Human induced pluripotent stem cells (hiPSCs) were thawed with mTeSR1 (STEMCELL, #85850) and 1 µM H1152 (Millipore, #555550), seeded on Matrigel (Corning, #354230) coated 6-well plates and subsequently adapted to further culture in StemFlex media (Gibco, #A3349401). hiPSCs at ~ 70% confluence (1.5x10 6 cells / well of a 6-well plate) were incubated in EDTA (Life Technologies #15575-020) for 4 min at room temperature (RT), the EDTA was aspirated, the cells dissociated in fresh StemFlex media and seeded onto Matrigel-coated plates at 3-5x10 5 cells/well. Media was replaced every other day for 4 days until the next passage. For freezing and shipping to partners, cells were dissociated with EDTA and subsequently frozen in Stemflex medium with 10% DMSO (Sigma, # D2650) at -80°C and transferred to liquid nitrogen for long-term storage. All hiPSCs were tested and are mycoplasma free.
Stemflex media
Manufactured by Thermo Fisher Scientific
Sourced in United States
StemFlex media is a cell culture medium designed for the growth and maintenance of human pluripotent stem cells. It provides a defined, feeder-free, and serum-free environment to support the self-renewal and pluripotency of stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (9 mentions)
Geltrex, by Thermo Fisher Scientific (7 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (6 mentions)
Accutase, by Thermo Fisher Scientific (4 mentions)
Versene, by Thermo Fisher Scientific (3 mentions)
Accutase, by STEMCELL (3 mentions)
Stempro accutase, by Thermo Fisher Scientific (3 mentions)
Matrigel, by Thermo Fisher Scientific (3 mentions)
Relesr, by STEMCELL (2 mentions)
Matrigel coated plate, by Corning (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Laminin, by BioLamina (2 mentions)
Rock inhibitor y 27632 dihydrochloride, by Bio-Techne (2 mentions)
Matrigel coated, by Corning (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Emission filter wheel, by Sutter Instruments (1 mentions)
Ixon x3 emccd camera, by Oxford Instruments (1 mentions)
41 protocols using stemflex media
1
Thawing and Culturing of Human iPSCs
2
Live-cell Confocal Imaging of Cells
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We imaged cells on a Nikon Eclipse Ti inverted microscope (Nikon Instruments) fitted with a CSU-X spinning disk confocal head (Yokogawa), four solid-state lasers (Nikon), IXon X3 EMCCD camera (Andor), and emission filter wheel (Sutter Instruments). The imaging area was kept at 37 °C with 5% CO2 (In Vivo Scientific, LCI). We used a 100 × 1.45 NA Plan Apo oil immersion objective (Nikon). Images were acquired using Nikon Elements. We generally imaged 3–7 z slices with 300 nm z spacing at 3 s time intervals. Cells were seeded on sterile 4-chambered or 8-chambered #1.5H (0.170 ± 0.005 mm) cover glasses (CellVis). We imaged the cells in media supplemented with HEPES and the antioxidant oxyrase (OxyFluor) with substrate lactate. For quantitative fluorescence experiments we limited the cells’ exposure to ambient light, brightfield light and 488 nm laser light prior to acquisition, and used the same laser power during acquisition to compare experiments (generally 10% acousto-optic tunable filter (AOTF) power). For most experiments, we used DMEM/F12 without phenol red (Thermo Fisher) supplemented with the protein supplement used in StemFlex media (Thermo Fisher), which gave similar fluorescence intensity results as cells imaged in StemFlex (which has phenol red).
3
Hepatic Lineage Differentiation and Organoid Culture
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A derivative of the reference human iPSC line Wt11c, which was extensively characterized to be pluripotent and genetically stable,26 (link) was obtained from Coriell Institute (Camden, NJ) and maintained with Cultrex basement membrane extract (R&D Systems, Minneapolis, MN) and StemFlex media (Thermo Fisher Scientific, Waltham, MA). Differentiation toward the hepatic lineage was initiated as monolayers on 30-fold-diluted Matrigel (Corning Life Science, Glendale, AZ) using established protocols27 (link),28 (link) and described in more detail in Supplemental Methods . To generate iHEC-derived organoids, cells were released by Accutase (MilliporeSigma, St. Louis, MO) and resuspended in Organoid Media that consisted of 50% HCM media (Lonza, Walkersville, MD) and 50% EBM2 media (Lonza) with 40 ng/mL HGF (PeproTech, Cranbury, NJ), 20 ng/mL OSM (PeproTech) and 1 μg/mL dexamethasone (Thomas Scientific, Swedesboro, NJ).29 (link) Cells were seeded either onto 24-well-plates coated with 2-fold-diluted Matrigel, or into 10 mL RWVs (Synthecon, Houston, TX) set to 10.5 rpm rotation. For RWV organoid plus Matrigel (RWV ORG + MTG) conditions, 50 μL of 2-fold-diluted Matrigel was added to RWVs at the start of culture. All organoid cultures were incubated in 37 oC with 20% O2 and 5% CO2. Cell densities for all conditions were 5 × 105/mL, and media were changed every 2 days.
4
Generating Human Neural Progenitor Cells
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All human cell line work was performed on de-identified cell lines and approved by the Columbia University Institutional Review Board (IRB). IMR90 cl.4 hiPSCs (WiCell) (Yu et al., 2007 (link); Yu et al., 2009 (link); Chen et al., 2011 (link); Hu et al., 2011 (link)) were grown in StemFlex media (Thermo Scientific) on Cultrex (Biotechne) and passaged with ReLeSR as described previously (Song et al., 2021 ). Human neural progenitor cells (NPCs) were generated using dual SMAD inhibition as non-adherent embryoid bodies, followed by plating on polyornithine (10 ug/mL, Sigma P4957)/laminin (10 ug/mL, R&D Systems 3400-010-02) and subsequent manual rosette selection and expansion, as described previously (Topol et al., 2015 ; Sun et al., 2019 ). NPCs were maintained on Matrigel (Corning 354230) and split 1:2 to 1:3 at every 5–7 days. Neuronal differentiations were carried out by plating 200,000 cells/12 well-well or 500,000 cells/6 well-well in DMEM/F12 base media (Gibco 11320–033) supplemented with B27 (Gibco 17504–044), N2 (Gibco 17502–048), penicillin/streptomycin (Gibco 15140–122), BDNF (20 ng/mL, R&D Systems 248-BDB), and laminin (1 ug/mL). After 1 week of differentiation, AraC (Tocris 4520) was added at 100 nM to reduce remaining NPC proliferation. Neurons were differentiated for 6–11 weeks post NPC stage.
5
Generation and Characterization of iPSC Lines
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The Institutional Review Boards at Brown University and Lifespan Healthcare approved the human subjects research protocol. Informed consent was obtained from all participants or guardians of participants. The generation and characterization of the isogenic WT and CRISPR KO iPSC lines, as well as the patient-derived CS NHE6 mutant and CRISPR-corrected WT isogenic control iPSC lines, has been previously described (Lizarraga et al., 2021 (link); Ma et al., 2021 (link)). iPSCs were plated on Matrigel (Corning). Cultures were maintained in Stemflex media (Thermo Fisher Scientific) and continually monitored for quality.
6
hESC and hiPSC Culture and Characterization
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hESC lines (H1 and H9) were obtained from WiCell Research Institute (Maddison, WI, USA). Several hiPSC lines were generated from healthy individuals in our lab. Those hiPSC lines were extensively characterized as described in our recently published article [20 (link)]. hESCs/hiPSCs were cultured in StemFlex media (Thermo Fisher Scientific, Waltham, MA, USA) and were passaged using ReLeSR (Stem Cell Technologies, Vancouver, BC, Canada) or EDTA. The cells were treated with 10 μM Y-27632 (Stemgent, USA) for 24 h after passaging.
7
Cortical Neuron Induction from iPSCs
8
Genome Editing of BAX and BAK
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On the day of the transfection, the cells were counted to 1 × 106 cells per reaction. 2 µl Cas9 Nuclease (1 µg/µl) and 6 µl of gRNA designed to target BAX and BAK with a sequence complementary to the target DNA were added to 1.5 ml tubes. Cells were mixed in Resuspension Buffer R and the mixture was incubated at room temperature for 5–10 min (this is the Cas9/gRNA complex solution). The Neon Transfection System was then set up by filling the Neon Tube with 3 ml of Electrolytic Buffer (Buffer E2 for 100 µl Neon Tip) and the Neon Tube inserted into the Neon Pipette station. The 100 µl of Cas9/gRNA cell mix was aspirated with the 100 µl Neon Tip. The cells were then electroporated and immediately transferred to a 6-well plate with 2 ml of pre-warmed StemFlex media (Thermo Fisher, Waltham, MA). Cells were incubated at 37 °C for 48–72 h. Cell population was then DNA sequenced to determine indels.Cells were flow-sorted by single cell into a 96-well plate. They were then cultured for two weeks; their DNA was collected and sequenced. Immunoblots were also conducted to confirm that the protein had been knocked out.
9
Cell Culture Media Protocols
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Airway cells: PneumaCult Ex Plus medium (StemCell Technologies #05040) was used for culturing ABCs. Differentiation media was purchased from the University of North Carolina core.
HSPC: StemSpan™ SFEM II supplemented with StemSpan™ CD34+ Expansion Supplement (10X) (StemCell Technologies, Catalog # 09720). IPSC: StemFlex™ media (ThermoFisher Scientific, A3349401). HUVEC: EGM™−2 Endothelial Cell Growth Medium-2 BulletKit™ (Lonza, CC-3162). T-cells: X-Vivo 15 media (Lonza; 02-060Q)
HSPC: StemSpan™ SFEM II supplemented with StemSpan™ CD34+ Expansion Supplement (10X) (StemCell Technologies, Catalog # 09720). IPSC: StemFlex™ media (ThermoFisher Scientific, A3349401). HUVEC: EGM™−2 Endothelial Cell Growth Medium-2 BulletKit™ (Lonza, CC-3162). T-cells: X-Vivo 15 media (Lonza; 02-060Q)
10
Differentiation of FOXA2 Knockout iPSCs into Pancreatic Progenitors
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iPSC lines (Ctr1-iPSCs and Ctr2-iPSCs) generated and fully characterized in our laboratory were used as we previously reported [32 (link)]. FOXA2 knockout iPSCs from Ctr1-and Ctr2-iPSCs were generated using CRISPR/Cas9 as we recently reported [5 (link)]. Both wild-type (WT) and FOXA2–/– iPSCs were cultured and maintained using Stemflex media (ThermoFisher Scientific) on Matrigel-coated plates (Corning). iPSC lines were differentiated into PPs using our established protocol (Supplementary Table 1 ) [33 (link)–35 (link)].
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