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StemSpan™ Serum-Free Expansion Medium (SFEM) has been developed and tested for the in vitro culture and expansion of human hematopoietic cells, when the appropriate growth factors and supplements are added. This allows users the flexibility to prepare medium that meets their requirements. When combined with the appropriate cytokines, SFEM has been used for the culture and expansion of hematopoietic cells isolated from other species, including mouse, non-human primate, and dog. SFEM has also been used for culture of various other hematopoietic and non-hematopoietic cell types. Using appropriate StemSpan™ Expansion Supplements, SFEM may be used to expand CD34+ cells isolated from human cord blood, mobilized peripheral blood, or bone marrow samples, or to expand and differentiate lineage-committed progenitors to generate populations of erythroid, myeloid, or megakaryocyte progenitor cells.
StemSpan™ SFEM II (Catalog #09605) is an improved version of StemSpan™ SFEM that is further enriched to promote and support higher rates of CD34+ expansion and/or cell differentiation.
Contains
• Iscove’s MDM
• Bovine serum albumin
• Recombinant human insulin
• Human transferrin (iron-saturated)
• 2-Mercaptoethanol
• Supplements
Figure 1. Expansion of CD34+ Human Cord Blood Cells Cultured in StemSpan™ Media Containing CC100 Cytokine Cocktail
Purified CD34+ human cord blood (CB) cells were suspended at a concentration of 10,000 per mL in StemSpan™ SFEM (dark gray bars), SFEM II (blue bars) and AOF (orange bars) media containing CC100 Cytokine Cocktail (Catalog #02690). Cultures were maintained for 7 days, after which the cells were counted and examined for CD34 and CD45 expression by flow cytometry. Shown are the fold expansion of total nucleated cells (TNC) (A) and CD34+ cells (B) per input CD34+ cell, and the percent CD34 + cells (C). Results represent the average results of 32 different CB samples. Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in StemSpan™ SFEM II were significantly higher than in StemSpan™ SFEM and StemSpan™-AOF (*p<0.001, paired t-test, n=32).
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
Figure 2. Expansion of CD34+ Human Cord Blood Cells Cultured in StemSpan™ Media Containing CD34+ Expansion Supplement
Purified CD34+ human cord blood (CB) cells were suspended at a concentration of 10,000 per mL in StemSpan™ SFEM (dark gray bars), SFEM II (blue bars) and AOF (orange bars) media containing CD34+ Expansion Supplement (Catalog #02691). Cultures were maintained for 7 days, after which the cells were counted and examined for CD34 and CD45 expression by flow cytometry. The number of colony-forming units (CFU) in the expanded population was determined by replating cells in MethoCult™ H4435 and counting the number of colonies produced 14 days later. Shown are the fold expansion of total nucleated cells (TNC) (A), CD34+ cells (B) and CFU numbers (C) per input CD34+ cell, and the percent CD34+ cells (D) in these cultures (n=6). Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in StemSpan™ SFEM II was significantly higher than in SFEM and AOF (*p<0.001, #p<0.05, paired t-test, n=6).
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
Figure 3. StemSpan™ Media Support Greater Expansion of Human CD34+ and CD34bright Cells than Other Commercial Media
Purified CB-derived CD34+ cells were cultured for 7 days in select StemSpan™ media (StemSpan™ SFEM, StemSpan™ SFEM II, StemSpan™-XF, or StemSpan™-AOF, orange bars), and in five xeno-free media formulations from other suppliers (Xeno-Free Commercial Alternative, grey bars) including (in random order) CTS™ StemPro™ HSC (Thermo), SCGM (Cellgenix), X-VIVO™ 15 (Lonza), Stemline™ II (Sigma), and StemPro™-34 (Thermo). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM171*. The (A) frequency and (B) cell expansion of viable CD34+ and CD34bright cells in culture were based on viable cell counts and flow cytometry results as shown in Figure 1. StemSpan™ showed significantly higher expansion of CD34+ and CD34bright cells (P < 0.05 when comparing StemSpan™ SFEM II to five media from other suppliers, calculated using a one-way ANOVA followed by Dunnett’s post hoc test) and StemSpan™-AOF, the only animal origin-free formulation, showed equivalent performance to all xeno-free commericals alternatives tested. Data shown are mean ± SEM (n = 8).
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al., 2014.
Figure 4. StemSpan™ Media Support Equal or Greater Expansion of Primitive Human CD34brightCD90+CD45RA- Cells Than Other Commercial Media
Purified CB-derived CD34+ cells were cultured for 7 days in select StemSpan™ media (StemSpan™ SFEM, StemSpan™ SFEM II, StemSpan™-XF, or StemSpan™-AOF, orange bars), and in five xeno-free media formulations from other suppliers (Commercial Alternative, grey bars) including (in random order) CTS StemPro HSC (Thermo), SCGM (Cellgenix), X-VIVO 15 (Lonza), Stemline II (Sigma), and StemPro 34 (Thermo). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM171*. The (A) frequency and (B) cell expansion of CD34+CD90+CD45RA- (solid) and CD34brightCD90+CD45RA-(dotted overlay) cells in culture were based on viable cell counts and flow cytometry results as shown in Figure 1. StemSpan™ media showed similar or significantly higher expansion of CD34brightCD90+CD45RA- cells (P < 0.05 compared to five media from other suppliers, calculated using one-way ANOVA followed by Dunnett’s post hoc test) and StemSpan™-AOF, the only animal origin-free formulation tested, showed equivalent performance to all xeno-free commercial alternatives tested. Data shown are mean ± SEM (n = 8).
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al. 2014.
Table 1. Production of Erythroid Cells From CD34+ Human Cord Blood Cells Cultured in StemSpan™ SFEM Serum-Free Expansion Medium Containing Erythroid Expansion Supplement
Numbers and percent of erythroid cells produced after 14 days of culture of enriched CD34 + cells from 14 different cord blood (CB) samples. Erythroid cells were characterized by flow cytometry on the basis of transferrin receptor (CD71) and glycophorin A (CD235) expression.*95% confidence limits, the range within which 95% of the results fall.
Figure 5. StemSpan™ SFEM II Serum-Free Expansion Medium Containing Erythroid Expansion Supplement Supports Greater Expansion of Erythroid Cells Than Other Media Tested
The numbers of erythroid cells, normalized relative to the values obtained in StemSpan™ SFEM medium (dark gray bar), obtained after culturing purified CD34+ CB cells for 14 days in StemSpan™ SFEM, SFEM II (blue bar) and AOF (orange bar), and six media from other commercial suppliers (light gray bars, commercial alternative 1-6, which included, in random order, X-Vivo-15 and HPGM (both from Lonza), StemLine II (Sigma), HP01 (Macopharma), StemPro34 (Life Technologies) and SCGM (Cellgenix). All media were supplemented with StemSpan™ Erythroid Expansion Supplement (Catalog #02692). Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in StemSpan™ SFEM II were significantly higher than in all other media (*p<0.05, paired t-test, n=6).
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
Table 2. Production of Megakaryocytes From CD34+ Human Cord Blood Cells Cultured in StemSpan™ SFEM Serum-Free Expansion Medium Containing Megakaryocyte Expansion Supplement
Numbers and percent of cells expressing the megakaryocyte marker CD41a produced after 14 days of culture of enriched CD34 + cells from 6 independent cord blood (CB) samples. *95% confidence limits, the range within which 95% of the results fall.
Figure 6. StemSpan™ SFEM II Serum-Free Expansion Medium Containing Megakaryocyte Expansion Supplement Supports Greater Expansion of Megakaryocytes Than Other Media Tested
The numbers of megakaryocytes, normalized relative to the values obtained in StemSpan™ SFEM medium (dark gray bar), obtained after culturing purified CD34+ CB cells for 14 days in StemSpan™ SFEM, SFEM II (blue bar) and AOF (orange bar), and six media from other commercial suppliers (light gray bars, Commercial Alternative 1-6, which included, in random order, StemLine II (Sigma), HPGM (Lonza), HP01 (Macopharma), SCGM (Cellgenix), StemPro34 (Life Technologies) and X-Vivo-15 (Lonza). All media were supplemented with StemSpan™ Megakaryocyte Expansion Supplement (Catalog #02696). Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in the StemSpan™ media were significantly higher than in the other media (*p<0.01 paired t-test, n=6).
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
This product is designed for use in the following research area(s) as part
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A profile of circulating vascular progenitor cells in human neovascular age-related macular degeneration.
T. Catchpole et al.
PloS one 2020
Abstract
BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion, the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media, and markers such as NG2 and $\alpha$SMA in mesenchymal media. However, analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However, nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease.
Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.
R. Liang et al.
Cell stem cell 2020
Abstract
Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.
Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations.
R. Shahbazi et al.
Nature materials 2019 may
Abstract
Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells, with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors.
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