contribute to mesenchymal progenitors to create early cells in the osteogenic lineage. Unfortunately, the study by Sueyama et al23 using a rat model found that implanting MSCs alone showed incomplete dentin bridges, while coimplantation of MSCs with endothelial cells resulted in pulp healing with complete dentin bridge formation.
As such, a viable strategy to allow osteogenic regeneration involves the use of cord blood MSCs. This avoids the ethical concerns of embryonic stem cells and the morbidity of bone marrow acquisition while retaining the multipotency required for the regeneration of complex endodontic tissue. In 2018, Chen et al24 showed that MSCs derived from cord blood were able to achieve successful osteogenic and angiogenic properties, in addition to density, when cocultured with human umbilical vein endothelial cells. These cord blood MSCs had similar capabilities and performance as human bone marrow–derived MSCs and human embryonic stem cells.24
Currently, the literature does not indicate any effect of cord blood stem cells used in combination with residual dental pulp tissue on the ability of a tooth to regenerate pulpal tissues. The authors propose that cord blood stem cells will enhance the ability of residual pulp tissue to regenerate.
MSC Isolation Methods
Cord blood–derived and WJ-derived MSCs are an excellent option for therapeutic use because they are easily collected, readily available, and highly proliferative. They can also be used as allografts. Procedures used to isolate MSCs from these sources are described in the following sections.
Cord blood
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).
Wharton’s jelly
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.
References
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