CLINICAL-SCALE HUMAN UMBILICAL-CORD BLOOD-CELL EXPANSION IN A NOVEL AUTOMATED PERFUSION CULTURE SYSTEM

Use of umbilical cord blood (CB) for stem cell transplantation has a n umber of advantages, but a major disadvantage is the relatively low ce ll number available. Ex vivo cell expansion has been proposed to overc ome this limitation, and this study therefore evaluated the use of per fusion culture systems for CB cell expansion. CB was cryopreserved usi ng standard methods and the thawed unpurified cells were used to initi ate small-scale cultures supplemented with PIXY321, flt-3 ligand, and erythropoietin in serum-containing medium. Twelve days of culture resu lted in the optimal output from most CB samples. Frequent medium excha nge led to significant increases in cell (93%), CFU-GM (82%) and LTC-I C (350%) output as compared with unfed cultures. As the inoculum densi ty was increased from 7.5 x 10(4) per cm(2) to 6.0 x 10(5) per cm(2), the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM out put reached a plateau at 6.0 x 10(5) per cm(2), whereas LTC-IC output continued to increase up to 1.2 x 10(6) per cm(2). Because the increas e in culture output did not increase linearly with increasing inoculum density, expansion ratios were greatest at 1.5 x 10(5) per cm(2) for cells (6.4-fold) and CFU-GM (192-fold). Despite the lack of adherent s troma, CB cultures expressed mRNA for many growth factors (G-CSF, GM-C SF, IL-1, IL-6, LIF, KL, FL, Tpo, TGF-beta, TNF-alpha, and MIP-1 alpha ) that were also found in bone marrow (BM) cultures, with the exceptio n of IL-11 (found only in BM) and IL-3 (found in neither). Culture out put was remarkably consistent from 10 CB samples (coefficient of varia tion 0.3) indicating that the procedure is robust and reproducible. Tw o commercial serum-free media were evaluated and found to support only approximately 25% of the culture output as compared with serum-contai ning medium. Implementation of optimal conditions in the clinical scal e, automated cell production system (CPS) showed that the process scal ed-up well, generating 1.7 x 10(7) CFU-GM (298-fold expansion) from 1. 2 x 10(8) thawed viable nucleated CB cells (n = 3). The ability to gen erate >10(7) CFU-GM from <15 mi of CB within this closed, automated sy stem without the need for extensive cell manipulations will enable cli nical studies to test the safety and efficacy of expanded CB cells in the transplant setting.