 |
 |
 |
 |
 |
 |
 |
||
 |
 |
 |
 |
|
||||
Cytomatrix |
|
Engraftment of CD34+ HPC’s Cultured in a 3-Dimensional Bioreactor (Cellfoam) in the Absence of Serum and Cytokines |
||
|
B. Ehring, D. Plosky, T. Upton, N. Banu, M. Pykett, M. Rosenzweig
Cytomatrix, Woburn, MA. |
||||
We have previously described the utility of our 3-dimensional matrix (CellfoamTM) for the extended culture of multipotent hematopoietic progenitors in vitro (Bagley, et al, Experimental Hematology 1999). This study was initiated to validate the in vitro observations in a pre-clinical in vivo model, the NOD/SCID mouse. This model permits in vivo analysis of engraftment and multipotency of human hematopoietic progenitors. We used cryopreserved cells as a control, as this is generally how cells are processed during a clinical bone-marrow transplant protocol. In general cells are frozen for 14 days during a period of chemotherapy and radiation treatment, and as our bioreactor provided optimal in vitro results after 14 days, this seemed to a be reasonable control as well as a clinically relevant comparison. We isolated CD34+ cells from human bone marrow and half of the cells were placed into CellfoamTM cultures and the other half were cryopreserved in PBS/1% Dextran 60/10% DMSO based on a protocol derived from the Placental Blood Project of the New York Blood Center. In the CellfoamTM matrix cells were cultured in serum-free medium without addition of exogenous cytokines. Cells were recovered from culture or cryopreservation after 2 weeks, and the cells from the two groups were transplanted in 6 week old sub-lethally irradiated female NOD/SCID mice. Each mouse received an equivalent number of cells, based on the input population. The mice were euthanized after 6 weeks, and peripheral blood (PB), bone marrow, spleen, heart, liver and kidney were harvested for further analyses. The mice were considered to be engrafted at a frequency of more than 0.1% human CD45+ cells as determined flow cytometry in bone marrow. The results from these studies demonstrated that a larger number of mice engrafted in both the bone marrow and peripheral blood when transplanted with cells cultured in a CellfoamTM bioreactor as compared to cryopreservation (p<0.005). As additional confirmation of the data generated using flow cytometry, we performed semi-quantitative PCR using primers specific for human globin sequences. The PCR data confirmed our observations made using flow cytometry. Autopsies were performed on all mice. No gross abnormalities were detected and samples were collected for histopathology. These samples have been examined and have determined no evidence of pathology in either group of transplanted mice. Additional studies are being performed and this data will be presented. This data demonstrates the efficacy of CellfoamTM as 3-dimensional tissue matrix that enables the serum-free, cytokine-free culture of multipotent hematopoietic progenitors that retain in vivo engratment capacity. This observation establishes a platform technology that is applicable to clinical bone marrow transplantation.   |
||||