Читать книгу PRF Applications in Endodontics - Mahmoud Torabinejad - Страница 18

Umbilical cords are collected from eligible donors at the time of delivery and transported to the processing facility on ice (2°C to 8°C) in Dulbecco’s Modified Eagle Media (DMEM). Cords are processed immediately under aseptic conditions, and MSCs are collected for culture according to the procedure described below.

To start a primary explant culture, a roughly 1 × 1–cm segment of cord is obtained using sterile forceps and scalpel. This segment is dissected to remove blood vessels and isolate the WJ. The resulting segments of WJ are then added to 10 mL of a collagenase-DMEM solution and incubated at 37°C for 4 hours. The collagenase-DMEM solution is prepared to a strength of 300 collagenase degrading units (CDU) per mL.Digested pieces of tissue are then collected using sterile forceps and transferred to a T-25 flask containing 10 mL of MSC-Brew Xeno-Free Media (Miltenyi Biotec). Cultures are then incubated for 72 hours at 37°C, at which point the media is replaced and the pieces of tissue are removed from the flask.

Once the primary culture has been established, regular media changes occur every 2 to 3 days, and cells are allowed to grow to 80% to 90% confluency. At this point, cells are passaged using trypsin-EDTA solution (0.25%) and reseeded into a T-75 flask. The culture is maintained this way until the target number of cells has been reached, at which point passaged cells are suspended in Stem-Cellbanker and frozen at –80°C.