The serum free media that were tested for myoblast proliferation were: the Fibroblast basal medium with the FGM-CD SingleQuots Kit™ (FBM, Lonza, Germany), StemPro™ MSC SFM (StemPro™, Thermo Fisher Scientific, The Netherlands), the Essential 8™ Medium (Essential 8™, Life technologies, USA), the STEMmacs™ HSC Expansion Media XF (STEMmacs™, Miltenyi Biotec, The Netherlands), mTeSR1™ (mTeSR1™, Stemcell Technologies, Canada), MesenCult™ ACF Culture Kit (Mesencult™, Stemcell Technologies, Canada) and TeSR™-E8™ (Stemcell Technologies, Canada).
Stempro msc sfm
Manufactured by Thermo Fisher Scientific
Sourced in United States
StemPro® MSC SFM is a serum-free, animal component-free medium designed for the expansion of human mesenchymal stem/stromal cells (hMSCs) in vitro. It supports the maintenance of multipotency and self-renewal of hMSCs.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Calibur flow cytometer, by BD (3 mentions)
Pe conjugated antibodies against cd44, by BD (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Mesencult acf culture kit, by STEMCELL (1 mentions)
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Stemmacs hsc expansion media xf, by Miltenyi Biotec (1 mentions)
Mtesr1, by STEMCELL (1 mentions)
Tesr e8, by STEMCELL (1 mentions)
Zetaview basic nta, by Particle Metrix (1 mentions)
Phycoerythrin, by BD (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Fibronectin from human plasma, by Merck Group (1 mentions)
Recombinant human albumin, by Merck Group (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
14 protocols using stempro msc sfm
1
Serum-Free Media for Myoblast Proliferation
2
Umbilical Cord MSC Isolation and Characterization
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MSCs were obtained from the discarded umbilical cord tissues and prepared and stored in the GMP laboratory of Shanghai East Hospital. The use of the human samples and cells has received approval from the Ethics Committee of Shanghai East Hospital. P1 UC-MSCs were provided free of charge by GMP facilities for validation experiments. Cells were expanded under 2D attachment condition with a serum free medium (StemPro® MSC SFM, Thermofisher, MA, United States) for up to 15 passages at a ratio of 1:3 every 4 days. Cells at passages P4 were used for the characterization of the ISCT-recommended cell surface characteristics of MSCs, including expression of CD73, CD90, and CD105 (>95%) and no presentation of CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR.
3
Exosome Isolation from Mesenchymal Stem Cells
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hMSCs or hAAT-MSCs at 70–80% of confluence were cultured in StemPro™ MSC SFM (Thermo Fisher, Waltham, MA, USA). Cell culture medium was collected 48 h later, centrifuged at 300× g for 4 min, and then at 2000× g for 10 min. Supernatant was collected and frozen at −80 °C for further use. Exosomes were harvested using ultracentrifugation at 10,000× g for 40 min at 4 °C. The collected supernatants were centrifuged again at 100,000× g for 90 min at 4 °C. The pellets were washed with PBS and then stored at −80 °C. The size distribution of exosomes was measured by ZetaView® BASIC NTA (Particle Metrix, Mebane, NC, USA).
4
Isolation and Characterization of Human Amniotic Fluid Stem Cells
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Institutional review board of our hospital approved this study (No. 201601690A3).
hAFSCs were obtained after written informed consent from freshly collected
amniotic fluid by routine amniocentesis in the second trimester of healthy
pregnant donors. Cells were cultured in StemPro® MSC SFM supplemented
with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and incubated at
37°C with 5% carbon dioxide. Specific surface antigens of hAFSCs had been
examined using flow cytometry in our previous study4 (link). Cultured cells were trypsinized and stained with
phycoerythrin-conjugated antibodies against CD44, CD45, CD73, CD90, CD105, and
CD117 (BD PharMingen, San Diego, CA, USA). Cells were analyzed using Calibur
flow cytometer (Becton Dickinson, Heidelberg, Germany). Passage 4 to 6 hAFSCs
were collected and prepared to a final concentration of 1 × 106cells/0.3 ml PBS. hAFSC dose was determined according to our previous work that
used hAFSCs to treat rat bladder dysfunction after focal cerebral ischemia4 (link).
hAFSCs were obtained after written informed consent from freshly collected
amniotic fluid by routine amniocentesis in the second trimester of healthy
pregnant donors. Cells were cultured in StemPro® MSC SFM supplemented
with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and incubated at
37°C with 5% carbon dioxide. Specific surface antigens of hAFSCs had been
examined using flow cytometry in our previous study4 (link). Cultured cells were trypsinized and stained with
phycoerythrin-conjugated antibodies against CD44, CD45, CD73, CD90, CD105, and
CD117 (BD PharMingen, San Diego, CA, USA). Cells were analyzed using Calibur
flow cytometer (Becton Dickinson, Heidelberg, Germany). Passage 4 to 6 hAFSCs
were collected and prepared to a final concentration of 1 × 106cells/0.3 ml PBS. hAFSC dose was determined according to our previous work that
used hAFSCs to treat rat bladder dysfunction after focal cerebral ischemia4 (link).
5
Isolation and Culture of Murine MSCs
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We isolated and purified bone marrow-derived MSCs from C57Black-6 mice by flushing the cavity of femurs as described previously [5] (link). Cells were cultured using serum-free medium (STEMPRO® MSC SFM, Invitrogen).
6
Amniotic Fluid Stem Cells for Bladder Dysfunction
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The hAFSCs were obtained from freshly collected amniotic fluid by routine amniocentesis from second-trimester healthy pregnant donors. Cells were cultured in StemPro® MSC SFM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C with 5% CO2. The specific surface antigens of hAFSCs were characterized using flow cytometry according to our previous work13 (link)–15 (link). The cells in culture were trypsinized and stained with phycoerythrin (PE)-conjugated antibodies against CD44, CD45, CD73, CD90, CD105 and CD117 (BD PharMingen, San Diego, CA, USA). Thereafter, the cells were analyzed using Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Passage 6–8 hAFSCs were collected and prepared to a final concentration of 1 × 106 cells/0.3 mL in PBS. In the hAFSCs-treated groups, 1 × 106 collected hAFSCs were injected into 5 sites of bladder in each rat (anterior, posterior, bilateral, and dome) under inhalation anesthesia according to our previous method14 (link),15 (link). The treatment dose of hAFSCs was determined according to our previous study that used hAFSCs to treat bladder dysfunction after focal cerebral ischemia in rats15 (link).
7
Amniotic Fluid Stem Cells for Stroke
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The hAFSCs were obtained from the freshly collected amniotic fluid by routine amniocentesis from healthy pregnant donors with 15–20 gestational weeks. Cells were cultured in StemPro MSC SFM (serum free medium) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and incubated at 37°C with 5% carbon dioxide. Culture medium was changed every 3–4 days. The specific surface antigens of hAFSCs were characterized by flow cytometry analyses as shown in our previous work 18 . The cells in culture were trypsinized and stained with phycoerythrin (PE)‐conjugated antibodies against CD44, CD73, CD90, CD105, CD117, and CD45 (BD PharMingen, CA). Thereafter, the cells were analyzed using the Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Passage 6‐8 hAFSCs were collected and prepared to a final concentration of 1 × 106 cells per 0.3 milliliter in PBS. In the hAFSCs‐treated groups, 1 × 106 collected hAFSCs were transplanted into each rat at 3 hours after MCAO by injection into the five sites of bladder (anterior, posterior, bilateral, and dome) under inhalation anesthesia. Before each local injection, the syringe was pushed backwards to confirm the needle was not present inside the vessel.
8
Culturing Human Bone Marrow MSCs
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Human bone marrow derived MSCs (LONZA, Basel, Switzerland) from 5 donors (Lot Number:0000296577, 0000310956, 0000318006, 0000296578, 0000307219) were cultured in human MSC culture medium (StemPro MSC SFM, Invitrogen, Carlsbad, USA) with antibiotics in humidified air containing 5% CO 2 at 37 C.
9
Serum-free Culture of Human Mesenchymal Stem Cells
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All methods regarding human subjects were approved by the Harvard University Committee On the Use of Human Subjects. All experimental methods were carried out in accordance with the approved protocols. Bone-marrow derived primary human MSC from two healthy donors were obtained from the Texas A&M Health Science Center, Institute for Regenerative Medicine (Temple, TX). Informed consent was obtained from all subjects. MSC at passage 3–5 were cultured in either serum-containing or serum-free medium. The serum-containing medium comprised αMEM (Life Technologies, Carlsbad, CA) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA), 1% L-Glutamine (Life Technologies, Carlsbad, CA) and 1% Penicillin-Streptomycin (Life Technologies, Carlsbad, CA). The serum-free medium consisted of STEMPRO® MSC SFM (Life Technologies, Carlsbad, CA) supplemented with 1% Penicillin-Streptomycin and 1% L-Glutamine. The mouse leukemic monocyte macrophage cell line RAW264.7 (ATCC, Manassas, VA) was used to verify the immunosuppressive effect of MSC14 (link). RAW264.7 was cultured in αMEM supplemented with 10% FBS, 1% L-Glutamine and 1% Penicillin-Streptomycin.
10
Optimizing Serum-free Media for Cell Culture
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In this study, we tested two different media: serum-free medium 1 (SFM 1) StemPro MSC SFM (Life Technologies, Carlsbad, CA, USA) (used according to the producer’s protocol), and serum-free medium 2 (SFM 2), composed according to the following protocol [21 (link)]. It contains DMEM/F12, 0.25% recombinant human albumin (Sigma-Aldrich, St. Louis, MO, USA), 1% chemically defined lipid concentrate (CDLC) (Life Technologies, Carlsbad, CA, USA), 1 x insulin-transferrin-selenium (Life Technologies, Carlsbad, CA, USA), 10 ng/mL platelet-derived growth factor-AB (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL basic fibroblast growth factor (Life Technologies, Carlsbad, CA, USA), 5 ng/mL transforming growth factor-b1 (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 1% Glutamax (Sigma-Aldrich, St. Louis, MO, USA), 1% antibiotic–antimycotic mixture and 0.1% fibronectin from human plasma (Sigma-Aldrich, St. Louis, MO, USA) for the cell dish covering. The cells were subcultured with CTS TrypLE Select Enzyme (Life Technologies, Carlsbad, CA, USA).
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