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Mesenchymal Stem Cell Culture Techniques for use in Composite Tissue Allotransplantion.
Eric Wimmers, Tim Nguyen, Gerald Brandacher, W.P. Andrew Lee
University of Pittsburgh
2010-03-31
Presenter: Eric Wimmers
Affidavit:
Director Name:
Author Category: Resident/Fellow
Presentation Category: Basic Science Research
Abstract Category: Hand
Composite tissue allotransplantation (CTA) is hampered by inadequate nerve regeneration and difficulties balancing immunosuppression. We propose a novel cell-based therapy using mesenchymal stem cells (MSC), which have been shown to improve nerve regeneration and also enhance immunomodulation. To date, MSC have not been tested in a CTA model to demonstrate both effects. Our study devises an efficient method of MSC culture and isolation, followed by testing of MSC on a sciatic nerve transection model and a hindlimb transplant model in the rat. The cultured MSC are obtained from Brown Norway rat femur and tibia bone marrow. Red blood cells are depleted with lysis buffer. Cells are expanded over 2 weeks, and then isolated with CD45 positive selection with magnetic columns. Isolated cells are fluorescently labeled with CD29 and CD90 to measure total purity. Each animal produces 8x10^7 cells after bone marrow isolation. After CD45+ isolation, 20 million cells remain as putative MSCs. Flow cytometry confirmed over 80% purity of the culture. In our study, we produced a reliable method to create 2x10^7 MSC within two weeks. These cells may be a novel method to enhance functional outcome in CTA.
