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F R A U N H O F E R I N S T I T U T E F O R I N T E R F A C I A L E N G I N E E R I N G A N D B I O T E C H N O L O G Y I G B

HUMAN ORGAN-LIKE THREE-DIMENSIONAL TEST SYSTEMSALTERNATIVES TO ANIMAL TESTING

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3D TEST SYSTEMS

NEW OPPORTUNITIES FOR DRUG DEVELOPMENT AND SUBSTANCE TESTING IN VITRO

New drugs and substances are required to be tes ted for qual i t y, safet y and ef f icac y before market autho -

r izat ion. Due to the lack of equivalent a l ternat ive methods, animal exper iments are an impor tant s tandard

ins t rument in drug research. Due to spec ies - spec i f i c d i f ferences , however, animal exper iment s are not

always sui table for the author izat ion of new substances or the adaptat ion of new therapies to humans.

Therefore, the Fraunhofer IGB has been increasingly engaged

in the development of alternative human test systems that

mimic the complex characteristics of the body and permit the

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-

mary cells, cell lines, induced-pluripotent stem cells or adult

stem cells. In order to ensure cell functionality in vitro, culture

conditions are created that are similar to the natural microen-

vironment of the cell in the body. For specific applications,

co-cultures with other cell types and custom-designed carrier

substrates must be employed.

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 dif-

ferentiation status and thus, ultimately, the function of the

tissue as a test model.

3D test systems for various applications for substance testing

or stem cell differentiation tests have already been devel-

oped:

3D test systems

� Skin equivalent

� Skin cancer model

� Caco-2 intestine model

� Trachea model

� Cardiac muscle model

� Blood vessel model

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Basic drug discovery research can identify chemical com-

pounds as effective drug candidates. However, questions

regarding compound safety must be answered. ADMET is

an acronym in pharmacokinetics and pharmacology for ab-

sorption, distribution, metabolism, excretion and toxicolo-

gy. These criteria are important for describing the proper-

ties 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 ex-

creted 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 de-

velopment to make relevant projections on the in vivo ef-

fects 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.

Why ADMET?

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After many years of development, the Fraunhofer IGB has a

patented three-dimensional two-layer human skin equivalent

(patent ID: EP 1 290 145B1 and US8 222 031B2), which very

closely matches natural skin. The dermis layer of the model is

composed of dermal fibroblasts. They are embedded into a

biomatrix consisting of tissue-typical matrix proteins and serve

as a scaffold for the epidermal keratinocytes seeded on them.

During a three-week culture in special culture conditions, the

keratinocytes differentiate into a multilevel epidermis with

Stratum basale, Stratum spinosum, Stratum granulosum and

Stratum corneum. The horny layer plays an important barrier

function for substance penetration. Due to the interaction of

fibroblasts and keratinocytes between dermal and epidermal

sections of the skin model, a functional basal membrane con-

sisting of matrix proteins develops in the model.

The defined two-layered structure of the skin equivalent

permits the analysis of a wide variety of interactions between

epidermal and cutaneous cells. The skin model is certified for

the examination of the biocompatibility of medical devices

(DIN ISO 10993-5) and can be extended – as required – by

other cells such as melanocytes, skin tumor cells or micro-

vascular endothelial cells. Like the skin equivalent, these ex-

tended models can be applied as a preliminary stage to animal

experiments for investigations of functional parameters such

as the penetration, distribution and metabolization of test

substances in various tissue layers. Additionally, effects con-

cerning the proliferation, differentiation, cell death (necrosis,

apoptosis) and the initiation and graduation of tumors of the

applied cell types can be examined.

In vitro model for human squamous cell carcinoma

With 400,000–600,000 new cases per year worldwide,

human squamous cell carcinoma (SCC) is one of the most

common types of skin cancer. SCC has its origin in the

development of atypical epidermal keratinocytes and may

result from so-called precancerous tissue changes associated

with an increased risk of cancer, such as actinic keratosis or

Bowen’s disease. Caused by chronic photo damage, white

skin cancer occurs mainly in fair-skinned people with light-

sensitive skin after years of exposure to UV. Despite high cure

rates, the early treatment of superficial skin cancer is recom-

mended because the formation of cancer metastases is still

a potential threat. A promising new therapeutic approach is

photodynamic therapy (PDT), in which a chemical compound

accumulates selectively in the tumor cells and makes them

more sensitive to light. Subsequently, the tumor and the

healthy tissue surrounding it are irradiated with light of a suit-

able wavelength. This photochemical process generates toxic

substances, leading to cell death.

At the Fraunhofer IGB, we have developed a white skin cancer

model for the testing of new photosensitizers that allow for

the optimization of novel therapy approaches [1]. We intro-

duced white skin cancer cells (cell line SCC-25) into our well-

established three-dimensional skin model creating the first

in vitro model for squamous cell carcinoma. When the SCC

cells are simultaneously introduced with healthy keratinocytes

in defined ratios, both cell types can be co-cultured in the

skin model producing a test system that is morphologically

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

solely of SCC cells. In addition, SCC cells were integrated into

the dermal part of the model to allow the development of

1 3D skin model.

2 Skin model with tumor nests.

SKIN EQUIVALENT

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tumor nests, which is similar to nests found in the very late

stage of squamous cell carcinoma. Using Raman spectroscopy,

we were able to non-destructively distinguish healthy kera-

tinocytes from tumor cells in non-fixed models without the

need to use of cell-specific markers.

Our model allows the investigation of new photosensitizing

substances and their effects on healthy and sick cells. We can

further use the model to test different irradiation protocols

for photodynamic therapy and develop comparative studies

of various radiation sources to apply to the tumor cells more

effectively. Furthermore, the model can be used for the devel-

opment of new photosensitizer formulations that reach tumor

cells located in the deep layers of the skin. In collaboration

with the University of Stuttgart, we have also developed a

skin model for skin melanoma.

In vitro pigmented skin model

Skin color is determined by a pigment known as melanin

which is made by melanocytes. A person’s skin color is

determined by the amount and type of melanin. Melanin

levels depend on race and amount of sunlight exposure, as

well as hormonal changes. Exposure to sunlight increases

melanin production to protect the skin against ultraviolet

rays. Skin pigmentation disorders such as postinflamma-

tory hyperpigmentation, melasma and vitiligo affect a great

number of people. Pharmaceuticals to treat skin disorders

are typically tested on animal models. At the Fraunhofer IGB,

we have developed a pigmented in vitro skin model for the

characterization of cosmetic and pharmaceutical substances

that modulate the pigmentation of the skin, as well as mela-

nogenic self-tanning agents used to increase the natural sun

protection of the skin.

In this project, human melanocytes and keratinocytes were

successfully combined to establish the test system. The

melanocyte cell markers melan-a and hmb-45 were positively

identified in the skin model and Fontana-Masson staining

showed that the tissue morphology of the epidermis models

was comparable with that of native human skin. Further-

more, a spectrometric melanin quantification method and a

L-dihydroxyphenylalanine (DOPA) test procedure for topically

applied melanogenic substances was successfully established

and validated. The melanin content of pigmented epidermis

models was significantly increased in a dose-dependent

manner by treatment with DOPA. The effect of melanogenic

sun protection agents was analyzed with a newly developed

parameter used to describe the UV radiation sensitivity of in

vitro epidermis models.

ArtiVasc – Vascularized and standardized artificial skin

equivalent

As part of the circulatory system, vascularization is one of the

most important and highly challenging issues in the develop-

ment of soft tissue [2]. The role of the circulatory system is to

transport nutrients, oxygen, carbon dioxide and blood cells

to and from cells throughout the body and within multilayer

tissues, such as the skin. Without this transport, tissues would

become ischemic and die. As part of the twenty-partner

collaboration within the EU FP7 funded project “ArtiVasc”,

engineers, physicists, chemists, biologists and physicians are

working on developing standardized processes for producing

vascularized scaffolds as well as processes to culture cells

within the scaffolds in order to rapidly and inexpensively pro-

duce standardized and vascularized artificial skin.

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The two main applications for the research into vascularized

artificial skin are in the fields of regenerative medicine and

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

uptake of the pharmaceuticals into the blood. The advantage

of an artificial mass produced test system is a significant in-

crease in standardization and decrease in cost and production

time when compared to animal based vascularized substrates.

Importantly, an animal does not need to be sacrificed to pro-

duce these test systems.

The main objective of the work package at the Fraunhofer IGB

is the development of a vascularized composite graft that is

comparable to native vascularized skin. It will consist of three

layers: subcutaneous fatty tissue, dermis and epidermis. We

have established standard operating procedures (SOPs) for the

isolation and culture of primary preadipocytes and adipocytes

from subcutaneous fat. The human primary adipocytes and

fibroblasts were seeded on synthetic and biological scaffolds

to develop an in vitro fatty tissue. This fatty tissue will be com-

bined 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.

The Skin Factory – Mass produced organ replicates:

tissue engineering on demand

The production of artificial skin is extremely complex. In

order to meet the growing demand for in vitro test systems

that are produced at an efficient cost and constant quality,

scientists from the Fraunhofer IGB, IPT, IPA and IZI, in a project

financed by the Fraunhofer Zukunftsstiftung (Fraunhofer

Future Foundation), set out to automate this manufacturing

process. Scientists and engineers were able to fully automate

the continuous process chain from cell extraction and cell pro-

liferation, up to the creation of a complete three-dimensional

tissue structure. The Fraunhofer IGB and colleagues have

demonstrated for the first time world-wide, the ability to fully

automate the production of two-layer skin models in a single

system.

www.tissue-factory.com

Contact

Dipl.-Biol. (t.o.) Sibylle Thude

Phone +49 711 970-4152

sibylle.thude@igb.fraunhofer.de

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2D accredited test model

The standardized in vitro model for the investigation of absorp-

tion mechanisms at the intestinal barrier is based on a 2D test

system with Caco-2 cells (colon carcinoma cell line), which is

cultured on an artificial PET insert membrane. This test system

was considerably improved in the Department of Cell and Tis-

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 environ-

ment, with a high-prismatic morphology. In comparison to

dynamic cultures, cells cultured under static conditions are

flattened. We further evaluated the transport of low perme-

able substances, such as fluorescein and desmopressin, which

increased within the dynamic bioreactor cultures. Immunohis-

tochemical staining showed a significantly higher expression

of the efflux transport p-glycoprotein (p-gp) under dynamic

culture conditions in comparison to static cultures [4].

Applications

The 3D intestine test system allows the investigation of resorp-

tion, toxicity and bioavailability of orally applied active sub-

stances and targeted improvement of formulations. It is also

a novel model for the exploration of basic science questions

regarding intestinal development and intestinal crypts.

INTESTINE TEST MODEL

1+2 Tissue factory.

3 Human intestinal villus.

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Treatments for the diseases and injuries of the musculoskeletal

system are currently focused on the development of new

composite materials. Many of these material developments

are aimed at improving the biodegradability and mechanical

properties of the load-bearing areas of the implants. To ensure

the utility of the new material, the establishment of biological

test systems for the analysis of ingrowth into the bone and

the degradation behavior of implant materials are of particular

relevance. However, there are no standardized systems for the

appropriate analysis of material resorption and osteoinduction,

which is the material’s ability to stimulate the formation of new

bone, analyzing both osteoblast and osteoclast function. The

establishment of such systems is part of the Fraunhofer joint

project “DegraLast” as an alternative or supplement to animal

experiments.

Standardized in vitro test systems with bone-forming

and bone-degrading cells

The standardization of cell-based test systems using osteo-

blasts and their precursor cells to simulate bone formation, as

well as bone-resorbing cells, the osteoclasts, to mimic bone

loss, is the goal of the work package at the Fraunhofer IGB. To

assess the ingrowth and the osteoinductive properties of a ma-

terial, we investigate the differentiation of human mesenchy-

mal stem cells (hMSCs) into osteoblasts by analyzing specific

differentiation markers on standard materials, as well as newly

developed materials and coatings. Cell adhesion, proliferation

and differentiation are characterized by the qualitative analysis

of type I collagen as well as the quantitative examination of

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

differentiated into osteoclasts. The characterization of dif-

ferentiated osteoclasts was demonstrated by polynuclear size,

the restructuring of the cytoskeleton and the expression of

specific marker proteins. Furthermore, the activity of the cells

was determined by the absorption of a bovine bone substitute

material.

IN VITRO TEST SYSTEMS FOR NOVEL BONE IMPLANTS

1 Histological staining of the vitronectin receptor and

the cell nuclei of human mesenchymal stem cells

and monocytes. In co-culture, the cells develop an

osteoclast phenotype.

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.

1 2 A 2 B

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Advanced model by the co-culture of osteoblasts and

osteoclasts

The recapitulation of the physiological process of bone remod-

eling is an effective method to obtain the desired properties

for bone replacement materials. While osteoclasts resorb the

material, osteoblasts form new bone. Current in vitro studies

focus only on one type of cell and investigate either bone re-

sorption or bone formation. Therefore, we aimed to establish a

co-culture of both cell types to simulate the bone remodeling

process and to develop as an extended test system. In the

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

without addition of differentiation factors, which allowed for

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.

Contact

Dipl.-Biol. Claudia Kleinhans

Phone +49 711 970-4073

claudia.kleinhans@igb.fraunhofer.de

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, embry-

onic, or induced-pluripotent stem cells (iPSC).

Depending on the application, we can create systems based

on natural or synthetic scaffolds using custom designed

bioreactor systems to mimic the biomechanical properties of

the heart and vessels, including electronic stimulation. We

can successfully culture embryonic or iPSC-derived and fetal

cardiomyocytes as well as other cell types of the cardiovascu-

lar system. Our numerous non-contact diagnosis techniques

allow for the continuous monitoring of the cardiovascular test

systems.

For more information, please read our cardiovascular

and non-invasive diagnostics brochures.

CARDIOVASCULAR TEST SYSTEMS

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achieve in culture. Moreover, interactions of the electrospun

scaffolds with immune-mediated mechanisms showed low

immunogenicity [5, 6].

Applications

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-

tion of aerosols or solids into the reactor can be investigated

using different methods. Penetrated particles can be

demonstrated with histological methods, while quantitative

methods for the analysis of inflammation markers or other

metabolic enzymes in the medium permit conclusions on

toxicity and biocompatibility.

Natural matrices

The airway is a substantial barrier for inhaled materi-

als 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-

etrated particles on the cells of the upper respiratory system.

Here, human trachea epithelial cells or appropriate cell lines

(Calu-3) were seeded upon decellularized small intestine seg-

ments. Calu-3 cells grew on the biological matrix and form

the highly prismatic morphology of trachea epithelial cells.

The trachea models are cultured in a bioreactor system that

simulates human respiration.

Synthetic electrospun scaffolds

Decorin is a structural and functional proteoglycan (PG)

residing in the complex network of extracellular matrix (ECM)

of the trachea. To biofunctionalize an electrospun synthetic

scaffold for tissue engineering applications, we introduced

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-inter-

actions, 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

TRACHEA MODEL

20 μm

Contact

Svenja Hinderer

Phone +49 711 970-4082

svenja.hinderer@igb.fraunhofer.de

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Prof. Dr. Katja Schenke-Layland

Head of Department

Cell and Tissue Engineering

Phone +49 711 970-4082

katja.schenke-layland@

igb.fraunhofer.de

Prof. Dr. Petra Kluger

Head of Department

Cell and Tissue Engineering

Phone +49 711 970-4072

petra.kluger@igb.fraunhofer.de

� Cell isolation from primary material (biopsies)

� Derivation of tissue-specific cell types from pluripotent

stem cells

� In-depth cell characterization

� Construction, establishment and validation of 3D static

and dynamic testing systems

� Studies and testing services

� Histology, molecular, cellular and biochemical analyses

of tissues and medium supernatants

RANGE OF SERVICES Contact

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

Medical Devices1 Native trachea tissue.

2 Electrospun decorin containing

scaffold seeded with hPAECs.

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 toxicity drug formulations

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

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Stay in contact:

Fraunhofer Institute

for Interfacial Engineering

and Biotechnology IGB

Nobelstrasse 12

70569 Stuttgart | Germany

Phone +49 711 970-4401

Fax +49 711 970-4200

info@igb.fraunhofer.de

www.igb.fraunhofer.de

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 stan-

dards with professional know-how in our competence areas of Interfacial Engineering and

Materials Science, Molecular Biotechnology, Physical Process Technology, Environmental Bio-

technology 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 Fraunhofer-

Gesellschaft, Europe’s largest organization for application-oriented research.

www.igb.fraunhofer.de

Director

Prof. Dr. Thomas Hirth

Phone +49 711 970-4400

thomas.hirth@igb.fraunhofer.de