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, different stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in published frequencies of LTCIC alternative CAFC.


METHODS
In order to identify the most suitable stromal source in terms of supportive capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma in secondary long-term culture set-ups.


RESULTS
Seeking an approximation to the supportive capacity of human BM stroma we found 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 reproducible (50.5 +/- 6.66 - 54.6 +/- 7.07/10(4) plated cells, p value > 0.95) and the assay was suitable for inter-individual comparison in a clinical setting. In fact, the week 5 CAFC results were even more reproducible than those of the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU assays). On the other hand, when extending the culture period to 8 weeks, the 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.


CONCLUSIONS
Among the stromal cell sources tested, the FBMD-1 line was found to be superior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.
Olesen
G
G
Department of Hematology, Aarhus University Hospital, Denmark.
Tønder
H
H
Holm
M S
MS
Hokland
P
P
D000328:Adult; D000818:Animals; D018952:Antigens, CD34; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay / Q000379:methods* / Q000592:standards; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D051379:Mice; D017154:Stromal Cells / Q000166:cytology* / Q000502:physiology
D018952:Antigens, CD34