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StemSpan™ Leukemic Cell Culture Kit

For culture, expansion, and drug screening of chronic and acute myeloid leukemia cells
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  • Optimized for culture, expansion, and drug screening of human myeloid leukemia cells.

  • ~65-fold expansion of CD34+ cells per input CD34+ CML cell; ~30-fold expansion of CD34+ cells per input CD34+ AML cells.

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    概述
    实验数据
    产品说明书及文档
    应用领域
    相关材料与文献
    StemSpan™ Leukemic Cell Culture Kit has been developed for the in vitro culture and expansion of malignant cells and has been tested on chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) samples. This optimized protocol allows users to expand, culture, and use malignant cells for drug screening. StemSpan™ Leukemic Cell Culture Kit includes the serum-free medium SFEM II (Catalog #09605), StemSpan™ CD34+ Expansion Supplement (10X; Catalog #02691), and small molecule UM729 (Catalog #72332).

    Data Figures

    The experiments in this Technical Bulletin were performed using cryopreserved cells, however similar results are expected when using fresh samples. Additionally in place of UM729, the small molecule UM171 was used to generate data in Figures 4 - 7. UM171 is no longer licensed for sale by STEMCELL Technologies, however similar results are expected when using UM729 prepared to a final concentration of 1 μM (data not shown). Further titration may be necessary to optimize cell fold expansion in specific conditions.
    For more information including data comparing UM171 and UM729, see Fares et al. 2014.

    Figure 1. Isolation of Leukemic CD34+ Cells

    Cryopreserved CML or AML PBMCs and BMMCs were thawed and prepared for isolation. CD34+ cells were isolated using EasySep™ Human Cord Blood CD34 Positive Selection Kit II. The percentage of CD34+ cells before (A, C) and after (B, D) CD34+ cell isolation was measured by flow cytometry. Dead cells were excluded by light scatter profile and viability staining. In this example the purity of CD34+ cells increased from 3% to 82% (CML) and from 16% to 93% (AML).


    Figure 2. Expansion of CD34+ CML Cells

    CD34+ CML cells were cultured in StemSpan™ SFEM II containing CD34+ Expansion Supplement (Exp) without or with UM171. After 7 and 14 days, the cultured cells were stained with fluorescently labeled antibodies against CD45, CD34, CD90, CD45RA, and with ALDEFLUOR™ (Catalog #01700) to measure ALDH activity, and analyzed by flow cytometry. Sequential gates were used to determine the percentages of viable CD45+, CD45+CD34+ and CD45+CD34+CD90+CD45RA- cells(based on “Fluorescence Minus One” (FMO) controls), and ALDHbr cells (based on DEAB control). (A) Representative flow cytometry profiles at day 7 are shown. The (B,D) frequency and (C,E) cell numbers of these subsets per initial CD34+ cell on (B,C) day 7 and (D,E) day 14 are shown. StemSpan™ SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of CML cells in culture. The addition of UM171 enhances expansion of all subsets shown (~10-fold expansion of CD34+ progenitor cells at day 7 and ~20-fold at day 14 compared to cultures without UM171). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.0001). All six CML samples tested were able to expand in culture.


    Figure 3. Expansion of CD34+ AML Cells

    CD34+ AML cells were cultured in StemSpan™ SFEM II containing CD34+ Expansion Supplement (Exp) alone, or with UM171. After 7 and 14 days, the cultured cells were stained with fluorescently labeled antibodies and with ALDEFLUOR™ Reagent as described in Figure 2. (A) Representative flow cytometry profiles at day 7 are shown. The (B, D) frequency and (C, E) cell numbers of these subsets per initial CD34+ cell on (B, C) day 7 and (D, E) day 14 are shown. SFEM II supplemented with CD34+ Expansion Supplement supports the expansion of AML cells in culture. The addition of UM171 further enhances expansion of all subsets shown (~3-fold expansion of all subsets at day 7 and ~7-fold at day 14 compared to cultures without UM171). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01). Six out of ten AML samples tested were able to expand in culture.

    Figure 4. Colony-Forming Potential of CD34+ CML Cells is Maintained During Culture

    CML cells were assayed in colony assays using MethoCult™ H4435 Enriched medium directly after CD34+ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 2). After 14 days of culture in StemSpan™ SFEM II with CD34+ expansion supplement (Exp) with or without UM171, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34+ cell. Numbers above each of the individual bars indicate the proportion of BCR-ABL positive colonies, measured by qRT-PCR on individual plucked colonies across 6 different samples (8-12 colonies were plucked for each sample per condition). SFEM II supplemented with CD34+ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. UM171 further promotes colony forming progenitor cell output (~3.5-fold expansion at day 7 and ~8-fold at day 14). Single-colony qRT-PCR analysis revealed that colonies generated from samples on day 0, and colonies generated from cells expanded for 7 and 14 days, were predominantly BCR-ABL+ but also that normal BCR-ABL- progenitor cells were present at low frequencies. Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).

    Figure 5. Colony-Forming Potential of CD34+ AML Cells is Maintained During Culture

    AML cells, after CD34+ cell isolation (day 0) or after 7 or 14 days of expansion without or with UM171 (as described in Figure 3), were plated in colony assays with MethoCult™ H4435 Enriched medium. After 14 days of incubation, colonies were (A) imaged with STEMvision™ and counted manually from digital images. (B) CFU output expressed as the total number of colonies per original input CD34+ cell. SFEM II supplemented with CD34+ Expansion Supplement (Exp) supports the expansion of colony-forming progenitor cells in culture. Addition of UM171 further promotes colony-forming progenitor cell output (~3-fold expansion at day 7 and ~4-fold at day 14). Data shown are mean ± SEM (n = 6). P-values were calculated using a two-tailed paired Student’s t-test (*P < 0.05).

    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
    Product Name
    StemSpan™ Leukemic Cell Culture Kit
    Catalog #
    09720
    Lot #
    All
    Language
    English
    Document Type
    Safety Data Sheet 1
    Product Name
    StemSpan™ Leukemic Cell Culture Kit
    Catalog #
    09720
    Lot #
    All
    Language
    English
    Document Type
    Safety Data Sheet 2
    Product Name
    StemSpan™ Leukemic Cell Culture Kit
    Catalog #
    09720
    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 (5)

    Hematopoietic Stem and Progenitor Cells - Products for Your Research
    Brochure
    Hematopoietic Stem and Progenitor Cells - Products for Your Research
    In Vitro Drug Screening with the StemSpan™ Leukemic Cell Culture Kit
    Technical Bulletin
    In Vitro Drug Screening with the StemSpan™ Leukemic Cell Culture Kit
    Culturing Leukemic Stem & Progenitor Cells with StemSpan™ Medium
    Technical Bulletin
    Culturing Leukemic Stem & Progenitor Cells with StemSpan™ Medium
    Bone Marrow Niches and HSC Fates
    Wallchart
    Bone Marrow Niches and HSC Fates
    Troubleshooting In Vitro Expansion of Leukemic Cells
    45:17
    Webinar
    Troubleshooting In Vitro Expansion of Leukemic Cells

    Related Products

    Related Products

    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
    Product Name
    StemSpan™ Leukemic Cell Culture Kit
    Catalog #
    09720
    Lot #
    All
    Language
    English
    Document Type
    Safety Data Sheet 1
    Product Name
    StemSpan™ Leukemic Cell Culture Kit
    Catalog #
    09720
    Lot #
    All
    Language
    English
    Document Type
    Safety Data Sheet 2
    Product Name
    StemSpan™ Leukemic Cell Culture Kit
    Catalog #
    09720
    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
    PRODUCTS ARE FOR RESEARCH USE ONLY AND 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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