In the laboratory, it has been shown that embedding stem cells into autologous fibrin clots may reduce the deposition of scar tissue and improve the quality of repair tissues. However, their survivability inside fibrin matrices has not been clearly demonstrated. Therefore, the purpose of this study was to evaluate the ability of human whole blood-derived fibrin clots to capture human muscle-derived stem cells (hMDSCs) and to preserve their viability and intrinsic biologic functions over time. We hypothesized that hMDSCs would be captured within the fibrin matrix, would survive inside the clot for at least 2 weeks, and would also maintain their intrinsic capacities for proliferation and migration without undergoing programmed cell death.
Materials And Methods
Institutional Review Board (IRB) approval and individual informed consents were obtained before initiation of this study. Patients who underwent elective arthroscopic knee surgery with platelet-rich plasma (PRP) augmentation were enrolled in this study. On the day of surgery, previously-prepared hMDSCs (6 x 105 cells) transduced to express green fluorescent protein (GFP) were suspended in 3 mL of phosphate-buffered saline (PBS) and transported to the operating room. Approximately 30 mL of whole blood was drawn from each patient using the same venous access (separate from PRP), placed into a 100-mL beaker, and the hMDSCs were added to the blood. Each beaker was swirled in a circular motion for 10 minutes at 95 rpm using a commercially-available blood agitation device. The final clot was separated from its residual and transported in saline gauze to our laboratory. Small sections of each hMDSC-seeded fibrin clot were digested sequentially with collagenase, dispase, and trypsin over a period of 48 hours and incubated in Dulbecco's Modified Eagle Medium (DMEM) for 48 hours, 72 hours, 4 days, 7 days, and 14 days. At each time point, GFP fluorescence of each digested clot fraction was evaluated for evidence of cell viability and proliferation. The culture media containing undigested clot fractions were also analyzed for GFP-positivity. In addition, multiple 5-µm cryosections were created and stained with either Ki67 (a marker for active cell division) or cleaved caspase-3 (a marker for active cell apoptosis).
Five hMDSC-seeded fibrin clots were analyzed in our laboratory. GFP fluorescence was absent in all residual sera after 48 hours of culture. GFP fluorescence of hMDSCs was observed after enzymatic digestion of each clot fraction, and the cells appeared to proliferate up to 2 weeks after clot formation. 5-µm clot cryosections were positive for Ki67 (active proliferation) and negative for cleaved caspase-3 staining (no active apoptosis) at 2 weeks. In the undigested clot fractions, GFP-positive hMDSCs were found outside of the clot matrix and within the culture media at up to 2 weeks.
Our results indicate that the viability and characteristic features of stem cell populations can be maintained within whole blood fibrin clots up to 2 weeks after clot formation, suggesting that autologous fibrin clot may represent an important biologic scaffold and delivery vehicle for future autologous stem cell treatments.