Bioengineered tissue constructs and cardiac uses thereof
A technology for constructs and hearts, applied in the field of implantation or attachment of cell-matrix constructs, implantation or attachment of bioengineered tissue constructs, and promotion of angiogenesis in tissues and organs
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Embodiment 1
[0153] Example 1: Formation of Collagen Matrix by Human Neonatal Foreskin Fibroblasts
[0154] Human neonatal foreskin fibroblasts (derived from Organogenesis, Inc.Canton, MA) were 5×10 5 cell / 162cm 2Inoculate into tissue culture treated flasks (Costar Corp., Cambridge, MA, cat # 3150) and grow in growth medium. The growth medium consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum (NBCS) (HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine (BioWhittaker, Walkersville, MD). ) (high glucose formulation, without L-glutamine, BioWhittaker, Walkersville, MD). Keep these cells at 37±1°C, 10±1% CO 2 atmosphere in the incubator. The medium was replaced with freshly prepared medium every 2-3 days. After 8 days in culture, cells have grown to confluence, that is, cells have formed a packed monolayer along the bottom of the tissue culture flask, and the culture medium is aspirated from the flask. Sterile filtered phosphate buffe...
Embodiment 2
[0165] Example 2: Full thickness skin constructs
[0166] Using the dermal construct formed using the method described in Example 1, normal human neonatal foreskin epidermal keratinocytes (derived from Organogenesis, Inc. Canton, MA) were seeded on the cell-matrix construct to form the surface of the skin construct. cortex.
[0167] Aseptically remove the medium from the culture insert and its surroundings. Normal human epidermal keratinocytes were expanded from frozen subcultured cell stocks to passage 4 to confluence. Cells were then detached from the dish using trypsin-verene, mixed, centrifuged to form a cell pellet, resuspended in epidermalization medium, counted and divided into 4.5 × 10 4 cells / cm 2 The density is seeded on top of the membrane. Then at 37±1℃, 10% CO 2 The constructs were incubated for 90 minutes at 90°C to allow keratinocytes to attach. After incubation, the constructs were submerged in epidermalization medium. The epidermization medium consists ...
Embodiment 3
[0171] Example 3: In vitro formation of a collagen matrix by human neonatal foreskin fibroblasts in a chemically defined medium
[0172] Using the method described in Example 1, human neonatal foreskin fibroblasts were expanded. Then resuspend the cells to 3 x 10 6 The concentration of cells / ml is 3.0×10 6 cell / TW(6.6×10 5 cells / cm 2 ) on 0.4 micron pore size, 24 mm diameter tissue culture treated membrane inserts in 6-well trays. These cells were then maintained as in Example 1 with medium omitting neonatal calf serum throughout. More specifically, the medium contained: DMEM, a 3:1 matrix mixture of HamsF-12 medium (Quality Biologics, Gaithersburg, MD), 4 mM GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng / mL human recombinant Epidermal growth factor (Upstate Biotechnology, Lake Placid, NY), 0.4 μg / ml hydrocortisone (Sigma St.Louis, MO), 1×10 -4 M ethanolamine (Fluka, Ronkonkoma, NY cat. #02400 ACS grade), 1 x 10 -4 M o-phosphoryl-ethanolamine (Sigma, St. Lou...
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