Rpmi b27 without insulin

Manufactured by Thermo Fisher Scientific

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RPMI/B27 without insulin is a cell culture medium supplement designed to support the growth and maintenance of various cell types, including neurons, stem cells, and other sensitive cell lines. It provides a defined and serum-free environment to cultivate cells while promoting their survival and differentiation.

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Lab products found in correlation

Chir99021, by Bio-Techne (5 mentions) Rock inhibitor y 27632, by Bio-Techne (5 mentions) Rpmi b27 without insulin, by Bio-Techne (4 mentions) Versene solution, by Thermo Fisher Scientific (3 mentions) Basic fibroblast growth factor (bfgf), by WiCell (3 mentions) Gfr matrigel, by BD (3 mentions) Tesr e8 medium, by STEMCELL (2 mentions) Matrigel, by BD (2 mentions) Activin a, by R&D Systems (2 mentions) Cd144 conjugated magnetic microbeads, by Miltenyi Biotec (2 mentions) Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions) Egm 2, by Lonza (2 mentions) Chir99021, by Selleck Chemicals (2 mentions) Matrigel, by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by Lonza (1 mentions) Geltrex, by Thermo Fisher Scientific (1 mentions) Rpmi b27, by R&D Systems (1 mentions) Oncostatin m, by R&D Systems (1 mentions) Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions) Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)

Close spelling variants of this product

Insulin (904 citations) RPMI/B27 (30 citations) B27 without insulin (17 citations) B27-Insulin (10 citations) RPMI/B27-insulin (9 citations)

13 protocols using rpmi b27 without insulin

Cardiac Differentiation of hiPSCs

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The primary hiPSC line used in this study was DF 19-9-11 (derived from neonatal skin fibroblast) and was maintained on feeder-free Matrigel (BD Biosciences) in mTeSR1 or TeSR-E8 medium (STEMCELL Technologies) (Figure S1A). For cardiac differentiation via small molecules, as adapted from Lian et al. (2012) (link), hiPSCs were maintained on Matrigel plates for 4 days or until they reached confluence. Cells were treated with 10 μM CHIR99021 (GSK-3 inhibitor, Selleck Bio) along with Matrigel in RPMI/B27 without insulin (Invitrogen) for 24 hr (day 0 to day 1). The next day, medium was changed to RPMI/B27 without insulin plus inhibitor of Wnt protein 4 (IWP4) at 5 μM (Stemgent) and removed during the medium change on day 2. Cells were maintained in the RPMI/B27 plus insulin starting from day 7, with media changes every 1–2 days (Figures S1A and S1B). The use of RPMI and B27+ insulin cocktail culture condition has the advantage of preventing fibroblast overgrowth compared to use of FBS. Regular media changes were performed to limit pH alterations.

Bedada F.B., Chan S.S., Metzger S.K., Zhang L., Zhang J., Garry D.J., Kamp T.J., Kyba M, & Metzger J.M. (2014). Acquisition of a Quantitative, Stoichiometrically Conserved Ratiometric Marker of Maturation Status in Stem Cell-Derived Cardiac Myocytes. Stem Cell Reports, 3(4), 594-605.

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Cardiac Fibroblasts Differentiation from iPSCs

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Human cardiac fibroblasts were differentiated from a human‐induced pluripotent stem cell (iPSC) line iPS‐DF19‐9‐11T (male) (WiCell) as described previously (Zhang et al., 2019 (link)). Briefly, iPSCs were dissociated with 1 ml/well Versene solution (Invitrogen) at 37℃ for 5 min, and seeded on Matrigel (GFR, BD Biosciences) ‐coated six‐well plates at the density of 2 × 10⁶ cells/well in mTeSR1 medium supplemented with 10 μM ROCK inhibitor (Y‐27632) (Tocris). Cells were cultured for 5 days in mTeSR1 medium with medium change daily until 100% confluence was reached and differentiation was started (day 0). At Day 0, the medium was changed to 2.5 ml RPMI+B27 without insulin and supplemented with 12 µM CHIR99021 (Tocris) and cells were treated in this medium for 24 h (day 1). At Day 1 medium was changed to 2.5 ml RPMI+B27 without insulin (Invitrogen) and cells were cultured in this medium for another day (day 2). At Day 2, the medium was changed to 2.5 ml of the defined fibroblast culture medium (CFBM) (Zhang et al., 2019 (link)) supplemented with 75 ng/ml bFGF (WiCell). Cells were fed every other day with CFBM supplemented with 75 ng/ml bFGF and cultured until day 20 for flow cytometry analysis and subculture of cardiac fibroblasts.

Napiwocki B.N., Stempien A., Lang D., Kruepke R.A., Kim G., Zhang J., Eckhardt L.L., Glukhov A.V., Kamp T.J, & Crone W.C. (2021). Micropattern platform promotes extracellular matrix remodeling by human PSC‐derived cardiac fibroblasts and enhances contractility of co‐cultured cardiomyocytes. Physiological Reports, 9(19), e15045.

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Differentiation of hPSCs into Hepatocytes

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hPSCs were differentiated as discussed previously (Si-Tayeb et al. 2010 (link); Mallanna and Duncan 2013 ; Yanagihara et al. 2016 (link)). Briefly, hPSCs were harvested using Accutase and plated at 600,000 cells per well in 24-well plates precoated with 300 µl/well Geltrex (Thermo Fisher Scientific, Waltham, MA). Approximately 24 h after seeding the cells with mTeSR1 (Stemcell Technologies), when the cells were 85–95% confluent, differentiation was initiated by culture for 5 d with 50 ng/ml Activin A (R&D Systems) in RPMI/B27 (without insulin) supplement (Invitrogen) under ambient oxygen/5% CO₂. In addition, we included 10 ng/ml BMP4 (R&D Systems) and 20 ng/ml FGF2 (R&D Systems) for the first 2 d. Then, the cells were cultured for 5 d with 20 ng/ml BMP4 (R&D Systems)/10 ng/ml FGF-2 (R&D Systems) in RPMI/B27 (containing insulin) under 4% O₂/5% CO₂, then for 5 d with 20 ng/ml HGF (R&D Systems) in RPMI/B27 (containing insulin) under 4% O₂/5% CO₂, and finally for 5 d with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO₂.

Yanagihara K., Hayashi Y., Liu Y., Yamaguchi T., Hemmi Y., Kokunugi M., Yamada K.U., Fukumoto K., Suga M., Terada S., Nikawa H., Kawabata K, & Furue M. (2024). Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells. In Vitro Cellular & Developmental Biology. Animal, 60(5), 521-534.

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Differentiation of hESCs into Cardiomyocytes

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Undifferentiated RUES2 human embryonic stem cells (hESCs) from Rockefeller University were maintained in mouse embryonic-fibroblast conditioned media, supplemented with basic fibroblast growth factor (R&D Systems). HESCs were differentiated into cardiomyocytes with a previously described directed differentiation protocol (Figure 1(a) [9 (link)]). Briefly, CHIR99021 (Cayman Chemical), activin A, bone morphogenetic protein 4 (BMP4; R&D Systems), and tankyrase inhibitor XAV 939 (Tocris Bioscience) were applied sequentially in defined, monolayer culture conditions on Matrigel™- (BD Biosciences) coated 6-well plates [10 ]. HESC-cardiomyocytes were differentiated in RPMI 1640 + 1X B27 supplement without insulin (RPMI/B27 without insulin; Life Technologies) and maintained in RPMI/B27 (with insulin) with medium replaced every two days. After two weeks of differentiation, hESC-cardiomyocytes were harvested with 0.25% trypsin in 0.5 M EDTA (Life Technologies) and either incorporated into engineered tissues (Figures 1–5) or cryopreserved [11 (link)] for later use in single-cell experiments (Figures 6 and 7).

Rupert C.E, & Coulombe K.L. (2017). IGF1 and NRG1 Enhance Proliferation, Metabolic Maturity, and the Force-Frequency Response in hESC-Derived Engineered Cardiac Tissues. Stem Cells International, 2017, 7648409.

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Cardiac Myocyte Differentiation Protocol

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Direct CM differentiation was carried as previously described.^{5 (link),29 (link),30 (link)} Briefly, after cell expansion on Plastic MC, cells/MC aggregates were re-plated on LN521-coated plates. The cells were treated with 12 μM Gsk3 inhibitor CHIR99021 (Selleck) in RPMI/B27 without insulin (Life Technologies) for 24 h, followed by a treatment with 5 μM inhibitor of Wnt production-2 (IWP2; Stemgent) at day 3. The medium was replaced on day 5 by fresh RPMI/B27 without insulin and cells were then maintained in this medium until day 10, when they were maintained in RPMI/B27 with insulin (B27^® Supplement; Life Technologies) until day 15. The differentiated cells were then trypsinized into a single-cell suspension and fixed with 4% paraformaldehyde, followed by staining with 1:40 diluted anti-Cardiac myosin heavy chain (MF20; Developmental Studies Hybridoma Bank) and 1:200 anti-troponin 1 cardiac (cTnT; Millipore) before FACS analysis, as described earlier.

Lam A.T., Li J., Chen A.K., Birch W.R., Reuveny S, & Oh S.K. (2015). Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures. BioResearch Open Access, 4(1), 242-257.

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Differentiation and Isolation of iPSC-Derived Endothelial Cells

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Patient-specific iPSCs were generated using the OSKM CytoTune-iPS 2.0 Sendai Reprogramming Kit viral particle factors (Life Technologies) as described previously (17 (link)). The iPSCs used for this study were at passages 22–25. iPSCs were cultured as described above until reaching 80% confluence. The medium was switched to RPMI-B27 without insulin (Life Technologies) with 6 μM CHIR99021 for 2 days, and then changed to 6 μM CHIR99021 for another 2 days. During the differentiation process, from day 4 to day 12, the medium was changed to EGM2 (Lonza) supplemented with 50 ng/ml VEGF (Peprotech), 20 ng/ml BMP4, and 20 ng/ml FGF2 (Peprotech). By day 12, cells were dissociated using TyrpLE for 5 min and sorted using CD144-conjugated magnetic microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD144-positive cells were seeded on 0.2% gelatin-coated plates and maintained in EGM2 medium supplemented with 10 μM TGFβ inhibitor (SB431542) (Selleckchem). After passage 2, iPSC-ECs were cultured in normal EGM2, without SB431542. The iPSC-ECs used for this study were all at passage 3.

Sayed N., Liu C., Ameen M., Himmati F., Zhang J.Z., Khanamiri S., Moonen J.R., Wnorowski A., Cheng L., Rhee J.W., Gaddam S., Wang K.C., Sallam K., Woo Y.J., Rabinovitch M, & Wu J.C. (2020). Lovastatin improves endothelial dysfunction and cellular crosstalk in LMNA-related dilated cardiomyopathy. Science translational medicine, 12(554), eaax9276.

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Cardiac Differentiation of Human iPSCs

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Cardiac differentiation was performed as described previously (17 (link)). Briefly, colonies of iPSCs were plated onto dishes pre-coated with matrigel and cultured in mTeSR1 medium in a hypoxic incubator (4% O₂, 5% CO₂). Before starting cardiac differentiation, iPSCs were singularized with Accutase and seeded on a matrigel-coated plate at the density of 100,000 cells/cm² in mTeSR1 medium supplemented with 10 μM ROCK inhibitor Y-27632 (EMD Millipore, Billerica, MA, USA). The medium was changed daily. When the cells reached 100% confluence (the day was referred to as day 0), the medium was replaced with RPMI/B27 without insulin (Life Technologies) supplemented with 12 μM CHIR-99021 (Selleck Chemicals, Houston, TX, USA) and the cells were cultured in a normoxic incubator (20% O₂, 5% CO₂). After 24 h, the culture medium was switched to RPMI/B27 without insulin and changed daily. On day 3, 5 μM Wnt Inhibitor IWP-2 (Stemgent, Cambridge, MA, USA) was added to the medium and cells were cultured for 48 h. On day 5 the culture medium was switched to RPMI/B27 without insulin and changed daily. On day 7 the culture medium was switched to RPMI/B27 with insulin (Life Technologies) and changed every two days. Daily microscopic observations were conducted to detect the appearance of contracting cells.

Kikuchi C., Bienengraeber M., Canfield S., Koopmeiner A., Schäfer R., Bosnjak Z.J, & Bai X. (2015). Comparison of cardiomyocyte differentiation potential between type 1 diabetic donor- and non-diabetic donor-derived induced pluripotent stem cells. Cell transplantation, 24(12), 2491-2504.

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Differentiation of iPSCs into ECs

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The iPSCs were cultured as described above until reaching 80% confluence. The medium was switched to RPMI-B27 without insulin (Life Technologies) with 6 μM CHIR99021 for 2 days and then changed to 2 μM CHIR99021 for another 2 days. During differentiation, from days 4 to 12, the medium was changed to EGM2 (Lonza) supplemented with vascular endothelial growth factor (VEGF) (50 ng/ml) (PeproTech), bone morphogenetic protein 4 (BMP4) (20 ng/ml), and fibroblast growth factor 2 (FGF2) (20 ng/ml) (PeproTech). On day 12, cells were dissociated using TrypLE for 5 min and sorted using CD144-conjugated magnetic microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD144-positive cells were seeded on 0.2% gelatin-coated plates and maintained in EGM2 medium supplemented with 10 μM transforming growth factor β (TGFβ) inhibitor (SB431542). (Selleckchem). After passage 2, iPSC-ECs were cultured in EGM2. The iPSC-ECs were analyzed at passage 3 post differentiation.

Wallner M., Meera P, & Toro L. (1999). Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: A transmembrane β-subunit homolog. Proceedings of the National Academy of Sciences of the United States of America, 96(7), 4137-4142.

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Cardiac Differentiation of Human iPSCs

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Cells were dissociated by Versene (Life technologies), then incubate the plate at 37°C, 5% CO2 and wait for 4 minutes. Aspirate the supernatant, resuspend the cells in mTeSR1 + 5 μM Y27632 and seeded onto Geltrex-coated plates at a density of 3 × 105 cells/cm2. Add mTeSR1 + 5 μM Y27632 medium to each well to make a final volume of 2 ml in each well of the 12-well plate. This time point corresponds to day −4. On day −3, day −2, and day −1, aspirate the medium and replace with 2 ml room temperature mTeSR1 per well of the 12-well plate. At on day 0, the cells were treated with 6 μM CHIR99021 (Selleckchem) in insulin-free RPMI/B27 without-insulin medium (Life Technologies) for 24 hours. The medium was replaced with basal medium for another 2 days. At on day 3, the culture medium was subsequently replaced with 5 μM IWP2 (Tocris) in insulin-free RPMI/B27 without-insulin for 48 hours. At On day 7, the culture medium was changed to RPMI/B27 with containing insulin (Life Technologies), and the culture medium was refreshed thereafter every 3 days.

Chien Y., Chien C.S., Chiang H.C., Huang W.L., Chou S.J., Chang W.C., Chang Y.L., Leu H.B., Chen K.H., Wang K.L., Lai Y.H., Liu Y.Y., Lu K.H., Li H.Y., Sung Y.J., Jong Y.J., Chen Y.J., Chen C.H, & Yu W.C. (2016). Interleukin-18 deteriorates Fabry cardiomyopathy and contributes to the development of left ventricular hypertrophy in Fabry patients with GLA IVS4+919 G>A mutation. Oncotarget, 7(52), 87161-87179.

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Generation of iPSC-derived Cardiac Fibroblasts

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For the generation of iPSC-CFs a protocol developed by Zhang et al. was used^{13 (link)}. Briefly, human iPSCs were dissociated with 1 mL/well 0.5 µM EDTA solution (Invitrogen) at RT for 5 min and seeded on Vitronectin XF (StemCell Technologies) coated 6-well plates at a density of 15.000–30.000 cells/cm² in TeSR-E8 medium (StemCell Technologies) supplemented with 5 μM ROCK inhibitor (Y-27632) (Tocris) for 24 h. Cells were cultured for 6–7 days in TeSR-E8 medium with medium changes every other day until they reached 100% confluency and differentiation started (day 0). At day 0, the medium was changed to 2.5 mL/well RPMI + B27 without insulin (Gibco) and supplemented with 12 µM CHIR99021 (Tocris) for 24 h (day 1). After day 1, the medium was changed to 2.5 mL RPMI + B27 without insulin for 24 h (day 2). Afterwards, the medium was changed to 2.5 mL/well of the CFBM medium (Table S1) supplemented with 75 ng/mL bFGF (StemCell Technologies). Cells were refreshed with 2 mL/well CFBM supplemented with 75 ng/mL bFGF every other day until day 20 when RNA was collected, and cells were dissociated using TrypLE Select (10x) (Thermo Fisher) for 10 min at 37 °C. After dissociation, cells were cultured in DMEM + 10% Fetal bovine serum. For the first two passages, 5 μM ROCK inhibitor was added for 24 h to help cell attachment. Cells between passage 3–6 were used for experiments.

Bekedam F.T., Smal R., Smit M.C., Aman J., Vonk-Noordegraaf A., Bogaard H.J., Goumans M.J., De Man F.S, & Llucià-Valldeperas A. (2024). Mechanical stimulation of induced pluripotent stem derived cardiac fibroblasts. Scientific Reports, 14, 9795.

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