StemSpan™-ACF Without Phenol Red
cGMP medium, for culture and expansion of human hematopoietic cells
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StemSpan™-ACF Without Phenol Red -
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Overview
StemSpan™-ACF Without Phenol Red is an animal component-free (ACF) medium that has been developed for the in vitro culture and expansion of human hematopoietic cells, when appropriate growth factors/cytokines and supplements (e.g. small molecules) are added. This allows users the flexibility to prepare medium that meets their requirements. StemSpan™-ACF Without Phenol Red contains only recombinant proteins and synthetic components, and does not contain serum or other human- or animal-derived components.
Using appropriate cytokines, StemSpan™-ACF Without Phenol Red 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 progenitor cells to generate populations of myeloid or megakaryocyte progenitor cells.
StemSpan™-ACF Without Phenol Red is manufactured and tested following relevant cGMPs under a certified quality management system. For additional quality information, visit www.STEMCELL.com/compliance.
Using appropriate cytokines, StemSpan™-ACF Without Phenol Red 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 progenitor cells to generate populations of myeloid or megakaryocyte progenitor cells.
StemSpan™-ACF Without Phenol Red is manufactured and tested following relevant cGMPs under a certified quality management system. For additional quality information, visit www.STEMCELL.com/compliance.
Advantages:
⦁ 20X expansion of human cord blood-derived CD34+ cells after 7 days of culture
⦁ Maintenance of primitive CD34brightCD90+CD45RA- population in culture
⦁ Suitable for use in genome editing protocols
⦁ Animal component- and phenol red-free formulation
⦁ Manufactured and tested under relevant cGMPs
⦁ Full traceability of raw materials
⦁ Maintenance of primitive CD34brightCD90+CD45RA- population in culture
⦁ Suitable for use in genome editing protocols
⦁ Animal component- and phenol red-free formulation
⦁ Manufactured and tested under relevant cGMPs
⦁ Full traceability of raw materials
Contains:
This product contains only recombinant proteins and synthetic components.
Subtype:
Specialized Media
Cell Type:
Hematopoietic Stem and Progenitor Cells
Species:
Human
Application:
Cell Culture; Differentiation; Expansion; Genome Editing
Brand:
StemSpan
Area of Interest:
Cell Therapy; Stem Cell Biology; Transplantation Research
Formulation:
Animal Component-Free; Serum-Free
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Scientific Resources
Product Documentation
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Educational Materials
(5)
Product 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.
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Data
Figure 1. Day 7 Immunophenotyping of CD34+ Cells Cultured in StemSpan™-ACF Without Phenol Red
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™-ACF Without Phenol Red (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.
Figure 2. 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 II, or StemSpan™-ACF, 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™ SFEM II 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™-ACF, the only animal component-free formulation, showed equivalent performance to all xeno-free competitors tested. Data shown are mean ± SEM (n = 8).
Note: Data for StemSpan™-ACF 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-ACF(Catalog #100-0130) was very similar.
*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 3. 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 select StemSpan™ media (StemSpan™ SFEM II, or StemSpan™-ACF, 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™-ACF, the only animal component-free formulation tested, showed equivalent performance to all xeno-free competitors tested. Data shown are mean ± SEM (n = 8).
Note: Data for StemSpan™-ACF 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-ACF(Catalog #100-0130) was very similar.
*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 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™ media (StemSpan™ SFEM II or StemSpan™-ACF, orange bars), or five 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™-ACF 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-ACF Without Phenol Red (Catalog #100-0130) was very similar.
*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.
Publications
(3)
Stem Cell Research 2019 oct
Detection of all adult Tau isoforms in a 3D culture model of iPSC-derived neurons
Abstract
Abstract
Tauopathies are a class of neurodegenerative diseases characterized by the presence of pathological intracellular deposits of Tau proteins. Six isoforms of Tau are expressed in the adult human brain, resulting from alternative splicing of the MAPT gene. Tau splicing is developmentally regulated such that only the smallest Tau isoform is expressed in fetal brain, contrary to the adult brain showing the expression of all 6 isoforms. Induced Pluripotent Stem Cell (iPSC) technology has opened up new perspectives in human disease modeling, including tauopathies. However, a major challenge to in vitro recapitulation of Tau pathology in iPSC-derived neurons is their relative immaturity. In this study, we examined the switch in Tau splicing from fetal-only to all adult Tau isoforms during the differentiation of iPSC-derived neurons in a new 3D culture system. First, we showed that iPSC-induced neurons inside Matrigel-coated alginate capsules were able to differentiate into cortical neurons. Then, using a new assay that allowed both the qualitative and the quantitative analysis of all adult MAPT mRNA isoforms individually, we demonstrated that BrainPhys-maintained neurons expressed the 6 adult MAPT mRNA transcripts from 25 weeks of maturation, making this model highly suitable for modeling Tau pathology and therapeutic purposes.
Nature methods 2017 JUN
Marker-free coselection for CRISPR-driven genome editing in human cells.
Abstract
Abstract
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.
Stem Cell Reports 2014 NOV
Scalable generation of universal platelets from human induced pluripotent stem cells
Abstract
Abstract
Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates�
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.