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The EpiDerm™ Skin Model (MatTek)
 

 
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The EpiDerm™ Skin Model

 

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피부 자극성 검사, 부식성 검사, 광독성 검사, 투과성 검사,  표피 분화 연구, 등

 

Features

EpiDermTM Tissue Model

  • Normal Human 3D (NHu-3D) Model of Epidermal Tissue
  • Produced From Normal Epithelial Keratinocytes
  • In Vivo-Like Lipid Profile
  • 15 YEARS of Publicly Available QC Data
  • Available in 24 and 96-Well High-Throughput Formats
  • Transdermal Drug Delivery/Percutaneous Absorption
  • Ideal for Gene Expression Analysis and RNAi/siRNA-Based Therapeutic Drug Screening
  • Full-Thickness Tissues (EpiDerm-FT) Available
  • “Underdeveloped” Tissues (EpiDerm-201) Available

General MatTek Tissue Features

  • Unsurpassed Long-Term Tissue Reproducibility
    • Lot-to-Lot, Year-to-Year
  • 3-Dimensional, Highly Differentiated Tissue
  • Metabolically, Mitotically Active
  • Produced from Normal (Non-Transformed) Human Cells - Ideal for Genomics Studies
  • Produced in Easily Handled Cell Culture Inserts
  • Grown in Completely Serum-Free Media System
  • Quantifiable, Objective Test Endpoints
  • Cost Effective Alternative to Animal and Human Clinical Testing

 

Applications

EpiDermTM Tissue Model

  • Skin Irritation (ECVAM Validated)
  • Skin Corrosion (ECVAM Validated)
  • Phototoxicity (ECVAM Pre-Validated)
  • Genotoxicity (COLIPA Pre-Validated)
  • Transdermal Drug Delivery
  • Nanoparticle Toxicity/Penetration
  • Inflammation
  • Basic Dermal Research

 

Data Sheet



MatTek's patented EpiDerm™ System consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. These "ready-to-use" tissues, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation on the cutting edge of in vitro skin technology. Ultrastructurally, the EpiDerm Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology.

The EpiDermTM Skin Model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. EpiDermTM consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. EpiDermTM
is mitotically and metabolically active.  Markers of mature
epidermis-specific differentiation such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase have been localized in the model.

Low magnification 100X

High magnification 400X

Ultrastructural analysis has revealed the presence of keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis.

The protocols for using the EpiDerm System are clear and straightforward. EpiDerm has been utilized with several common tests of cytotoxicity and irritancy, including MTT, IL-1α, PGE2, LDH, and sodium fluorescein permeability. Technicians find EpiDerm's rigid substrate design easier to handle in routine repetitive testing environments and scientists find that they are able to perform discriminating tests due to low background interference.

Various industrial and toxicology laboratories are actively seeking alternatives to whole animal testing. Cosmetic, household product, pharmaceutical and petrochemical companies have initiated in vitro toxicology testing to evaluate their raw materials and final product formulations A growing body of data indicates that EpiDerm effectively provides a non-animal means to assess dermal toxicology and skin research issues.

 

Barrier Function Properties


Recent results indicate that MatTek's in vitro skin model, EpiDerm, reproduces many of the barrier function properties of normal human skin. RuO4 stained transmission electron micrographs reveal intercellular lamellar sheets with the characteristic broad-narrow-broad spacing previously observed only in normal stratum corneum. Quantitative thin layer chromatographic analysis shows that EpiDerm contains all the lipids and ceramides present in vivo and a good quantitative correspondence between levels of ceramide l, the linoleate rich acylceramide strongly implicated in the maintenance of normal epidermal barrier function.

 

Permeability Characteristics


An initial evaluation of EpiDerm's permeability characteristics was made using a side-by-side diffusion chamber. A permeability coefficient, kp, for tritiated water flux was determined for standard EpiDerm (EPI-100) and somewhat more mature EpiDerm (EPI-612). Kp's of 5.0 x 10-3 and 3.6 x 10-3 cm/hr were calculated for EPI-100 and EPI-612 respectively, compared with 2.5 x 10-3 cm/hr for cadaver skin using the same diffusion apparatus. From these initial tests, EpiDerm appears more reproducible than cadaver skin, possibly due to EpiDerm's highly regular structure. Also, permeability studies were facilitated since steady state is reached more quickly due to EpiDerm's uniform thickness. Finally, similar to living epidermis, EpiDerm is metabolically and mitotically active and hence studies using EpiDerm should be more accurate than those obtained using non-viable cadaver or animal skin.

For these reasons, EpiDerm also appears to be a useful substrate for percutaneous absorption, transdermal drug delivery, and other studies relating to the barrier function of human skin.

 

 

Transmission Electron Micrograph of Intercellular
Lamellar Lipid Sheets Broad-Narrow-Broad Spacing
(Magnification 150,000x)

(See also EpiDerm-201™ Under-developed Skin Model)

 

EpiDerm
 

EpiDerm™ Technical Specifications

 

I. Ordering

  1. New Orders: MatTek Scientists will consult on your application at no additional charge. It is STRONGLY RECOMMENDED that you have this consultation BEFORE placing your first order to help ensure that all media and accessories needed to perform your application are included in the initial order.
  2. ALL Tissue Orders: Please allow 3-4 weeks from order date to delivery of tissues. Please contact MatTek Customer Service for additional details.

 

II. Cells

  1. Type: Normal human epidermal keratinocytes (NHEK).
  2. Genetic make-up: Single donor.
  3. Derived from: Neonatal-foreskin tissue.
  4. Alternatives: NHEK from adult breast tissue.
  5. Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma.

 

III. Medium

  1. Base medium: Dulbecco's Modified Eagle's Medium (DMEM).
  2. Growth factors/hormones: Epidermal growth factor, insulin, hydrocortisone and other proprietary stimulators of epidermal differentiation.
  3. Serum: None.
  4. Antibiotics: Gentamicin 5 µg/ml (10% of normal gentamicin level).
  5. Anti-fungal agent: Amphotericin B 0.25 µg/ml.
  6. pH Indicator: Phenol red.
  7. Other additives: Lipid precursors used to enhance epidermal barrier formation (proprietary).
  8. Alternatives: Phenol red-free (EPI-200-PRF), antibiotic-free (EPI-200-ABF), anti-fungal-free (EPI-200-AFF), or hydrocortisone-free medium and tissue (EPI-200-HCF) are available. Agents are removed at least 3 days prior to shipment.
    Please discuss your specific media formulation and tissue needs with MatTek scientific personnel before placing your order.
  9. Maintenance medium: Three maintenance media are available. EPI-100-MM is equivalent to the assay medium used for short term toxicological testing. Long Life Maintenance Medium, EPI-100-LLMM, contains α-MSH and β-FGF. New Maintenance Medium, EPI-100-NMM, contains Keratinocyte Growth Factor (KGF), but does not contain α-MSH and β-FGF. Although all media will result in good retention of epidermal morphology for up to 3 weeks (5.0 ml changed every other day), use of EPI-100-NMM will give the optimal histology for long term studies.

 

IV. Tissue

  1. Kit: Standard EpiDerm™ kit (EPI-200) consists of 24 individual tissues, each tissue 9 mm in diameter. (Tissue "kits" contain tissues, a small amount of culture medium, and plasticware; contact MatTek for specific kit contents.)
  2. Substrate: Chemically modified, collagen-coated, 9 mm ID single well tissue culture plate inserts are used (e.g. Millicell CM or Nunc polycarbonate single well tissue culture plate inserts).
  3. Culture: At air liquid interface.
  4. Histology: 8-12 cell layers (basal, spinous, and granular layers).
  5. Stratum Corneum: 10-15 layers (based on Transmission Electron Microscopy (TEM)).
  6. Lot Numbers: Tissue lots produced by each technician for each week are assigned a specific lot number. Typically, there are multiple lot numbers for any given week's tissue production. A letter of the alphabet is appended to the end of the lot number to differentiate between individual kits within a given lot of tissues. All tissue kits within a lot are identical in regards to cells, medium, handling, culture conditions, etc.
  7. Shipment: At 4°C on medium-supplemented, agarose gels in 24-well plate.
  8. Shipment day: Every Monday. Shipment on Thursday also possible upon special request.
  9. Delivery: Outside US: Tuesday-Thursday depending on location.
  10. Shelf life: Including time in transit, tissues may be stored at 4°C for up to 6 days prior to use. However, extended storage periods are not recommended unless necessary. In addition, the best reproducibility will be obtained if tissues are used consistently on the same day, e.g. Tuesday afternoon or following overnight storage at 4°C (Wednesday morning).
  11. Length of experiments: Cultures can be continued for up to 3 weeks with good retention of normal epidermal morphology. Cultures must be fed every other day with 5.0 ml of long life maintenance medium (EPI-100-LLMM), standard maintenance medium (EPI-100-MM), or new maintenance medium (EPI-100-NMM). Cell culture inserts are placed atop culture stands (MEL-STND; click on this link for photo) or washers (EPI-WSHR) in 6-well plates to allow use of 5.0 ml.

  12. Alternative Tissues/Forms:

    Alternative Media/Tissues: As also stated in section III. Medium above, Phenol red-free (EPI-200-PRF), antibiotic-free (EPI-200-ABF), anti-fungal-free (EPI-200-AFF), or hydrocortisone-free medium and tissue (EPI-200-HCF) are available. Agents are removed at least 3 days prior to shipment.
    Please discuss your specific media formulation and tissue needs with MatTek scientific personnel before placing your order.

    EPI-200-1S, -3S, -4S, -5S: Undifferentiated EPI-200 tissue. Tissue has not been cultured at the Air-Liquid Interface. Customer receives required media to continue cultures and produce EPI-200. Discussion with a MatTek technical representative is required prior to ordering due to proprietary nature of this product. Designed for study of broad range of skin phenomena in which experimental design dictates use of undifferentiated tissue.

    EPI-200-SIT: Kit for ECVAM-validated EpiDerm Skin Irritation Test (SIT). Irritant/Non-Irritant determination. Same as EPI-200 except contains items specific to this test protocol.

    EPI-200-CORR: Kit for Dermal Corrosivity determination. Same as EPI-200 except contains reagents specific to Dermal Corrosivity Assay.

    EPI-200-EACH: Same as EPI-200 except only 1 tissue, not 24 tissues.

    EPI-200-FRZN-EA: One EPI-200 tissue that has been subjected to 3 freeze/thaw cycles. Designed to serve as control for MTT auto-reduction test.

    EPI-200-PHO: Standard EPI-200 tissue kit except customer receives 2x60 mL bottles of assay medium. Designed for phototoxicity experiments utilizing Zebet protocol.

    EPI-200-X: Same as EPI-200, but with enhanced barrier function--designed for permeability studies.

    EPI-201: Cultured 3 days less than EPI-200, less differentiated than EPI-200. Customer receives 250 ml of medium to differentiate EPI-201 into EPI-200. Designed for studying modulators of epidermal differentiation.

    EPI-212: Same as EPI-200 except 12 tissues instead of 24.

    EPI-212-SIT: Same as EPI-200-SIT except 12 tissues instead of 24.

    EPI-212-X: Same as EPI-200-X except 12 tissues instead of 24.

    EPI-224: Same as EPI-200 except 24 tissues in one 24 well plate configuration.

    EPI-296: Same as EPI-200 except 96 tissues in one 96 well plate configuration.

    EPI-606: Same as EPI-200, except 6 tissues, each tissue cultured in a larger diameter cell culture insert (22 mm diameter).

    EPI-606-X: Same as EPI-606, but with enhanced barrier function--designed for permeability studies.

    ========================

    EpiDerm Total RNA & Protein Lysate:

    EPI-200-PROT: EpiDerm Total Protein Lysate prepared from EPI-200, 100 ug. Inquire about specific treatments (PMA, UV, etc.).

    EPI-RNA: Purified Total RNA from control (untreated) or treated EpiDerm tissues (EPI-200 and EPI-200-HCF). Each lot of RNA is characterized and provided with a quality control spec sheet including histology and Triton ET50 of the specific tissue lots, agarose gel electrophoresis of total RNA, RNA concentration, 260/280 ratio, G3PDH RT-PCR and TAQ-only PCR amplifications. Control (untreated), simulated solar UVA, solar UVA+UVB and phorbol ester treated EpiDerm RNAs are available.

 

V. Quality Control and Sterility

  1. Visual inspection: All tissues are visually inspected and if physical imperfections are noted, tissues are rejected for shipment.
  2. End-use testing: Tissues are exposed to 1% Triton X-100 for 4, 6, 8, and 12.5 hours. The time of exposure required to reduce the tissue viability (ET-50) using the MTT assay is determined (See MatTek EpiDerm™ ET-50 protocol) for each lot of tissue. ET-50's must fall within the range of the 1996 EpiDerm™ database: 4.77-8.72 hours. ET-50's in customers' lab may differ slightly from the MatTek results.
  3. Sterility: All media used throughout the production process is checked for sterility. Maintenance medium is incubated with and without antibiotics for 1 week and checked for sterility. The agarose gel from the 24-well plate used for shipping is also incubated for 1 week and checked for any sign of contamination.
  4. Screening for pathogens: All cells are screened and are negative for HIV, hepatitis B and hepatitis C using PCR. However, no known test method can offer complete assurance that the cells are pathogen free. Thus, these products and all human derived products should be handled at BSL-2 levels (biosafety level 2) or higher as recommended in the CDC-NIH manual, “Biosafety in microbiological and biomedical laboratories,” 1998. For further assistance, please contact your site Safety Officer or MatTek technical service.
  5. Notification of lot failure: If a tissue lot fails our QC or sterility testing, the customer will be notified and the tissues will be replaced without charge when appropriate. Because our QC and sterility testing is done post-shipment, notification will be made as soon as possible (Under normal circumstances, ET-50 failures will be notified by Wednesday 5 p.m.; sterility failures will be notified within 8 days of shipment).

 

EpiDerm Shipment and Dimension Information

Part
Number

Ship
Day

Tissue Diameter

Culture
Area

Number of Cultures

EPI-200

Monday

8 mm

0.6 cm2

24

EPI-200-1S

Tuesday

8 mm

0.6 cm2

24

EPI-200-3S

Monday

8 mm

0.6 cm2

24

EPI-200-5S

Wednesday

8 mm

0.6 cm2

24

EPI-200-FRZN

Monday

8 mm

0.6 cm2

1

EPI-200-PHO

Monday

8 mm

0.6 cm2

24

EPI-201

Monday

8 mm

0.6 cm2

24

EPI-212

Monday

8 mm

0.6 cm2

12

EPI-606

Monday

22mm

4.2 cm2

6

EPI-606X

Monday

22mm

4.2 cm2

6

EPI-612

Wednesday

8 mm

0.6 cm2

12

EPI-EACH

Monday

8 mm

0.6 cm2

1

Notes:
For phenol red-free medium add suffix –PRF to part number
For antibiotic-free medium add suffix –ABF to part number
For anti-fungal-free medium, add suffix –AFF to part number
For anti-fungal-free, anti-biotic-free medium, add suffix –AFAB to part number
For hydrocortisone-free medium, add suffix –HCF to part number

 

Guide to In Vitro Tissue Model Basics

 

EpiDerm™ Applications



Skin Irritation

EpiDerm Skin Irritation Test (SIT) 

Dermal Corrosion

Dermal Phototoxicity

Skin Inflammation

Percutaneous Absorption/Penetration

Transdermal Drug Delivery

EpiDerm - Long Term Reproducibility (MatTek TR-355)

EpiDerm Use In Psoriasis Research (MatTek TR-432)

EpiDerm High Throughput Screening (HTS) Formats


 

 

 

 

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