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StemSpanā„¢ SFEM

Serum-free medium for culture and expansion of hematopoietic cells

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StemSpanā„¢ SFEM

Serum-free medium for culture and expansion of hematopoietic cells

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Serum-free medium for culture and expansion of hematopoietic cells
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Overview

StemSpanā„¢ 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 StemSpanā„¢ 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.

StemSpanā„¢ SFEM II (Catalog #09605) is an improved version of StemSpanā„¢ SFEM that is further enriched to promote and support higher rates of CD34+ expansion and/or cell differentiation.
Contains
ā€¢ Iscoveā€™s MDM
ā€¢ Bovine serum albumin
ā€¢ Recombinant human insulin
ā€¢ Human transferrin (iron-saturated)
ā€¢ 2-Mercaptoethanol
ā€¢ Supplements
Subtype
Specialized Media
Cell Type
Hematopoietic Stem and Progenitor Cells
Species
Human, Mouse, Non-Human Primate, Rat
Application
Cell Culture, Expansion
Brand
StemSpan
Area of Interest
Stem Cell Biology, Transplantation Research
Formulation Category
Serum-Free

Data Figures

Expansion of CD34 + Human Cord Blood Cells Cultured in StemSpan™ Media Containing CC100 Cytokine Cocktail

Figure 1. Expansion of CD34+ Human Cord Blood Cells Cultured in StemSpanā„¢ Media Containing CC100 Cytokine Cocktail

Purified CD34+ human cord blood (CB) cells were suspended at a concentration of 10,000 per mL in StemSpanā„¢ 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 StemSpanā„¢ SFEM II were significantly higher than in StemSpanā„¢ SFEM and StemSpanā„¢-AOF (*p<0.001, paired t-test, n=32).

Note: Data for StemSpanā„¢-AOF shown were generated with the original phenol red-containing version StemSpanā„¢-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpanā„¢-AOF (Catalog #100-0130) was comparable.

StemSpan™ SFEM II Serum-Free Expansion Medium Containing CC100 Cytokine Cocktail Supports Greater Expansion of Human CD34 + Cells Than Other Media Tested

Figure 2. Expansion of CD34+ Human Cord Blood Cells Cultured in StemSpanā„¢ Media Containing CD34+ Expansion Supplement

Purified CD34+ human cord blood (CB) cells were suspended at a concentration of 10,000 per mL in StemSpanā„¢ 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 MethoCultā„¢ 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 StemSpanā„¢ SFEM II was significantly higher than in SFEM and AOF (*p<0.001, #p<0.05, paired t-test, n=6).

Note: Data for StemSpanā„¢-AOF shown were generated with the original phenol red-containing version StemSpanā„¢-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpanā„¢-AOF (Catalog #100-0130) was comparable.

Expansion of CD34 + Human Cord Blood Cells Cultured in StemSpan™ Media Containing CD34 + Expansion Supplement

Figure 3. StemSpanā„¢ 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 StemSpanā„¢ media (StemSpanā„¢ SFEM, StemSpanā„¢ SFEM II, StemSpanā„¢-XF, or StemSpanā„¢-AOF, orange bars), and in five xeno-free media formulations from other suppliers (Xeno-Free Commercial Alternative, grey bars) including (in random order) CTSā„¢ StemProā„¢ HSC (Thermo), SCGM (Cellgenix), X-VIVOā„¢ 15 (Lonza), Stemlineā„¢ II (Sigma), and StemProā„¢-34 (Thermo). All media were supplemented with StemSpanā„¢ 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. StemSpanā„¢ showed significantly higher expansion of CD34+ and CD34bright cells (P < 0.05 when comparing StemSpanā„¢ SFEM II to five media from other suppliers, calculated using a one-way ANOVA followed by Dunnettā€™s post hoc test) and StemSpanā„¢-AOF, the only animal origin-free formulation, showed equivalent performance to all xeno-free commericals alternatives tested. Data shown are mean Ā± SEM (n = 8).

Note: Data for StemSpanā„¢-AOF shown were generated with the original phenol red-containing version StemSpanā„¢-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpanā„¢-AOF (Catalog #100-0130) was comparable. *Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1Ī¼M. For more information including data comparing UM171 and UM729, see Fares et al., 2014.

StemSpan™ SFEM II Serum-Free Expansion Medium Containing Megakaryocyte Expansion Supplement Supports Greater Expansion of Megakaryocytes Than Other Media Tested

Figure 4. StemSpanā„¢ 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 StemSpanā„¢ media (StemSpanā„¢ SFEM, StemSpanā„¢ SFEM II, StemSpanā„¢-XF, or StemSpanā„¢-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 StemSpanā„¢ 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. StemSpanā„¢ 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 Dunnettā€™s post hoc test) and StemSpanā„¢-AOF, the only animal origin-free formulation tested, showed equivalent performance to all xeno-free commercial alternatives tested. Data shown are mean Ā± SEM (n = 8).

Note: Data for StemSpanā„¢-AOF shown were generated with the original phenol red-containing version StemSpanā„¢-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpanā„¢-AOF (Catalog #100-0130) was comparable.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1Ī¼M. 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 StemSpanā„¢ SFEM Serum-Free Expansion Medium Containing Erythroid Expansion Supplement

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.

StemSpan™ SFEM II Serum-Free Expansion Medium Containing Megakaryocyte Expansion Supplement Supports Greater Expansion of Megakaryocytes Than Other Media Tested

Figure 5. StemSpanā„¢ 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 StemSpanā„¢ SFEM medium (dark gray bar), obtained after culturing purified CD34+ CB cells for 14 days in StemSpanā„¢ 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 StemSpanā„¢ 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 StemSpanā„¢ SFEM II were significantly higher than in all other media (*p<0.05, paired t-test, n=6).

Note: Data for StemSpanā„¢-AOF shown were generated with the original phenol red-containing version StemSpanā„¢-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpanā„¢-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

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.

StemSpan™ SFEM II Serum-Free Expansion Medium Containing Megakaryocyte Expansion Supplement Supports Greater Expansion of Megakaryocytes Than Other Media Tested

Figure 6. StemSpanā„¢ 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 StemSpanā„¢ SFEM medium (dark gray bar), obtained after culturing purified CD34+ CB cells for 14 days in StemSpanā„¢ 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 StemSpanā„¢ 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 StemSpanā„¢ media were significantly higher than in the other media (*p<0.01 paired t-test, n=6).

Note: Data for StemSpanā„¢-AOF shown were generated with the original phenol red-containing version StemSpanā„¢-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpanā„¢-AOF (Catalog #100-0130) was comparable.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Name
Catalog #
09600, 09650
Lot #
All
Language
English
Document Type
Product Name
Catalog #
09600, 09650
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area
Workflow Stages

Resources and Publications

Educational Materials (11)

Brochure
Brochure
StemSpanā„¢ Media and Supplements for the Expansion and Differentiation of Erythroid Progenitor Cells
Technical Bulletin
StemSpanā„¢ Media and Supplements for the Expansion and Differentiation of Erythroid Progenitor Cell...
StemSpanā„¢ Serum-Free Expansion Media
1:51
Video
StemSpanā„¢ Serum-Free Expansion Media
Engineering the Hematopoietic System to Treat Non-Hematological Disorders
1:03:01
Webinar
Engineering the Hematopoietic System to Treat Non-Hematological Disorders
Qualification of Ancillary/Raw Materials for Clinical Use
54:39
Webinar
Qualification of Ancillary/Raw Materials for Clinical Use
New Tools for the Ex Vivo Expansion of Human Hematopoietic Stem and Progenitor Cells
1:06:52
Webinar
New Tools for the Ex Vivo Expansion of Human Hematopoietic Stem and Progenitor Cells
The Impact of Ancillary Materials on Your Translational Research
17:18
Webinar
The Impact of Ancillary Materials on Your Translational Research
CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells
55:51
Webinar
CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays
Mini Review
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays
Scientific Poster

Publications (227)

Enhancement of proliferation of human umbilical cord blood-derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules. Y. S. Park et al. Biochemistry and biophysics reports 2022 mar

Abstract

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.
Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging. J. Qiu et al. STAR protocols 2022 dec

Abstract

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here, we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs, which are the most potent subsets, it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol, please refer to Liang etĀ al. (2020) and Qiu etĀ al. (2021).
Reducing TGF-$\beta$1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling. X. Zhu et al. Cell proliferation 2021 mar

Abstract

OBJECTIVE As an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34- cell numbers. These CD34- cells influenced the ex vivo expansion of CD34+ cells. In this work, the effects of periodically removing CD34- cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated. MATERIALS AND METHODS CD34- cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF-$\beta$1 contents. RESULTS After 10-day culture, the expansion of CD34+ cells in the CD34- cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34- cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self-renewal symmetric division of CD34+ cells. In addition, the concentration of total TGF-$\beta$1 and activated TGF-$\beta$1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT-qPCR results showed that the periodic removal of CD34- cells with cooperation from SR1 further reduced the expression of AhR-related genes. CONCLUSIONS Periodic removal of CD34- cells plus cooperation with SR1 improved the expansion of CD34+ cells, maintained better biological function of expanded CD34+ cells and reduced the TGF-$\beta$1 contents by downregulating AhR signalling.
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