Long-term culture of hematopoietic stem cells - validating the stromal component of the CAFC assay

Background The stroma-based long-term culture is the assay of choice when a functional detection of primitive hematopoietic cells in vitro is sought. However, di fferent stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in publish ed frequencies of LTCIC alternative CAFC. Methods In order to identify the most suitable stromal source in terms of supportiv e capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma i n secondary long-term culture set-ups. Results Seeking an approximation to the supportive capacity of human BM stroma we f ound the FBMD-1 cell line supplemented with G-CSF and IL-3 superior to FBMD -1 cells alone, and to M2-10B4 and Sl/Sl cells. Moreover, in co-cultures of CD34(+) cells and the FBMD-1 line, we found week 5 CAFC content highly rep roducible (50.5+/-6.66-54.6+/-7.07/10(4) plated cells, p value >0.95) and t he assay was suitable for inter-individual comparison in a clinical setting . In fact, the week 5 CAFC results were even more reproducible than those o f the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU a ssays). On the other hand, when extending the culture period to 8 weeks, th e cobblestone area formation was best maintained by human BM stroma and the high reproducibility in CAFC enumeration in cultures supported by the FBMD -1 was lost. Discussion Among the stromal cell sources tested, the FBMD-1 line was found to be supe rior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.