Geltrex

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, Germany, United Kingdom, New Zealand, Australia, Switzerland, Denmark, Sweden

Geltrex is a soluble basement membrane extract of extracellular matrix proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is used as a cell culture substrate to support the growth and differentiation of various cell types, including stem cells, primary cells, and cell lines.

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760 protocols using geltrex

Geltrex Coating Protocol for Cell Culture

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To prepare the Geltrex coating, the stock solution of Geltrex (12–18 mg/ml, (Thermo Fisher)) was gently thawed on ice at 4°C overnight. Geltrex working solution of 0.4 mg/ml in 4°C cold DMEM/F12 (Thermo Fisher) was prepared in a 50 ml tube and introduced into the StemCellFactory. 6- or 24-well plates were automatically coated by adding 1000 or 300 μl of the Geltrex solution, respectively. Coated plates were incubated at RT for 1 h before use.

Elanzew A., Nießing B., Langendoerfer D., Rippel O., Piotrowski T., Schenk F., Kulik M., Peitz M., Breitkreuz Y., Jung S., Wanek P., Stappert L., Schmitt R.H., Haupt S., Zenke M., König N, & Brüstle O. (2020). The StemCellFactory: A Modular System Integration for Automated Generation and Expansion of Human Induced Pluripotent Stem Cells. Frontiers in Bioengineering and Biotechnology, 8, 580352.

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Time-lapse Imaging of N2a Cells

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Standardized imaging plates (Eppendorf; 170 µm glass thickness) were coated with matrigel (GelTrex™; Thermo Fisher) according to the manufacturer’s instructions using a working concentration of 0.1 mg GelTrex^TM diluted in 1 mL N2a culture medium. One hundred microlitre matrigel working solution was added per well and incubated for 60 min until hardening of the substrate. N2a cells were seeded with a density of 57 cells/mm² and treated with the inhibitor DZNep after 24 h as described above. To avoid phototoxic effects during imaging, culture medium was exchanged to DMEM without phenol red (Invitrogen) with 10% FBS (Biowest), 100 U/mL penicil-lin (Gibco), 100 µg/mL streptomycin (Gibco). Forty-eight hours after seeding, N2a cells were imaged every 15 min for 20 h at 37 °C and 5% CO₂.

Symmank J., Bayer C., Reichard J., Pensold D, & Zimmer-Bensch G. (2020). Neuronal Lhx1 expression is regulated by DNMT1-dependent modulation of histone marks. Epigenetics, 15(11), 1259-1274.

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Subcutaneous Xenograft Tumor Model

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2 × 10⁵ cells were suspended in 50 μL of Geltrex (Invitrogen, Auckland NZ) and injected sub-cutaneously into NOD/SCID mice, 3–5 mice per group. Tumour growth was recorded at 3–6 days, and when ~200 mm³, mice were culled by cervical dislocation and tumours excised. Tumours from each animal in the group were pooled, mechanically dissociated and filtered through 100, 70 and 40 μm filters into a single cell suspension, then plated into tissue culture plates in 20 mL of cell culture media. The following day, media was replaced, leaving adherent melanoma cells. Cultures were expanded by passage as required. Cells were lifted with 0.05 % Trypsin-EDTA (Life Technologies, NZ), and washed 3 times before being resuspended in Geltrex for the next injection.

Grasso C., Anaka M., Hofmann O., Sompallae R., Broadley K., Hide W., Berridge M.V., Cebon J., Behren A, & McConnell M.J. (2016). Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations. BMC Cancer, 16(1), 726.

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Cardiomyocyte Differentiation from hESCs

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The hESCs were cultured on tissue culture dishes coated with Geltrex (Life Technologies, Thermo Fisher Scientific) in mTeSR1 culture medium (STEMCELL Technologies, Vancouver, BC, Canada) with daily media changes. The cells were passaged every 3–4 days using Accutase (STEMCELL Technologies). The undifferentiated phenotype of the hESCs was checked daily using a light microscope. In order to differentiate hESCs to cardiomyocytes, they were dissociated by Versene (Life Technologies, Thermo Fisher Scientific), resuspended in mTeSR1 + 5 μM Y27632 and seeded onto Geltrex-coated plates at a density of 3 × 10⁵ cells/cm² and grown for the next four days with daily medium change. Following that, the cells were treated with 6 µM CHIR99021 (Selleckchem, Houston, TX, USA) in insulin-free RPMI/B27 medium (Life Technologies) for 24 h. The medium was replaced with basal medium for another 2 days. At day 3, the culture medium was subsequently replaced with 5 µM IWP-2 (Tocris Bioscience, Minneapolis, MN, USA) in insulin-free RPMI/B27 for 48 h. On day 7, the culture medium was changed to RPMI/B27 containing insulin (Life Technologies, Thermo Fisher Scientific), and the culture medium was refreshed every 3 days thereafter.

Song H.Y., Chien C.S., Yarmishyn A.A., Chou S.J., Yang Y.P., Wang M.L., Wang C.Y., Leu H.B., Yu W.C., Chang Y.L, & Chiou S.H. (2019). Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy. Cells, 8(4), 327.

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Angiogenic Potential of Endothelial Cells

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Endothelial cell tube-forming method A Geltrex (Life Technologies, Grand Island, NY) assay was used to assess the angiogenic capability of normal and AML BMECs. Calcein (20 μmol/L, Thermo Fisher Life Technologies, Pittsburgh, PA)-stained BMECs (1.5 × 10 6 cells/well) were seeded on Geltrex-coated plates and incubated at 37°C/5% CO 2 for 2-6 hours. In vitro tube formation was photographed using fluorescence phase-contrast microscopy (Nikon, Melville, NY).

Pizzo R.J., Azadniv M., Guo N., Acklin J., Lacagnina K., Coppage M, & Liesveld J.L. (2016). Phenotypic, genotypic, and functional characterization of normal and acute myeloid leukemia-derived marrow endothelial cells. Experimental hematology, 44(5).

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Kidney Organoid Differentiation from iPSCs

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The CMC11 iPSCs were obtained from The Catholic University of Korea (male donor). The cells were used between passages 20 and 40. The kidney organoid differentiation was performed as described previously [10 (link),11 (link)]. In brief, human iPSCs were plated at a density of 5,000 cells/well on a 24-well plate in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) plus 10 μM Y27632 (LC Laboratories, https://www.lclabs.com/) on glass plates (LabTek, Queensland, Australia) coated with 3 % GelTrex (Thermo Fisher Scientific, Waltham, MA, USA) (day 3). The medium was exchanged for 1.5% GelTrex in mTeSR1 (day 2), mTeSR1 (day 1), RPMI (Thermo Fisher Scientific) plus 12 μM CHIR99021 (Tocris, Seongnam, Korea) (day 0), or RPMI plus B27 supplement (Thermo Fisher Scientific) (day 1.5) and the cells were fed every 2 to 3 days to promote kidney organoid differentiation.

Kim J.W., Nam S.A., Seo E., Lee J.Y., Kim D., Ju J.H., Lim S.W., Kim H.L., Kim H.W., Yang C.W., Kim J., Kim D.S, & Kim Y.K. (2021). Human kidney organoids model the tacrolimus nephrotoxicity and elucidate the role of autophagy. The Korean Journal of Internal Medicine, 36(6), 1420-1436.

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3D Culture Model for Cell-Protein Interactions

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The 3D culture model that was developed for the purposes of this study involved a modification of the 3D-on-top type culture technique [49 (link)] that creates differential surfaces for cell contact. Geltrex (Thermofisher) depleted of growth factors using ammonium sulfate precipitation was used as the gel substrate and is referred to here as growth factor-reduced (GFR) Geltrex. Using this modified technique, the test (AMBN, AMEL) and control proteins (heat denatured AMBN, BSA) were restricted to one surface of the cells (along the Z axis), with the opposing Z surface contacting the top 10% Geltrex coat. Briefly, ice-cold Geltrex was added to the wells of the 96-well plate that were pre-coated with 20μg/ml of test (AMBN, AMEL, AMBNΔ5 and AMBNΔ6) or control (bovine serum albumin, heat denatured AMBN and Geltrex alone) proteins and allowed to polymerize into a gel at 37°C for 30 min. The cells were seeded on top of the gel at a density of 3.5 × 10⁴ cells/well [28 (link),49 (link),50 (link)]. A 10% Geltrex coat in culture media was added atop the attached cells to complete the 3D-on-top type culture and the cultures were maintained for 24-72h under standard conditions. The 3D cell culture experiment was repeated using TCMK-1 and LS-8 cells.

Visakan G., Su J, & Moradian-Oldak J. (2021). Ameloblastin promotes polarization of ameloblast cell lines in a 3-D cell culture system. Matrix biology : journal of the International Society for Matrix Biology, 105, 72-86.

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iPSC Kidney Organoid Differentiation Protocol

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WTC11 induced pluripotent stem cell (iPSC) between passages 30 and 60 were used. Kidney organoid differentiation was induced as described previously [7 (link)]. In brief, iPSCs were plated at a density of 5,000 cells/well in a 24-well plate in mTeSR1 medium (Stem Cell Technologies) + 10 µM Y27632 (LC Laboratories) on plates (SPL Life Sciences) coated with 1% GelTrex (Thermo Fisher Scientific) (day –3). The medium was exchanged with 1.5% GelTrex in mTeSR1 (day –2), mTeSR1 (day –1), RPMI (Thermo Fisher Scientific) + 12 µM CHIR99021 (Tocris, Bristol, UK) (day 0), or RPMI + B27 supplement (Thermo Fisher Scientific) (day 1.5) and cells were fed every 2–3 days to promote kidney organoid differentiation. Organoids were analyzed on day 18.

Park K., Lee J.Y., Lee S.Y., Jeong I., Park S.Y., Kim J.W., Nam S.A., Kim H.W., Kim Y.K, & Lee S. (2022). Deep learning predicts the differentiation of kidney organoids derived from human induced pluripotent stem cells. Kidney Research and Clinical Practice, 42(1), 75-85.

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Kidney Organoid Differentiation from iPSCs

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The CMC11 iPSC cell line was obtained from The Catholic University of Korea (male donor). Cells were used between passages 30 and 60. Kidney organoid differentiation was performed as described previously (Freedman et al.^{3 (link)}). In brief, hPSCs were plated at a density of 5000 cells/well of a 24-well plate in mTeSR1 medium (Stem Cell Technologies)+10 µM Y27632 (LC Laboratories) on glass plates (LabTek) coated with 3% GelTrex (Thermo Fisher Scientific) (day −3). The medium was exchanged for 1.5% GelTrex in mTeSR1 (day −2), mTeSR1 (day −1), RPMI (Thermo Fisher Scientific)+12 µM CHIR99021 (Tocris) (day 0), or RPMI+B27 supplement (Thermo Fisher Scientific) (day 1.5) and cells were fed every 2–3 days to promote kidney organoid differentiation. Organoids were fixed or transplanted into NOD-SCID mice on day 18.

Nam S.A., Seo E., Kim J.W., Kim H.W., Kim H.L., Kim K., Kim T.M., Ju J.H., Gomez I.G., Uchimura K., Humphreys B.D., Yang C.W., Lee J.Y., Kim J., Cho D.W., Freedman B.S, & Kim Y.K. (2019). Graft immaturity and safety concerns in transplanted human kidney organoids. Experimental & Molecular Medicine, 51(11), 1-13.

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Spheroid Invasion Assay Protocol

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The spheroid drop invasion assay was conducted in accordance with previously published methods [68 (link)]. A total of 1.5 × 10⁵ cells were centrifuged at 1000× g in a 1.5 mL Eppendorf tube. The supernatant was removed, and the cell pellet resuspended in 30 μL of Geltrex (#A1413202, Thermo Fisher Scientific). An amount of 10 μL of the suspended Geltrex cell solution was seeded onto the well of a 48-well plate (#150687, Thermo Fisher Scientific). The well plate was placed upside down into the incubator to incubate for 20 min at 37 °C and 5% CO₂. Once removed from the incubator, 2 mL of DMEM/F12 with 10% FBS medium was added to each well. The medium was changed every 48 h. The spheroids were imaged daily and the area of the Geltrex spheroid and invading cells were measured in ImageJ.

Ellis K, & Wood R. (2023). The Comparative Invasiveness of Endometriotic Cell Lines to Breast and Endometrial Cancer Cell Lines. Biomolecules, 13(6), 1003.

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