Name: An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model (Video) | JoVE Journal
Uploaded: 2009-07-13T21:00:00.000+00:00
Duration: 21 min 16 s
Description: Learn the EpiDerm Skin Irritation Test for assessing chemical safety using reconstructed human epidermis. This validated in vitro method replaces animal testing for cosmetics and pharmaceuticals.

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5 minutes

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 ± 5 min pre-incubation at 37 ± 1° C, 5 ± 1% CO2, 95 % RH(Relative Humidity).</li>
 <li>At the end of the 60 ± 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 ± 1° C, 5 ± 1% CO2, 95 % RH) for an overnight pre-incubation (18 ± 3 hr).</li>
 <li>Place the rest of the assay medium into the refrigerator (5 ± 3 °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 μL 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 μL 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 μL 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 ± 1 minutes to the humidified incubator (37 ± 1° C, 5 ± 1% CO2, 95 % RH).</li>
 <li>After the 35 ± 1 minute incubation at 37°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 ± 2 hours (37 ± 1° C, 5 ± 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 ± 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 ± 1° C, 5 ± 1% CO2, 95 % RH) for another 18 ± 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 μL 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 ± 1° C, 5 ± 1% CO2, 95 % RH) and incubate for 3 hours ± 5 min. Strictly adhere to the 3 hours ± 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 μL 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 ≥1.0 and ≤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="In vitro experiment data table, EpiDerm SIT, mean OD, viability percentages, test classifications." data-altupdatedat="1761735611000" data-alt="Figure 1"> <img src="/files/ftp_upload/1366/1366fig1_2.jpg" alt="Bar chart of relative viability percentages; statistical data analysis results; experimental comparison." data-altupdatedat="1761735611000" data-alt="Figure 2"> <img src="/files/ftp_upload/1366/1366fig1_3.jpg" alt="Viability assay results table; sample codes, raw data, corrected data, mean, and viability percentage." data-altupdatedat="1761735611000" data-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="EpiDerm SIT and MTT assay kit components, storage conditions, and usage details table." data-altupdatedat="1761735611000" data-alt="Materials List">

<ol><li>Kandárová, H., Liebsch, M., Genschow, E., Gerner, I. ngrid, Traue, D., Slawik, B., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+EpiDerm+Test+Protocol+for+the+upcoming+ECVAM+Validation+Study+on+In+Vitro+Skin+Irritation+Tests%5BTitle%5D)+AND+%22ALTEX%22%5BJournal%5D)">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>Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EpiDerm+Skin+Irritation+Test+Protocol+-+Assessment+of+the+performance+of+the+optimized+test%5BTitle%5D)+AND+%22ATLA%22%5BJournal%5D)">EpiDerm Skin Irritation Test Protocol - Assessment of the performance of the optimized test.</a> ATLA. 33, 351-367 (2005).</li>
<li>Kandárová, H., Hayden, P., Klausner, M., Kubilus, J., Kearney, P., Sheasgreen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vitro+Skin+Irritation+Test%3A+Improving+the+Sensitivity+of+the+EpiDerm+Skin+Irritation+Test+Protocol.Accepted+for+publication%5BTitle%5D)+AND+%22ATLA%22%5BJournal%5D)">In Vitro Skin Irritation Test: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol.Accepted for publication.</a> ATLA. , Forthcoming (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., Kandárová, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ECVAM+International+Validation+Study+on+In+Vitro+Tests+for+Acute+Skin+Irritation%3A+Report+on+the+Validity+of+the+EPISKIN+and+EpiDerm+Assays+and+on+the+Skin+Integrity+Function+Test%5BTitle%5D)+AND+%22ATLA%22%5BJournal%5D)">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>Follow-up validation of the modified EpiDerm Skin Irritation Test (SIT): results of a multicentre study of twenty reference test substances. 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-Schäfer, M. R., Schäfer, C., Spiller, E., Hayden, P., Kandárová, H. 15th Congress on Alternatives to Animal Experimentation, 2008 September 19-21, Linz Austria, , (2008).</li>
<li>United Nations. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Globally+Harmonized+System+of+Classification+and+Labeling+of+Chemicals+%28GHS%29%22">Globally Harmonized System of Classification and Labeling of Chemicals (GHS)</a>. , Second revised edition, UN. New York and Geneva. (2007).</li>
<li>ESAC. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Statement+on+the+Scientific+Validity+of+In-Vitro+Tests+for+Skin+Irritation+Testing%22">Statement on the Scientific Validity of In-Vitro Tests for Skin Irritation Testing</a>. , ESAC. (2008).</li>
<li>Gupta, K., Rispin, A., Stitzel, K., Coecke, S., Harbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ensuring+quality+of+in+vitro+alternative+test+methods%3A+Issues+and+answers%5BTitle%5D)+AND+%22Regul+Toxicol+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Ensuring+quality+of+in+vitro+alternative+test+methods%3A+Current+practice%5BTitle%5D)+AND+%22Regul+Toxicol+Pharmacol%22%5BJournal%5D)">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

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).

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.

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 ...

Epiderm is a normal human cell derived reconstructed in vitro model of the human epidermis developed by the Matt Tech Corporation. The EC VM validated epiderm skin irritation test protocol replaces traditional animal-based testing of products such as cosmetic, raw materials and industrial agents that will come in contact with human skin. This test begins on day zero when the skin irritation kit and epiderm tissue arrives in the lab and is subsequently preconditioned an assay medium following an overnight incubation.On day one, the atypical surface of the epiderm tissue is exposed for one hour with a putative irritant compound and then the irritant is removed with a washing step on day two. After a 24 hour incubation, the culture insert is placed in fresh media and the old media can be used for analysis of released cytokines and chemokines. The tissue is reacted with a compound known as MTT, which is converted by mitochondrial reductase to a dark purple for maoz and salt.Providing a readout for cell viability. A lack of dark purple color in the sample is an indicator of decreased viability. And when the calor metric readout for a test compound is less than 50%of negative controls, the test compound can be classified as an irritant.Hi, I am Helena Kava from Matt Corporation. Today we will show you procedure for in vitro skin irritation testing using reconstructed human skin model epiderm. This procedure, depending on regulatory requirements, can be used as full or partial replacement of in vivo rapid skin irritation test.This new in vitro procedure can be used for hazard identification and labeling of chemicals and raw cosmetic materials. So let's get started. Epiderm is derived from human epidermal keratinocytes culture to form a three-dimensional, multi-layered and highly differentiated model of the human epidermis.The tissues are mitotically and metabolically active, closely mimic their in vivo counterparts both structurally and biochemically, and do so with guaranteed reproducibility like human epidermis. Epiderm consists of organized basal spinous and granular layers and a multi-layered stratum corneum. The latter contains intercellular lam Miller lipid layers arranged in patterns analogous to those found in vivo.These tissues are cultured in serum free medium. Each tissue grown in an individual cell culture insert. Other configurations are available including a 96 well high throughput format.Tissue production takes approximately three weeks to complete at a specific point in this process, the tissue is raised to the air liquid interface and all liquid is removed from the APIC surface of the tissue. The tissues of N Fed only through the basolateral surface, which remains in contact with mat tech's proprietary culture medium. A typical Epiderm kit contains 24 tissues each tissue, nine millimeters in diameter in its own tissue culture insert plus sufficient assay medium to perform this skin irritation test.The 24 inserts are firmly seated on medium infused AGA rose gel one tissue in each well of a standard 24 well plate shipment is by overnight carrier at a temperature of three to four degrees centigrade before embarking on testing chemicals. Using the EPIDERM test system, prepare and sterilize all the instruments and tools used in the assay as described in the written protocol. A portion of the materials like cotton swabs should come in sterile packaging or be autoclaved.70%ethanol should be used to sterilize instruments such as forceps and UV or gamma irradiation should be used to sterilize the washing bottles. Two critical solutions used in the assay should be pre-prepared DPBS and a 5%SDS solution. In distilled water, approximately two liters of sterile DPBS pH seven will be used in the assay with one kit containing 24 epiderm tissues.Sterile DPBS serves as a negative control as well as washing solution. 5%SDS should be prepared and used as the positive control. The skin irritation assay starts upon receipt of the EPIDERM EPI 200 SIT kit.Make sure to check all the kit components for integrity and contact the Mat Tech corporation immediately with any concern if everything is there, proceed to the next step. To begin the test, let the assay medium supplied with the kit reach room temperature. Do not preheat in the laminar flow hood.Prepare one sterile six well plate for every three epiderm tissue inserts and pipette 0.9 milliliters of the assay medium into each well under sterile conditions. Open the plastic bag holding the 24 well plate containing the epidermal tissues under sterile airflow. Remove the sterile gauze and use sterile forceps to carefully take out each insert containing the epidermal tissue.Remove any remaining aros that adheres to the outer sides of the insert by gentle blotting on sterile gauze or filter paper. And then place the tissues in the empty sterile 24 well plate within the next five minutes. Visually inspect the inserts.Record any tissue defects and excess of moisture on the surface. Do not use defective tissues or tissues. Completely covered with liquid.Optimal tissue inserts. Should have a dry surface and be free of detachments, uneven parts or excess liquid at the surface. Next, 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 surface after drying the surface. Transfer three tissues to the top row of every six.Well plate prefilled with 0.9 milliliter assay medium. Release any air bubbles trapped underneath the inserts. Next place the six well plates containing the inserts into the incubator for one hour.Pre incubation at 37 degrees Celsius. At the end of the first pre incubation period, transfer the inserts from the upper wells into the lower wells of the six well plate. Place the plates back into the incubator for an overnight pre incubation.Place the rest of the assay medium into the refrigerator. Following the overnight pre incubation the test chemicals can be applied to the epiderm tissue inserts. Before starting the chemical exposure assay, place all the necessary devices, solutions and test chemicals at room temperature.In the sterile hood, wipe all non-sterile material bottles with 70%ethanol before placing them inside the hood. The negative control sterile DPBS positive control and 5%SDS and distilled water should be placed in the hood and room temperature as well. A large vial should be set aside for waste.Prepare one six. Well plate per each chemical by filling the upper row only with 0.9 milliliters of assay medium per well. Label each plate with tissue number and chemical name.Approximately five minutes before the planned exposure to chemicals. Remove the six well plates containing the precondition tissues from the incubator. Then visually evaluate the surface of tissues.Remove any moisture using sterile cotton tips and do not press on the tissue surface. Finally, label the six well plate lids with the test material codes or names. Do not test more than six chemicals, including negative and positive controls each in triplicate in a single testing run.Use a chemical application and washing interval of one minute for handling known test substances. Follow the instructions given in the material safety data sheet or MSDS for coded or unknown samples. Follow chemical safety guidelines and use a ventilated cabinet.Wear gloves and protect your eyes and face dose tissues with chemicals at one minute time intervals to facilitate rinsing of the test chemicals after exposure, keep the plates with dose tissues in the laminar hood until the last tissue is dosed. A bulb headed past your pipette will be used throughout the procedure to manipulate the tissue and test compounds. Bunsen burner flaming can be used to generate the bulb shaped tip to test liquid chemicals.Dispense 30 microliters of the liquid test article directly atop the tissue and immediately start the timer so that it counts up. Place the nylon mesh on the surface of the tissue When the timer reaches one minute, add the second liquid compound. Use three tissues per test chemical remembering to add each compound at one minute intervals.For testing semi solids, use a positive displacement pipette to dispense 30 microliters directly atop the tissue. If necessary, spread the chemical with pasture pipette to match the size of the tissue. Shortly before application of the solid substance, moisten the tissue surface with 25 microliters of sterile DPBS to improve contact of the tissue surface with the test chemical fill a 25 milligram sharp application spoon with the fine ground test material.Apply the solid test chemical to the tissue surface. If necessary, use a sterile bulb headed past your pipette to empty the spoon completely. Gently shake the inserts to improve the spreading of the solid on the surface.You may also use the bulb headed past your pipette to match the tissue size. Do not press on the tissue surface. Finally, apply DPBS as negative control and 5%SDS as positive control to the tissue inserts also in triplicates after dosing the last tissue, transfer all the plates for 35 plus or minus one minutes to the humidified 37 degrees Celsius incubator.During this incubation, make sure to place all materials needed for the washing steps in the hood. Following the 35 minute incubation, remove all the plates from the incubator. Place them into the sterile hood and wait until a period of 60 minutes for chemical exposure to be completed for the first dose tissue.Next, start the washing procedure. Rinse the tissues with sterile DPBS 15 times. To remove any residual test material, fill the tissue insert by using a constant stream of DPBS supplied 1.5 centimeters away from the tissue surface and empty it.Make sure that the DPBS stream is not too weak or the test substance will not be removed. Remember to maintain the one minute time interval for washing four tissues with a nylon mesh wash five times and remove the mesh carefully with the pointed sharp forceps. Then continue with the remaining 10 washes after the 15th, rinse using the wash bottle completely submerged the insert three times and 150 milliliters of DPBS and shake to remove the remainder of the test material.Finally, rinse the tissue. Insert once from the inside and once from the outside with sterile DPBS. Remove any excess of DPBS from the tissue by gently shaking the insert and blotting it on the sterile blotting paper.Next, transfer the blotted tissue inserts to the new six well plate previously prefilled with assay medium. Again, remember that all these steps must be done within the one minute interval to maintain the exposure time of 60 minutes for each exposed. After the last tissue is rinsed, carefully dry the surface of each tissue with a sterile cotton tipped swab.If traces of the chemical are still present on the surface, try removing them using the sterile wedded cotton swab. Last incubate tissues in the incubator for the next 24 hours. Record the start time of the incubation in the methods documentation sheet.Also record all the different applications in the method documentation sheet. Annex to the standard operating procedure provided by Mat Tech Corporation moving the plates in the hood. Transfer the insert into the lower part of the six well plate prefilled with 0.9 milliliter media.Collect the media from the upper row and pipe edit into a pre-labeled 24 well plate, which is then placed at minus 20 degrees Celsius. Until analysis, analysis of cytokines and or chemokines released into the culture media is an optional step. Continue with the incubation for another 18 to plus or minus three hours for the MTT assay label 2 24 Well plates with the chemical names or codes.Remember that MTT is toxic, so be sure to wear protective gloves. When handling. Prepare the MTT solution by thawing the MTT concentrate and diluting it with the MTT dilutant.Protect from light as MTT is light sensitive and use within two to three hours. Since MTT degrades over time pipette 300 microliters of MTT medium in each well of the 24 well plate. Now remove the six well plates from the incubator.Blot the bottom of the inserts on a blotting paper and transfer them into the 24 well plate prefilled with MTT. Place the 24 well plate in the incubator and incubate for three hours. Do not forget to record the start time of MTT incubation in the MDS strictly adhere to the three hours incubation time to avoid getting different MTT readings When the MTT incubation is complete, use a suction pump with a toxic waste trap to gently aspirate the MTT medium from all the wells.Refill the wells with DPBS and aspirate. Again, repeat this rinsing two more times and make sure the tissues are dry after the last aspiration. After the washes transfer the inserts to a new 24 well plate immerse the inserts by gently pipetting two milliliters of extract solution into each insert.The level will rise above the upper edges of the insert, thus completely covering the tissues from both sides. Seal the 24 well plate using a heat sealer or paraform to inhibit extract and evaporation. Record the start time of the extraction in the methods documentation sheet and extract for at least two hours at room temperature with gentle shaking on a plate shaker After the extraction period is complete, pierce the inserts with an injection needle or bulb headed past your pipette and allow the extract to run into the well from which the insert was taken.Discard the punctured inserts and pipette the solution in the well up and down three times until it is homogenous. A plate shaker set to 120 RPMs can also be used per each tissue. Transfer two 200 microliter aliquots of the blue forma and solution into a 96 well flat bottom micro titer plate.Transfer the duplicates according to the fixed plate design given in the spreadsheet accompanying this video protocol for blanks. Use ISO propanol, read the optical density or OD and a 96 well plate spectrophotometer using a wavelength of 570 nanometers without using a reference filter. Enter the results in the spreadsheet for automatic calculation of the results.Account for corrections due to test chemical interference as outlined in the accompanying protocol, a test chemical is labeled irritant. If the percent tissue viability is equal to or less than 50%following spectro photometric analysis, the OD five 70 of the NC tissues is greater than or equal to one and less than or equal to 2.5. And the mean viability of PC tissues expressed as a percentage of the negative control tissues is less than 20%And SD calculated from individual percentage tissue variabilities of three identically treated replicants is less than 18%Compounds yielding cell viability values less than 50%can be considered irritants.We have just shown you how to perform the validated 60 minutes, 42 hours skin irritation test with reconstructed human skin model.Epiderm. When performing this procedure, it is important to remember that major part of the test is performed under the sterile or as septic conditions. If there is a need for any method modification or if you need a better explanation of any of the procedure steps, please do not hesitate to contact us for help and advice.So that's it. Thank you for watching and good luck with your experiments.

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Research

JoVE Journal

Biology

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Generation of Self-assembled Vascularized Human Skin Equivalents

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

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