Melanoma, In Vitro Disease Tissue Model

3D Tissue Models

Tissue:
Kit: Full thickness melanoma skin construct (MLNM-FT-A375 kit) consists of 24 tissues. (Tissue "kits" contain tissues, a small amount of culture medium, and plasticware; contact us for specific kit contents.)
Substrate: Costar SnapwellTM single well tissue culture plate inserts are used. Pore size = 0.4 µm, Diameter = 1.2 cm, Surface Area = 1.0 cm2.
Culture: Initially submerged and then at the air liquid interface.
Histology: Epithelium: 8-12 epithelial cell layers plus stratum corneum (basal, spinous, and granular layers); Dermis: Collagen gel containing fibroblasts. At early stage melanoma cells form nodes at dermal/epidermal junction. At later stages melanomas progress to RGP (radial growth phase), VGP (vertical growth phase) and consecutively invade dermal component.
Lot numbers: Tissue lots produced each week are assigned a specific lot number. 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.
Shelf life: Including time in transit, tissues may be stored at 4°C for up to 3 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 4 weeks for melanoma development and progression with good retention of normal epidermal morphology. Cultures must be fed every other day with 5.0 ml of MLNM-FT-MM.
Alternative tissues:
MLNM-FT-A375 (Day 7): Early stage MLNM-FT-A375 tissue. Tissue has not been cultured at the Air-Liquid Interface. Customer receives required media (MLNM-FT-GM) to continue cultures and produce MLNM-FT-A375. Discussion with a technical representative is required prior to ordering due to proprietary nature of this product. Designed for study of early stages of melanoma development in which experimental design dictates use of early stage tissue.
MLNM-FT-EXP: Same as MLMN-FT-A375 except A375 cells are replaced with customer-supplied melanoma cells.

Cells:
Type: Human melanoma cells (A375); Normal human epidermal keratinocytes (NHEK); Normal human dermal fibroblasts (NHDF).
Derived from: Malignant melanoma cell line (A375); Neonatal-foreskin tissue (NHEK); Neonatal skin (NHDF);
Alternatives: Human malignant melanoma cells SK-Mel-28.
Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma.

Medium:
Base medium: DMEM Basal Medium.
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).
Assay/Maintenance medium: MLNM-FT-MM is utilized for assays as well as long term maintenance of the MLNM-FT-A375 tissues.

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 from the production lot are retained, grown for additional 7 days, fixed for histology, and analyzed.
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.
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 sterility failures will be notified within 8 days of shipment).

Tissues are shipped at 4°C on medium-supplemented, agarose gels in 6-well plates.

Tumor Invasion and Anti-Melanoma Drug Screening
MatTek's Melanoma tissue exhibits radial growth phase, vertical growth phase, and metastatic melanoma phenotypes, paralleling the progression of melanoma in vivo. This model provides a valuable tool for oncologists and cancer researchers to study, understand, and develop preventative and therapeutic treatments for one of the most serious cutaneous malignancies.