Читать книгу PRF Applications in Endodontics - Mahmoud Torabinejad - Страница 17

Cord blood is collected from eligible donors at the time of delivery and transported to the processing facility on ice (2°C to 8°C) in a blood bag. Upon arrival, the blood is processed immediately under aseptic conditions (Fig 1-1a).

FIG 1-1 The different stages of cord blood processing. (a) Blood bag as it was received being processed under aseptic conditions. (b) Conical tube after centrifugation. Note plasma layer (top), buffy coat layer (middle), and red blood cells (bottom).(c) PBMCs suspended in Stem-Cellbanker ready for storage in a cryovial.

The blood bag is first drained into conical tubes and centrifuged at 1500 × g to separate components. This results in distinct layers in the conical tube, with plasma rising to the top and red blood cells forming a pellet at the bottom of the tube. There is also a distinct buffy coat layer below the plasma that contains the cells of interest. The buffy coat layer is then collected and diluted with a phosphate-buffered saline (PBS) solution before undergoing density gradient centrifugation. The buffy coat and PBS mixture is added to Ficoll-Paque (GE Healthcare) density gradient media and then centrifuged at 409g. This results in an isolation of peripheral blood mononucleated cells (PBMCs) in the resulting buffy coat (Fig 1-1b).

The PBMCs are then collected and diluted once again with PBS before being centrifuged at 409 × g. This wash step is repeated until the resulting supernatant is no longer cloudy or hazy, and the resulting pellet is then resuspended in Stem-Cellbanker (Amsbio). Cell count, viability, and surface marker profile are then tested using flow cytometry. This information is used to aliquot the appropriate cell number into cryovials, and the cells are immediately stored at –80°C (Fig 1-1c).