For spheroid formation, a suspension containing 2 × 106 cells in 120 µL of agarose-supplemented DMEM was pipetted into micromolds (MicroTissues® 3D Petri Dish®, Sigma–Aldrich) and incubated at 37 °C in a humidified atmosphere with 5% CO2. Caco-2 and HT-29 cells were used. Treatments were as follows: MRK-107 at GI50 and DGI50 (1.13 and 2.26 µM for Caco-2 and 2.4 and 4.8 µM for HT-29, respectively), DOX at 3.68 µM, and untreated spheroids [81 (link)]. The proportion of cell aggregation was monitored at different time points by light microscopy The collection of spheroids for testing was carried out with pipettes and cell dissociation was performed with enzymatic action (0.25% trypsin solution) [82 (link)].
Microtissues 3d petri dish
Manufactured by Merck Group
Sourced in United States
The MicroTissues 3D Petri Dish is a laboratory equipment product developed by Merck Group. It is a specialized cell culture dish designed to facilitate the growth and study of three-dimensional (3D) cellular structures. The product provides a controlled environment for the cultivation of complex tissue models, allowing researchers to investigate cellular behavior and interactions more accurately than traditional two-dimensional cell culture methods.
Automatically generated - may contain errors
Lab products found in correlation
Methocult gf m3434, by STEMCELL (1 mentions)
Agarose gel, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Merck Group (1 mentions)
Collagen 1, by Corning (1 mentions)
Msc medium, by ScienCell (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Ncm460, by INCELL (1 mentions)
8 protocols using microtissues 3d petri dish
1
Spheroid Formation in 3D Culture
2
Engineered Human Heart Tissues
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Human iPSC-CMs, HCVFBs, hADSCs, and HUVECs were constructed into EHHTs as adapted from Richards et al.50 (link) Briefly, agarose hydrogel molds were generated from the MicroTissues 3D Petri Dish (Z764051, Sigma) according to the manufacturer’s protocol and transferred to 24-well plates. Cell suspensions with 50% hiPSC-CMs and 50% non-myocyte (including FBs, HUVECs, and hADSCs at a 4:2:1 ratio) were prepared in culture medium (F12/DMEM with 10% FBS, 1% glutamine, and 1% non-essential amino acids) (Gibco) at a final concentration of 2 × 106 cells/mL, and then 75 μL of the cell suspension was pipetted into each agarose mold and incubated at 37°C for 15 min to let the cells settle into the recesses of the mold. One mL culture medium was then added to submerge the molds and exchanged every 2 days for a total of 10 days.
3
Isolated Urine-Derived Stem Cell Culture
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Human urine samples were collected from 12 healthy male donors aged from 17 to 65 years. Cell pellets were washed with PBS following urine samples being centrifuged. The cells were plated in culture plates with USC medium. USC were cultured at 37 °C in a humidified atmosphere of 5% CO2. Cells at passage (p) 3 were used for all the groups and tests. To assess cell morphology, proliferation and live/dead, USC were seeded into 96-well plates in three culture conditions: i) 2D culture (4 × 10 [3 ] cells/well); ii) 3D sphere in 96-well plates with ULA (4 × 10 [3 ] cells/well); iii) 3D s-SFM in 96-well plates with ULA (4 × 10 [3 ] cells/s-SFM/well), respectively. To generate larger numbers of cells for Western-blot analysis, USC were cultured either in l-SFM for 3D USC-SFM in 12-well or 6-well ULA plates (5 × 10 [5 (link)] cells/l-SFM/well) or in Micro-molds (Microtissues 3D Petri Dish (Sigma, USA)) for 3D spheres with 88 wells. Culture media were changed every other day.
4
Clonal Analysis of Hematopoietic Progenitors
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GMPs were plated in MethoCult GF M3434 (Stem Cell Technologies) for colony forming, following manufacturer’s instructions. For serial colony forming, colonies were scored after 7 days from initial plating, cells were collected, and replated as single-cell suspension.
For tracking colony formation at clonal level, micro-wells were casted using 1.2% low gelling temperature argarose (Sigma, A9045) with MicroTissues® 3D Petri Dish® (Sigma, Z764043). The micro-well gels were submerged in PBS for equilibration at 37 °C for 20 min, followed by x-vivo15 overnight. Medium was removed the next day, and GMPs resuspended at 2,500 cells/mL in 75 μl were loaded into each gel chamber, which were then incubated at 37 °C for 15 min to allow cells settling down into individual wells. Afterward, extra medium supplemented with cytokines was added to cover the entire gels in order to provide sufficient volume and nutrients for cell growth. After 2 days in liquid culture, medium was replaced with MethoCult GF M3434 for colony growing. Schema is shown in Supplementary Fig.3a .
For tracking colony formation at clonal level, micro-wells were casted using 1.2% low gelling temperature argarose (Sigma, A9045) with MicroTissues® 3D Petri Dish® (Sigma, Z764043). The micro-well gels were submerged in PBS for equilibration at 37 °C for 20 min, followed by x-vivo15 overnight. Medium was removed the next day, and GMPs resuspended at 2,500 cells/mL in 75 μl were loaded into each gel chamber, which were then incubated at 37 °C for 15 min to allow cells settling down into individual wells. Afterward, extra medium supplemented with cytokines was added to cover the entire gels in order to provide sufficient volume and nutrients for cell growth. After 2 days in liquid culture, medium was replaced with MethoCult GF M3434 for colony growing. Schema is shown in Supplementary Fig.
5
Isolated Urine-Derived Stem Cell Culture
6
3D Spheroid Invasion Assay
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For invasion assay, spheroids formed by the B16 and A375 cells in 2% agarose gel (Sigma Aldrich Co.) using MicroTissues 3D Petri Dish (Sigma Aldrich Co.) were put in 96-well plate with 10% FBS-containing DMEM medium and collagen I (Corning, Corning, NY, USA), 10 × Dulbecco’s Phosphate Buffered Saline (Sigma Aldrich Co.) and 1 M NaOH containing gel. After solidifying the gel, it was covered with medium. After 48 h, the spheroids were photographed in each well. The extent of invasion was estimated from perimeter/area ratio of spheroids using ImageJ software.
7
Agarose-Embedded MSC Spheroid Formation
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Agarose solution (2%) was melted and then slowly added to a MicroTissues 3D Petri Dish (Sigma). Then, the agarose was solidified into the cell culture mold. MSC (ScienceCell) were inoculated in the agarose mold. After 12 h culture in the mold with MSC medium (ScienceCell), the cells were agglomerated into cell spheres.
8
3D Tumor Spheroid Formation Protocol
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Colorectal cancer cells, SW480 (ATCC, USA) and Caco-2 (ATCC) and the normal colon mucosal cell line NCM460 (INCELL, USA) were grown in Dulbecco's Modified Eagle's Medium (DMEM; GE Hyclone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, GE Hyclone, USA) and 1% penicillin-streptomycin (GE Hyclone, USA) cocktail. Standard culture conditions (37 °C, 5% CO 2 ) were used and the cells were passaged when they reached 80-90% confluence. For 2D cell culture studies, the cells were seeded one day prior to any treatment with initial seeding densities of 37 500 cells per cm 2 , 112 500 cells per cm 2 and 60 000 cells per cm 2 for SW480, Caco-2 and NCM460, respectively. These seeding densities were applied to the following experiments unless otherwise stated. 3D tumor spheroids were formed with the help of nonadherent agarose micro-molds as previously described. 47 Briefly, non-adherent micro-molds were formed by casting 2% agarose solution in a MicroTissues® 3D Petri Dish (array 5 × 7; Sigma-Aldrich). The formed micro-molds thereafter were placed in a 24-well plate, sterilized with UV irradiation for 30 min and soaked overnight with complete DMEM. Thereafter, Caco-2 (1 000 000 cells) or SW480 (500 000 cells) cells were added and allowed to incubate for 24 h to generate 3D tumor spheroids.
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