GMP-Conform Generation and Cultivation of Unrestricted Somatic Stems Cells (USSC) from Cord Blood Using the SEPAX©-Separation Method a Closed Culture System Applying Cell Stacks.

Generation and characterization of unrestricted somatic stem cells (USSC) from cord blood (CB) was described by our group and has been well established under laboratory conditions [Koegler et al 2004, 2005 and 2006; Sensken et al 2007]. Due to their proliferative and differentiation capacity, USSCs are an interesting candidate for the future development of cellular therapy for tissue repair and tissue regeneration as well as a supportive cell layer to support hematopoietic reconstitution. Since generation and expansion under GMP-grade conditions is mandatory for use in clinical application, the automated cell processing system Sepax (BIOSAFE) with the CS900 separation kit was used for mononuclear cell separation and the subsequent generation of the USSC colonies in the presence of 30% GMP-grade fetal calf serum (Perbio), low-glucose DMEM-medium/10-7M dexamethasone. Expansion of USSC was performed in a closed system (Macopharma) applying cell stacks (Costar Corning). Results achieved so far indicate that the generation frequency and quality of generated USSC under GMP conditions are equal or even superior (45%) to manual generation under laboratory conditions (43%). 20 cord-blood units (mean volume 88,5 +− 15,8 ml; mean number of mononuclear cells 3,1 +−0,6 *108 MNC) have been processed, resulting in 9 USSC-colony formations and lines within 14–28 days. Growth kinetics is equal to the previously established USSC-lines (∼36–48 h / population doubling). Analysis of the immunophenotype as well as the differentiation potential towards the mesenchymal, neural and endodermal lineages also showed no difference to those lines generated manually using Ficoll-separation and normal cell culture flasks (Costar Corning T225). The closed system applied here is perfectly suitable to ensure safe and easy handling of the USSC, including seeding, trypsination and harvesting. In combination with the cell stack system (1, 2, 5 and 10 layers), cell amounts of more than 1.0×109 USSC can be achieved within 4 passages. These USSC products were temperature controlled cryopreserved in the presence of 10% DMSO, HSA and dextran. USSC can be thawed and further expanded in clinical grade quality. On the basis of their pluripotency and expansion under GMP-conditions into large quantities, these USSC from cord blood, when pretested for infectious agents and matched for the major transplantation antigens, may serve as a universal allogeneic stem cell source for tissue repair and tissue regeneration.