the fields of regenerative medicine and
[email protected]
pharmaceutical testing. For patients requiring major soft tissue
implants after traumatic injury, our scaffolds represent a safe,
fast and inexpensive tissue substitute. In the pharmaceutical
industry, our scaffold will be used as a substitute for expensive
and ethically disputed pharmaceutical tests on animals. The
synthetic vascularized test beds will be used to simulate the
The Skin Factory – Mass produced organ replicates:
uptake of the pharmaceuticals into the blood. The advantage
tissue engineering on demand
of an artificial mass produced test system is a significant increase in standardization and decrease in cost and production
The production of artificial skin is extremely complex. In
time when compared to animal based vascularized substrates.
order to meet the growing demand for in vitro test systems
Importantly, an animal does not need to be sacrificed to pro-
that are produced at an efficient cost and constant quality,
duce these test systems.
scientists from the Fraunhofer IGB, IPT, IPA and IZI, in a project
financed by the Fraunhofer Zukunftsstiftung (Fraunhofer
The main objective of the work package at the Fraunhofer IGB
Future Foundation), set out to automate this manufacturing
is the development of a vascularized composite graft that is
process. Scientists and engineers were able to fully automate
comparable to native vascularized skin. It will consist of three
the continuous process chain from cell extraction and cell pro-
layers: subcutaneous fatty tissue, dermis and epidermis. We
liferation, up to the creation of a complete three-dimensional
have established standard operating procedures (SOPs) for the
tissue structure. The Fraunhofer IGB and colleagues have
isolation and culture of primary preadipocytes and adipocytes
demonstrated for the first time world-wide, the ability to fully
from subcutaneous fat. The human primary adipocytes and
automate the production of two-layer skin models in a single
fibroblasts were seeded on synthetic and biological scaffolds
system.
to develop an in vitro fatty tissue. This fatty tissue will be combined with a dermal and an epidermal layer to create a 3D
skin equivalent. Finally, the vascular system will be integrated
in the fatty tissue to supply the artificial skin with nutrients.
When complete, the vascularized 3D scaffolds will be a
cutting-edge component based product for both patients and
industry, with the foundation vascularized construct allowing
for the creation of many different organs and tissues.
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www.tissue-factory.com
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INTESTINE TEST MODEL
2D accredited test model
Applications
The standardized in vitro model for the investigation of absorp-
The 3D intestine test system allows the investigation of resorp-
tion mechanisms at the intestinal barrier is based on a 2D test
tion, toxicity and bioavailability of orally applied active sub-
system with Caco-2 cells (colon carcinoma cell line), which is
stances and targeted improvement of formulations. It is also
cultured on an artificial PET insert membrane. This test system
a novel model for the exploration of basic science questions
was considerably improved in the Department of Cell and Tis-
regarding intestinal development and intestinal crypts.
sue Engineering by modifying the cell culture conditions. It is
currently accredited for transport studies across the intestinal
barrier.
3D test system
We developed a dynamic 3D cell co-culture of human Caco-2
cells with primary-isolated human microvascular endothelial
cells (hMECs) on decellularized porcine jejunal segments within
a custom-made dynamic bioreactor system resembling the
apical and basolateral side of the intestine. After 14 days,
histological analyses revealed that the Caco-2 cells resembled
normal primary enterocytes within their native environment, with a high-prismatic morphology. In comparison to
dynamic cultures, cells cultured under static conditions are
flattened. We further evaluated the transport of low permeable substances, such as fluorescein and desmopressin, which
increased within the dynamic bioreactor cultures. Immunohistochemical staining showed a significantly higher expression
of the efflux transport p-glycoprotein (p-gp) under dynamic
culture conditions in comparison to static cultures [4].
1+ 2 Tissue factory.
3 Human intestinal villus.
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2B
IN VITRO TEST SYSTEMS FOR
NOVEL BONE IMPLANTS
Treatments for the diseases and injuries of the musculoskeletal
Standardized in vitro test systems with bone-forming
system are currently focused on the development of new
and bone-degrading cells
composite materials. Many of these material developments
are aimed at improving the biodegradability and mechanical
The standardization of cell-based test systems using osteo-
properties of the load-bearing areas of the implants. To ensure
blasts and their precursor cells to simulate bone formation, as
the utility of the new material, the establishment of biological
well as bone-resorbing cells, the osteoclasts, to mimic bone
test systems for the analysis of ingrowth into the bone and
loss, is the goal of the work package at the Fraunhofer IGB. To
the degradation behavior of implant materials are of particular
assess the ingrowth and the osteoinductive properties of a ma-
relevance. However, there are no standardized systems for the
terial, we investigate the differentiation of human mesenchy-
appropriate analysis of material resorption and osteoinduction,
mal stem cells (hMSCs) into osteoblasts by analyzing specific
which is the material’s ability to stimulate the formation of new
differentiation markers on standard materials, as well as newly
bone, analyzing both osteoblast and osteoclast function. The
developed materials and coatings. Cell adhesion, proliferation
establishment of such systems is part of the Fraunhofer joint
and differentiation are characterized by the qualitative analysis
project “DegraLast” as an alternative or supplement to animal
of type I collagen as well as the quantitative examination of
experiments.
alkaline phosphatase and calcium, which showed a significant
increase in differentiated cells relative to control cells.
Loss of bone substance
Osteoclasts are largely responsible for the resorption of
bone. For the osteodegradation test system, monocytes
were isolated from human peripheral blood and successfully
1 Histological staining of the vitronectin receptor and
the cell nuclei of human mesenchymal stem cells
ferentiated osteoclasts was demonstrated by polynuclear size,
and monocytes. In co-culture, the cells develop an
the restructuring of the cytoskeleton and the expression of
osteoclast phenotype.
specific marker proteins. Furthermore, the activity of the cells
2 Actin cytoskeleton (red) and cell nuclei (blue)
of undifferentiated (A) and differentiated (B)
monocytes.
3 Embryonic stem cell derived cardiomyocytes. F-actin
is represented in blue, DAPI in yellow, and cardiac
troponin in green.
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differentiated into osteoclasts. The characterization of dif-
was determined by the absorption of a bovine bone substitute
material.
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Contact
Dipl.-Biol. Claudia Kleinhans
CARDIOVASCULAR
TEST SYSTEMS
Phone +49 711 970-4073
[email protected]
Cardiovascular disease remains one of the leading causes of
death in the world. In Europe alone, an estimated 10 million
people are affected each year. New pharmaceutical and
regenerative therapies are being developed all over the world.
The Fraunhofer IGB is developing both a cardiac muscle and
blood vessel test systems based on human primary, embryAdvanced model by the co-culture of osteoblasts and
onic, or induced-pluripotent stem cells (iPSC).
osteoclasts
Depending on the application, we can create systems based
The recapitulation of the physiological process of bone remod-
on natural or synthetic scaffolds using custom designed
eling is an effective method to obtain the desired properties
bioreactor systems to mimic the biomechanical properties of
for bone replacement materials. While osteoclasts resorb the
the heart and vessels, including electronic stimulation. We
material, osteoblasts form new bone. Current in vitro studies
can successfully culture embryonic or iPSC-derived and fetal
focus only on one type of cell and investigate either bone re-
cardiomyocytes as well as other cell types of the cardiovascu-
sorption or bone formation. Therefore, we aimed to establish a
lar system. Our numerous non-contact diagnosis techniques
co-culture of both cell types to simulate the bone remodeling
allow for the continuous monitoring of the cardiovascular test
process and to develop as an extended test system. In the
systems.
development of in vitro co-culture system, we first identified
optimal culture conditions for the two cell types. We then
developed a method that leads to osteoclast differentiation
For more information, please read our cardiovascular
without addition of differentiation factors, which allowed for
and non-invasive diagnostics brochures.
the co-culture of both cell types.
Applications
Our multipotent stem cell bone test system is designed for
the investigation of material resorption and osteoinduction,
analyzing both osteoblast and osteoclast function.
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20 μm
TRACHEA MODEL
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Contact
Svenja Hinderer
Natural matrices
Phone +49 711 970-4082
[email protected]
The airway is a substantial barrier for inhaled materials such as fine dust, nanoparticles and also pathogenic
microorganisms. It has a special cleaning mechanism, with
which the foreign particles are trapped by means of mucus
and transported out of the respiratory pathway by specific
movements of the cilia. A trachea model was developed at
the Fraunhofer IGB for the investigation of the effect of pen-
achieve in culture. Moreover, interactions of the electrospun
etrated particles on the cells of the upper respiratory system.
scaffolds with immune-mediated mechanisms showed low
Here, human trachea epithelial cells or appropriate cell lines
immunogenicity [5, 6].
(Calu-3) were seeded upon decellularized small intestine segments. Calu-3 cells grew on the biological matrix and form
the highly prismatic morphology of trachea epithelial cells.
Applications
The trachea models are cultured in a bioreactor system that
simulates human respiration.
With our trachea model, various issues concerning the
absorption, biocompatibility and toxicity of a wide variety of
materials can be examined. Cellular reactions after introduc-
Synthetic electrospun scaffolds
tion of aerosols or solids into the reactor can be investigated
using different methods. Penetrated particles can be
Decorin is a structural and functional proteoglycan (PG)
demonstrated with histological methods, while quantitative
residing in the complex network of extracellular matrix (ECM)
methods for the analysis of inflammation markers or other
of the trachea. To biofunctionalize an electrospun synthetic
metabolic enzymes in the medium permit conclusions on
scaffold for tissue engineering applications, we introduced
toxicity and biocompatibility.
decorin into the polymer solution before electrospinning
the scaffold. Scanning electron microscopy, atomic force
microscopy, contact angle measurements and dynamic
mechanical analysis were used to analyze the spun scaffolds.
PG functionality was confirmed with immunostaining and
1,9-dimethylmethylene blue. To determine cell-matrix-interactions, tracheal cells (hPAECs) were seeded and analyzed
using an FOXJ1-antibody. Our analysis showed a significant
increase in cell proliferation, which is extremely difficult to
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3D TEST SYSTEM
APPLICATION AREAS
ADMET
skin equivalent penetration, irritation and toxicity studies
absorption, distribution, toxicity
intestine test system resorption and toxicity studies, testing of
drug formulations
toxicity
trachea model
resorption, biocompatibility and toxicity studies
absorption, toxicity
bone model
material resorption and osteoinduction
cardiac muscle
cell function, cytotoxicity, pace testing
cardiac blood vessel
biocompatibility, functionality
RANGE OF SERVICES
Contact
Prof. Dr. Katja Schenke-Layland
Cell isolation from primary material (biopsies)
Head of Department
Derivation of tissue-specific cell types from pluripotent
Cell and Tissue Engineering
Phone +49 711 970-4082
stem cells
In-depth cell characterization
katja.schenke-layland@
Construction, establishment and validation of 3D static
igb.fraunhofer.de
and dynamic testing systems
Studies and testing services
Prof. Dr. Petra Kluger
Histology, molecular, cellular and biochemical analyses
Head of Department
Cell and Tissue Engineering
of tissues and medium supernatants
Phone +49 711 970-4072
[email protected]
References
[1] Brauchle, E.; Johannsen, H.; et Int, and Schenke-Layland K.
(2013) Design and analysis of a squamous cell carcinoma in vitro
model system. Biomaterials
[2] Novosel, E. C.; Kleinhans, C. and Kluger, P. J. (2011)
Vascularization is the key challenge in tissue engineering. Advanced
Drug Delivery Reviews
[3] Novosel, E. C.; Meyer, W. M.; et Int, and Kluger, P. J. (2011)
Evaluation of Cell-Material Interactions on Newly Designed,
Printable Polymers for Tissue Engineering Applications. Advanced
Engineering Materials
[4] Pusch, J.; Votteler, M.; et Int, and Schenke-Layland K. (2011)
The physiological performance of a three-dimensional model that
mimics the microenvironment of the small intestine. Biomaterials
[5] Hinderer, S.; Schesny, M.; et Int, and Schenke-Layland K. (2012)
Engineering of fibrillar decorin matrices for a tissue-engineered
trachea. Biomaterials
[6] Hinderer, S.; Schenke-Layland, K. (2013) Tracheal tissue
engineering: building on a strong foundation. Expert Review of
1 Native trachea tissue.
Medical Devices
2 Electrospun decorin containing
scaffold seeded with hPAECs.
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Fraunhofer Institute
for Interfacial Engineering
Phone +49 711 970-4401
Director
and Biotechnology IGB
Fax
Prof. Dr. Thomas Hirth
Nobelstrasse 12
[email protected]
Phone +49 711 970-4400
70569 Stuttgart | Germany
www.igb.fraunhofer.de
[email protected]
+49 711 970-4200
Fraunhofer IGB
The Fraunhofer IGB develops and optimizes processes and products in the fields of medicine,
pharmacy, chemistry, the environment and energy. We combine the highest scientific standards with professional know-how in our competence areas of Interfacial Engineering and
Materials Science, Molecular Biotechnology, Physical Process Technology, Environmental Biotechnology and Bioprocess Engineering, as well as Cell and Tissue Engineering – always with
a view to economic efficiency and sustainability. Our strengths are to offer complete solutions
from laboratory scale to pilot plant. Customers also benefit from the constructive interplay
of the various disciplines at our institute, which opens up new approaches in areas such as
medical engineering, nanotechnology, industrial biotechnology, and environmental technology.
The Fraunhofer IGB is one of 67 institutes and independent research units of the FraunhoferGesellschaft, Europe’s largest organization for application-oriented research.
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