iPSCs were generated from dermal fibroblasts from two patients with wet AMD associated with the homozygous CFH Y402H polymorphism (high‐risk AMD RPE) and two age matched donors with no clinical or genotypic indication of ocular disease (low‐risk control RPE), as reported previously (Hallam et al., 2017 (link)). Two additional control cell lines (WT1 and WT3) were used to confirm the reproducibility of our iPSC and iPSC‐RPE culture (Buskin et al., 2018 ). iPSCs were cultured in mTeSR1 (Stem Cell Technologies) growth media on Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning) in humidified, 5% CO2, 37°C conditions. iPSCs were passaged every 4–6 days using Versene (EDTA) 0.02% (Lonza).
Versene edta 0.02
Manufactured by Lonza
Versene (EDTA) 0.02% is a laboratory reagent produced by Lonza. It is a solution of ethylenediaminetetraacetic acid (EDTA) in water, with a concentration of 0.02%. EDTA is a chelating agent that binds to metal ions, making it a useful tool in various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel growth factor reduced basement membrane matrix, by Corning (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
E8 flex medium, by Thermo Fisher Scientific (1 mentions)
Revitacell, by Thermo Fisher Scientific (1 mentions)
Primocin, by InvivoGen (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
E6 medium, by Thermo Fisher Scientific (1 mentions)
4 protocols using versene edta 0.02
1
Generation of iPSC-derived RPE from AMD Patients
2
Culturing Human Induced Pluripotent Stem Cells
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iPSCs were cultured in Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning) coated plates and mTeSR1 (STEMCELL Technologies) growth media in humidified, 5% CO2, 37°C environment. iPSCs were passaged in ratio 1:6 every 4 to 6 days using Versene (EDTA) 0.02% (Lonza).
3
Fibroblast-derived induced pluripotent stem cells
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A punch biopsy from the inner side of the upper arm was taken from the patient. The biopsy was cut in smaller pieces and incubated with collagenase and trypsine for 1 h in 37 °C. Afterwards fibroblasts were cultured in RPMI medium (Life Technologies) supplemented with 15% FBS (Life Technologies), 1% sodium pyruvate (Life Technologies), 100 U/mL Pen/Strep (Life Technologies) and 0,1% primocin (InvivoGen Europe). Fibroblasts were plated in one well of a 6-well plate and after two days, the cells were transduced with the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Life Technologies) following the manufacturer’s protocol. After seven days, cells were plated on Matrigel (Corning). One day later, the medium switched from RPMI to E8 flex medium (Life Technologies). Colonies were manually picked and seeded on Matrigel coated 24-well plates and incubated at 37 °C/5%CO2/5%O2. After five rounds of picking, cells were chemically passaged as small clumps with Versene (EDTA 0,02%) (Lonza) every 4–5 days and expanded in a 1:5 ratio. Cells were supplemented with 1x Revitacell (Life Technologies) for 24 h after a picking/passage.
4
iPSC Differentiation into Embryoid Bodies
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iPSCs (p16-BBANTWi006-A, p16-BBANTWi007-A) were collected using Versene (EDTA 0,02%) (Lonza) for 5 min at room temperature (RT) followed by washing of the cells. 500.000 cells/well were seeded onto a 24-well low-attachment plate with E6 medium (Life Technologies) and incubated at 37 °C/5%CO2/5%O2 and half a medium change was performed every other day. After 14 days EBs were collected for RNA extraction.
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