EpiDerm™ Technical Specifications
I. Ordering- 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.
- 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- Type: Normal human epidermal keratinocytes (NHEK).
- Genetic make-up: Single donor.
- Derived from: Neonatal-foreskin tissue.
- Alternatives: NHEK from adult breast tissue.
- Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma.
III. Medium - Base medium: Dulbecco's Modified Eagle's Medium (DMEM).
- Growth factors/hormones: Epidermal growth factor, insulin, hydrocortisone and other proprietary stimulators of epidermal differentiation.
- Serum: None.
- Antibiotics: Gentamicin 5 µg/ml (10% of normal gentamicin level).
- Anti-fungal agent: Amphotericin B 0.25 µg/ml.
- pH Indicator: Phenol red.
- Other additives: Lipid precursors used to enhance epidermal barrier formation (proprietary).
- 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. - 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 - Kit: Standard EpiDerm™ kit (EPI-200) consists of 24 individual tissues, each tissue 9 mm in diameter.
- 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).
- Culture: At air liquid interface.
- Histology: 8-12 cell layers (basal, spinous, and granular layers).
- Stratum Corneum: 10-15 layers (based on Transmission Electron Microscopy (TEM)).
- 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.
- Shipment: At 4°C on medium-supplemented, agarose gels in 24-well plate.
- Shipment day: Every Monday. Shipment on Thursday also possible upon special request.
- Delivery: Tuesday morning via FedEx priority service (US). Outside US: Tuesday-Thursday depending on location.
- 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).
- 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.
- 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-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-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.
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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 - Visual inspection: All tissues are visually inspected and if physical imperfections are noted, tissues are rejected for shipment.
- 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.
- 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.
- 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.
- 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).
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