Colony Assay Media (for CFC assays)
Long-Term Culture Media (for LTC-IC assays)
Sera & Serum-Free Media
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Contract Assay Services
L-Calc software for limiting dilution analysis
Hematopoietic culture assays are used to detect the proliferation and differentiation ability of hematopoietic cells at distinct and successive stages of differentiation and to measure the frequency of these cells in hematopoietic tissues and purified cell populations. Long-term culture (LTC) assays are usually performed on pre-established layers of stromal cells that provide essential stimulatory and inhibitory signals to regulate hematopoietic cell outgrowth. Variants of this assay differ in the nature of the stromal cell types used (e.g. primary BM-derived fibroblasts or transformed cell lines), the readout system, and the approach used to obtain frequency information from the culture data (usually by limiting dilution analysis). Irrespective of the method used, the cells that develop within 1-3 weeks after initiation of a long-term culture are considered to represent the progeny of relatively mature progenitor cells, whereas those that develop at later time points (e.g. after 5-8 weeks) are thought to arise from very primitive cells, called LTC-initiating cells (LTC-IC).1,2
The most common approaches to quantify multi-lineage- or single lineage-committed hematopoietic progenitors, called colony-forming cells (CFCs) or colony-forming units (CFUs), utilize viscous or semi-solid matrices and culture supplements that promote their proliferation and differentiation and allow the clonal progeny of a single progenitor cell to stay together and thus form a colony of more mature cells. Culture media have been optimized for outgrowth of erythroid, monocyte/macrophage, granulocytic, megakaryocytic and multipotent progenitor cells. Colony assays are used to quantitate and characterize hematopoietic progenitors from different sources (e.g., BM, umbilical cord blood (UCB), mobilized peripheral blood, fetal tissues, patient samples) and for quality control of clinical stem cell collection, processing and cryopreservation. They are also used to investigate progenitor responses to growth factors, inhibitors and drugs, as readout for LTC-IC assays, to quantitate progenitor cell numbers after ex vivo expansion and to assess gene transfer efficiencies to stem cells and progenitor cells. Use of functional in vivo assays and in vitro LTC-IC and CFC assays are essential, together with phenotypic studies, to identify stem cell markers and to develop stem cell isolation strategies.
The ability to maintain, expand and assay hematopoietic stem cells and progenitors in vitro has been instrumental in advancing our understanding of hematopoiesis. Historically, medium containing fetal bovine serum (FBS) has been used for culture of hematopoietic cells. However, the concentrations of inhibitors including transforming growth factor-?and other constituents vary between batches of sera. In order to minimize these variables, particularly for studies of the effects of specific regulatory or growth factors, there has been a move towards the use of serum-free media.
1. Miller CL, Eaves CJ: Long-term culture-initiating cell assays for human and murine cells. In: Hematopoietic Stem Cell Protocols. Methods in Molecular Medicine. Klug CA and Jordan CT eds, Human Pres, Totowa, NJ, pp123-141, 2002.
2. de Haan G, Ploemacher R: The cobblestone-area-forming cell assay. In: Hematopoietic Stem Cell Protocols. Methods in Molecular Medicine. Klug CA and Jordan CT eds, Human Pres, Totowa, NJ, pp143-151, 2002.