Membrane-Separated Cocultivation of Cord Blood Hematopoietic Stem Cells with Stromal Cell Lines

Cord blood hematopoietic stem and progenitor cells (HSPC) represent an important cell source for transplantation of cancer patients after high dose chemotherapy if autologous cells are not suitable (e.g. patients with hematopoietic malignancies) and no adult allogeneic donor is available. The main disadvantage of cord blood is the low number of cells obtained due to the small volume of blood collectable from umbilical cords. Without an expansion of the HSPC a transplantation is mostly limited to juvenile patients.We developed a novel small scale membrane bioreactor for parallelized membrane-separated cocultivation of HSPC with stromal cells. This system imitates the natural hematopoietic environment with stromal growth factors, ECM components and direct cell-cell contact between HSPC and stromal cells while maintaining a physical separation of the cells.The mini membrane bioreactor consists of a turnable insert for conventional 12-well plates, wherein a thin, porous polyethylene membrane is fixed. Murine stromal cells adhere to the underside on the membrane and HSPC are cocultured on top of it. Soluble factors and stromal extensions travers the membrane, whereas cell bodies are retained. Thus, stroma directly supports HSPC expansion without contaminating the future transplant. Purified CD34+ cells are cultured for 7 days in X-Vivo10 supplemented with four cytokines (Tpo, SCF, IL-3 and FL). Many important parameters have been investigated using this system (membrane material, pore size, type of stromal cells, cell density, agitation, feeding, etc.). With the novel membrane reactor we are able to expand HSPC to high cell densities. On average, after 12 days total cells and progenitor cells (CFC) expand 242 and 29fold. Early progenitors (CAFC) could even be expanded 39fold.