5 × 105 AF‐MSCs were detached using TrypLE (Life Technologies, Paisley, UK), centrifuged at 1,200 rpm for 5 minutes then electroporated as described previously 49 but without the pCXWB‐EBNA1 episomal plasmid. After transfection, cells were plated onto one 0.1% gelatin (Sigma‐Aldrich)‐coated well of a 6‐well plate. Twenty‐four hours post‐transfection, cells were detached and seeded into one 6 cm2 dish precoated with Matrigel (Scientific Laboratory Supplies ltd, Nottingham, UK) in Essential 8 medium (Life Technologies). The medium was then replenished every 2 days until around day 20, when iPSC colonies were passaged manually with a needle into a fresh Matrigel‐coated well of a 6‐well plate in Essential 8 medium. Cells were passaged with 0.5 mM EDTA (Life Technologies) thereafter.
Matrigel
Manufactured by Scientific Laboratory Supplies
Sourced in United Kingdom
Matrigel is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. It is commonly used as a substrate for the culture of various cell types, including stem cells, and in the study of cell-matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
Essential 8 medium, by Thermo Fisher Scientific (2 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
3 protocols using matrigel
1
iPSC Generation from AF-MSCs
2
Characterization of Human Stem Cell Lines
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The human pluripotent stem cell-lines used in this study were obtained with full Ethical/Institutional Review Board approval by the University of Edinburgh and validated using standard methods including chromosomal analysis, pluripotency and absence of plasmid integration. The iPSC lines CS02iCTR-NTn1 (hPSC1, male) and CS25iCTRL-18n2 (hPSC2, male) were obtained from Cedars-Sinai. For the Nfasc155-/- lines, fibroblasts were obtained by RS with ethical approval granted by the Institutional Review Board of Warsaw Medical University (Smigiel et al., 2018 (link)). The CS00iNK-n1 (Nfasc155-/-clone 1, female) and CS00iNK-n2 (Nfasc155-/-clone 2, female) iPSC lines were generated by Cedars-Sinai. The human embryonic stem cell-line SHEF4 (hPSC3, male) was obtained from the UK Stem Cell Bank. iPSCs were maintained on Matrigel (Scientific Laboratory Supplies)-coated 6-well plates in Essential 8 medium (Thermo Fisher Scientific) at 37 °C and 5% CO2. iPSC colonies were passaged by incubating in Dispase (0.5 mg/ml, Thermo Fisher Scientific)/Collagenase (1 mg/ml, Thermo Fisher Scientific) for 15-25 minutes at 37 °C, washed in DPBS and resuspended in Essential 8 medium before being redistributed in new 6-well plates. Cultures were regularly tested and maintained mycoplasma free. These cell-lines have not been authenticated.
3
Evaluation of VEGF Inhibitor Efficacy
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Pre-chilled, 96-well, flat-bottomed plates were coated with Matrigel (growth factor reduced, phenol red free; Scientific Laboratory Supplies, Nottingham, UK) and left to polymerize for 1 h at 37°C and 5% CO2. HUVECs were seeded onto the Matrigel at 1.5 × 104 cells/well. Vector-derived aflibercept, recombinant aflibercept, and rhIgG1 were diluted in untransduced D17 culture supernatant containing 20 ng/mL of rhVEGFA165 to a concentration of 0.5 nM and added to the plated HUVECs. Plates were incubated for 18 h at 37°C and 5% CO2, after which bright-field images were taken using a digital camera mounted on a Carl Zeiss microscope. Analysis of the images was carried out by Onimagin Technologies (Cordoba, Spain) using the Wimasis software, and percentage inhibition of vector-derived aflibercept on HUVEC total tubule length relative to inhibition with untransduced D17 control supernatant was calculated.
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