Human iPSCs were routinely cultured in mTeSR1 on Matrigel (growth factor reduced) (BD Biosciences, Bedford, MA, USA)-coated plates for the TKDA3-4 iPSC clone and in StemFit (AK03N, Ajinomoto, Tokyo, Japan) on Laminin-511 E8 (Nippi, Tokyo, Japan)-coated dishes for the clinical xeno-free iPSC clones including D2, 1383D6, 1231A3, and Ff06. To generate definitive endoderm cells, we plated monolayers of pluripotent cells harvested using Accutase (Millipore, Billerica, MA, USA) on 6-well plates (Corning Costar #3516) (Corning, NY, USA) pre-coated with 1:30 diluted Matrigel (growth factor reduced; BD Bioscience) or 0.5 μg/cm2 Laminin-511 E8 (Nippi) at a density of 6 × 105 cells per well with 100 ng/mL activin A (R&D Systems, Minneapolis, MN, USA), 50 ng/mL Wnt3a, and 10 μM Rock inhibitor Y27632 in RPMI/B27 (Invitrogen, Carlsbad, CA, USA) medium for 1 day. Differentiation was initiated by culture with 100 ng/mL activin A and 50 ng/mL Wnt3a in RPMI/B27 medium for 1 day under 5% CO2, followed by 2 days with 100 ng/mL activin A, 0.5 ng/mL BMP4 (Peprotech, Rocky Hill, NJ, USA), 5 ng/mL FGF2 (Invitrogen), and 10 ng/mL VEGF (Gibco, Carlsbad, CA, USA) in RPMI/B27 under 5% CO2, then followed by another 2 days with 100 ng/mL activin A, 0.5 ng/mL BMP4 (Peprotech), 5 ng/mL FGF2 (Invitrogen), and 10 ng/mL VEGF (Gibco) in SFD-based medium under 5% CO2.
Rpmi b27
Manufactured by Thermo Fisher Scientific
Sourced in United States
RPMI/B27 is a cell culture medium that supports the growth and maintenance of various cell types, including neurons, stem cells, and other sensitive cell lines. It is a combination of the RPMI 1640 medium and the B27 supplement, which together provide a balanced formulation of essential nutrients, growth factors, and other components required for optimal cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Selleck Chemicals (6 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (6 mentions)
Matrigel, by BD (5 mentions)
Activin a, by R&D Systems (4 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (4 mentions)
Matrigel, by Corning (3 mentions)
Matrigel, by Thermo Fisher Scientific (3 mentions)
Mtesr1 media, by STEMCELL (3 mentions)
Recombinant human activin a, by R&D Systems (3 mentions)
Rpmi b27, by R&D Systems (3 mentions)
Accutase, by Merck Group (3 mentions)
Recombinant human hgf, by Thermo Fisher Scientific (3 mentions)
Iwr 1, by Selleck Chemicals (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions)
Matrigel coated plate, by BD (2 mentions)
Chir99021, by Bio-Techne (2 mentions)
Rock inhibitor y 27632, by Bio-Techne (2 mentions)
Oncostatin m, by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (2 mentions)
30 protocols using rpmi b27
1
Definitive Endoderm Differentiation from iPSCs
2
Cardiac Differentiation of iPSCs
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iPSCs were cultured in human embryonic stem cell medium (20% knockout serum replacement in DMEM/F12 medium supplemented with 10 ng/ml basic fibroblast growth factor, 1% nonessential amino acid [vol/vol], 1% penicillin/streptomycin [vol/vol], 0.1 mM β-mercaptoethanol [Sigma], and 2 mM L-glutamine; Thermo Fisher Scientific). 70–80%-confluent (4–5-d-old) iPSCs cultured on MEFs were treated with 1 mg/ml dispase (Thermo Fisher Scientific) for 7–9 min. The iPSCs were mechanically dissociated using a 5-ml pipette, collected in a 15-ml Falcon tube, and spun down at 200 rpm for 3 min to deplete the MEF cells. The iPSCs were then plated on a Matrigel (1:60 dilution; Corning)–coated well for culture for 3–4 d in mTeSR medium (05850; STEMCELL Technologies) until ∼90% confluency and treated with 20 μM CHIR99021 (Selleckchem) on day 0 for 24 h and 5 µM IWP4 (Stemgent) on day 3 in RPMI/B27 (Gibco) minus insulin media for 48 h. During the cardiac differentiation, we changed the media every other day with RPMI/B27 minus insulin media. After beating, CMs were cultured in regular RPMI/B27 (including insulin) media. CMs at day 14 were treated with 4 mM lactate (Sigma) in glucose-free medium for 4 d to get enriched CMs.
3
Cardiomyocyte Differentiation from hiPSCs
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The Gi(I/M)Wi differentiation protocol was conducted as described in the Results section with the reagents listed in Supplemental Table 1 . hiPSCs were seeded into each well of a Matrigel-coated 6-well plate (0.2 million/well). When monolayers of hiPSCs are 85–90% confluent, cardiac differentiation were performed. For the cell-suspension system, the hiPSCs were dissociated, suspended in mTeSR1 medium supplemented with 5 μM Y-27632, transferred into a low-binding 24-well plate (MBL International; NCP-LH24-2) (1 × 106 cells/mL, 1 mL/well), and maintained in a horizontal shaker for 24 hr. Aggregates of cells were typically observed after 24 hours of shaking, and the medium was replaced with mTeSR1 only for 48 hr before differentiation was initiated.
CHIR99021 (Stemcell technologies; 72054) and IWR1 (Stemcell technologies; 72562) were added at the indicated concentrations and time points in RPMI1640/B27 without insulin (Fisher Scientific; 11-875-119 and A1895601). After the 48 hr IWR1 treatment period, the medium was replaced with RPMI/B27 minus insulin for 48 hours (i.e., from Day 5 to Day 7); then, the cells were maintained in RPMI/B27 with insulin (Fisher Scientific; 11-875-119 and 17-504-044) with medium changes every 2-3 days. Beating cardiomyocytes were typically observed 7–10 days after differentiation was initiated.
CHIR99021 (Stemcell technologies; 72054) and IWR1 (Stemcell technologies; 72562) were added at the indicated concentrations and time points in RPMI1640/B27 without insulin (Fisher Scientific; 11-875-119 and A1895601). After the 48 hr IWR1 treatment period, the medium was replaced with RPMI/B27 minus insulin for 48 hours (i.e., from Day 5 to Day 7); then, the cells were maintained in RPMI/B27 with insulin (Fisher Scientific; 11-875-119 and 17-504-044) with medium changes every 2-3 days. Beating cardiomyocytes were typically observed 7–10 days after differentiation was initiated.
4
Differentiation of hiPSCs to Hepatocytes
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hiPSC lines were passaged with Accutase (Innovative Cell Technologies, San Diego, CA) for 5 minutes at room temperature (RT) and plated for differentiation on Geltrex coated dishes following Stephen Duncan’s protocol with modifications.(5 ) For efficient DE differentiation, the DE kit (STEMCELL Technologies) was used for the first 4 days in ambient O2/5% CO2. Later, cells were cultured in RPMI‐B27 (with insulin) supplemented with BMP4 (20 ng/mL; PeproTech, Rocky Hill, NJ) and FGF2 (10 ng/mL; R&D Systems) for 5 days in 5% O2/5% CO2, followed by RPMI‐B‐27 (with insulin) supplemented with recombinant human HGF (20 ng/mL; PeproTech) for 5 days in 5% O2/5% CO2. The last stage required culturing cells in HCM Hepatocyte Culture Medium (Lonza Group AG, Basel, Switzerland) that was supplemented with recombinant human Oncostatin M (20 ng/mL; R&D Systems) for 5 days in ambient O2/5% CO2.(9 )
5
Directed Differentiation of hiPSCs to Hepatocyte-like Organoids
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hiPSC lines were passaged with Accutase for 5 minutes at RT and plated in 96‐well U‐bottom low adherent plates using the DE kit with 10 µM Rock inhibitor and continued in ambient O2/5% CO2 throughout the differentiation. Approximately 3,000‐5,000 cells per 100 µL per 96‐well plate gave efficient aggregation to organoids with no to minimal evidence of central darkening suggestive of cell death. However, standardization from line to line is necessary to make sure the organoid size does not become limiting, in which case the potential for central cell death is higher. After 24 hours, half the medium was removed, and fresh medium with 4% matrigel and CultureX was added and cultured for 2 more days with daily medium addition. At day 5, aggregates were harvested and cultured in low‐adherent 6‐well plates in RPMI‐B27 (with insulin) supplemented with BMP4 (20 ng/mL), FGF2 (10 ng/mL), and KGF (20 ng/mL; PeproTech) for 5 days, followed by RPMI‐B‐27 (with insulin) supplemented with recombinant human HGF (20 ng/mL; PeproTech) and 0.5% DMSO for 5 days. Later, aggregates were cultured in HCM Hepatocyte Culture Medium and supplemented with recombinant human Oncostatin M (20 ng/mL) and Dexamethasone for 5 days.
6
Directed Cardiac Differentiation of hiPSCs
7
Differentiation and Maintenance of hiPSC-Cardiac Fibroblasts
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Mouse embryonic fibroblast cell line (NIH3T3), purchased from the Bioresource Collection and Research Center, Taiwan, was cultured in Dulbecco's Modified Eagle Medium, high glucose (Gibco) supplemented with 10% bovine serum (Gibco) and 1x penicillin/streptomycin (Corning). Differentiation and maintenance of human iPSC-Cardiac fibroblasts (hiPSC-CFs) were carried out as described in Zhang et al 32 (link). In brief, human iPSCs were dissociated and seeded on Matrigel (GFR, BD Biosciences) coated 6-well plates in mTeSR1 medium supplemented ROCK inhibitor (Y-27632) (Tocris). Cells were cultured for 5-6 days in mTeSR1 medium with medium changes daily until they reached 100% confluence when differentiation started. The medium was then changed to RPMI+B27 (Gibco) without insulin and supplemented with CHIR99021 (Tocris) for 24 h, followed with CFBM medium supplemented with bFGF until day 20 when they were used for flow cytometry analysis and passaged. The hiPSC-CFs were fed every other day with the FibroGRO+2% FBS medium and passaged every 4-6 days using 0.05% Trypsin-EDTA. For the hypoxia experiments, cells were incubated in a hypoxia chamber supplemented with 1% O2 and 5% CO2 for 48 h.
8
Differentiation of hiPSC-Derived Cardiomyocytes
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hiPSCs were reprogrammed from human cardiac fibroblasts and maintained under optimized conditions (Zhang et al., 2014 (link); Zhu et al., 2017 (link)) in mTeSR Plus (STEMCell) maintenance media on 6-well plates coated with Geltrex (Gibco). hiPSC-CM differentiation was conducted as previously reported (Lian et al., 2013 (link); Burridge et al., 2014 (link); Ye et al., 2014 (link)). Briefly, when iPSCs reached 80-100% confluency, referred to as day 0, media was changed to 2.5 mL RPMI 1640 with B27 without insulin (RPMI/B27-) (Gibco) supplemented with 10 μM CHIR99021. Subsequently, on days 1, 3, and 5 media was changed to 3 mL RPMI/B27- and supplemented with 5 μM IWR on day 3. Media was changed to RPMI 1640 with B27 with insulin (RPMI/B27+) on day 7. Spontaneous contractions were typically observed 7 to 10 days after differentiation was initiated, and the number of beating cells continued to increase until day 12. The differentiated hiPSC-CMs were purified by culturing them in the absence of glucose (RPMI 1640 without glucose, supplemented with sodium DL-lactate and B27+, Gibco) for 3-6 days beginning on day 9; at least 95% of the purified cells expressed cardiac troponin T (cTnT) as shown in Figure S1 . Cells were maintained in RPMI/B27+ until used for fabrication and dissociated using STEMdiff™ Cardiomyocyte Dissociation media as needed.
9
Directed Differentiation of iPSCs into iCMs
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iPSCs (Coriell Institute for Medical Research, AICS-0048-039) were cultured in mTeSR1media (STEMCELL Technologies, 85850) on Matrigel (Gibco, A1413302)-coated plates. At 80% of confluency, iPSCs were differentiated into iCMs as previously described 26 (link). Briefly, the iPSCs were treated with 8μM CHIR-99021 (SelleckChem, S2924) in RPMI (Gibco, 11875093)-B27(no insulin) (Gibco, A1895601) from day 0–1. Media was changed on day 2 and the cells were treated with 5 μM IWR1 (SelleckChem, S7086) in RPMI-B27(no insulin) from day 3–4. Starting from day 7, RPMI-B27(with insulin) (Gibco, 17504044) media were given to iPSC-derived cardiomyocytes (iCMs). Two rounds of glucose starvation from day 12–15 and from day 20–23 were performed to eliminate non-cardiomyocyte cells. At day 30, iCMs were transferred to Matrigel-coated 24-well plates and maintained for further use. For co-culture of iCM and HCF, iCM cells were seeded at 2.5×104 cells per well in a Matrigel-coated 24-well plate on day 1, and HCF cells were seeded at 1.5×104 cells per well on day 2. Cells were cultured in RPMI-B27 (with insulin). Co-culture was used on day 3 for experiments and analysis.
10
Cardiomyocyte Differentiation and Purification
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As described previously [42 (link)], we differentiated ventricular cardiomyocytes (CMs) from human induced pluripotent stem cells (WTC human male iPSCs, Gladstone Institutes) in cardiac differentiation media 3 (CDM3) by modulating the Wnt signaling pathway. Briefly, high-density hiPSC monolayer cultures (80–90%) were treated with a Wnt activator that inhibits glycogen synthase kinase 3 (4–5 μM Chiron 99021, Tocris) for 24 ± 1 hour, followed by a chemical Wnt inhibitor (5 μM IWP2, Tocris) at day 3. Differentiated cardiomyocytes began to beat between days 8 and 12, and once beating, were cultured in RPMI 1640 medium with B27 supplement (RPMI+B27; Gibco). Cardiomyocytes were then harvested and replated onto plates coated with Matrigel (Corning) for metabolic-based lactate purification [43 (link)]. Leveraging their unique ability to use lactate as an energy source, CMs were cultured in 4 mM sodium L-lactate (Sigma) in sodium pyruvate- and glucose-free DMEM for 4 days, effectively reducing non-cardiomyocyte population to improve cardiomyocyte purity.
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