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Human Fibroblast-Derived Temporary Skin Substitute
» Human Fibroblast-Derived Temporary Skin Substitute is a nonliving monolayer skin substitute derived from neonatal foreskins. It is composed of fibroblasts, an extracellular matrix, and a nylon mesh bonded to a transparent, semi-permeable silicone membrane. Human fibroblasts are seeded onto the nylon mesh. The fibroblasts proliferate within the nylon mesh and secrete human dermal collagen, matrix proteins, growth factors, and cytokines. Following freezing, no cellular metabolic activity remains; however, the tissue matrix and bound growth factors remain. Human Fibroblast-Derived Temporary Skin Substitute does not contain macrophages, lymphocytes, blood vessels, hair follicles, muscle fibers, or keratin. The fibroblast-cell banks, from which Human Fibroblast-Derived Temporary Skin Substitute is derived, test negative for human and animal viruses and retroviruses and are also tested for normal cell morphology, human karyology, and isoenzymes. Maternal blood sera are tested for evidence of infection with human immunodeficiency virus types 1 and 2, hepatitis B and C viruses, syphilis, and human T-lymphotropic virus type 1 and is found negative for the purpose of donor selection. Reagents used in the manufacture of Human Fibroblast-Derived Temporary Skin Substitute are tested and found free from viruses, retroviruses, endotoxins, and mycoplasma before use. Human Fibroblast-Derived Temporary Skin Substitute is manufactured with sterile components under aseptic conditions within the final package. All materials derived from bovine sources originate from countries free of bovine spongiform encephalopathy. During subsequent screening of the fibroblast cell strain at various stages in the manufacturing process, testing for these same viruses, as well as Epstein-Barr virus and human T-lymphotropic virus type 2, is carried out and found to be negative. The final product tests negative for the presence of mycoplasma.
Packaging and storage— Human Fibroblast-Derived Temporary Skin Substitute is aseptically packaged and supplied frozen in a clear plastic cassette containing two, approximately 12.5- × 19-cm, units. The solution within the cassette is a phosphate-buffered cryoprotectant solution used to facilitate long-term storage. A clear plastic bag surrounds the cassette for its protection. Human Fibroblast-Derived Temporary Skin Substitute should be stored at a temperature of –70 to –20 for no longer than 18 months.
Labeling— The label indicates the dimensions of the Human Fibroblast-Derived Temporary Skin Substitute material enclosed. It contains the expiry date, required storage conditions, and the lot number. The label cautions that Human Fibroblast-Derived Temporary Skin Substitute is not to be used if the package shows signs of damage. Additional labeling requirements include instructions on the proper thawing and handling of Human Fibroblast-Derived Temporary Skin Substitute and the time frame for use after package opening.
USP Authentic Visual References 11— USP Human Fibroblast-Derived Skin Substitute Reference Photomicrographs. These three photomicrographs represent examples of passing units, prepared as directed in Hematoxylin–eosin staining, Collagen staining, and Distribution of fibronectin. They are specified to assist in ascertaining histological quality. The fibroblasts are embedded in an extracellular matrix that they have secreted (USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 1). The collagen (USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 2) and fibronectin (USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 3) are to be found throughout the extracellular matrix. The nylon fibers (yellow in USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 1) and the silicone backing (grey in USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 1) are frequently visible although easily lost during processing. However, at this magnification, the presence of these components is the only visible difference between the Cryopreserved Human Fibroblast-Derived Dermal Substitute and the Human Fibroblast-Derived Temporary Skin Substitute.
Sterility tests 71: meets the requirements.
Test solution— Thaw Human Fibroblast-Derived Temporary Skin Substitute by placing the tissue, still in its polycarbonate cassette contained in a plastic covering bag, in a water bath heated to a maximum of 37 for 15 to 20 minutes until no visible ice remains in the cassette. The minimum amount of water in the water bath is 2 L per Human Fibroblast-Derived Temporary Skin Substitute unit. Perform the test on 20 mL of the cryopreservative.
Bacterial endotoxins 85— Thaw Human Fibroblast-Derived Temporary Skin Substitute by placing the tissue, still in its polycarbonate cassette contained in a plastic covering bag, in a water bath heated to a maximum of 37 for 15 to 20 minutes until no visible ice remains in the cassette. The minimum amount of water in the water bath is 2 L per Human Fibroblast-Derived Temporary Skin Substitute unit. Remove the unit from the polycarbonate cassette, and immerse in 25 mL of LAL Reagent Water. Extract for 60 minutes at 37 with shaking on an orbital shaker set at 125 revolutions per minute. Remove a 4-mL aliquot of the extract for testing: it contains not more than 0.5 USP Endotoxin Unit per mL.
Histological characterization—
Buffered formalin and Preparation of tissue for staining— Proceed as directed in the test for Histological characterization under Cryopreserved Human Fibroblast-Derived Dermal Substitute, substituting Human Fibroblast-Derived Temporary Skin Substitute for Cryopreserved Human Fibroblast-Derived Dermal Substitute. The fibroblasts appear elongated and spindle shaped. The tissue contains about 106 cells per cm2 and about 500 cells per mm along the section.
HEMATOXYLIN–EOSIN STAINING—
Hematoxylin–alcohol solution, Hematoxylin staining solution, 10% Acid alcohol, Eosin solution, and Procedure— Proceed as directed for Hematoxylin–eosin staining in the test for Histological characterization under Cryopreserved Human Fibroblast-Derived Dermal Substitute. Using USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 1 (hematoxylin–eosin stained) for comparison, the nylon-scaffold mesh, silicone membrane, and secreted collagen-based matrix are present and the tissue contains normal human fibroblast distributed throughout the secreted matrix and resembles normal human papillary dermis.
COLLAGEN STAINING—
Bouins's solution, Weigert's iron hematoxylin working solution, Gomori's trichrome solution, 1% Acetic acid, and Procedure— Proceed as directed for Collagen staining in the test for Histological characterization under Cryopreserved Human Fibroblast-Derived Dermal Substitute. Using USP Human Fibroblast-Derived Skin Reference Photomicrograph 2 for comparison, collagen is found throughout the extracellular matrix in a manner indistinguishable from Cryopreserved Human Fibroblast-Derived Dermal Substitute.
DISTRIBUTION OF FIBRONECTIN—
Tris-saline buffer, 3% Hydrogen peroxide, Diaminobenzidine solution, Hematoxylin staining solution, and Procedure— Proceed as directed for Distribution of fibronectin in the test for Histological characterization under Cryopreserved Human Fibroblast-Derived Dermal Substitute. Using USP Human Fibroblast-Derived Skin Substitute Reference Photomicrograph 3 (diaminobenzidine–hematoxylin stained) for comparison, fibronectin binds to collagen and is found throughout the extracellular matrix in a manner indistinguishable from Cryopreserved Human Fibroblast-Derived Dermal Substitute.
Metabolic activity assessment—
DPBS working solution, Assay stock medium, MTT-assay solution, MTT formazan calibration solutions, and Procedure— Proceed as directed in the test for Metabolic activity assessment under Cryopreserved Human Fibroblast-Derived Dermal Substitute, substituting Human Fibroblast-Derived Temporary Skin Substitute for Cryopreserved Human Fibroblast-Derived Dermal Substitute: the absorbance value of individual Human Fibroblast-Derived Temporary Skin Substitute sections at 540 nm is less than 0.1.
DNA content—
Cell culture water, Working DNA extraction buffer, Dilution buffer, DPBS without Ca++, Mg++ solution, Calf thymus DNA calibration solutions, DNA staining solution, and Procedure— Proceed as directed in the test for DNA content under Cryopreserved Human Fibroblast-Derived Dermal Substitute, substituting Human Fibroblast-Derived Temporary Skin Substitute for Cryopreserved Human Fibroblast-Derived Dermal Substitute. The amount of DNA in individual Human Fibroblast-Derived Temporary Skin Substitute 11- × 11-mm sections is between 6 and 14 µg.
Total collagen content—
DPBS without Ca++, Mg++ solution, DPBS with Ca++, Mg++ solution, Collagenase extraction solution, 2% Acetic acid solution, Collagen calibration standards, Sirius red solution, 1% (p-tert-Octylphenoxy)polyethoxyethanol solution, and Procedure— Proceed as directed in the test for Total collagen content under Cryopreserved Human Fibroblast-Derived Dermal Substitute, substituting Human Fibroblast-Derived Temporary Skin Substitute for Cryopreserved Human Fibroblast-Derived Dermal Substitute: the amount of collagen in individual Human Fibroblast-Derived Temporary Skin Substitute 11- × 11-mm samples is between 0.50 and 4.0 mg.
Residual solvents 467: meets the requirements.
(Official January 1, 2007)
Auxiliary Information— Staff Liaison : Anita Y. Szajek, Senior Scientist
Expert Committee : (BBCGT05) Biologics and Biotechnology - Cell and Gene Therapy
USP29–NF24 Page 1967
Pharmacopeial Forum : Volume No. 30(4) Page 1221
Phone Number : 1-301-816-8325