Ex vivo-Expansion mesenchymaler Stammzellen (MSC) des Knochenmarks mittels geschlossenem Bioreaktor

Abstract

Introduction: The expanding potential of mesenchymal stem cells (MSCs) for tissue repair and regeneration stimulates clinical research to develop novel and improved therapeutical strategies. However, due to low numbers of MSCs e.g. within bone marrow these progenitor cells may be expanded for clinical use if necessary. The maintenance and propagation of culture cells are crucially dependent on the cellular pattern of the inoculum and cell culture conditions including exogenous medium supplements. Materials and methods: For validation of a clinical study density-gradient isolated mononu-clear cell (MC) fractions of bone marrow (BM) taken from the iliac crest of healthy volunteers (n=4) were inoculated into the automated AastromReplicell™ Cell Production System (Aastrom Biosciences, Inc, Ann Harbor, MI) which encompasses a closed and sterile cell production bioreactor for clinical use. The MC cells (255 × 10E6) were cultured for 12 days within a single-use disposable cell cassette which contained the cell-chamber using supplemented medium (IMDM containing FCS, HS, EPO, PIXY321, Flt-3-ligand, L-glutamine, gentamicin, and vancomycin). BM-aspiration, MC isolation and cell expansion protocol were the same as intended for patients treatment. To evaluate the quality of the expanded cell products inoculum and harvest cells were characterized by flow cytometry (FACSCalibur), immunohistochemistry (Vector Stain), and magneto bead separation (Miltenyi). Results: Total cell expansion was in the range from 906 × 10E6 to 1440 × 10E6 cells. The respective expression of CD45, CD90, CD105 and the occurrence of lineage (Lin)-specific markers (CD3, CD11b, CD14, CD15, CD20, CD235a-GlyA) were analyzed. The cell culture conditions led to an expansion of CD45−CD90+Lin− up to 63.8 fold and CD45−CD105+Lin− up to 21.2 fold in absolute cell numbers. The expansion of myeloids was maximal 6.0 fold and of erythroids was maximal 11.2 fold. Compared to inoculum cells lymphocytes analyzed as CD3+-cells were decreased more than 100 fold in the harvest cell fraction. In addition, harvest cells which were conventionally cultured (6 months) without further addition of exogenous growth factors showed an increasing number of CD90+ cells and a decreasing number of CD45+ cells. Conclusions: The used ex vivo cell production system is applicable for clinical use, it requires about 30 ml BM aspirate for a broad expansion of cells bearing MSC-typical markers concomitant with large numbers of harvest cells.