Murine HSCs or lineage negative (Lin−) cells were cultured under 37°C, 5%CO2, 3%O2 conditions in IMDM containing 10% FBS & PS precalibrated for distinct pH in the presence of cytokines (SCF, TPO (Prospec CTY275, CYT‐346) and G‐CSF (Neupogen 300 μg/ml, Amgen)) at 50 ng/ml concentration each. Cells were cultured at pH 6.9 or pH 7.4 +/− DFMO (Sigma) 5 mM or +/− SPN (Sigma) 1 μM for 2 or 6 days in 96 well‐round bottom plates (TPP Tissue culture plates, Midwest Scientific TP92097). In some experiments, murine HSCs were also cultured in Serum‐free STEMspan SFEM medium (Stemcell Technologies; 09650) with distinct pHs or in the presence of DFMO. Human HSPCs (sorted linneg CD34pos SSClow cells from BM, anti‐CD34 clone 581 (BD #555824), human hematopoietic lineage antibody cocktail, FITC, eBioscience (#22‐7778‐72)) were incubated in serum‐free STEMspan SFEM medium (Stemcell Technologies; 09650) supplemented with cytokines (huIL3 (10 ng/ml), huIL6 (50 ng/ml), huFlt3 (20 ng/ml), huSCF and huTPO (both 100 ng/ml), all Stemcell Technologies) under hypoxic (3%O2) conditions in 96‐well flat bottom plates (suspension Sarstedt #83.3924). Half of the cultivation media was replaced every 24 h of culture with medium of the same precalibrated pH and also containing all the cytokines and the compounds like DFMO or SPN.
Stemspan sfem medium
Manufactured by STEMCELL
Sourced in Canada, Germany
StemSpan SFEM medium is a serum-free culture medium designed for the in vitro expansion and maintenance of hematopoietic stem and progenitor cells. It provides the necessary components to support the growth and differentiation of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (5 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
M3434 methylcellulose medium, by STEMCELL (4 mentions)
Doxycycline, by Merck Group (4 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (4 mentions)
Human cd34 microbead kit, by Miltenyi Biotec (4 mentions)
Lipid mixture 1, by Merck Group (4 mentions)
Ficoll paque plus, by GE Healthcare (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Heparin, by Merck Group (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (3 mentions)
Stemspan cd34 expansion supplement, by STEMCELL (3 mentions)
Stemspan cc100, by STEMCELL (3 mentions)
Rmscf, by Thermo Fisher Scientific (3 mentions)
Rhflt3 ligand, by Thermo Fisher Scientific (3 mentions)
Rhil 6, by Thermo Fisher Scientific (3 mentions)
Glutamine, by Corning (2 mentions)
88 protocols using stemspan sfem medium
1
Murine and Human Hematopoietic Stem Cell Culture
2
Expansion and Cryopreservation of CD34+ Cord Blood Stem Cells
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1×106 CD34+ cord blood stem cells were thawed in a 37°C water bath and pipetted into 20 mL pre-warmed StemSpan SFEM medium (STEMCELL Technologies, #09650). Cells were centrifuged and resuspended into StemSpan medium containing 10% fetal bovine serum (FBS) (Sigma, #F4135). Cells were adjusted to 2.5×104 cells/mL, supplemented with 20 ng/mL huIL-3 (PeproTech, #200–03), 100 ng/mL huSCF (PeproTech, #300–07), 100 ng/mL huFLT3L (PeproTech, #300–19), 50 ng/mL huTPO (PeproTech, #300–18), plated at 0.5×104 cells per well in a 96-well U bottom plate and cultured at 37°C in a CO2 incubator. After 7 days of expansion, cells were harvested and frozen in 80% MEM-α (Gibco, #12 561–056), 10% DMSO (Sigma, #D2650), 10% FBS at 2.6×106 cells/mL. Cells were stored in liquid nitrogen.
3
Expansion of Healthy and Diabetic CD34+ Cells
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CD34+ cells were prepared as previously described 10 , 11 , 12 . The CD34+ cell fraction had a purity of 80%–90% as determined by fluorescence‐activated cell sorting (FACS) analysis. Freshly isolated CD34+ (pre‐QQc) cells from healthy and diabetic patients were placed in an ex vivo serum‐free expansion culture QQc as previously described 13 . In brief, the cells were seeded at a density of 1 × 104 cells per well in 24‐well Primaria plates (BD Falcon, Franklin Lakes, NJ), with 0.5 ml/well StemSpan SFEM medium (Stem Cell Tech., Vancouver, BC, Canada) supplemented with recombinant human (rh) VEGF (50 ng/ml), rhIL‐6 (20 ng/ml), rhFlt3‐ligand (100 ng/ml), rh thrombopoietin (20 ng/ml), rhSCF (100 ng/ml) (all from PeproTech, Rocky Hill, NJ), and antibiotic cocktail (Invitrogen, Carlsbad, CA), and cultured for 7 days at 37°C in 5% CO2.
4
Cultivation and Differentiation of Hematopoietic Cell Lines
5
Cytokine-Modulated Expansion of Human Fetal CD34+ Cells
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CD34+ cells were isolated from human fetal liver tissues. Then the cells were cultured in StemSpan SFEM medium (Stem Cell Technologies) with heparin (10 μg/ml, Sigma), recombinant human SCF (20 ng/ml, R&D), thrombopoietin (40 ng/ml, Cell Sciences) and CHIR99021 (GSK3 inhibitor, 250 nM, STEMGENT) for 48 hours in the presence of IFN-α or IFN-β at the dose of 20 IU/ml and 200 IU/ml, respectively. The cells were counted and collected for the detection of CD38 expression on HPCs.
6
Culturing U937 and CD34+ Cells
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The U937 cell line (ATCC, Manassas, VA, USA) was cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies). Primary CD34+ cells were obtained from human umbilical cord blood collected at Skåne University Hospital. The mononuclear cell population was isolated by separation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) and CD34+ cells were subsequently selected using the Indirect CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were grown in StemSpan SFEM medium (Stemcell Technologies, Vancouver, Canada) supplemented by 20% fetal bovine serum and the CC100 cytokine cocktail (Stemcell Technologies).
7
Isolation and Expansion of HSCs and MSCs
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HSCs were isolated from the BM of mice by immunomagnetic beads (Lineage Cell Depletion Kit, Miltenyi Biotec). Cell suspension was expanded in StemSpan SFEM medium (STEMCELL Technologies) supplemented with SCF (50 ng/mL), TPO (15 ng/mL), IL3 (30 ng/mL), FLT3 ligand (50 ng/mL), and IL6 (20 ng/mL).
MSCs were purified from the bone and the trabecular fraction. Initially cells were incubated with collagenase I (Sigma-Aldrich; 1 mg/mL) for 1 hour at 37°C. Then, cell suspension was incubated with the following PE-conjugated antibody: CD45, B220, CD3, CD11b, CD11c, Gr1, F4/80, Ter119 (all markers of hematopoietic cells). After separation with anti-PE microbeads (Miltenyi Biotec), MSCs were phenotypically characterized by flow cytometry analysis with the following markers: CD45, Ter119, CD31, Sca-1, CD29, CD44.
For in vitro experiments, BM-derived mesenchymal cells were expanded and differentiated with MesenCult MSC Basal Medium supplemented with Mesenpure cocktail (STEMCELL Technologies).
HSCs were stimulated for 24 hours with 25 ng/mL of recombinant IL1B (211–11B, PeproTech). Anti-IL1B neutralizing antibody (B122, BioXCell) and relative Isotype control (Hamster IgG, BioXCell) were added to the medium prior to coculture experiments at the dose of 5 ug/mL. HSCs were also differentiated toward monocytes in the presence of macrophage colony-stimulating factor (M-CSF; 40 ng/mL).
MSCs were purified from the bone and the trabecular fraction. Initially cells were incubated with collagenase I (Sigma-Aldrich; 1 mg/mL) for 1 hour at 37°C. Then, cell suspension was incubated with the following PE-conjugated antibody: CD45, B220, CD3, CD11b, CD11c, Gr1, F4/80, Ter119 (all markers of hematopoietic cells). After separation with anti-PE microbeads (Miltenyi Biotec), MSCs were phenotypically characterized by flow cytometry analysis with the following markers: CD45, Ter119, CD31, Sca-1, CD29, CD44.
For in vitro experiments, BM-derived mesenchymal cells were expanded and differentiated with MesenCult MSC Basal Medium supplemented with Mesenpure cocktail (STEMCELL Technologies).
HSCs were stimulated for 24 hours with 25 ng/mL of recombinant IL1B (211–11B, PeproTech). Anti-IL1B neutralizing antibody (B122, BioXCell) and relative Isotype control (Hamster IgG, BioXCell) were added to the medium prior to coculture experiments at the dose of 5 ug/mL. HSCs were also differentiated toward monocytes in the presence of macrophage colony-stimulating factor (M-CSF; 40 ng/mL).
8
Analyzing TGFβ and Irradiation Signaling
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Whole cell lysates were prepared using RIPA cell lysis buffer (9803, Cell Signaling) and 1 mM PMSF (8553s, Cell Signaling). Lin- cells, cells were cultured for 24 h in StemSpan SFEM medium (09600, StemCell Technologies) containing 2% L-glutamine (25030-081GIBCO), 1% penicillin/streptomycin (15140–122, GIBCO), 100 ng/ml SCF (250-03-10UG, Peprotech) and 100 ng/ml TPO (315-14-10UG, Peprotech). Lin- cells were exposed to TGFβ1 (5 ng/ml) or TGFβ3 (5 ng/ml) along with LSN3301240 (5 μM) during 2 h. Western blots were performed using the following antibodies SMAD2/3 (86855, Cell Signalling), phospho-SMAD2 (ab3849, Millipore), phospho-SMAD2/3 (8828s, Cell Signaling) and Vinculin (sc-25336, Santa Cruz) antibodies.
Bone marrow stromal cell lines were exposed to irradiation (5 or 10 Gy), MEK inhibitor PD0325901 (10 μM) or TGFβ inhibitor LSN3301240 (5 μM). Western blots were performed with the following antibodies DNA-PKcs, 53BP1, RAD51, phospho-SMAD3 (9502s, Cell Signaling Technology, Danvers, MA, USA), p-ERK and actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Antibodies for analysis of fetal liver were DNA-PKcs, 53BP1, pERK1/2, total ERK1/2, CD45 CD41 and Vinculin.
Bone marrow stromal cell lines were exposed to irradiation (5 or 10 Gy), MEK inhibitor PD0325901 (10 μM) or TGFβ inhibitor LSN3301240 (5 μM). Western blots were performed with the following antibodies DNA-PKcs, 53BP1, RAD51, phospho-SMAD3 (9502s, Cell Signaling Technology, Danvers, MA, USA), p-ERK and actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Antibodies for analysis of fetal liver were DNA-PKcs, 53BP1, pERK1/2, total ERK1/2, CD45 CD41 and Vinculin.
9
Ex Vivo Expansion of CD34+ Hematopoietic Cells
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CD34-positive cells from the BM aspirate were obtained using antibody-mediated positive selection followed by purification. CD34+ cells were then cultured in StemSpan SFEM Medium (Stem Cell Technology) supplemented with 10% FBS (Stem Cell Technology), 10 ng/mL IL-3 (Stem Cell Technology), 50 ng/mL SCF (Stem Cell Technology), and 2 U/mL recombinant human EPO (Amgen Inc.) from day 0 to day 6. After day 6, cells were cultured in StemSpan SFEM Medium supplemented with 30% FBS and 2 U/mL EPO.
10
Erythroid Differentiation of HUDEP-2 Cells
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HUDEP-2 cells28 (link) were cultured in StemSpan SFEM medium (STEMCELL Technologies) supplemented with 100 ng/mL SCF, 3 IU/mL EPO, 10−6 M dexamethasone, and 1 μg/mL DOX. Erythroid differentiation was induced in IMDM containing 5% human AB serum, 100 ng/mL SCF, 3 IU/mL EPO, 10 μg/mL insulin, 330 μg/mL transferrin, 2 U/mL heparin, and 1 μg/mL DOX for 6 days.
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