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StemSpan™-AOF

cGMP培养基，用于培养和扩增人造血细胞
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产品号 #（选择产品）

产品号 #100-0130_C

cGMP培养基，用于培养和扩增人造血细胞

产品优势

  • 培养7天后，人脐带血来源的 CD34+ 细胞扩增20倍；
  • 可维持原始 CD34brightCD90+CD45RA- 群体的培养；
  • 适用于基因组编辑方案；
  • 配方无动物源和酚红；
  • 按照相关 cGMP 标准生产和检测；
  • 原材料的全面可追溯性
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总览
实验数据
产品说明书及文档
应用领域
相关材料与文献
相关产品

总览

StemSpan™-AOF是一种无动物源（AOF）培养基，专为体外培养和扩增人造血细胞而开发，可添加适当的生长因子/细胞因子以及添加物（例如小分子）。这使得用户可以根据实验需求灵活地配制培养基。StemSpan™-AOF仅含有重组蛋白和合成成分，不含血清或其他人体或动物来源的成分。

StemSpan™-AOF在扩增富含造血干细胞的CD34⁺CD90⁺CD45RA⁻ 和CD34brightCD90⁺CD45RA⁻ 细胞亚群方面表现出比其他培养基更高的扩增能力：脐带血CD34+ 细胞在StemSpan™-AOF扩增7天后，移植到亚致死剂量照射的NSG小鼠体内，并在短期（3周）和长期（20周）植入时间点进行检测，均表现出与未培养细胞相似或更高的扩增率。StemSpan™-AOF推荐用于培养CD34+造血干祖细胞，或用于分化为髓系祖细胞。如需培养或分化为其他细胞类型，请访问StemSpan™产品检索工具，找到最适合您应用的StemSpan™培养基。

StemSpan™-AOF按照相关的cGMP标准生产，确保质量和一致性，从而获得可重复的实验结果。如需了解更多质量信息，请访问www.STEMCELL.com/compliance。

包含
• Phenol Red-Free Iscove’s MDM • Recombinant human albumin • Recombinant human insulin • Recombinant human transferrin • 2-Mercaptoethanol • Supplements
 
亚型
专用培养基
 
细胞类型
造血干/祖细胞
 
种属
人
 
应用
细胞培养，分化，扩增，基因组编辑
 
品牌
StemSpan
 
研究领域
干细胞生物学，移植研究，细胞治疗开发
 
制剂类别
Animal Origin-Free，无血清，Xeno-Free
 

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Data Figures

Analysis of the 7 days expanded Cord Blood CD34<sup>+</sup> cells by Flow Cytometry

Figure 1. Day 7 Immunophenotyping of CD34+ Cells Cultured in StemSpan™-AOF

CD34+ cells were purified from cord blood (CB) using the EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896) and cultured in StemSpan™-AOF (Catalog #100-0130) supplemented with StemSpan™ CD34+ Expansion Supplement (Catalog #02691) (A) without or (B) with the addition of UM729 (Catalog #72332). After 7 days, the cultured cells were stained with fluorescently labeled antibodies against CD34, CD90, and CD45RA, in addition to viability dye 7-AAD, and analyzed by flow cytometry. The horizontal dotted line in the CD34 vs FSC plots indicates the boundary between CD34- and CD34+ cells as based on a fluorochrome minus one (FMO) control for CD34 expression. Orange gates on these plots indicate the population of CD34bright cells used to generate data in Figures 2 and 3. Sequential gates were used to determine the percentages of viable CD34+ cells, CD34bright cells, and CD34brightCD90+CD45RA- cells.

Analysis of the 7 days expanded Mobilized Peripheral Blood CD34+ cells by Flow Cytometry

Figure 2. Analysis of the 7 days expanded Mobilized Peripheral Blood CD34+ cells by Flow Cytometry

Purified CD34+ cells derived from G-CSF mobilized peripheral blood (mPB) were cultured for 7 days in StemSpan™ AOF (Catalog #100-0130) supplemented with StemSpan™ CD34+ Expansion Supplement (Catalog #02691) and UM729 (Catalog #72332). After 7 days, the cultured cells were stained with fluorescently labeled antibodies against CD34, CD45RA, CD90, EPCR and CD133, in addition to viability dye Zombie YellowTM, and analyzed by flow cytometry. The horizontal dotted line on the CD34 and FSC plot indicated the fluorescence minus one (FMO) control for CD34 marker expression. Sequential gates (orange gates ) were used to determine the percentages of viable CD34bright cells, CD34brightCD90+CD45RA- cells and CD34bright CD45RA-CD90+CD133+EPCR+ cells and used to generate data in Figures 3.

StemSpan™-AOF Supports Equal or Greater Expansion of Mobilized Peripheral Blood HSPCs Compared to Other Commercial Media

Figure 3. StemSpan™-AOF Supports Equal or Greater Expansion of Mobilized Peripheral Blood HSPCs Compared to Other Commercial Media

Purified CD34+ cells derived from G-CSF mobilized peripheral blood (mPB) were cultured at a concentration of 10000 cells per mL in StemSpan™-AOF medium (orange bars) or in four xenofree media from other suppliers (Commercial Alternatives, grey bars). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM729 (1uM). After 7 days of culture, the (A) frequency and (B) cell expansion of viable CD34bright, CD34bright CD45RA-CD90+, and CD34bright CD45RA-CD90+CD133+EPCR+ cells were analyzed by flow cytometry, as described in Figure 2, and fold expansion normalized to StemSpanTM-AOF (above bar expansion value ± SD). The performance of StemSpan™-AOF, the only animal origin-free formulation, was similar to the performance of the xeno-free alternative media. Data shown are mean ± SEM (n=5).

StemSpan™-AOF Supports Equivalent or Greater Expansion of Human CD34+ and CD34<sup>bright</sup> Cells than Other Commercial Media

Figure 4. StemSpan™-AOF Supports Equivalent or Greater Expansion of Human CD34+ and CD34bright Cells than Other Commercial Media

Purified cord blood (CB)-derived CD34+ cells were cultured for 7 days in StemSpan™-AOF (orange bar), and in four xeno-free media formulations from other suppliers (Xeno-Free Commercial Alternative, grey bars), including (in random order) SCGM (Cellgenix), X-VIVO™ 15 (Lonza), Stemline™ II (Sigma), and StemPro™-34 (Thermo). 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. StemSpan™-AOF, the only animal origin-free formulation, showed equivalent performance to all xeno-free alternative media tested. All media were supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM171*. 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.

StemSpan™ Media Support Greater Expansion of Human CD34+CD90+CD45RA- and CD34<sup>bright</sup>CD90+CD45RA- Cells than Other Commercial Media

Figure 5. StemSpan™ Media Support Greater Expansion of Human CD34+CD90+CD45RA- and CD34brightCD90+CD45RA- Cells than Other Commercial Media

Purified CB-derived CD34+ cells were cultured for 7 days in StemSpan™-AOF medium (orange bar), and in four xeno-free media formulations from other suppliers (Commercial Alternative, gray bars) including (in random order) 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™-AOF showed similar or significantly higher expansion of CD34brightCD90+CD45RA- cells (P 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.

StemSpan™ Media Support Better CD34+ and Primitive CD34+CD90+CD45RA- HSPC Expansion for Genome Editing Applications Compared with Alternative Commercial Media

Figure 6. StemSpan™ Media Support Better CD34+ and Primitive CD34+CD90+CD45RA- HSPC Expansion in a Genome Editing Application Compared with Alternative Commercial Media

Purified CB-derived CD34+ cells were cultured for 2 days in select StemSpan™-AOF, (orange bar), or four xeno-free media formulations from other suppliers (gray bars). All media were supplemented with StemSpan™ CD34+ Expansion Supplement and UM171*. Cells were then electroporated using Arcitect™ CRISPR-Cas9 RNP complexes containing crRNA:tracrRNA targeting beta-2-microglobulin (B2M), and cultured for an additional 4 days in the same conditions. Knockout efficiency as measured by staining for MHC-I and analyzing by flow cytometry, was similar in all media tested, ~70-80%. (A) The percentage of CD34+ cells and (B) CD34+CD90+CD45RA- cells were quantified by flow cytometry 4 days post-electroporation. Data shown are mean + SD (n = 4 donors; **P < 0.01).

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

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1 μM. For more information including data comparing UM171 and UM729, see Fares et al., 2014.

Data showing engraftment of StemSpan™-AOF expanded CD34⁺ cells in NSG mouse recipients

Figure 7. StemSpan™-AOF-Expanded Cord Blood CD34⁺ Cells Engraft in NSG Mouse Recipients

Purified cord blood-derived CD34⁺ cells were cultured for 7 days in StemSpan™-AOF supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM729 (1 μM). After 7 days of expansion, progeny of 10,000 fresh or uncultured CD34⁺ cells were transplanted in sub-lethally irradiated NSG mice. (A-D) The number of human platelets and the frequency of human cells expressing the pan-leukocyte marker CD45 were measured in peripheral blood at 3 and 18 weeks post-transplantation. Data shown are mean ± SEM (n = 3 - 5 mice). (A) At 3 weeks, engraftment of human platelets was lower in recipients of cells cultured in StemSpan™-AOF than in recipients of uncultured cells. (C) At week 18, there were no significant differences in platelet engraftment between the expanded and uncultured cells. (B,D) Human CD45⁺ cell frequencies in recipients of cells expanded in StemSpan™-AOF were similar to those in recipients of uncultured cells. (E-H) At week 20, long-term multilineage engraftment was measured in bone marrow of transplanted NSG mice. Data shown are mean ± SEM (n = 3 - 4 mice). (E,F) Recipients of StemSpan™-AOF expanded cells showed similar frequencies of human CD45⁺ and CD34⁺ cells in the mouse bone marrow compared to recipients of uncultured cells. (G,H) Cells expanded with StemSpan™-AOF showed similar levels of myeloid (CD45⁺ CD33⁺ ) and lymphoid (CD45⁺ 19⁺ B cells and CD45⁺ CD3⁺ T cells) engraftment relative to uncultured cells.

Figure 8. Estimation of Long-term SCID Repopulating Cell in Cord Blood CD34⁺ cells expanded in either StemSpan™-AOF or StemSpan™ SFEM II using Limiting Dilution Transplantation assay

Purified cord blood-derived CD34⁺ cells were cultured for 7 days in either StemSpan™-AOF or StemSpan™ SFEM II supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM729 (1 μM). After 7 days of expansion, progeny of 10, 100, 250 and 2500 initial CD34⁺ cells were injected intravenously into sub-lethally irradiated NSG mice. For uncultured CD34⁺ cells, 250, 500 and 2500 cells were transplanted. The frequency of human cells expressing the pan-leukocyte marker CD45 was measured in bone marrow at ~20 weeks post-transplantation. A threshold of >0.1% of CD45⁺ cells was used to consider if mice were positive or negative for engraftment. Limiting dilution analysis was performed using the ELDA software from the Walter and Eliza Hall Institute of Medical Research software. LT-HSC frequencies (red lines) and 95% confidence intervals (boxes) are presented as 1/number of original CD34⁺ cell (day 0 equivalent) for each condition; n = 2 independent experiments performed, 2-7 mice per group per experiment. Significance level * p< 0.001 (Chi-square test). StemSpan™ SFEM II and StemSpan™-AOF expanded cells results in ~36 and ~17-fold increase in LT-HSC compared to fresh CD34⁺ cells, with SRC frequencies of 1/23, 1/48, 1/835 for StemSpan™ SFEM II, StemSpan™-AOF and fresh control respectively.

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 Information Sheet 1
Product Name
StemSpan™-AOF
Catalog #
100-0513
Lot #
1000152166 or higher
Language
English
Document Type
Product Information Sheet 2
Product Name
StemSpan™-AOF
Catalog #
100-0513
Lot #
1000126365 or lower
Language
English
Document Type
Product Information Sheet 1
Product Name
StemSpan™-AOF
Catalog #
100-0130
Lot #
1000167366 or higher
Language
English
Document Type
Product Information Sheet 2
Product Name
StemSpan™-AOF
Catalog #
100-0130
Lot #
1000167365 or lower
Language
English
Document Type
Safety Data Sheet
Product Name
StemSpan™-AOF
Catalog #
100-0513
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
StemSpan™-AOF
Catalog #
100-0130
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
Workflow Stages for Genome Editing
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更多信息
Species Human
Contains • Phenol Red-Free Iscove’s MDM • Recombinant human albumin • Recombinant human insulin • Recombinant human transferrin • 2-Mercaptoethanol • Supplements
Formulation Category Animal Origin-Free, Serum-Free, Xeno-Free
THIS PRODUCT IS MANUFACTURED AND TESTED FOLLOWING RELEVANT CGMPs UNDER A CERTIFIED QUALITY MANAGEMENT SYSTEM. PRODUCT IS FOR FURTHER MANUFACTURING OR RESEARCH USE. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE
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