Matrigel

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Canada, United Kingdom, China, Ireland

Matrigel is a basement membrane extract derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It provides a complex mixture of extracellular matrix proteins, growth factors, and other components that support the growth and differentiation of various cell types in cell culture systems.

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696 protocols using matrigel

Maintenance of Human iPSC Line

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The human iPSCs line hFSiPS1, which was derived from male dermal fibroblasts,
⁵³ (link) was acquired from the National Stem Cell Bank of Korea. The iPSCs were routinely maintained for 5–6 days on a dish coated with Matrigel (Corning) using Essential 8 (E8) feeder‐free medium (Invitrogen). To prepare the Matrigel‐coated dish, Matrigel was diluted in Dulbecco's Modified Eagle's Medium (DMEM)/F12 medium (Invitrogen) to create a 1% Matrigel solution. The dish was then coated with this 1% Matrigel solution at 4°C for 18 h. Before conducting the experiments, the Matrigel‐coated dish was thoroughly washed with PBS. Next, the iPSCs were dissociated using 5 mM EDTA (Invitrogen) at 37°C for 4 min. Following the EDTA treatment, iPSC colonies were dissociated into clumps within the culture medium. These clumps were then seeded onto the newly Matrigel‐coated dish, which was supplemented with E8 medium and 3 μM y‐27632 (Tocris). The culture medium was refreshed daily, starting from the second day of seeding.

Kim J., Kim J., Kim D., Bello A.B., Kim B.J., Cha B, & Lee S. (2023). Therapeutic potential of mesenchymal stem cells from human iPSC‐derived teratomas for osteochondral defect regeneration. Bioengineering & Translational Medicine, 9(2), e10629.

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Extracellular Matrix Coating Protocol

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Culture plates were coated with ECM, Matrigel (BD Biosciences) or laminin (Sigma) by the following procedure. A Matrigel bottle was thawed on ice in a 4°C refrigerator overnight until Matrigel liquified. Matrigel was divided into 300 μl aliquots and stored at −20°C until use. For preparation of Matrigel-coated plates, working Matrigel solution was prepared by diluting 300 μl of Matrigel with 29 ml of DMEM/F12 medium (Invitrogen) and thorough mixing. This solution was added to 12-well plates (0.5 ml per well) or 6-well plates (1 ml per well) to cover the whole surface of the wells. The plates were allowed to sit for 1 h at room temperature or overnight at 4°C. Excess Matrigel solution was then removed, and the plates were washed once with DMEM/F12. For laminin coating, laminin stock solution (1 mg/ml) was divided into 50 μl aliquots and stored at −20°C. laminin working solutions (20 μg/ml) were prepared by diluting the stock solution with Dulbecco's phosphate-buffered saline (DPBS), and added to 12-well plates (0.5 ml per well) or 6-well plates (1 ml per well). Plates were incubated with laminin solution for at least 2 h in a 37°C in a cell culture incubator, then excess laminin solution was removed and the plates were washed twice with DPBS.

Choi N.Y., Park Y.S., Ryu J.S., Lee H.J., Araúzo-Bravo M.J., Ko K., Han D.W., Schöler H.R, & Ko K. (2014). A Novel Feeder-Free Culture System for Expansion of Mouse Spermatogonial Stem Cells. Molecules and Cells, 37(6), 473-479.

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In vitro Tube Formation Assay for Angiogenesis

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The capillary-like structure (CLS) formation of angiogenesis from ECs can be modelled in vitro by a Matrigel-based tube formation assay (Thermo Fisher).^{19 (link),20 (link)} ECs plated on Matrigel at low densities form a network of branching structures, which can be photographed and quantified by measuring the length, perimeter or area of the CLS.^{19 (link),20 (link)} Matrigel in vitro tube formation was performed in 48-well plates. Briefly, Matrigel (80 μL per well, Thermo Fisher) was added to a 48-well plate and incubated at 37 °C for 30 min. hDMVECs (n = 4 for each cell model prepared as described above) were trypsinized and resuspended in EC basal medium without supplements. Each type of cell (2·5–4 9 10⁴ in 200 μl) was then added into each well and incubated at 37°C with 5% CO₂ for 12–16 h to form a CLS. The cells were fixed with 4% buffered formalin at the end of the experiments. Images were acquired using a Nikon Eclipse Ti-E system. The CLS wall thickness, perimeter and area of branching point were outlined from the acquired images and directly measured with Nikon NIS-Elements software. The total sample sizes for capillary wall thickness were 47, 46 and 18 CLSs, perimeters were 35, 33 and 12 CLSs, and area of branching points were 155, 79 and 24 CLSs.

Tan W., Wang J., Zhou F., Gao L., Yin R., Liu H., Sukanthanag A., Wang G., Mihm MC J.r., Chen D.B, & Nelson J.S. (2017). Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation. The British journal of dermatology, 177(6), 1601-1611.

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3D Matrigel Cell Culture Protocol

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A total of 12 µL Matrigel (Corning, 356234) was placed in a 24-well plate and incubated for 20 minutes at 37°C to solidify Matrigel. A total of 5,000 cells were resuspended in 6 µL of a 1:1 mixture of Matrigel and DMEM/F12 media (Gibco, 14170-112). The cells were then placed on top of the solidified Matrigel and incubated for an additional 20 minutes at 37°C to solidify. A final 12 µL Matrigel was placed on top and allowed to incubate 20 minutes longer at 37°C. The resulting Matrigel “plug” was then covered with 1 mL warm DMEM/F12 w/GlutaMAX (Gibco, 10565018) media.

Freeburg N.F., Peterson N., Ruiz D.A., Gladstein A.C, & Feldser D.M. (2023). Metastatic Competency and Tumor Spheroid Formation Are Independent Cell States Governed by RB in Lung Adenocarcinoma. Cancer Research Communications, 3(10), 1992-2002.

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Fibroblast-derived Induced Pluripotent Stem Cell Differentiation Protocol

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A matched set of male human foreskin fibroblasts (HFF) and reprogrammed induced pluripotent stem cells (iPSC) were purchased from System Biosciences. iPSCs were grown on Matrigel (Corning). Plates were coated with Matrigel in accordance with manufacturer protocols, Matrigel was diluted in DMEM/F12 (+ L-glutamine, + 15 mM HEPES, - phenol red; Gibco, Dublin, Ireland). iPSCs were cultured in either exclusively mTesR1 media (STEMCELL Technologies) or a combination of mTesR1 and StemFlex (Thermo Fisher Scientific) media at 37°C in 5% CO₂. Briefly, the combination culture consisted of passaging iPSCs in StemFlex for the first 1–2 d, to increase yield, then switch back to mTesR1 for the remainder of the culturing period. HFFs were cultured in DMEM (4.5 g/L glucose) supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids and 10% NBCS (Peak Serum) at 37°C in 5% CO₂.
Neural induced differentiation of iPSCs was performed using STEMdiff Neural Induction Medium (STEMCELL Technologies) in accordance with manufacturer's recommendations. Briefly, iPSCs were passaged onto new Matrigel coated plates in StemFlex and left to attach overnight. Approximately 24 h after passaging, media was removed and replaced with STEMdiff Neural Induction Medium. Cells were cultured in STEMdiff Neural Induction Medium for up to 7 d with media changed daily.

Heck A.M., Russo J., Wilusz J., Nishimura E.O, & Wilusz C.J. (2020). YTHDF2 destabilizes m6A-modified neural-specific RNAs to restrain differentiation in induced pluripotent stem cells. RNA, 26(6), 739-755.

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Cyst Formation from Bronchial Epithelial Cells

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To generate the cysts structure, BECs were seeded on the honeycomb microwell (on PDMS-bottom 96-wells O₂ permeable plates) (Fig. 1e) and centrifuged at 30 g for 5 min with minimum acceleration and deceleration to retain the BECs inside the microwell. Cultures were incubated at 37 °C in 5% CO₂. A series of Matrigel (Gibco) supplementations were conducted 1 day after inoculation (Fig. 1f) to determine the optimum condition for cyst formation: 0, 150, 300, 600 μg/ml and 1, 3, 6 mg/ml, and original Matrigel concentration (9.2 mg/ml). Culture medium was changed every other day with Matrigel-supplemented culture medium. To investigate the aggregation and conformation of BECs into cysts, a real-time recording (JuliBr Live Cells Movie Analyzer) was performed within 24 h with 1 h intervals (Fig. 2d).

Rizki-Safitri A., Shinohara M., Miura Y., Danoy M., Tanaka M., Miyajima A, & Sakai Y. (2018). Efficient functional cyst formation of biliary epithelial cells using microwells for potential bile duct organisation in vitro. Scientific Reports, 8, 11086.

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3D LUAD Tumor Sphere Culture

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Primary 3D tumor sphere cultures were generated from tumors isolated
from 30–34 week-old mice bearing 17–22 week old LUAD tumors.
The entire primary culture was used in the downstream experiments as
described in the manuscript.
Mouse cultures were plated on Matrigel as previously described
(Tammela et al., 2017 (link)). Briefly,
350–1000 KP primary mouse LUAD cells were mixed in
50% Matrigel (Corning, catalog #CB-40230C) and 50% Advanced DMEM/F12 (Gibco,
catalog #12634028) and plated on 10–12 μl of Matrigel on an 8
chambered coverglass (Thermofisher, catalog #155379). The solution was
allowed to solidify at 37° C and then Advanced DMEM/F12 supplemented
with Gentamicin, Penicillin-Streptomycin (Gibco, catalog #15140163), 10 mM
HEPES (Gibco, catalog #15630080), and 2% heat-inactivated fetal bovine serum
was added to fully cover the Matrigel plug. Cultures were grown in standard
tissue culture conditions at 37° C. Media was refreshed every
1–3 days.

Marjanovic N.D., Hofree M., Chan J.E., Canner D., Wu K., Trakala M., Hartmann G.G., Smith O., Kim J., Evans K.V., Hudson A., Ashenberg O., Porter C.B., Bejnood A., Subramanian A., Pitter K., Yan Y., Delroy T., Phillips D.R., Shah N., Chaudhary O., Tsankov A., Hollmann T., Rekhtman N., Massion P.P., Poirier J.T., Mazutis L., Li R., Lee J.H., Amon A., Rudin C.M., Jacks T., Regev A, & Tammela T. (2020). Emergence of a high-plasticity cell state during lung cancer evolution. Cancer cell, 38(2), 229-246.e13.

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Inhibition of Tumor Growth by eLIFR-Fc in Xenograft Model

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KP4 cells (5 × 10⁶) were engrafted into the right flank of 6-7 weeks old nude, female mice (NU/J, 002919, The Jackson Laboratory; n = 25 mice). Cells were resuspended in PBS before being mixed 1:1 with Matrigel (CB-40234A, Fisher Scientific) and injected using 100 μL of cell/Matrigel mixture per mouse. Tumors were allowed to grow for 20 days before mice were separated into treatment groups (n = 7 mice per arm). Only mice with firmly engrafted tumors >130 mm³ were separated into treatment groups. Tumor sizes were evenly distributed between groups. Mice were treated with PBS or eLIFR-Fc (10 mg/kg), given 2×/week for 2.5 weeks. Mice were weighed prior to compound administration and throughout the study. Tumors were measured with digital calipers 2×/week at the same time as dosing. Study was concluded when tumor measurements surpassed euthanasia criteria. At the conclusion of the study, tumors were excised, weighed, and measured using digital calipers.

Hunter S.A., McIntosh B.J., Shi Y., Sperberg R.A., Funatogawa C., Labanieh L., Soon E., Wastyk H.C., Mehta N., Carter C., Hunter T, & Cochran J.R. (2021). An engineered ligand trap inhibits leukemia inhibitory factor as pancreatic cancer treatment strategy. Communications Biology, 4, 452.

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Proliferation and 3D Growth Assays

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For cell proliferation assays, PDAC-stable cells overexpressing FLAG-WT-p65 and mutant p65 constructs were seeded in triplicate at 2 × 10⁴ cells/well in a 6-well plate. PANC1 cells were counted at days 3, 5, 7, 9, and 11 days, while MIA PaCa2 cells were counted at days 3, 5, 7, and 9 after seeding using a cell counting chamber. For 3D growth assays, Matrigel (Fisher scientific, Waltham, MA, USA) was used to prepare 3% Matrigel and media cell suspension. 250 cells were seeded and cultured for 5 days at 37 °C and 5% CO₂. On day 5, formed spheroids were imaged and captured using a Canon EOS Rebel T3i Digital SLR camera and treated with Alamar Blue (Sigma-Aldrich, St. Louis, MO, USA) at 10% culture volume. Fluorescence intensity was read using the Synergy H1 Multi-Mode Reader (BioTek Instruments Inc., Winooski, VT, USA) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

Motolani A., Martin M., Wang B., Jiang G., Alipourgivi F., Huang X., Safa A., Liu Y, & Lu T. (2023). Critical Role of Novel O-GlcNAcylation of S550 and S551 on the p65 Subunit of NF-κB in Pancreatic Cancer. Cancers, 15(19), 4742.

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Establishing Colorectal Tumor Organoid Cultures

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Colorectal tumor organoid cultures were performed as described previously^{21 (link)} with minor modifications. Briefly, tumor tissues were mechanically separated from surrounding normal tissues and washed in PBS containing 100 U/ml penicillin and 100 µg/ml streptomycin. These antibiotics were added to all solutions used in the following procedure. After washing with PBS, tumor tissues were cut into small pieces and digested with 200 U/ml type IV collagenase for 2 hours. The digested tumor tissue was passed through a 70-µm cell strainer. Then, 10000 tumor cells were mixed with Matrigel (Fisher Scientific) and seeded in a 24-well plate. 15 minutes after the Matrigel polymerization, DMEM/F12 medium (ThermoFisher Scientific) contains 1× Glutamax (ThermoFisher Scientific), 1 M HEPES (Life Technologies), 1× N2 supplement (ThermoFisher Scientific), 1× B27 supplement (ThermoFisher Scientific), and 50 ng/µl epidermal growth factor (EGF; Sigma) was added to the plate.

Sun D., Wang W., Guo F., Pitter M.R., Du W., Wei S., Grove S., Vatan L., Chen Y., Kryczek I., Fearon E.R., Fang J.Y, & Zou W. (2022). DOT1L affects colorectal carcinogenesis via altering T cell subsets and oncogenic pathway. Oncoimmunology, 11(1), 2052640.

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