This protocol allows for the production of filaments of bulk extracellular matrix from cultured cell lines, allowing for research into cell matrix interactions, as well as research into the potential of this biomaterial for wound repair. These synthetic fibers can avoid the foreign body response directed against synthetic materials, while still drawing from the rich repertoire of textile fabrication techniques to create a wide range of woven implantables. Demonstrating the procedure will be Cassandra Reed, a graduate student from my lab.
Before seeding the cells, autoclave the hollow fiber membranes at 121 degrees Celsius for 30 minutes, followed by treatment with 50 milliliters of bovine plasma fibronectin in PBS at 37 degrees Celsius in 5%carbon dioxide for one hour. At the end of the incubation, use sterile micro scissors to cut the fibers to six centimeters or less, and use a one milliliter syringe equipped with a 21 gauge needle to seed one times 10 to the fifth fibroblast cells directly into the lumina of the hollow fiber membranes. Place six seeded fibers into a six inch diameter Petri dish, and briefly incubate the seeded fibers at 37 degrees Celsius in 5%carbon dioxide.
After five minutes, transfer the fibers from the incubator into a new six centimeter Petri dish containing fibroblast culture medium supplemented with L-ascorbic acid, L-ascorbic acid 2 phosphate, and transforming growth factor beta 1 for up to three weeks in the cell culture incubator. At the end of the incubation, use forceps o transfer the cultured fibers into individual scintillation vials, and tilt each vial to allow the addition of up to five milliliters of N-Methyl-2-pyrrolidone down the side of the vials. Then use a serological pipette to slowly aspirate the N-Methyl-2-pyrrolidone from each fiber, and transfer the fibers into new scintillation vials for a second immersion in fresh N-Methyl-2-pyrrolidone.
After the third immersion, rinse the resulting extracellular matrix threads three times in deionized water in a similar manner, and place a three-inch-long, one-inch wide 1/23rd-inch-thick piece of silicone rubber with a central eight-by-four millimeter rectangular mold onto a standard three-by-one inch glass microscope slide. After autoclaving, lay each extracellular matrix fiber side-by-side in the eight-by-four millimeter silicone mold until there is no visible open space. Place the mold into a 50 milliliter conical tube, and freeze the fibers at negative 80 degrees Celsius until completely frozen.
For decellularization, incubate the mold in 1%sodium dodecyl sulfate for 24 hours at room temperature on a rocker with gentle agitation. The next day, rinse the extracted extracellular matrix with three washes in three milliliters of PBS per wash, and fill the mold with freshly prepared DNAs/RNAs digestion buffer for a six hour incubation at four degrees Celsius. At the end of the digestion, aspirate the digestion solution and rinse the mold three times in sterile PBS as demonstrated.
After the third wash, incubate the scaffolds in 10%Penicillin-Streptomycin in PBS at four degrees Celsius overnight before freezing in a 50 milliliter conical tube at negative 80 degrees Celsius for about one hour. When the mesh has completely frozen, lyophilize the decellularized extracellular mesh overnight, or until completely dry, and transfer the lyophilized scaffolds to a sterile container within a bio safety hood and store at four degrees Celsius until use. Here a transverse cross-section of a polysulfone hollow fiber membrane fabricated using this protocol is shown, exhibiting outer and inner layers of finger-like pores characteristic of an asymmetric membrane.
After fibroblast cell seeding and culture, N-Methyl-2-pyrrolidone rinsing as demonstrated, translucent threads of extracellular matrix are produced. The extracellular matrix producing cells remain viable inside the hollow fiber membranes throughout the entire three week culture period. The extracted matrix has a translucent appearance when hydrated, exhibiting an off-white color and fibrous appearance with a gross longitudinal alignment upon mesh assembly and lyophilization.
The most critical step is the dissolving process of the cultured hollow fiber membranes in the N-Methyl-2-pyrrolidone solvent in order to extract the extracellular matrix. The material produced by this method can be used for fabricating tissue engineered implants for investigating the repair of tissues in pre-clinical studies. The N-Methyl-2-pyrrolidone used in this protocol is a skin and eye irritant, and therefore it is recommended that you handle the chemical using nitrile gloves, goggles, a lab coat, and handle it within a fume hood.
