DEVELOPMENT OF NOVEL PERFUSION CHAMBER TO RETAIN NONADHERENT CELLS AND ITS USE FOR COMPARISON OF HUMAN MOBILIZED PERIPHERAL-BLOOD MONONUCLEAR CELL-CULTURES WITH AND WITHOUT IRRADIATED BONE-MARROW STROMA

Perfusion and static cultures of peripheral blood (PB) mononuclear cel ls (MNCs), obtained from patients following stem cell mobilization, we re supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-st imulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal lay er. Perfusion cultures without a stromal layer effectively retained no nadherent cells through the use of a novel ''grooved'' perfusion chamb er, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allow ed easy and efficient culture inoculation and cell recovery. Average m aximum expansion of CFU-GM (colony-forming unit granulocyte-macrophage ) cells was observed on day 10 for all cultures. Perfusion cultures ha d a maximum CFU-GM expansion of 17- and 19-fold with and without a str omal layer, respectively. In contrast, static cultures had a maximum C FU-GM expansion of 18- and 13-fold with and without a stromal layer, r espectively. Average long-term-culture initiating cell (LTC-IC) number s on day 15 were 34% and 64% of input in stroma-containing and stroma- free perfusion cultures and 12% and 11% of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhance d CFU-GM expansion and LTC-IC maintenance more for the stroma-free cul tures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications. (C) 1996 J ohn Wiley & Sons, Inc.