TCPS plates were coated with 0.61 μg of ECM protein per cm2 ECM (fibronectin (Sigma, St. Louis, MO), laminin 1,1,1 (Sigma), or collagen type 1 (Nutragen, Advanced Biomatrix, San Diego, CA)) in phosphate buffered saline (PBS) and placed in an incubator overnight at 37°C. For Synthemax plates, TCPS plates were coated with 3.3 μg per cm2 Synthemax (Corning) in PBS and incubated overnight at 37°C. Prior to cell seeding, plates were washed with sterile PBS to remove any non-adsorbed coating.
Synthemax
Manufactured by Corning
Sourced in United States
Synthemax is a cell culture surface coating designed to support the attachment and growth of a variety of mammalian cells, including stem cells. It provides a defined, animal-component-free substrate that helps maintain cell functionality and phenotype. Synthemax is suitable for use in a range of cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (2 mentions)
Tesr e8 medium, by STEMCELL (2 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Evom2 resistance meter, by World Precision Instruments (1 mentions)
Endohm electrodes, by World Precision Instruments (1 mentions)
Transwell culture insert, by Corning (1 mentions)
Vitronectin, by Thermo Fisher Scientific (1 mentions)
Nicotinamide, by Thermo Fisher Scientific (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Su5402, by Merck Group (1 mentions)
Activin a, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Activin a, by Merck Group (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Noggin, by R&D Systems (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
Igf 1, by Merck Group (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
16 protocols using synthemax
1
ECM and Synthemax Coating Protocols
2
Differentiation of RPE from IMR90-4 Cells
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RPE was derived from IMR90-4 cells and cultured as described earlier.44 (link),45 (link) hfRPE was cultured as described earlier.44 (link) In each case, cells were seeded on Transwell culture inserts (Corning Inc., Corning, NY, USA) that were coated with 10 µg Synthemax (Corning). Six weeks post-confluence, cells were adapted to SFM. The transepithelial electrical resistance (TER) was monitored using an EVOM2 resistance meter with EndOhm electrodes (World Precision Instruments, Sarasota, FL, USA). One week after plating on GCH-521, RPC cultures were layered cell-side down onto differentiated RPE (Supplementary Fig. S1 ). Co-cultures were maintained up to 9 months.
3
Efficient Differentiation of hiPSCs into RPE Cells
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The protocol for differentiation of pluripotent hiPSCs into RPE was adapted from Buchholz et al., 2013 [14 (link)]. For differentiation, iPSCs were plated at 70–80% confluence on 9.6cm2 wells coated with 1X Matrigel (Corning) or Synthemax (3535XX1 Corning, Corning NY) or vitronectin (PeproTech) in basal neural induction medium (DMEM/F12+ 1X N2+ 1X B27 + 1X Non-essential amino acids, Thermo Fisher Scientific) supplemented for 2 days with Nicotinamide (10mM) (Sigma-Aldrich, St. Louis, MO), Noggin (50ng/ml) (PeproTech), Dkk-1 (10ng/ml) (R&D Systems, Minneapolis, MN.) and IGF-1 (10ng/ml) (R&D Systems) with a medium change on day 1. On days 3 and day 4, the Noggin concentration was reduced to 10ng/ml and bFGF was added at 5ng/ml (R&D Systems). The Nicotinamide, bFGF and Noggin were removed from the media on day 5 and on days 5 and 6 the basal medium was supplemented with only Dkk-1 (10ng/ml), IGF1 (10ng/ml) and Activin A (100ng/ml) (PeproTech). The DKK and IGF 1 were then removed and from day 7 to day 14 the basal medium was supplemented with Activin A (100ng/ml), and SU5402 (10μm) (Sigma-Aldrich). CHIR 99021(3μM) (TOCRIS, Bristol, UK.) was also added from day 8 to day14. (See Fig 1 ). All media supplements were removed for the expansion of iPSC-derived RPE.
4
Cell Culture Protocols for Retinal Research
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Human fibroblasts and human retinal pigment epithelial (RPE) cell line ARPE-19 (American Type Culture Collection, Manassas, VA, USA) were cultivated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 4 mM L-glutamine, 10% fetal bovine serum (FBS), 100 U/mL penicillin G, 100 µg/mL streptomycin sulphate, and 1 ng/mL basic fibroblast growth factor (Gibco, Karlsruhe, Germany). The murine photoreceptor cell line 661W was provided by Dr. Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA) and cultured in DMEM supplemented with 4 mM L-glutamine, 10% FBS (Gibco), 32 mg/L putrescine, 40 µg/L progesterone, 30 µg/L hydrocortisone, and 20 µL/L β-mercaptoethanol (Sigma-Aldrich). Cells were grown at 37°C with 5% CO2 and passaged using 0.05% trypsin/0.02% ethylenediamine tetra-acetic acid (EDTA; T/E; Gibco) twice a week or at a confluency of ca. 80%. Human fetal RPE cells were provided by the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR, Newcastle, UK) and cultured on Millicell cell culture inserts (50,000 cells/well, growth area: 0.33 cm2, pore size: 1 µm; Merck) coated with Synthemax (2.5 µg/well; Corning, Corning, NY, USA) in modified Minimum Essential Medium (see Supplementary Table S1 ). Medium was changed every two to three days.
5
Defined Substrate Coatings for Cell Culture
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Six- and 12-well TCPS plates were coated with a range of defined substrates according to the manufacturer’s recommendations. Vitronectin (provided by the lab of Dr. Jamie Thomson, University of Wisconsin-Madison) or laminin-521 (BioLamina) were diluted in PBS and coated onto TCPS plates at a density of 0.61 μg/cm2 overnight at 37°C. Synthemax (Corning) was coated onto TCPS at a density of 3.3 μg/cm2 in PBS and incubated overnight at 37°C. StemAdhere coatings were achieved by diluting 240 μl StemAdhere (STEMCELL Technologies) in 6 ml of StemAdhere dilution buffer, with 1 ml of this solution (1.02 μg/cm2) placed into each well of a 6-well non-tissue culture polystyrene plate (BD Falcon) and incubated overnight at 37°C. Prior to cell seeding, all plates were washed with sterile PBS to remove any non-adsorbed coating.
6
Feeder-free Episomal iPSC Generation
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RCS-iPSCs were established by transfection of patient PMNCs with episomal vectors encoding OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA as previously described34 (link). iPSCs were maintained with feeder-free cultures using StemFit AK02N medium (ReproCELL, Tokyo, Japan) on cell culture plates (CellBIND; Corning, NY, USA) coated with Synthemax (Corning). The medium was changed daily, and passage was performed at 80–90% confluence. iPSCs were routinely monitored for mycoplasma contamination. Three iPSC cell lines (585A1, 585B1, and 648A1), which were generated from PMNCs of 30 s healthy Japanese men using the same method, were used as control iPSCs.
7
Feeder-independent Maintenance of Human Stem Cells
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For feeder-independent maintenance of human ESCs and iPSCs, basal mTeSR1 medium (STEMCELL Technologies) supplemented with 5X mTeSR1 supplement (STEMCELL Technologies) was used. Culture plates were pre-coated with growth factor reduced matrigel (BD Biosciences). Cells were passaged mechanically or enzymatically using 200 units/ml of collagenase IV or dispase (STEMCELL Technologies), washed and replated at a dilution of 1∶2 to 1∶5. Differentiated cells were removed and/or cleaned under a laminar flow dissection hood. Cultures were maintained at 37°C and 5% CO2 (for reprogramming experiments hypoxic conditions were applied) and medium changed every other day for fibroblast lines and every day for hESC and iPSC lines. For GMP conversion, cells were first gradually adapted to a 1∶1 blend of mTeSR1 medium (STEMCELL Technologies) supplemented with 5X mTeSR1 supplement (STEMCELL Technologies) and Nutristem (Stemgent) before they were fully converted towards a 1∶1 blend of TeSR2 (STEMCELL Technologies) and Nutristem (Stemgent). Cultures initially derived on Synthemax (Corning) or CELLstart (Invitrogen) were directly converted towards a 1∶1 blend of TeSR2 (STEMCELL Technologies) and Nutristem (Stemgent).
8
Cryopreservation of Cardiomyocytes
9
Embryoid Body Formation and Cardiomyocyte Differentiation
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Embryoid body (EB) formation was performed using the aggrewell system (StemCell Technologies), harvesting the cells using TrypLE with 1-h ROCKi pre-treatment and seeding an average of 2,000 cells per EB and with ROCKi addition. The EBs were then kept in GMEM 20% FBS and suspension for 2 weeks and then plated onto Matrigel (Corning) or Synthemax (vitronectin, Corning) for 2 more weeks. Beating cardiomyocytes areas were filmed after 3–4 weeks of growth using a light-phase microscope coupled to a NikonE990 camera.
10
Maintenance of Pluripotent Stem Cells
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WA09 hESC (WiCell) and A04 hiPSC, a clonal derivative of HPSI0114i-kolf_2 [95 (link)], were provided by Dr. Bill Skarnes, JAX. Cells were maintained in StemFlex defined culture medium (Thermo Fisher Scientific, Waltham, MA, USA) as adherent cultures on a substrate of a recombinant fragment of human vitronectin (Synthemax, Corning, Corning, NY, USA). Cells were routinely passaged with a non-enzymatic dissociation reagent (ReLeSR, Stem Cell Technologies, Vancouver, CB, Canada). Cells were used within 15 passages from diploid master stocks, and undifferentiated cultures expressed cell surface markers and transcription factors characteristic of the pluripotent state. Bimonthly testing confirmed the absence of contamination by mycoplasma or other adventitious microbial agents. Human embryonic stem cell research was approved by the University of Connecticut Stem Cell Research Oversight Committee on behalf of JAX.
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