StemSpan™ SFEM II

用于培养和扩增造血细胞的无血清培养基

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产品号 #09605_C

用于培养和扩增造血细胞的无血清培养基

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总览

StemSpan™ 无血清扩增培养基 II（SFEM II）是StemSpan™ SFEM的优化版本，专为体外培养和扩增人造血细胞而开发。该培养基在Iscove’s MDM培养基的基础上包含经过测试的牛血清白蛋白、胰岛素、转铁蛋白以及其他添加物。StemSpan™ SFEM II 中未添加造血细胞生长和扩增所需的重组造血生长因子，这使得用户可以根据实验需求灵活地配制培养基。

搭配合适的细胞因子（例如 StemSpan™ CC100、StemSpan™ CC110 或 StemSpan™ CD34+ 扩增添加物），StemSpan™ SFEM II 可用于扩增来自脐带血、骨髓或其他细胞来源的总有核细胞和 CD34+ 细胞。当与 StemSpan™ 红系扩增添加物（产品号 #02692）、StemSpan™ 髓系扩增添加物（产品号 #02693）、StemSpan™ 髓系扩增添加物 II（产品号 #02694）或 StemSpan™ 巨核细胞扩增添加物（产品号 #02696）分别搭配使用时，StemSpan™ SFEM II 可用于扩增和分化谱系定向祖细胞，以生成有核红细胞、粒细胞、单核细胞或巨核细胞。

实验数据

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.

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 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 CD34^bright 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 CD34^bright 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 CD34^bright 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.

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

Figure 4. StemSpan™ Media Support Equal or Greater Expansion of Primitive Human CD34^brightCD90⁺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 CD34^brightCD90⁺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 CD34^brightCD90⁺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.

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.

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.

Table 1. Production of Myeloid Cells from Human CB CD34⁺ Cells Cultured in SFEM II Containing Myeloid Expansion Supplement or Myeloid Expansion Supplement ll

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

Shown are numbers of total nucleated cells (TNCs) produced per input human CB-derived CD34⁺ cell and percentages of cells positive for myeloid markers CD13, CD14 and CD15 after 14 days of culture in SFEM II containing Myeloid Expansion Supplement (n = 14) or Myeloid Expansion Supplement II (n = 16). *95% confidence limits (CL); the range within which 95% of results typically fall.

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Document Type

Product Information Sheet

Product Name

StemSpan™ SFEM II

Catalog #

09605, 09655

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English

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StemSpan™ SFEM II

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09605, 09655

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English

文献 (18)

Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth. C. H. Man et al. Blood 2022 jan

Abstract

Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.

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Establishment of a human iPSC line (SUTCMi001-A) derived from a healthy donor. L. Min et al. Stem cell research 2022 aug

Abstract

This study describes the characterization of one induced pluripotent stem cell line (iPSC) from a healthy female. It is crucial to use iPSCs derived from healthy individuals as controls in genetic disease studies. Thus, we established a human iPSC cell line derived from healthy people. The iPSC cell line was generated in our lab from the peripheral blood mononuclear cells (PBMCs) of a 28-year-old girl. The generated hiPSC line is free of episomal vectors, has a normal karyotype, expresses pluripotency markers and can differentiate into three germ layers in vivo.

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CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells. Q. Cheng et al. Annals of translational medicine 2022 aug

Abstract

BACKGROUND Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has become an increasingly vital tool for modifying gene expression in a variety of cell types. Lentiviral transduction and electroporation are the two main approaches used to deliver CRISPR/Cas9 into cells. However, the application of CRISPR/Cas9 in primary hematopoietic cells has been limited due to either low transduction efficiency in terms of viral-based delivery or difficult selection and enrichment of transfected and edited cells with respect to electroporation of CRISPR/Cas9 ribonucleoprotein (RNP). METHODS In this study in vitro transcription was used to synthesize the guide RNA (gRNA), and plasmid pL-CRISPR.EFS.GFP was used as its DNA template. Then the in vitro transcribed gRNA was labeled with pCp-Cy5 via T4 ligase before incubating with Cas9 protein. Furthermore, CRISPR/Cas9 RNP was electroporated into primary CD34+ cells isolated from cord blood, and cell survival rate and transfection efficiency were calculated and compared to that of lentiviral transduction. RESULTS Here, we show that electroporation of CRISPR/Cas9 RNP resulted in higher cell viability compared to electroporation of CRISPR/Cas9 all-in-one plasmid, providing important findings for further studies in hematology via CRISPR/Cas9 technology. Moreover, we established a method for labeling in vitro-transcribed gRNA with fluorophore and the sorted fluorescent cells displayed higher knockout efficiency than nonsorted transfected cells. CONCLUSIONS Electroporation of fluorescence labeled CRISPR/Cas9 RNP is a perspective approach of gene editing. Our study provides an efficient and time-saving approach for genome-editing in hematopoietic cells.

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种属	Human
Contains	• Iscove’s MDM • Bovine serum albumin • Recombinant human insulin • Human transferrin (iron-saturated) • 2-Mercaptoethanol • Supplements
配方类别	Serum-Free

StemSpan™ SFEM II

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产品号 #09605_C

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StemSpan™ SFEM II

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产品号 #09605_C

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