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

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.