Conventional HUVEC‐spheroids (~200 μm diameter) were prepared using non‐adherent agarose molds in accordance with the manufacturer's protocols to confirm the efficacy of generated one‐step spheroid printing (One‐SP)‐spheroids.43 Briefly, the agarose molds were prepared by casting 2% agarose (in DPBS; Invitrogen, USA) into a 3D Petri dish (Sigma‐Aldrich, USA). Then, the HUVEC suspension (190 μl; 1.4 × 106 cells/ml) was seeded into the agarose mold and cultured with GM.
3d petri dish
Manufactured by Merck Group
Sourced in United States
The 3D Petri Dish is a lab equipment product designed for culturing cells and tissues. It provides a three-dimensional environment for cell growth and development. The product features a circular, dome-shaped structure to facilitate the cultivation of cells in a more natural, physiologically relevant setting.
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5 protocols using 3d petri dish
1
HUVEC Spheroid Formation using Agarose Molds
2
Agarose 3D Petri Dish for Cell Spheroids
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To produce the MDS and CS, we used agarose 3D Petri Dish® made in 12-series micromolds (sigma-aldrich). The desired cells in suspension (1.5 × 106 cells in 190 μl and 3.5 × 105 cells in 75 μl of culture medium respectively for MDS and CS) were added drop-wise to the center of the 3D Petri Dish®. When necessary, 0.5 μm fluorescent beads (Invitrogen Inspeck Green 505/515 100% relative intensity) were added to the cells. After incubation at 37° in 5% CO2 atmosphere for indicated time, MDS and CS of 400–450 μm in diameter were collected by pipetting, washed three times with PBS and then fixed with 10% neutral buffered formalin (Sigma–Aldrich) at room temperature for two hours.
3
3D Spheroid Invasion Assay
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To obtain spheroids, cells were grown in gelled agarose microwells using a 3D Petri Dish (Sigma Aldrich) in DMEM supplemented with 10% FBS and penicillin and streptomycin for 48-72 h. Buffered solution composed of DMEM, 0.375% (w/v) NaHCO3, 8.5 mM NaOH, and 15 mM HEPES was mixed in the 3:1 ratio with 1 mg/mL of collagen solution (Cultrex 3-D Culture Matrix Rat Collagen I; R&D Systems, Minneapolis, MN, USA) and spheroids were embedded into 3D collagen matrix. The polymerized collagen matrix was overlaid with culture media supplied with DMSO or the MO-I-1151 inhibitor. Images of the embedded spheroid were taken at 0 and 72 h. The area of control and treated spheroids before and after invasion were calculated by the ImageJ software and at least three spheroids per condition were analyzed in each experiment.
4
Fabrication of Minispheroid-laden Structures
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According to the manufacturer's protocols, agarose molds were used to prepare the conventional hASC-minispheroids (∼100 μm diameter) to verify the efficacy of generated E-MS-printed minispheroids. Briefly, a 2% agarose hydrogel (in DPBS; Invitrogen, USA) was cast into a 3D Petri dish (Sigma-Aldrich, USA) to fabricate agarose molds. Then, the hASCs (190 μl; 1.7 × 104 cells/ml) were seeded into the prepared molds and cultured for 3 days with the DMEM-L.
To fabricate the conventionally prepared spheroid-laden structures (CS-structure), we printed ColMA (2.5% wt) bioink mixed with a photoinitiator (3% w/v). We prepared hASC spheroids using a normal bioprinting process under the same fabrication conditions as the E-MS-printing without E-field stimulation.
To fabricate the conventionally prepared spheroid-laden structures (CS-structure), we printed ColMA (2.5% wt) bioink mixed with a photoinitiator (3% w/v). We prepared hASC spheroids using a normal bioprinting process under the same fabrication conditions as the E-MS-printing without E-field stimulation.
5
Micromolded Agarose Hydrogels for 3D Cell Culture
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Silicone templates 3D Petri Dish® (Sigma-Aldrich, USA) were utilized to prepare micropatterned agarose hydrogels consisting of multiple recesses with desired geometries [32] . Ultrapure© Agarose (Invitrogen, Carlsbad, USA) powder was autoclaved and dissolved by heating in sterile Mili-Q water to form a 2.3% (w/w) solution which was subsequently added into each silicon template and allowed to solidify at room temperature for 25 min.
Afterwards, solidified micromolded agarose hydrogels were detached from the silicone template using a spatula and transferred to a sterilized 6-well tissue culture plate. Each micromolded agarose hydrogel was equilibrated with 3 mL of culture medium overnight before cell seeding. Toroid-fabricating recesses, arranged in a 6 × 6 array per agarose gel, were each 750 µm deep and 1400 µm wide. Their central pegs and surrounding circular tracks had diameters of 600 µm and 400 µm respectively. Rod-fabricating recesses, arranged in a 15 × 6 array per agarose gel, were each 2200 μm long, 400 μm wide and 750 μm deep.
Sphere-fabricating recesses, arranged in a 9 × 9 array per agarose gel, were each 800 μm deep with a round bottom of 800-μm diamter.
Afterwards, solidified micromolded agarose hydrogels were detached from the silicone template using a spatula and transferred to a sterilized 6-well tissue culture plate. Each micromolded agarose hydrogel was equilibrated with 3 mL of culture medium overnight before cell seeding. Toroid-fabricating recesses, arranged in a 6 × 6 array per agarose gel, were each 750 µm deep and 1400 µm wide. Their central pegs and surrounding circular tracks had diameters of 600 µm and 400 µm respectively. Rod-fabricating recesses, arranged in a 15 × 6 array per agarose gel, were each 2200 μm long, 400 μm wide and 750 μm deep.
Sphere-fabricating recesses, arranged in a 9 × 9 array per agarose gel, were each 800 μm deep with a round bottom of 800-μm diamter.
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