Serum-Free Expansion of CD34+ Cells with SFEM II and UM171 or UM729
CD34+ cells may be obtained as cryopreserved cells, isolated from CB or bone marrow (BM) prior to freezing, or from whole CB or fresh whole BM using methods for column-free cell isolation.
- Should I use fresh or frozen primary cells?
Both fresh or cryopreserved CD34+ cells can be used for expansion experiments. Favourable results can be achieved with either of these cell sources, provided that their content of hematopoietic progenitor cells, and their overall quality and viability, is good. Differences in cell quality are often larger between individual CB samples from different donors than would be observed between fresh or frozen samples from the same donor. Typically, one can obtain better results with frozen cells from “good” CB samples than with fresh cells from “poor” CB samples.
- I have fresh or frozen whole blood or BM. Which EasySep™ kit should I use to select for CD34+ cells?
For more information about choosing the correct cell separation product for your cell source please read our previous tech tip "Isolating Hematopoietic Progenitors”. If you are isolating CD34+ cells from fresh CB, find out why the EasySep™ Human Cord Blood CD34 Positive Selection Kit II is the best choice for this cell type.
CD34+ cells can be expanded in SFEM II medium using any type or size of cultureware, including flasks. Table 1 below can be used to determine how many CD34+ cells to plate in each well of 3 recommended tissue culture plate formats. These numbers are meant as guidelines only, as the optimal culture format and cell concentrations are dependent on the objectives of your experiment and the quality of the cells.
Table 1. General Recommendations for CD34+ Cell Plating Concentrations in Different Culture Plate Formats
*Both tissue culture-treated and non-tissue culture-treated plates are suitable for culture and have not been found to affect CD34+ cell expansion.
**StemSpan™ CD34+ Expansion Supplement is supplied as 10 mL of 10X concentrate, which is sufficient for use at 1X when combined with 90 mL of SFEM II.
Day 0 - Plate CD34+ cells (refer to the recommendations in Table 1 above) in SFEM II containing StemSpan™ CD34+ Expansion Supplement (by diluting 10 mL of 10X concentrate in 90 mL of SFEM II), with or without adding UM729 or UM171 (to a final concentration of 1.75μM or 175 nm, respectively. See expansion data in Figure 1 below).
Day 7 - Harvest cells for evaluation or downstream application.
After culture, it is recommended to count the number of both total and viable cells, e.g., using trypan blue and a hemocytometer, or using an automated cell counting method, and to measure CD34+ expression by flow cytometry. Additional immunophenotyping may be done to identify CD34+ cell subsets and/or differentiated CD34- cells produced in the cultures. Request our new wallchart for more information on human hematopoietic stem and progenitor cell (HSPC) cell surface markers for immunophenotyping or fluorescence-activated cell sorting (FACS).
SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of CD34+ cells in culture. The addition of UM729 or UM171 further enhances expansion of CD34+cells, in addition to more primitive subsets such as CD34+CD45RA-CD90+cells (~10 fold at day 7 compared to cultures without UM171).
Figure 1. Expansion of CD34+Cells Cultured in StemSpan™ SFEM II
CD34+cells were cultured in StemSpan™ SFEM II containing CD34+Expansion Supplement (Exp) alone, or with UM171 (to a final concentration of 175 nM). Shown is the average fold expansion of the total nucleated cells (TNC), CD34+, CD34+CD45RA-and CD34+CD45RA-CD90+cell subsets after 7 days of culture. Data shown are mean ± SEM (n = 7). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01).