MelanoDerm™ 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- Types: a) Normal human epidermal keratinocytes (NHEK); b) Normal human melanocytes (NHM).
- Genetic make-up: NHEK and NHM are from individual, but different donors. Tissue containing matched NHEK and NHM are available upon special request.
- Ethnic origin: MelanoDerm containing NHM from Caucasian (MEL-300-C), Black (MEL-300-B), and Asian (MEL-300-A) donors are available.
- Derived from: Neonatal-foreskin tissue.
- Alternatives: A limited number of strains of NHEK & NHM from adult breast tissue.
- Screened for: HIV, Hepatitis B, and Hepatitis C.
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 (MEL-300-B-PRF), antibiotic-free (MEL-300-B-ABF), anti-fungal-free (MEL-300-B-AFF), and hydrocortisone-free medium and tissue (MEL-300-B-HCF) are available. Agents are removed at least 3 days prior to shipment.
- Maintenance medium: Three maintenance media are available. EPI-100-MM is equivalent to 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 KGF, but does not contain α-MSH or β-FGF. Tissues will darken most in EPI-100-LLMM, moderately in EPI-100-NMM, and least in EPI-100-MM. All media will result in good retention of epidermal morphology for up to 3 weeks (5.0 ml changed every other day).
IV. Tissue - Kit: Standard MelanoDerm™ kit (MEL-300) 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.
- Seeding ratio: NHM:NHEK = 1/10. Ratio of 1/30 available upon request.
- Histology: 8-12 cell layers (basal, spinous, and granular layers).
- Stratum Corneum: 10-15 layers (based on 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 available upon special request.
- Delivery: Tuesday morning via FedEx priority service (US). Outside US: Tuesday-Wednesday 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 either long life maintenance medium (EPI-100-LLMM) or standard maintenance medium (EPI-100-MM). Cell culture inserts are placed atop culture stands (MEL-STND; see photo below) or washers (EPI-WSHR) in 6-well plates to allow use of 5.0 ml.
- Alternative tissues:
MEL-300-EACH: Same as MEL-300 except only 1 tissue, not 24 tissues.
MEL-300-B-FRZN-EA: MEL-300-B tissue that has been subjected to 3 freeze/thaw cycles. Designed to serve as control for melanin assay.
MEL-301: Cultured 3 days less than MEL-300, less differentiated than MEL-300
MEL-312 (-A, -B, -C): Identical to MEL-300 (-A, -B, -C) except 12 tissues per kit instead of 24.
MEL-606 (-A, -B, -C): Same as MEL-300 (-A, -B, -C) except 6 tissues per kit, each tissue cultured in a larger diameter cell culture insert (ID = 22 mm).
 |
|
MEL-STND Accessory For Extended Culture Periods: Shown: 6-well plate with cell culture insert atop a culture stand (Part #MEL-STND). For experiments extending beyond 24 hours, tissues need to be fed with 5.0 ml every other day. Use of the culture stand allows feeding with 5.0 ml from the basolateral side of the tissue. Use of culture stands is necessary in order to maintain normal tissue morphology. Click on photo to see larger image.
|
V. Quality Control and Sterility - Visual inspection: All tissues are visually inspected and if physical imperfections are noted, tissues are rejected for shipment. Select tissues are examined microscopically to insure that melanocytes are visible (for MEL-300-B and MEL-300-A tissues) and that they are evenly distributed throughout the tissue.
- End-use testing: Tissues are continued in EPI-100-LLMM to insure that macroscopic darkening of tissues occurs. Tissues are exposed to 1% Triton X-100 for 5 hrs. The viability at 5 hrs must exceed 45% as determined by the MTT assay (See MatTek EpiDerm™ ET-50 protocol) for each lot of tissue.
- 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, % viability failures will be notified by Wednesday 5 p.m.; sterility failures will be notified within 8 days of shipment).
|
|
|
