Underdeveloped EpiDerm™ Skin Model (EPI-201) – 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, antibiotic-free, anti-fungal-free, or hydrocortisone-free medium and tissue are available. Agents are removed at least 3 days prior to shipment.
- Maintenance medium: EPI-201 kits are provided with differentiation medium (EPI-100-DM). After 3 days of culture using EPI-100-DM (5.0 ml changed every other day), EPI-200 tissues are produced from the EPI-201 tissues. Beyond that point, maintenance media usable for EPI-100 can be used (EPI-100 is a previous version of EPI-200). See EPI-200 technical specifications for more details.
IV. Tissue - Kit: Under-developed EpiDermTM Skin Model (EPI-201) consists of 24 tissues which have been cultured for 3 days less than EPI-200.
- Substrate: Chemically modified, collagen-coated, 9 mm ID single well tissue culture plate inserts are used (e.g. Millicell CM, Nunc polycarbonate single well tissue culture plate inserts).
- Culture: At air liquid interface.
- Histology: 4-6 cell layers (basal, spinous, and granular layers).
- Stratum Corneum: <10 layers (based on TEM).
- Lot #'s: Tissue lots produced by each technician for each week are assigned a specific lot #. Typically, there are multiple lot #'s for any given week's tissue production. A letter of the alphabet is appended to the end of the lot # 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: Monday or Thursday.
- Lead Time: 3 weeks (for Monday shipment) maximum. Call Customer Service to see if shorter lead time is possible.
- 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 absolutely 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: EPI-201 cultures can be continued 3 days to produce EPI-200 tissues. After that, cultures can be continued for up to 3 weeks with good retention of normal epidermal morphology. Initially, EPI-201 cultures must be fed every other day with 5.0 ml of EPI-100-DM to produce EPI-200 tissue. After that point, the tissues are maintained in either long life maintenance medium (EPI-100-LLMM) or standard maintenance medium (EPI-100-MM). Cell culture inserts are placed atop washers (EPI-WSHR) or culture stands (MEL-STND) in 6-well plates to allow us of 5.0 ml.
- Alternative tissues:
EPI-201-PRF: Cultured using phenol red-free medium. All EPI-201 tissues are cultured for 3 days less than standard EPI-200 tissue.
EPI-201-AFAB: Cultured in antibiotic (gertamicin)-free and anti-fungal agent (Amphotericin B)-free medium. All EPI-201 tissues are cultured for 3 days less than standard EPI-200 tissue.
EPI-201-HCF: Cultured in hydrocortisone-free medium. All EPI-201 tissues are cultured for 3 days less than standard EPI-200 tissue.
EPI-200-1S: These undifferentiated tissues are grown for 1 day in submerged culture similar to the initial stages of standard EPI-200 production. These cultures are not exposed to the air-liquid inferface.
EPI-200-3S: These undifferentiated tissues are grown for 3 days in submerged culture similar to the initial stages of standard EPI-200 production. These cultures are not exposed to the air-liquid inferface.
EPI-200-5S: These undifferentiated tissues are grown for 5 days in submerged culture similar to the initial stages of standard EPI-200 production. These cultures are not exposed to the air-liquid inferface.
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: EPI-201 tissues are continued in culture for 3 additional days and tested in an identical manner to EPI-200 tissue lots. The EPI-200 tissue is 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.
- 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. Because our QC and sterility testing is done post-shipment, notification will be made as soon as possible.
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