A variety of highly specialised in vitro testing services (CRO activity) are offered by ATERA and its partnering laboratories.

Testing services are either performed on ATERA’s currently available tissue models (> see products), but also on advanced human tissue models that are currently not commercialized, including:

  • Reconstructed human vascularised skin model
  • Reconstructed human full-thickness skin
  • Reconstructed human intestinal epithelium
  • Reconstructed human lung epithelium
  • Ex-vivo human corneal model

The following standardized in vitro test protocols for toxicity and efficacy are established at ATERA’s CRO laboratories:

  • Toxicity tests
  • Efficacy tests

EFFICACY TESTING

ATERA TEST METHODS

Title Description Support
Antioxidant property SOD, Catalase immunostainings ATERA Tissue Models
Gene expression
Cellular viability assay (MTT)
mtDNAQuant index ATERA Tissue Models
SOD (Superoxide Dismutase) activity measurement by spectrophotometric assay
Protein quantification assay (ELISA) in supernatants or lysates
Bioavailability Chemical quantification in culture medium by HPLC ATERA Tissue Models
Hydration effect H&E Histology ATERA Tissue Models
Immunohistochemistry (Aquaporin,Claudin,Loricrin,…)
Gene expression
Photoprotective activity following UVA/UVB stress Protein quantification assay (ELISA) in supernatants or lysates ATERA Skin and Eye models
Cellular viability assay (MTT) ATERA Skin and Eye Models
mtDNAQuant index
Immunohistochemistry
Gene expression
Pigmentation/depigmentation activity Melanic index (Mexameter) ATERA Skin Model RHPE Phototype II, IV, VI
H&E and Fontana Masson Stainings
HMB45 Immunostaining
Anti-microbial properties Bacterial growth (metabolic activity) ATERA RHE
Cellular viability assay (MTT)
Histology H&E
Immunohistochemistry

> Skin-VaSc-TERM® – Vascularized Skin Testing

The vascular system in the human skin is a critical and essential element in the development of both healthy skin (exchange of nutritional elements as well as detoxification) and skin diseases, the penetration of substances, and the progression of skin cancer. The objective of the model is to replace classical animal experiments for scientific questions that comprise an interaction between the mesenchymal, epithelial, and endothelial cells. As an advantage to currently in vivo or ex vivo systems, the models allow a close control of the experimental setup.

Skin-VaSc-TERM®

The Skin-VaSc-TERM® is an advanced human skin model, consisting of dermal and epidermal part with a perfused vascular network in vitro closely resembling native human skin. The model was developed by Florian Groeber and Heike Walles of the Fraunhofer IGB and is available for advanced testing applications at ATERA. To generate the vascularized skin equivalent, skin-specific cells, such as primary human keratinocytes and fibroblasts, are seeded into the so called biological vascularized scaffold, known as BioVaSc-TERM®. As a unique feature, the BioVaSc-TERM® allows the additional seeding of primary human microvascular endothelial cells into the vasculature structures within the BioVaSc-TERM®. The matrix is then placed in a bioreactor system that is capable of perfusing the vasculature of the Skin-VaSc-TERM® and to culture the model at the air-liquid interface. Due to the perfusion of the vasculature the endothelium is cultured under physiological conditions and different cell types such as immune cells can be introduced in the skin model. Thereby the Skin-VaSc-TERM® can be used as a model system in various skin diseases, in which the interplay between the immune system and the endothelium is vital.

Please note that the human skin vascularized skin equivalent is not available for sale and only proposed for R&D and safety assessment.

Hydrophilic and hydrophobic ingredients or finished products. Additional the model can be adapted to specific scientific questions e.g. the interaction with other cell types such as melanoma cells, immune cells or microorganisms.

Depending on the applications a wide variety of test methods can be employed for the analysis. These methods include non-destructive impedance measurements (ImpSpec), histology, protein quantitation, immunolabelling and/or gene expression at different time points depending on specific needs.

——

Impedance spectrometry

To measure the epithelial integrity a highly sensitive, non-invasive method called impedance spectroscopy (ImpSpec) can be performed within the culture reactor. For this, the impedance is measured over a frequency range from 1 Hz to 100 kHz at 40 logarithmically distributed sampling points. From these spectra, electrical characteristics such as the capacitance and the ohmic resistance are extrapolated that are predictive for the status of the skin barrier.
——

H&E Histology

Tissues are fixed in a buffered 4% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin and eosin (H&E) allowing microscopic evaluation.
——

Viability assay

The MTT assay is used to quantify tissue viability. The tissue viability assay is based on the enzymatic conversion of the yellow dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue Thiazolyl tetrazolium bromide) by the viable cells of the tissue construct into a blue formazan salt that is subsequently quantitatively measured after extraction with isopropanol.
——

Protein quantitation

The enzyme-linked immunosorbent assay (ELISA) is used to quantify proteins present in supernatants or lysates, including connective tissue components as collagenous or elastic components, or cell signaling molecules as interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors (TNF).
——

Immunohistochemistry

Type IV or VII Collagens, laminins, cytokeratins or other targeted proteins are visualized on fixed tissue sections by indirect immunofluorescence labelling and microscopic analysis.
——

Gene expression

Gene expression profile analysis after compound exposure of ATERA 3D tissue models can be performed: total RNAs are extracted, processed for retro-transcription, amplification and labeling, microarrays hybridized and regulated transcripts are analyzed to correlate the modulation of gene expression with specific cellular events.

General characteristics

The vascularized skin model has a surface area of 2 x 4 cm² and is fixed in a polycarbonate frame that is placed in a custom bioreactor system. For experimental studies, test products can be applied topically on the epidermal skin surface or systemically through the vasculature system. Moreover, the experimental setting allows sampling from the vasculature or the basolateral side. Thus, multiple application scenarios are possible depending on the scientific question. As an advantage to other currently available full thickness skin models, the unique physiological collagen composition of the SkinVaSc-TERM® allows long term stability of the model and thus testing strategies over several weeks.
——

Epidermis

The surface of the model is covered homogenously by a well-stratified epidermal layer. Within the epidermis keratinocytes differentiate naturally and form a clear stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG) and stratum corneum (SC). Moreover, keratinocytes express differentiation markers such as cytokeratin 10 (CK-10), cytokeratin 14 (CK-14), Filaggrin (Filagg.) and Involucrin (Invol.) comparable to human skin.

Efficacy-Vascularized Skin Testing-results-epidermis

Figure 1: Schematic of the generation process of the vascularized skin model.

——

Vasculature

Within the dermal part (D) clear vascular structures can be seen that form a highly branched network. During culture these vessels are perfused with culture medium with a physiological pulsatile pressure profile between 80 and 120 mmHg. Histological analysis revealed that the vessels are lined with a single layer of endothelial cells that express PECAM-1 and von Willebrand Factor (vWF).

 

Efficacy-Vascularized Skin Testing-results-epidermis-vasculature

Figure 2: Characterization of the vascularized skin model using histology (H&E), immunostaining and MTT staining.

Scheller K. et al.

Upcyte® microvascular endothelial cells repopulate decellularized scaffold.

Tissue Eng Part C. 2013. doi: 10.1089/ten.TEC.2011.0723.

——

Groeber F. et al.

A bioreactor system for interfacial culture and physiological perfusion of vascularized tissue equivalents.

Biotechnol J. 2013. doi: 10.1002/biot.201200160

——

Groeber F. et al.

A first vascularized skin equivalent as an alternative to animal experimentation.

ALTEX. 2016 May 15. doi: 10.14573/altex.1604041.

> Anti-ageing effect

To evaluate the efficacy of cosmetic ingredients and finished products intended to rejuvenate the skin or to prevent or slowing down ageing processes.

RHFTS

The ATERA – Reconstructed Human Full Thickness Skin Models (RHFTS) is an advanced human skin model, consisting of dermal and epidermal part closely resembling native human skin.
——

RHE

The ATERA – Reconstructed Human Epidermis model (RHE) is proposed in its standard configuration e.g. cultured for 17 days. Depending on specific needs, customized tissues can be reconstructed with epidermal cells of different donors/ age groups or cultured under specific ageing-induced conditions.
——

Other models

Other ATERA – Human Reconstructed Human Tissue models, including gingival, corneal etc. can be used to studying anti-ageing effects.

Hydrophilic and hydrophobic ingredients or finished products

Multiple Endpoint Analysis (MEA) including histology, protein quantitation, immunolabelling, gene expression can be performed to assess the effects of a topical or systemic treatment on ATERA Reconstructed Human Tissue models. Criteria of study can be adapted depending on specific needs.

Antiageing-effect-principleH&E Histology

Tissues are fixed in a balanced 4% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin and eosin (H&E) allowing microscopic evaluation.

——

Viability assay

The MTT assay is used for quantifying tissue viability. The tissue viability assay is based on the enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide) by the viable cells of the tissue construct into a blue formazan salt that is subsequently quantitatively measured after extraction with isopropanol .

——

Protein quantitation

The enzyme-linked immunosorbent assay (ELISA) is used to quantify proteins present in supernatants or lysates, including connective tissue components as collagenous or elastic components, or cell signaling molecules as interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors (TNF).

——

Immunohistochemistry

Type IV or VII Collagens, laminins, cytokeratins or other targeted proteins are visualized on fixed tissue sections by indirect immunofluorescence labelling and microscopic analysis.

——

Gene expression

Gene expression profile analysis after compound exposure of ATERA 3D tissue models can be performed: total RNAs are extracted, processed for retro-transcription, amplification and labeling, microarrays hybridized and regulated transcripts are analyzed to correlate the modulation of gene expression with specific cellular events.

> Anti-inflammatory effect

To evaluate the efficacy of cosmetic ingredients and finished products intended to prevent or slow down the inflammatory condition of the skin or the targeted tissue.

RHE

The ATERA – Reconstructed Human Epidermis model (RHE) is proposed in its standard configuration e.g. cultured for 17 days to study the anti-inflammatory effects.
——

RHO

The ATERA – Reconstructed Human Oral epithelium model (RHO) is proposed to study the effects of substances on inflammatory pathway of multilayer mucosal equivalent.
——

Other models

Other ATERA – Human Reconstructed Human Tissue models, including gingival, corneal etc. can be used to studying anti-inflammatory effects.

Hydrophilic and hydrophobic ingredients or finished products

Multiple Endpoint Analysis (MEA) including histology, protein quantitation, immunolabelling, gene expression can be performed to assess the effects of a topical or systemic anti-inflammatory treatment on ATERA Reconstructed Human Tissue models. Hydrocortisone is proposed as positive control. Criteria of study can be adapted depending on specific needs.

Anti-inflammatory-effect-principleH&E Histology

Tissues are fixed in a balanced 4% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin and eosin (H&E) stain allowing microscopic evaluation.

——

Viability assay

The MTT assay is used for quantifying tissue viability. The tissue viability assay is based on the enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide) by the viable cells of the tissue construct into a blue MTT formazan salt that is subsequently quantitatively measured after extraction with isopropanol.

——

Protein quantitation

The enzyme-linked immunosorbent assay (ELISA) is used to quantify proteins present in supernatants or lysates, including cell signaling molecules as interleukins i.e. IL-8, interferons, chemokines, lymphokines, and tumor necrosis factors (TNF) or targeted inflammatory cytokines.

——

Immunohistochemistry

NOSi, COX-2, CD-54 or other targeted proteins are visualized on fixed tissue sections by indirect immunofluorescence labelling and microscopic analysis.

——

Gene expression

Gene expression profile analysis after compound exposure of ATERA 3D tissue models can be performed: total RNAs are extracted, processed for retro-transcription, amplification and labeling, microarrays hybridized and regulated transcripts are analyzed to correlate the modulation of gene expression with specific cellular events.

> Sub-clinical irritation testing (Stinging, itching, burning sensations)

Cosmetic or pharmaceutical formulations can sometimes induce adverse health effects called sub-clinical irritation. These transient effects such as a stinging, itching or burning sensations cannot be detected by standard in vitro skin irritation tests based on MTT, cytokine release and/or histological evaluation. The objective of this test procedure is to measure electrical properties of epidermal tissue models to assess potential of substances or formulations to elicit the sub clinical irritation potential.

RHE

The ATERA – Reconstructed Human Epidermis model (RHE) is proposed in its standard configuration e.g. cultured for 17 days. Depending on specific needs, customized tissues can be reconstructed with epidermal cells of different donors/ age groups or cultured under specific ageing-induced conditions.

——

Other models

Other ATERA – Human Reconstructed Human Tissue models, including gingival, corneal etc. can be used to study sub clinical effects in the respective organ.

Hydrophilic and hydrophobic ingredients or finished products

To assess sub clinical irritation a highly sensitive method called impedance spectroscopy (ImpSpec) is applied to investigate effects on the layers of reconstructed human skin models (RHE) or other epithelial models that contribute to the electrical resistance (Figure 1). Thus, the ImpSpec values are the result of the two distinct electrical barriers; one for the stratum corneum and one for the tight junctions in the vital cell layers. Tissue models are treated with the test formulations for different time periods, after which ImpSpec will be used to quantify the effects on the epidermal barrier properties. 2-Propanol or PBS are respectively used as positive or negative controls.

Figure 1: Principle of ImpSpec measurements. After being exposed to a test formulation/compound, RHE is placed between a working- (WE) and a counter electrode (CE) and both compartments of the used insert system (I) are filled with an conductive solution (S). Subsequently, impedance spectra are recorded and used to extract electrical parameters of the sample such as the capacitance (Kc) or Ohmic (Rc) resistance. U: electrical current.

Figure 1: Principle of ImpSpec measurements. After being exposed to a test formulation/compound, RHE is placed between a working- (WE) and a counter electrode (CE) and both compartments of the used insert system (I) are filled with an conductive solution (S). Subsequently, impedance spectra are recorded and used to extract electrical parameters of the sample such as the capacitance (Kc) or Ohmic (Rc) resistance. U: electrical current.

 

Additionally, Multiple Endpoint Analysis (MEA) including histology, protein quantitation, immunolabelling, gene expression can be performed to assess the effects on ATERA Reconstructed Human Tissue models. Criteria of study can be adapted depending on specific needs.

——

H&E Histology

Tissues are fixed in a buffered 4% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin and eosin (H&E) allowing microscopic evaluation.
——

Viability assay

The MTT assay is used to quantify tissue viability. The tissue viability assay is based on the enzymatic conversion of the yellow dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue Thiazolyl tetrazolium bromide) by the viable cells of the tissue construct into a blue formazan salt that is subsequently quantitatively measured after extraction with isopropanol.
——

Protein quantitation

The enzyme-linked immunosorbent assay (ELISA) is used to quantify proteins present in supernatants or lysates, including connective tissue components as collagenous or elastic components, or cell signaling molecules as interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors (TNF).
——

Immunohistochemistry

Type IV or VII Collagens, laminins, cytokeratins or other targeted proteins are visualized on fixed tissue sections by indirect immunofluorescence labelling and microscopic analysis.
——

Gene expression

Gene expression profile analysis after compound exposure of ATERA 3D tissue models can be performed: total RNAs are extracted, processed for retro-transcription, amplification and labeling, microarrays hybridized and regulated transcripts are analyzed to correlate the modulation of gene expression with specific cellular events.

For each tissue model, the impedance is measured over a frequency range from 1 Hz to 100 kHz at 40 logarithmically distributed sampling points. From these spectra, electrical characteristics such as the capacitance and the ohmic resistance are extrapolated that are predictive for mild effects on the skin. In contrast to standard trans-epithelial electrical resistance values, the used system shows a higher sensitivity, since the values are not biased by the electrical setup and electrode orientation (Figure 2). Furthermore, trans-epithelial electrical resistance measurements are limited to the ohmic resistance and extraction of further electrical parameters such as the capacitance is not possible. The impedance of each RHE will be measured before the test compound application, directly after the application and following a 42 hour post incubation time. Thereby, a direct comparison of effects on each model is possible. Additionally for each RHE model, tissue morphology (H&E staining) and viability (MTT assay) will be performed.

Figure 2: Change of the impedance of reconstructed human epidermis (RHE) following chemical treatment with PBS, SDS and 2-propanol. The mean viability of RHE and standard deviation were determined using a quantitative MTT assay, in which the viability of the PBS group is set to 100 % (left). The mean values and standard deviations of ohmic resistance (right) were determined before the application of the test substances (UT), after a 30 minute incubation phase followed by eight washing steps (30 m), and after a 42 hour recovery phase (42 h). Stars indicate statistical relevant differences (P-value ≤ 0.5). Each group comprises 3 individual samples.

Figure 2: Change of the impedance of reconstructed human epidermis (RHE) following chemical treatment with PBS, SDS and 2-propanol. The mean viability of RHE and standard deviation were determined using a quantitative MTT assay, in which the viability of the PBS group is set to 100 % (left). The mean values and standard deviations of ohmic resistance (right) were determined before the application of the test substances (UT), after a 30 minute incubation phase followed by eight washing steps (30 m), and after a 42 hour recovery phase (42 h). Stars indicate statistical relevant differences (P-value ≤ 0.5). Each group comprises 3 individual samples.

Principle of impedance spectroscopy measurements in human skin:

Yamamoto, T. and Yamamoto, Y.; Analysis for the change of skin impedance;

Med & Biol Eng & Comput; 1977; 15 (3): 219-227.
——

Test procedure:

Groeber, F. et al.; Impedance Spectroscopy for the Non-Destructive Evaluation of In Vitro Epidermal Models;

Pharm Res 2015; 32(5): 1845–1854.

> Skin barrier regeneration

To evaluate the efficacy of cosmetic ingredients and finished products intended to strengthen the skin barrier of stressed skin.

RHE

The ATERA – Reconstructed Human Epidermis model (RHE) is proposed in its standard configuration e.g. cultured for 17 days. Depending on specific needs, customized tissues can be reconstructed with epidermal cells of different donors/ age groups or cultured under specific ageing-induced conditions.

Test can be performed either via topical application for hydrophilic and hydrophobic ingredients or finished products or in case of a systemic application for hydrophilic ingredients.

To measure the barrier regeneration, RHE with a stressed phenotype are created and the re-formation of the barrier is monitored using a highly sensitive method called impedance spectroscopy (ImpSpec) in a non-invasive monitoring assay combined with endpoint viability assay and histology analysis.

 

Figure 1: Principle of barrier regeneration studies.

Figure 1: Principle of barrier regeneration studies.

——

Stressed skin models

RHE mimicking a stressed phenotype can be created by disrupting the skin barrier either mechanically or chemically. Thereby, the barrier can be reduced up to 80 % without a loss of viability.

——

Viability assay

The MTT assay is used to quantify tissue viability. The tissue viability assay is based on the enzymatic conversion of the yellow dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue Thiazolyl tetrazolium bromide) by the viable cells of the tissue construct into a blue formazan salt that is  subsequently quantitatively measured after extraction with isopropanol .

——

Impedance spectroscopy

ImpSpec will be performed by placing each RHE model between a working and a counter electrode in a newly developed measuring chamber allowing a precise positioning of multiple ImpSpec measurements (Figure 1 and Figure 2). The space between the sample and the electrodes is then filled with tissue culture medium. For each model, the impedance is measured over a frequency range from 1 Hz to 100 kHz at 40 logarithmically distributed sampling points. From these spectra, electrical characteristics such as the capacitance and the ohmic resistance are extrapolated that are predictive for the repair of the skin barrier. The impedance values of each model is measured before the disruption of the skin barrier, directly after the disruption and every 24 hours over a time course of several days. Thereby, for each model the re-epithelialization can be assessed by monitoring the increase of impedance over time. Additionally, Multiple Endpoint Analysis (MEA) including histology, protein quantitation, immunolabelling, gene expression can be performed to assess the effects on the ATERA RHE. Criteria of study can be adapted depending on specific needs.

 

Figure 2: Principle of ImpSpec measurements. A biological sample e.g. a RHE is placed between a working- (WE) and a counter electrode (CE) and both compartments of the used insert system (I) are filled with an conductive solution (S). Subsequently, impedance spectra are recorded and used to extract electrical parameters of the sample such as the capacitance (Kc) or Ohmic (Rc) resistance. U: electrical current

Figure 2: Principle of ImpSpec measurements. A biological sample e.g. a RHE is placed between a working- (WE) and a counter electrode (CE) and both compartments of the used insert system (I) are filled with an conductive solution (S). Subsequently, impedance spectra are recorded and used to extract electrical parameters of the sample such as the capacitance (Kc) or Ohmic (Rc) resistance. U: electrical current

 

Figure 3: System for ImpSpec measurements.

Figure 3: System for ImpSpec measurements.

For each RHE model, the impedance is measured over a frequency range from 1 Hz to 100 kHz at 40 logarithmically distributed sampling points. From these spectra, electrical characteristics such as the capacitance and the ohmic resistance are extrapolated that are predictive for the re-formation of the skin barrier. Additionally for each skin model, tissue morphology (H&E staining) and viability (MTT assay) can be performed at specific sampling points.

 

Figure 4: Recovery of skin barrier over time: Following the disrupting of the RHE barrier without harming viable cell layers measured via MTT (left) the barrier regeneration is measured using ImpSpec over 4 days

Figure 4: Recovery of skin barrier over time: Following the disrupting of the RHE barrier without harming viable cell layers measured via MTT (left) the barrier regeneration is measured using ImpSpec over 4 days

Principle of impedance spectroscopy measurements in human skin:

Yamamoto, T. and Yamamoto, Y.; Analysis for the change of skin impedance;

Med & Biol Eng & Comput 1977; 15(3): 219-227.

——

Test procedure:

Groeber, F. et al.; Impedance Spectroscopy for the Non-Destructive Evaluation of In Vitro Epidermal Models;

Pharm Res 2015; 32(5): 1845–1854.

——

Skin barrier regeneration:

Refinement of RHE-based testing strategies:
Impedance spectroscopy as new tools for hazard identification and efficacy testing.
Groeber F, Engelhardt L, Guillon F, Nikoyan-Ginosian A, Le Varlet B, De Wever B , Walles H.
ESTIV 17-20 October 2016, Juan-les-Pins, France

> Wound healing

The purpose of this study is to evaluate the effect of topically or systemically applied actives or finished products on cutaneous wound healing of ‘pre-wounded’ ATERA RHE or full thickness skin models. Wound closure is monitored over the course of several days by employing histology, MTT staining or a highly sensitive non-destructive method called impedance spectroscopy (ImpSpec) that allows precise and objective measurements of re-epithelialization in the individual skin models over time.

RHFTS

The ATERA – Reconstructed Human Full Thickness Skin Model (RHFTS) is an advanced human skin model, consisting of dermal and epidermal part closely resembling native human skin. Please note, for the RHFTS, ImpSpec measurements are currently not available.

RHE

The ATERA – Reconstructed Human Epidermis model (RHE) is proposed in its standard configuration e.g. cultured for 17 days. Depending on specific needs, customized tissues can be reconstructed with epidermal cells of different donors/ age groups or cultured under specific ageing-induced conditions.

Hydrophilic and hydrophobic ingredients or finished products

Wound healing can be assessed using different methods including non-destructive impedance measurements. Additionally, histology, protein quantitation, immunolabelling and/or gene expression can be performed at different time points depending on specific needs.

——

Wounding of skin models

In RHFTS models ‘wounds’ with a diameter of 2 mm will be introduced by using an automated specially designed drilling device. Thereby, very precise wounds can be generated that allow an accurate assessment of wound closure. In all experiments the wound depth can be adjusted to specific needs between 200 µm and 2 mm. For RHE epithelial wounds thorough the full depth of the model will be manually generated using a specifically developed procedure. The diameter of the wound can be adjusted to 1 or 2 mm.

 

Figure 1: Principle of wound healing studies.

Figure 1: Principle of wound healing studies.

——

Impedance spectroscopy

ImpSpec will be performed by placing each RHE model between a working and a counter electrode in a newly developed measuring chamber allowing a precise positioning of multiple ImpSpec measurements. The space between the sample and the electrodes is then filled with tissue culture medium. For each model, the impedance is measured over a frequency range from 1 Hz to 100 kHz at 40 logarithmically distributed sampling points. From these spectra, electrical characteristics such as the capacitance and the ohmic resistance are extrapolated that are predictive for the repair of the skin barrier and thus for reepithelialisation. The impedance of each model is measured before the wounding, directly after the wounding and every 24 hours over a time course of several days. Thereby, for each model the re-epithelialization can be assessed by monitoring the increase of impedance over time.

Figure 2: Principle of ImpSpec measurements. After being exposed to a test formulation/compound, the RHE is placed between a working- (WE) and a counter electrode (CE) and both compartments of the used insert system (I) are filled with an conductive solution (S). Subsequently, impedance spectra are recorded and used to extract electrical parameters of the sample such as the capacitance (Kc) or Ohmic (Rc) resistance. U: electrical current

Figure 2: Principle of ImpSpec measurements. After being exposed to a test formulation/compound, the RHE is placed between a working- (WE) and a counter electrode (CE) and both compartments of the used insert system (I) are filled with an conductive solution (S). Subsequently, impedance spectra are recorded and used to extract electrical parameters of the sample such as the capacitance (Kc) or Ohmic (Rc) resistance. U: electrical current

——

H&E Histology

Tissues are fixed in a buffered 4% formalin solution and embedded in paraffin. Four micron vertical sections are stained with hematoxylin and eosin (H&E) allowing microscopic evaluation.

——

Viability assay

The MTT assay is used to quantify tissue viability. The tissue viability assay is based on the enzymatic conversion of the yellow dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue Thiazolyl tetrazolium bromide) by the viable cells of the tissue construct into a blue formazan salt that is subsequently quantitatively measured after extraction with isopropanol.

——

Protein quantitation

The enzyme-linked immunosorbent assay (ELISA) is used to quantify proteins present in supernatants or lysates, including connective tissue components as collagenous or elastic components, or cell signaling molecules as interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors (TNF).

——

Immunohistochemistry

Type IV or VII Collagens, laminins, cytokeratins or other targeted proteins are visualized on fixed tissue sections by indirect immunofluorescence labelling and microscopic analysis.

——

Gene expression

Gene expression profile analysis after compound exposure of ATERA 3D tissue models can be performed: total RNAs are extracted, processed for retro-transcription, amplification and labeling, microarrays hybridized and regulated transcripts are analyzed to correlate the modulation of gene expression with specific cellular events.

For each RHE model, the impedance is measured over a frequency range from 1 Hz to 100 kHz at 40 logarithmically distributed sampling points. From these spectra, electrical characteristics such as the capacitance and the ohmic resistance are extrapolated that are predictive for the re-formation of the skin barrier. Additionally for each skin model, tissue morphology (H&E staining) and viability (MTT assay) can be performed at specific sampling points.

 

Figure 3: Exemplary data of a RHE wound healing assay with and without a treatment. Statistic relevant differences are indicated with stars (*: p=0,05 and ****p=0,001).

Figure 3: Exemplary data of a RHE wound healing assay with and without a treatment. Statistic relevant differences are indicated with stars (*: p=0,05 and ****p=0,001).

Test procedure:

Groeber, F. et al.; Impedance Spectroscopy for the Non-Destructive Evaluation of In Vitro Epidermal Models;

Pharm Res 2015; 32(5): 1845–1854.

——

Wound-healing test procedure:

Rossi, A. et al.; Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding. J Vis Exp 2015; 96 e52576, doi:10.3791/52576. See the video >

Pharm Res 2015; 32(5): 1845–1854.

SAFETY TESTING

ATERA TEST METHODS

Title Description Support
Cytotoxicity MTT assay, LDH, GLU, XTT, NR, SRB ATERA Tissue Models, cellular monolayers
Eye irritation MTT assay ATERA Eye Model RHC
Gingival irritation MTT assay ATERA Mucosal Model RHG
Skin corrosion MTT assay ATERA Skin Model RHE
Skin irritation OECD TG 439 (Ongoing Performance Standard ECVAM Validation) MTT assay ATERA Skin Model RHE
Skin absorption HPLC assay ATERA Skin Model RHE
Oral irritation MTT assay ATERA Mucosal Model RHO
Phototoxicity MTT assay following UVA/UVB exposure ATERA Skin Model RHE
Vaginal irritation MTT assay ATERA Mucosal Model RHV

Contact information

Depending on the specific needs, adapted protocols can be developed, please contact us.