Placenta Derived Adherent Cell (PDAC) Interaction and Response on Extracellular Matrix Isolated from Human Placenta
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Methods
Isolation of pECM. The pECM was isolated from frozen placentas, qualified as described previously.7,8 A frozen human placenta was thawed in 0.5 M sodium chloride, ground in an Anvil meat-grinder (Model MIN0012), and repeatedly washed in 0.5 M sodium chloride and water mixture using a New Brunswick Scientific (Model C25; Edison, NJ) incubator shaker at 23°C. This was followed by a detergent wash with either 1% SDS or deoxycholic acid. The blood-free placental tissue was treated with 0.1–0.5 N sodium hydroxide (3–24 hours), followed by rinsing with PBS to neutralize the pH. The material produced was a stable paste and stored at 4°C.
Biochemical analysis. To determine the biochemical composition of the isolated pECM,a sample of known dry weight was freeze-dried and the dry weight was determined.The pECM was solubilized by either dissolving in 10 mM HCl at 100°C or by treating with pepsin (1 mg/g) in 10 mM HCl at 23°C for 18 hours.The tissue dissolved in 10 mM HCl was used to determine content of fibronectin, laminin, glycosaminoglycans (GAGs), and elastin. The pepsin-solubilized tissue was used to determine collagen content using the SIRCOL dye based assay kits from Accurate Chemical and Scientific (Westbury, NY). Fibronectin and laminin concentrations were determined using a sandwich ELISA obtained from Takara Bio USA (Madison,Wis). Elastin and GAG content were determined using the FASTIN and BLYSCAN dye based assay kits (Accurate Chemical and Scientific).For collagen I, III, and IV, sandwich ELISAs were developed using primary and HRP conjugated secondary antibodies from Rockland Immunochemicals, Inc. (Gilbertsville, Pa).
Preparation of pECM constructs. To prepare sheets of this pECM, an even layer of hydrated pECM paste was sandwiched between 2 medical grade Tyvek sheets from Tolas Health Care (Feasterville, Pa).This construct was loaded into a gel drier (BioRad Model 583) and vacuum was applied overnight at 23°C until the pECM film was dry. Sheets were cut to an appropriate size for cell culture studies.To prepare 3-D structures of the pECM, the pECM paste was filled into various molds and freeze-dried using a benchtop Labconco Freezone 4.5 freeze dryer (Labconco,Kansas City,Mo).To study the stability of the pECM sheets and 3-D molds in cell culture media or water.The constructs were incubated at 37°C up to 1 week in water, saline, or cell culture media.
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