The authors would like to thank to ZEBET at the BfR (Germany), BASF SE (Germany), IIVS Inc. (USA), Zet-LSL (Austria) for participation in Multi-center International Validation Study of the Modified EpiDerm SIT (5).

I. Tissue conditioning - Day 0
 <ol>
 <li>Upon receipt of the EpiDerm EPI-200-SIT kit, check all the kit components for integrity (for kit details see Table of specific reagents and equipment). </li>
 <li>For every three EpiDerm tissues, prepare one sterile 6-well plate pre-filled with 0.9 ml of assay medium. </li>
 <li>Under sterile conditions, open the plastic bag containing the 24-well plate containing the EpiDerm tissues and remove the sterile gauze. </li>
 <li>Use sterile forceps to remove each insert containing the EpiDerm tissue and place the insert in the empty 24-well plate. During this step, remove any remaining shipping agarose that adheres to the outer sides of the insert by gentle blotting on sterile filter paper.</li>
 <li>Within the next 5 minutes, visually inspect the tissues. Do not use defective tissues or tissues which are completely covered with liquid. </li>
 <li>Using a sterile cotton tip swab, carefully dry the surface of the tissues. Try not to touch the tissue directly - capillary effects are sufficient to remove the moisture from the tissue surface. </li>
 <li>After drying the tissue surface, transfer three tissues to the top row of the 6-well plates pre-filled with 0.9 mL of assay medium. Release any air bubbles trapped underneath the inserts. </li>
 <li>Place the 6-well plates containing the inserts into the incubator for a 60 &plusmn; 5 min pre-incubation at 37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH(Relative Humidity).</li>
 <li>At the end of the 60 &plusmn; 5 min pre-incubation period, transfer the inserts from the upper wells into the lower wells of the 6-well plate. Place the plates back into the incubator (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH) for an overnight pre-incubation (18 &plusmn; 3 hr).</li>
 <li>Place the rest of the assay medium into the refrigerator (5 &plusmn; 3 &deg;C). Following the overnight pre-incubation, the test chemicals can be applied to the EpiDerm tissues.
 Note: The pre-incubation procedure can be shortened in case of late tissue delivery (e.g. if tissues arrive on Wednesday instead of Tuesday). 
</li>
 </ol>
 II. Chemical exposure - Day 1
 <ol>
 <li>Prepare one 6-well plate per test chemical by filling the upper row only with 0.9 ml per well of assay medium.</li>
 <li>Approximately 5 minutes before the planned exposure to chemicals, remove the 6-well plates containing the pre-conditioned tissues from the incubator. </li>
 <li>Evaluate the surface of tissues and remove any moisture using sterile cotton tips.</li>
 <li>Label all 6-well plate lids with the test material codes or names.</li>
 <li>Dose the tissues with the test chemicals at 1 minute intervals to facilitate rinsing of the test chemicals after exposure. Keep the plates with dosed tissues in the sterile hood, until the last tissue is dosed. </li>
 <li>To test liquid chemicals, dispense 30 &mu;L of the solution directly atop the tissue and place the nylon mesh on the tissues surface. If necessary, gently position the mesh using the bulb headed glass Pasteur pipette. Do not press on the tissue surface.</li>
 <li>For testing semisolids, use a positive displacement pipette to dispense 30 &mu;L directly atop the tissue. If necessary spread the chemical with Pasteur pipette to cover the tissue surface as much as possible.</li>
 <li>For solid materials, shortly before application of the test substance, moisten the tissue surface with 25 &mu;L of sterile DPBS. This will improve contact of the tissue surface with the test chemical. Fill a 25 mg calibrated sharp application spoon (or any other appropriate tool) with approximately 25 mg of finely ground test material. Apply the solid test chemical to the tissue surface. If necessary, use a bulb headed Pasteur Pipette to empty the spoon completely. Gently shake the inserts to improve the spreading of the solid on the surface.</li>
 <li>For test substances with waxy consistency, try to form a flat "cookie like" piece about 8 mm in diameter and place it atop the tissue, which was previously wetted with sterile DPBS. To improve the contact between test substance and tissue, weigh down the "cookie" with a stainless steel or plastic aid.</li>
 <li>Apply DPBS as the negative control and 5% SDS as the positive control to the control tissues.</li>
 <li>After dosing the last tissue, transfer all the plates for 35 &plusmn; 1 minutes to the humidified incubator (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH).</li>
 <li>After the 35 &plusmn; 1 minute incubation at 37&deg;C remove all the plates from the incubator. Place the plates into the sterile hood and wait until a period of 60 minutes chemical exposure is completed for the first dosed tissue.</li>
 <li>Afterwards start the washing procedure using the one insert per minute time interval (to insure that the total exposure time for every insert is 60 minutes).</li>
 <li>Use a wash bottle to rinse the tissues with sterile DPBS. Fill the tissue insert 15 times by using a constant stream of DPBS and empty it (Note: for tissues with a nylon mesh, wash the tissue surface 5 times and remove the mesh carefully with the pointed, sharp forceps. Afterwards, continue with the remaining 10 washes).</li>
 <li>After the 15th rinse using the wash bottle, completely submerge the insert 3 times in 150 ml DPBS and shake to remove the remainder of the test material.</li>
 <li>Finally, rinse the tissue insert once from the inside and once from the outside with sterile DPBS. </li>
 <li>Remove any excess of DPBS from the tissue by gently shaking the insert, and blotting it on the sterile blotting paper. </li>
 <li>Transfer the blotted tissue inserts to the new 6-well plate previously pre-filled with assay medium. All of these steps (14-18) must be done within 1 min for each insert.</li>
 <li>After the last tissue is rinsed, carefully dry the surface of each tissue with a sterile cotton tipped swab. </li>
 <li>Incubate the tissues in the incubator for the next 24 &plusmn; 2 hours (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH).
 Note: Each assay technician should not test more than 6 test substances including the negative and positive controls in a single testing run (set), to be able follow the protocol as described below</li>
</ol>
III. Medium exchange - Day 2
 <ol>
 <li>After the 24 &plusmn; 2 hour post-incubation, transfer the inserts into the lower part of the 6-well plate, pre-filled with 0.9 ml of media. Media from upper rows can be collected for analysis of the additional endpoints (cytokines, chemokines etc.). Analysis of the secondary endpoints is optional.</li>
 <li>Continue with the post-incubation (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH) for another 18 &plusmn; 3 hours.</li>
 </ol>
IV. MTT Viability Assay - Day 3
 <ol>
 <li>Label two 24-well plates with the chemical names or codes. One plate will serve for tissue incubation with MTT and the other for the extraction step.</li>
 <li>Prepare the MTT solution by thawing the MTT concentrate (5 mg/ml) and diluting it with the MTT diluent. Final concentration of MTT solution is 1 mg/ml. </li>
 <li>Pipette 300 &mu;L of MTT solution into each well of the 24 well plate.</li>
 <li>Remove the 6-well plates from incubator, blot the bottom of the inserts on a blotting paper, and transfer them into the 24-well plate pre-filled with MTT.</li>
 <li>Place the 24-well plate in the incubator (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH) and incubate for 3 hours &plusmn; 5 min. Strictly adhere to the 3 hours &plusmn; 5 min incubation time to avoid deviation between different MTT readings. </li>
 <li>When the MTT incubation is complete, use a suction pump to gently aspirate the MTT medium from all the wells. </li>
 <li>Refill the wells with DPBS and aspirate again. Repeat this rinsing two more times and make sure that tissues are dry after the last aspiration. </li>
 <li>After the washes transfer the inserts to a new 24-well plate. Immerse the inserts by gently pipetting 2 mL extractant solution (Isopropanol) into each insert. The level will rise above the upper edges of the insert, thus completely submerging the tissues. </li>
 <li>Seal the 24-well plate (e.g. with Parafilm or using sealing bag) to inhibit extractant evaporation. Extract the MTT from the tissues for at least 2 hours at room temperature with gentle shaking on a plate shaker (~ 120 rpm). Overnight extraction without shaking can also be used. </li>
 <li>After the extraction period is complete, pierce the inserts with an injection needle or bulb beaded Pasteur pipette and allow the extract to run into the well from which the insert was taken.</li>
 <li>Discard the punctured insert and pipette the solution in the well up and down three times until it is homogenous. </li>
 <li>For each tissue, transfer two 200 &mu;L aliquots of the purple formazan solution into a 96-well flat bottom microtiter plate. Transfer the duplicates according to the fixed plate design given in the spreadsheet accompanying this video protocol. For blanks, use isopropanol. </li>
 <li>Read the optical density (OD) of the MTT extracts in a 96-well plate spectrophotometer using a wavelength of 570 nm (540-580) without a reference filter.</li>
 <li>Enter the results in the spreadsheet for automatic calculation of the results (Figure 1). </li>
 <li>A test chemical is labeled 'irritant' (R38 or GHS category 2) if the percent tissue viability determined by MTT assay is &lt;50% relative to the negative control. 
 Note: MTT is toxic, so be sure to wear protective gloves when handling. Protect MTT and its solutions from light, since MTT is light sensitive. Use MTT solution within a few hours after preparation because it degrades over time.</li></ol>
 V. Assay Quality Controls
<ul>
<li> EpiDerm EPI-200-SIT kit Acceptance Criteria: 
In vitro RHE models should provide consistent, high quality data over time, not just during the validation process (8). Recommended guidelines to ensure long-term assay reproducibility and performance include "full characterization of cells or tissues, sampling of each lot ... for performance, and regular use of controls and benchmark chemicals to provide assurance of consistency of assay performance" (9). In addition to assay acceptance criteria outlined below, each production lot of EpiDerm is quality control tested by the manufacturer against an additional benchmark chemical (1% Triton X-100). The exposure time needed to reduce tissue viability to 50% (ET-50) for Triton X-100 is determined. For EpiDerm, yearly average ET-50 values have ranged from 5.9 hrs to 7.5 hrs. The lot to lot CV for negative control tissues (i.e. the variability in baseline viability) has averaged under 7.5% for every year since 1996. The ET50 value of each production lot must fall within 2 standard deviations of the historical average Triton X-100 ET50 established in 1996 (i.e. ET-50 = 6.7 hours; Lower acceptance limit = 4.8h ; upper acceptance limit = 8.7h) in order to be released for use by the manufacturer.</li>
<li>EPI-200-SIT Assay Acceptance Criterion 1: Negative Control: 
The absolute OD of the negative control (NC) tissues (treated with sterile DPBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is &ge;1.0 and &le;2.5.</li>
<li>EPI-200-SIT Assay Acceptance Criterion 2: Positive Control 
A 5% SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means that the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95% confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is 20%.</li>
<li>EPI-200-SIT Assay Acceptance Criterion 3: Standard Deviation (SD) 
The skin irritancy is predicted from the mean viability determined on 3 single tissues and therefore the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is &lt;18%.</li>
</ul>
Representative Results
<img src="/files/ftp_upload/1366/1366fig1.jpg" alt="Figure 1" /> <img src="/files/ftp_upload/1366/1366fig1_2.jpg" alt="Figure 2" /> <img src="/files/ftp_upload/1366/1366fig1_3.jpg" alt="Figure 3" /> Figure 1. Results obtained for 6 test articles, NC and PC control - Part of the automated spreadsheet for calculation of results. Test chemicals which reduced tissue viability below 50% referenced to NC are classified as irritants. The test is optimized to provide results that are sufficiently far from the classification cut-off (50% viability) thus increasing the robustness of the assay.
<img src="/files/ftp_upload/1366/materialstable.jpg" alt="Materials List" />

<ol>
	<li>Kand&#225;rov&#225;, H., Liebsch, M., Genschow, E., Gerner, I. n. g. r. i. d., Traue, D., Slawik, B., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+EpiDerm+Test+Protocol+for+the+upcoming+ECVAM+Validation+Study+on+In+Vitro+Skin+Irritation+Tests.">Optimization of the EpiDerm Test Protocol for the upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests.</a> ALTEX. 21, 107-114 (2004).</li><li>Kand&#225;rov&#225;, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpiDerm+Skin+Irritation+Test+Protocol+-+Assessment+of+the+performance+of+the+optimized+test.">EpiDerm Skin Irritation Test Protocol - Assessment of the performance of the optimized test.</a> ATLA. 33, 351-367 (2005).</li><li>Kand&#225;rov&#225;, H., Hayden, P., Klausner, M., Kubilus, J., Kearney, P., Sheasgreen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Skin+Irritation+Test:+Improving+the+Sensitivity+of+the+EpiDerm+Skin+Irritation+Test+Protocol.Accepted+for+publication.">In Vitro Skin Irritation Test: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol.Accepted for publication.</a> ATLA. , (2009).</li><li>Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovi, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kand&#225;rov&#225;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ECVAM+International+Validation+Study+on+In+Vitro+Tests+for+Acute+Skin+Irritation:+Report+on+the+Validity+of+the+EPISKIN+and+EpiDerm+Assays+and+on+the+Skin+Integrity+Function+Test.">The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test.</a> ATLA. 35, 559-601 (2007).</li><li>Liebsch, M., Gamer, A., Curren, R., Frank, J., Genschow, E., Tharmann, J., Remmele, M., Bauer, B., Raabe, H., Barnes, N., Hilberer, A., Wilt, N., Lornejad-Sch&#228;fer, M. R., Sch&#228;fer, C., Spiller, E., Hayden, P., Kand&#225;rov&#225;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follow-up+validation+of+the+modified+EpiDerm+Skin+Irritation+Test+(SIT):+results+of+a+multicentre+study+of+twenty+reference+test+substances.">Follow-up validation of the modified EpiDerm Skin Irritation Test (SIT): results of a multicentre study of twenty reference test substances.</a> , (2008).</li><li>United Nations. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Globally Harmonized System of Classification and Labeling of Chemicals (GHS). , (2007).</li><li>ESAC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Statement on the Scientific Validity of In-Vitro Tests for Skin Irritation Testing. , (2008).</li><li>Gupta, K., Rispin, A., Stitzel, K., Coecke, S., Harbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+quality+of+in+vitro+alternative+test+methods:+Issues+and+answers.">Ensuring quality of in vitro alternative test methods: Issues and answers.</a> Regul Toxicol Pharmacol. 43, 219-224 (2005).</li><li>Rispin, A., Stitzel, K., Harbell, J., Klausner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+quality+of+in+vitro+alternative+test+methods:+Current+practice.">Ensuring quality of in vitro alternative test methods: Current practice.</a> Regul Toxicol Pharmacol. 45, 97-103 (2006).</li></ol>

The authors are employees of MatTek corporation which produce reagents and tools used in this article.

In this video, we have demonstrated the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. When performing this method, it is important to work in aseptic conditions and to strictly follow the validated protocol, since deviation from the protocol may cause different outcomes. Some modifications of the assay are possible, however, changes to the protocol should be discussed directly with the authors.
The only limitation of this method is a possible interference of the test substance with the MTT endpoint. A colored test substance or one that directly reduces MTT (and thereby mimics dehydrogenase activity of the cellular mitochondria) may interfere with the MTT endpoint. However, these test substance are a problem only if at the time of the MTT test (i.e. 42 hours after test substance exposure) sufficient amounts of the test substance are still present on (or in) the tissues. In case of this unlikely event, the (true) metabolic MTT reduction and the contribution by a colored test material or (false) direct MTT reduction by the test material can be quantified by a procedure described in detail in the assay Standard Operation Procedure provided by MatTek Corp.

an in vitro skin irritation test (sit) using the epiderm reconstructed human epidermal (rhe) model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

Watch this Scientific Journal Video about An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model at JoVE.com

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

an-vitro-skin-irritation-test-sit-using-epiderm-reconstructed-human

Research

JoVE Journal

Biology

67.6K Views.  MatTek Corporation.  日本語 

Video: An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Bioengineering

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Chemistry

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Developmental Biology

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...