MethoCult™ for Rat Hematopoietic Colony-Forming Unit (CFU) Assays
- Document # 28732
- Version 4.0.0
- Mar 2017
Hematopoietic cell 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. The colony-forming unit (CFU) assay is an in vitro functional assay for enumerating multipotential and lineage-committed hematopoietic progenitor cells through the use of semi-solid culture media. These culture media are supplemented with cytokines to promote the proliferation and differentiation of hematopoietic progenitors and their viscosity constrains the clonal progeny of a single progenitor cell to a small area, thus forming a colony of mature cells. CFU assays are used to quantitate and characterize hematopoietic progenitors from tissues such as bone marrow (BM), spleen and peripheral blood (PB), and to investigate their responses to growth factors, inhibitors and drugs. Optimized culture media (MethoCult™) are available for CFU assays for the detection of erythroid (CFU-E, BFU-E), granulocyte/macrophage (CFU-G, CFU-M, CFU-GM) and multipotent progenitor cells (CFU-GEMM).
Rat models have been used for evaluation of hematopoietic progenitor responses in a number of protocols, including the assessment of drug-induced hematotoxicity,1 effect of seizures on hematopoiesis2 and adeno-associated viral gene transfer into hematopoietic cells.3 In addition, rats are commonly used as an in vivo model for safety evaluations of new drugs. Several different cytokine combinations have been published to promote rat CFU-GM development.1,4,5,6
MethoCult™ Methylcellulose-Based Medium for Rat CFU-GM Assays
As shown in Figure 1 the combination of granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF) and interleukin-3 (IL-3) provides optimal growth of rat CFU-G, CFU-M and CFU-GM colonies from rat BM cells. Colonies could be identified and counted after 9 - 14 days of culture. A complete medium, MethoCult™ GF R3774 (Catalog #03774), containing this cytokine combination is available as a ready-to-use formulation from STEMCELL Technologies.
Representative examples of rat CFU-GM-derived colonies cultured in MethoCult™ GF R3774 are shown in Figure 2. The optimal plating density for culture of rat CFU-GM colonies from BM cells is ~1 x 104 to 2 x 104 cells per 35 mm dish (Table 2). For spleen and PB cells, the number of cells plated per 35 mm dish should be at least 1 x 105. Plating spleen and PB cells at two or even three different plating densities, e.g. 1 x 105, 3 x 105 and 1 x 106 per 35 mm dish, is recommended to ensure that optimal colony numbers (50 - 100 per dish) are obtained with at least one of the plating densities.
Figure 1. Cytokine Responsiveness of CFU-GM in Rat BM
The combination of GM-CSF, SCF and IL-3 provides optimal CFU-GM colony numbers from rat BM cells.
Figure 2. Examples of Rat BM CFU-GM-Derived Colonies Cultured in MethoCult™ GF R3774
Photographed at 40X magnification after 11 days of culture.
MethoCult™ Methylcellulose-Based Medium for Rat BFU-E Assays
A complete erythropoietin (EPO)-containing methylcellulose medium, MethoCult™ SF M3436 (Catalog #03436, Table 1), originally developed for the culture of mouse burst-forming unit-erythroid (BFU-E) progenitors, is also suitable for colony assays of rat erythroid progenitor cells from BM, spleen and PB. As shown in Figure 3, > 90% of colonies cultured from rat BM in MethoCult™ SF M3436 were erythroid. Two types of erythroid colonies were identified: small colonies (~100 - 200 cells per colony) containing several distinct clusters of 10 - 20 erythroblasts (mature BFU-E, Figure 4A) and large colonies (> 1000 cells per colony) with a dense core, containing multiple erythroblast clusters (immature BFU-E, Figure 4B). A small proportion of colonies were classified as non-erythroid (Figure 3). These colonies consisted of a single cluster (Figure 4C) or multiple clusters of cells that were larger and more distinct from each other than the erythroblasts in the BFU-E colonies and did not show evidence of hemoglobinization. These colonies could not be classified as myeloid, since they did not develop in the absence of EPO (data not shown). This suggested they were derived from immature erythroid progenitors that may require more time than the standard 9 - 14 day culture period for full erythroid differentiation.
The optimal plating density for culture of erythroid progenitors from rat BM is 2.5 - 5 x 104 cells per 35 mm dish (Table 2). Erythroid colonies can also be cultured from rat spleen and PB cell preparations, but plating densities need to be higher, i.e. 3 x 105 - 1 x 106 cells per 35 mm dish (Table 2).
Figure 3. Colony Types in BFU-E Assay of Rat BM with MethoCult™
Rat BM cells were plated in 35 mm dishes at the indicated cell concentrations in MethoCult™ SF M3436 and colonies (orange = non-erythroid, grey = primitive BFU-E, gold = mature BFU-E) were counted after 14 days of culture.
Figure 4. Examples of colonies cultured from rat BM in MethoCult™ M3436.
(A) Mature BFU-E-derived colony; (B) Primitive BFU-E-derived colony; (C) Non-erythroid colony. Photographed at 20X magnification after 14 days of culture.
Table 1. MethoCult™ Media Validated for use in Rat CFU Assays
MethoCult™ GF R3774 allows quantitation of total myeloid colonies.
Plating Densities for Rat CFU Assays
Rat BM, spleen and PB cells were isolated from three- to seven month- old Noble rats or eleven-week old Sprague-Dawley rats. Nucleated cells were counted according to standard procedures. Duplicate or triplicate cultures containing BM, spleen or PB cells in 1.1 mL of MethoCult™ GF R3774 or M3436 medium were plated in 35 mm culture dishes, and incubated for 9 - 14 days at 37°C, 5% CO2 and >95% humidity. Colony numbers were assessed using an inverted microscope equipped with 2X, 4X and 10X planar objective lenses. Recommended plating densities for rat CFU assays determined by this procedure are shown in Table 2.
Applications of the Rat CFU Assay
- In vitro model for drug toxicity testing
- Assessing hematopoiesis in transgenic models
- Optimization of gene transfer protocols
Table 2. Recommended Plating Densities for Rat CFU Assays
These plating densities were established using healthy Noble and Sprague-Dawley rats. For other strains, transgenic animals or cytotoxicity testing, it is recommended to plate cells at two to three different plating densities.
- Matsumura-Takeda K, Kotosai K, Ozaki A, Hara H, Yamashita S. Rat granulocyte colony-forming unit (CFU-G) assay for the assessment of drug-induced hematotoxicity. Toxicol In Vitro 16: 281-288, 2002
- Bhatt R, Rameshwar P, Goldstein K, Siegel A. Effects of kindled seizures upon hematopoiesis in rats. Epilepsy Res 54: 209-219, 2003
- Shah R, Jindal RM. Stable transfection of rat preporinsulin II gene into rat hematopoietic stem cells via recombinant adeno-associated virus. Life Sci 65: 2041-2047, 1999
- Gowing H, Lawler M, Hagenbeek A, McCann S, Pamphilon D, Hudson J, van Weelden H, Braakman E, Mertens ACM. Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved. Blood 87: 1635-1643, 1996
- Bristol LA, Bachovchin W, Takacs L. Inhibition of CD26 enzyme activity with pro-boropro stimulates rat granulocyte/macrophage colony formation and thymocyte proliferation in vitro. Blood 85: 3601-3609, 1995
- Lautraite S, Parent-Massin D, Rio B, Hoellinger H. In vitro toxicity induced by deoxynivalenol (DON) on human and rat granulomonocytic progenitors. Cell Biol Toxicol 13: 175-183, 1997