B27 insulin

Manufactured by Thermo Fisher Scientific

Sourced in United States

B27-Insulin is a supplement for cell culture media that supports the growth and differentiation of neural cells. It contains insulin, a critical component for neuronal development and survival. The product is designed to provide a defined, animal-component free supplement for primary neuron cultures and neural stem/progenitor cell cultures.

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Lab products found in correlation

M 199, by Sartorius (2 mentions) Roswell park memorial institute (rpmi), by Bio-Techne (2 mentions) B27 insulin, by Bio-Techne (2 mentions) Roswell park memorial institute (rpmi), by Sartorius (2 mentions) L glutamine, by Bio-Techne (2 mentions) Chir99021, by Bio-Techne (2 mentions) Vitamin b12, by Merck Group (2 mentions) L glutamine, by Sartorius (2 mentions) Fetal bovine serum (fbs), by Sartorius (2 mentions) Activin a, by Thermo Fisher Scientific (2 mentions) Extracellular matrix gel, by Merck Group (1 mentions) Penicillin, by Avantor (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) N2 supplement, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Matrigel basement membrane matrix, by BD (1 mentions) Nutristem, by Sartorius (1 mentions) Accutase, by STEMCELL (1 mentions)

10 protocols using b27 insulin

Cardiomyocyte Differentiation of hiPSCs

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The
Ethics Committee of Pirkanmaa Hospital District gave approval to conduct
research on hiPSC lines (Aalto-Setälä R08070). The hiPSC
line UTA.04602.WT was cultured and characterized at the stem cell
state, as previously described.^{40 (link)} The cardiomyocyte
differentiation was done by modulating Wnt signaling with small molecules,
according to the protocol published by Lian et al. 2012.^{41 (link)} In short, differentiation was initiated by plating
700 000 hiPSCs/well in a Nunc 12-multiwell plate (Thermo Fisher
Scientific, USA) in feeder-free condition on Geltrex-coating (Thermo
Fisher Scientific, USA) in mTeSR1 medium (STEMCELL Technologies, Canada)
supplemented with 50 U mL^–1 Pen/Strep for 4 days.
Ten days after initiation, the medium was changed to RPMI (Thermo
Fisher Scientific, USA) supplemented with B27(−insulin) (Thermo
Fisher Scientific, USA) and 50 U mL^–1 Pen/Strep.
During this time, on day one, 8 μM CHIR99021 (REPROCELL, United
Kingdom) was applied to the cells. After 24 h, CHIR99021 was removed.
On day3, 5 μM IWP-4 (R&D Bio-Techne, USA) was added for
48 h. From day 10 onwards, B27(−insulin) was changed to B27(+insulin)
(Thermo Fisher Scientific, USA), and the cells were cultured in this
medium until they were used for the hydrogel experiments.

Koivisto J.T., Gering C., Karvinen J., Maria Cherian R., Belay B., Hyttinen J., Aalto-Setälä K., Kellomäki M, & Parraga J. (2019). Mechanically Biomimetic Gelatin–Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes. ACS Applied Materials & Interfaces, 11(23), 20589-20602.

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Cardiac Differentiation of hPSCs

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When hPSCs maintained on a Matrigel-coated surface achieved confluence, cells were singularized with 0.5 mM EDTA in DPBS at 37 °C for 5 min and then seeded onto a Matrigel-coated cell culture dish at 100,000 cells/cm² in LaSR supplemented with 5 μM Y-27632 (Selleckchem) (day -2) for 24 hours. Cells were then cultured in LaSR without Y-27632, changed daily. Cardiac differentiation of hPSCs was performed according to previously published GiWi method^{5 (link), 6 (link), 22 (link)}. Briefly, at day 0, cells were treated with 9 µM CHIR99021 (Selleckchem) for 24 hour in RPMI medium supplemented with 2% B27-insulin (Gibco), followed by a change with RPMI + B27-insulin medium at day 1. At day 3, 2 µM Wnt-C59 (Tocris) was added, followed by a medium change with RPMI plus 2% B27 (Gibco) at day 5. Cells were then cultured in RPMI plus 2% B27 with medium change every three days.

Randolph L.N., Bao X., Zhou C, & Lian X. (2017). An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives. Scientific Reports, 7, 1549.

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Directed Differentiation of iHIOs from hESCs

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Matrigel basement membrane matrix (BD Biosciences, cat. 356234), and extracellular matrix gel (Sigma, cat. E1270) were used to embed the iHIOs in order to support development of 3-dimensional architecture. Gut media for iHIO culture was prepared using advanced Dulbecco's Modified Eagle Medium/Ham's F-12 (DMEM/F12) (Gibco, Invitrogen, cat. 12634–028) supplemented with B27 insulin (Invitrogen, cat. 17504044), N2 supplement (Invitrogen, cat. 17502048), 2 mM L-glutamine (Fisher, cat. SH3003401), 15 mM HEPES (Invitrogen, cat. 15630080), 100 ng ml^-1epidermal growth factor (R&D Systems, cat. 236-EG-200), and either 2 mM penicillin/streptomycin (Invitrogen, cat. 15140–122) or penicillin alone (Amresco, cat. E480-20ML). iHIOs were maintained in tissue culture treated Nucleon delta 4-well (Nunc, cat. 176740) and 2-well (Lab-Tek, cat. 155380) dishes.
iHIOs prepared by directed differentiation of H1 human embryonic stem cell line (NIH registry number 0043) [12 (link)] were obtained from Pluripotent Stem Cell Facility and Organoid Core at Cincinnati Children’s Hospital and Medical Center. iHIOs were maintained in reconstituted gut media, which was changed twice weekly.

Karve S.S., Pradhan S., Ward D.V, & Weiss A.A. (2017). Intestinal organoids model human responses to infection by commensal and Shiga toxin producing Escherichia coli. PLoS ONE, 12(6), e0178966.

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Cardiomyocyte Differentiation Protocol

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Prior to differentiation, cells were dissociated with Accutase™ (StemCell Technologies, Vancouver, BC, Canada) and passaged to six-well plates. Until 100% confluence of iPSCs, NutriStem™ (Biological Industries) was refreshed. On day 0, the medium was changed to 3 mL of RPMI (Biological Industries), supplemented with 0.5% L-glutamine (Biological Industries), B27-Insulin (Invitrogen, Carlsbad, CA, USA) and 4.5 µM CHIR-99021 (Tocris, Bristol, UK). On day 2, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine, B27-Insulin, and 5 µM IWP-2 (Tocris). On day 4, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27-Insulin. This medium was refreshed on day 6. On day 8, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27, and this medium was refreshed on day 10. From day 12, the medium was changed to M-199 (Biological Industries), supplemented with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological Industries), 0.6 mM CuSO₄·5 H₂O, 0.5 mM ZnSO₄·7 H₂O, and 1.5 mM Vitamin B12 (Sigma-Aldrich, Darmstadt, Germany). This medium was refreshed every other day.

Shilo M., Baruch E.S., Wertheim L., Oved H., Shapira A, & Dvir T. (2023). Imageable AuNP-ECM Hydrogel Tissue Implants for Regenerative Medicine. Pharmaceutics, 15(4), 1298.

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Cardiac Differentiation of Induced Pluripotent Stem Cells

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Prior to differentiation,
cells were passed to 6-well plates. NutriStem was refreshed daily
until iPSCs reached 100% confluence. At that point (Day 0), the medium
was changed to 3 mL of RPMI (Biological Industries), supplemented
with 0.5% l-glutamine (Biological Industries), B27-Insulin
(Invitrogen, Carlsbad, California), and 4.5 μM CHIR-99021 (Tocris,
Bristol, UK). On Day 2, the medium was changed to 3 mL of RPMI supplemented
with 0.5% l-glutamine, B27-Insulin, and 5 μM IWP-2
(Tocris). On Day 4, the medium was changed to 3 mL of RPMI supplemented
with 0.5% l-glutamine and B27-Insulin, and this medium was
refreshed on Day 6. On Days 8 and 10, the medium was changed to 3
mL of RPMI supplemented with 0.5% l-glutamine and B27. From
Day 12, the medium was changed to M-199 (Biological Industries), supplemented
with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological
Industries), 0.6 mM CuSO₄·5H₂O, 0.5 mM
ZnSO₄·7H₂O, and 1.5 mM Vitamin B12 (Sigma-Aldrich).
This medium was refreshed every other day.^{74 (link)}

Omar R., Saliba W., Khatib M., Zheng Y., Pieters C., Oved H., Silberman E., Zohar O., Hu Z., Kloper V., Broza Y.Y., Dvir T., Grinberg Dana A., Wang Y, & Haick H. (2024). Biodegradable, Biocompatible, and Implantable Multifunctional Sensing Platform for Cardiac Monitoring. ACS Sensors, 9(1), 126-138.

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Directed Differentiation of hiPSCs into Cardiomyocytes

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Cardiomyocyte differentiation was induced as previously reported, with a few modifications [11] (link). hiPSCs were detached by incubation with 0.5 mM EDTA in PBS for 7 min and seeded onto cell culture plates coated with laminin-521 or Matrigel at a density of 50,000 cells/cm² in Essential 8 or mTeSR1, respectively, for 3–4 days before cardiomyocyte induction. Confluent hiPSCs were treated with 12 μM CHIR99021 (Stemgent), a GSK3 inhibitor, in RPMI1640 medium (Sigma–Aldrich) supplemented with B27-insulin (Invitrogen) (RPMI/B27-insulin) for 24 h. At day 1, the medium was changed to RPMI/B27-insulin. At day 3, cells were treated with 5 μM IWP4 (Stemgent), a Wnt inhibitor, in RPMI/B27-insulin for 48 h. At day 5, the medium was changed to RPMI/B27-insulin. The cells were transferred to RPMI/B27 without insulin at day 7, and the medium was changed every 3 days. Differentiated cells were harvested at the indicated time for further analysis.

Kuroda T., Yasuda S., Matsuyama S., Tano K., Kusakawa S., Sawa Y., Kawamata S, & Sato Y. (2015). Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells. Regenerative Therapy, 2, 17-23.

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Pancreatic Differentiation Protocol with Activin A and Dorsomorphin

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For pancreatic differentiation, we followed Yuya Kunisada’s protocol with slight modification^{19 (link)}. Briefly stem cells were passaged on Matrigel-coated 24-well plates at a density of 10 × 10⁴ cells per well. Stem cells were cultured with mTeSR1 including Y27632 for 24 h and then 3 days with mTeSR1 to nearly reach confluence. For differentiation, the cells were cultured in RPMI1640 (Gibco) containing B27 insulin (Gibco), 100 ng/mL activin A (Pepro Tech) and 3 μM CHIR99021 (Stemgent) for 24 h and for 48 h in RPMI 1640 containing B27 minus insulin and 100 ng/mL activin A. Subsequently, the media was changed with DMEM/F12 containing B27 (Gibco), 1 μM dorsomorphin (Calbiochem), 10 μM SB431542 (Sigma) and 2 μM retinoic acid (Sigma) for 7 days and medium was replaced every other day. For insulin-producing cell differentiation, the medium was changed to DMEM/F12 containing B27, 10 μM forskolin (Stemgent), 10 μM dexamethasone (Enzo Life Sciences), 5 μM Alk5 inhibitor II (Calbiochem) and 10 mM nicotinamide (Stemcell) for 12 days and provided with fresh medium every other day.

Guo D., Liu H., Ruzi A., Gao G., Nasir A., Liu Y., Yang F., Wu F., Xu G, & Li Y.X. (2017). Modeling Congenital Hyperinsulinism with ABCC8-Deficient Human Embryonic Stem Cells Generated by CRISPR/Cas9. Scientific Reports, 7, 3156.

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Cardiomyocyte Maturation and Selection

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To select and maturate cardiomyocytes, the differentiated cells were cultured for 10 days under one of two conditions: RPMI1640 without glucose (Life Technologies, U.S.A) supplemented with 4 mM sodium DL-lactate (Sigma-Aldrich, U.S.A) and B27-insulin (GIBCO, U.S.A); or RPMI1640 without glucose (Life Technologies, U.S.A) supplemented with 5 mM sodium DL-lactate (Sigma-Aldrich, U.S.A), 500 µg/mL human albumin (Sigma-Aldrich, U.S.A), and 211 µg/mL L-ascorbic acid (Sigma-Aldrich, U.S.A). The medium was replaced every other day.

Kim J.E., Kim E.M., Lee H.A, & Kim K.S. (2023). Effective derivation of ventricular cardiomyocytes from hPSCs using ascorbic acid-containing maturation medium. Animal Cells and Systems, 27(1), 82-92.

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Sphere Formation Assay of Sorted Cells

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MIX- and MIX+ sorted-cells were seeded in ultra-low attachment 96-wells plates (Falcon Corning brand, France) in increasing cell densities, i.e., 600, 1250, 2500, 5000, 10000 cells. For each condition, cells were seeded in 3 wells in 200µL of defined medium composed of 1X Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (HAM 1:1) medium (Gibco - ThermoFisher Scientific, France) supplemented in 1X B27/Insulin (ThermoFisher Scientific, France), 10ng/mL Fibroblast Growth Factor (FGF; Peprotech, France) and 20ng/mL Epidermal Growth Factor (EGF; Peprotech, France). Twenty microliters of medium were added per well every week, for 4 weeks. The number of spheres formed per well and per condition was counted under optical microscope (Olympus CKX53; magnification, x100).

Blondy S., Durand S., Lacroix A., Christou N., Bouchaud C., Peyny M., Battu S., Chauvanel A., Carré V., Jauberteau M.O., Lalloué F, & Mathonnet M. (2022). Detection of Glycosylated Markers From Cancer Stem Cells With ColoSTEM Dx Kit for Earlier Prediction of Colon Cancer Aggressiveness. Frontiers in Oncology, 12, 918702.

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Feeder-Free iPSC Differentiation into Cardiomyocytes

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WT iPSCs were provided by Dr. Gordon Tomaselli (Albert Einstein College of Medicine) [24 ] , and LQT8 iPSCs were a generous gift from Dr. Masayuki Yazawa (Columbia Stem Cell Institute) [25 (link)]. IPSCs were cultured and differentiated in a feeder-free system using a previously described protocol [26 (link)]. IPSCs were cultured using Essential 8 medium (ThermoFisher), on Geltrex (ThermoFisher) coated tissue culture plates. When cells reached confluency, differentiation into cardiomyocytes was initiated. E8 media was exchanged with RPMI-1640 (Sigma-Aldrich) supplemented with B27 -insulin (ThermoFisher). 6 μM CHIR99021 (Sigma) was added on day 0, and 5 μM IWR-1 was added on day 3. Media was exchanged every 2 days throughout the differentiation process. Media was changed to RPMI-1640 +insulin (ThermoFisher) after cells demonstrated spontaneous contraction, and lactate was added on day 13 to select for healthy ventricular myocytes. 25-30 day-old cells were replated onto coverslips coated in Geltrex at a low density to facilitate recording from single cells. WT and LQT8 iPSCs were age-matched and recordings were performed on cells between 28-35 days post differentiation.

Bamgboye M.A., Traficante M.K., Owoyemi J., DiSilvestre D., Vieira D.C, & Dick I.E. (2022). Impaired CaV1.2 inactivation reduces the efficacy of calcium channel blockers in the treatment of LQT8. Journal of molecular and cellular cardiology, 173, 92-100.

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