Cells are thawed and seeded on 75 or 150 cm2 cell culture flasks at a density of 10 000 cells per cm2 (Techno Plastic Products AG, Switzerland). All cell culture was performed in humidified incubators at 37 °C, and an HEPA‐filtered atmosphere of air and 5% of CO2. Medium changes were performed every 2–3 days, except where indicated otherwise. Human neonatal dermal fibroblasts (HNDFs, Lonza Wakersville Inc., USA), or red fluorescent protein‐expressing HNDF (HNDF‐RFP, Angio‐Proteomie, USA) were cultured in endothelial cell medium (ScienceCell) supplemented with 5% fetal bovine serum (FBS, ScienceCell) and endothelial cell growth bullet kit (ScienceCell). MSCs (Lonza) were cultured in Nutristem medium (Biological Industries). Cells were harvested for experiments during passages 4–7 at ≈70% of confluence. For live cell tracking of MSCs or HNDF, cells were labeled with either DiI, DiD, or DiO Vybrant cell labeling solution (Thermo Fisher). For osteogenic differentiation of MSCs, DMEM low‐glucose medium was supplemented with 10% FBS, 1% Pen‐Strep, 100 × 10−9m dexamethasone, 10 × 10−3m β‐glycerol phosphate, and 50 × 10−6m ascorbic acid.
Nutristem medium
Manufactured by Sartorius
Sourced in United States
Nutristem medium is a cell culture medium designed for the growth and maintenance of human pluripotent stem cells. It provides the necessary nutrients and growth factors to support the proliferation and undifferentiated state of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Hndfs, by Lonza (1 mentions)
Matrigel, by Corning (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Matrigel, by BD (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Dmem f12, by Sartorius (1 mentions)
Dispase, by STEMCELL (1 mentions)
Htb 38, by American Type Culture Collection (1 mentions)
Forma steri cycle i160 co2 incubator, by Thermo Fisher Scientific (1 mentions)
Imdm medium, by Biowest (1 mentions)
Glutamine, by Lonza (1 mentions)
Roswell park memorial institute (rpmi), by Lonza (1 mentions)
7 protocols using nutristem medium
1
Culturing and Differentiating Stem Cells
2
Hypoxic Culture of hESC on HFFs
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hESC colonies were cut into small pieces with a fine glass needle and transferred onto freshly prepared gamma-irradiated HFFs. The cells were then cultured in NutriStem medium (Biological Industries, USA) under hypoxic conditions and were subcultured every 4–5 days when their colonies reached optimal size. For the feeder-free system, hESC colonies were cultured on a Matrigel-coated plate in NutriStem media under hypoxic conditions and subcultured every 4–5 days using Versene treatment (Life Technologies, USA).
3
Maintaining Pluripotent H9 hESC Line
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The H9 human embryonic stem cell line obtained from WiCell was maintained in Nutristem medium (Biological Industries, 05-100-1A) on 1% Matrigel (Corning, 354230) pre-coated plates and passaged when colonies reached about 90% confluency using TrypLE (Gibco, 12604021) at the ratio 1:5. Cells were checked for normal karyotype and were mycoplasma-free. Unless otherwise specified, organoids shown were generated from H9 cells below passage 60. All cultures were maintained in 5% CO2 incubators at 37°C.
4
Establishment and Maintenance of Human Pluripotent Stem Cells
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VAL9 hESCs were obtained from the Spanish National Stem Cell Bank (http://www.isciii.es/ISCIII/es ) after approval of the InnovaLiv project by the following ethics committees: Spanish “Comision Nacional Medicina Regenerativa” on 21 May 2012 and French Agency of Biomedicine on 25 June 2012. VAL9 hESCs were cultured in feeder-free conditions on culture dishes pre-coated with 0.05 mg/ml Geltrex (Life technologies) in Nutristem medium (Biological industries) supplemented with 8 ng/ml fibroblast growth factor (FGF)2 (CellGenix) at 37 °C/5 % CO2 in animal-free conditions [30 (link)].
The hiPSC line was already established in the laboratory from human foreskin fibroblasts. hiPSCs were maintained on MEF feeders in DMEM/F12 medium supplemented with 20 % knockout serum replacement, 1 mM L-glutamine, 1 % non-essential amino acids, 0.1 mM β-mercaptoethanol and 4 ng/ml FGF2 at 37 °C/5 % CO2. Prior to differentiation, hiPSCs were plated on culture dishes pre-coated with Geltrex and maintained in the same conditions as VAL9 hESCs for a few passages before starting differentiation.
The hiPSC line was already established in the laboratory from human foreskin fibroblasts. hiPSCs were maintained on MEF feeders in DMEM/F12 medium supplemented with 20 % knockout serum replacement, 1 mM L-glutamine, 1 % non-essential amino acids, 0.1 mM β-mercaptoethanol and 4 ng/ml FGF2 at 37 °C/5 % CO2. Prior to differentiation, hiPSCs were plated on culture dishes pre-coated with Geltrex and maintained in the same conditions as VAL9 hESCs for a few passages before starting differentiation.
5
Generation of FMR1 Knockin Cell Lines
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A P2a peptide-hygromycin-HSV thymidine kinase-stop codon-SV40 poly(A)(Hyg-TK) cassette was inserted into exon 4 of FMR1 using CRISPR-mediated homologous recombination. For the details of construct generation, validation, and selection procedure, see Supplemental Experimental Procedures . Knockin lines were maintained in Nutristem medium (Biological Industries) supplemented with 25 μg/mL hygromycin. Positive-negative selection was performed with 10 μM ganciclovir without hygromycin.
6
Omentum-Derived iPSC Generation
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iPSCs were generated from omental stromal cells and were a kind gift from Dr. Rivka Ofir, Ben Gurion University. The undifferentiated cells were cultivated on culture plates, pre‐coated with Matrigel (BD, New Jersey), diluted to 250 µg mL−1 in DMEM/F12 (Biological Industries), or cultured within the omentum hydrogel. All cells were cultured at 37 °C with 5% CO2. Undifferentiated iPSCs were maintained in NutriStem medium containing 0.1% Penicillin/Streptomycin (Biological Industries). Medium was replaced daily and cells were passaged weekly with 1 U mL−1 dispase (Stemcell Technologies, Vancouver, Canada) followed by mechanical trituration. iPSCs were seeded in small colonies in the presence of Y‐27632 (10 µm ; Tocris, UK).
7
Culturing Cell Lines for Biomedical Research
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All cell lines mentioned below were cultured at 37 °C and 5% CO2 in a humidified Forma Steri-cycle i160 CO2 incubator (Thermo Fischer Scientific) and were grown in the described media unless otherwise specified. HFF (ATCC® CRL-2429™, male), passage 3–6, were grown in IMDM medium (Biowest) supplemented with 10% Gibco™ Fetal Bovine Serum, Qualified, US Origin, Standard (Sterile-Filtered) (Gibco). RKO cells (ATCC® CRL-2577™, female), passage 3–5, were grown in DMEM supplemented with 2 mM glutamine (Lonza) and 10% FBS. HT29 (ATCC® HTB-38™, female), passage 3–5 were grown in RPMI (Lonza) supplemented with 10% FBS. ESCs, wild-type H9 (WiCell WA-09, female)61 (link), passage 35–40 and HS980, passage 9–25 were grown on LN-521-coated dishes in NutriStem medium (Biological Industries).
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