DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Keep isolated organoids on ice up to 2 h before seeding for 3D culture Seed 200, 400, or 1000 organoids per dome Use of 24-well culture plate	Collect organoids for histology, gene expression, and Western blot analysis

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Keep isolated organoids on ice up to 2 h before seeding for 3D culture Seed 200, 400, or 1000 organoids per dome Use of 24-well culture plate
Downstream tips
Collect organoids for histology, gene expression, and Western blot analysis

Publication protocol

Isolation of Primary Mammary Epithelial Organoids: Primary mammary organoids were prepared from 7- to 10-week-old female mice (ICR or C57/BL6) as previously described (Koledova, 2017b; Supplementary Figure 1A). ICR strain was used for the branching morphogenesis and time-lapse imaging, cell viability and replating assays, and confocal imaging. C57/BL6 strain was used for the rest of the experiments. The animals were obtained from the Central Animal Facility of the Institut Pasteur and the Laboratory Animal Breeding and Experimental Facility of the Faculty of Medicine, Masaryk University. Experiments involving animals were approved in accordance with French legislation in compliance with European Communities Council Directives (A 75-15-01-3), the regulations of Institut Pasteur Animal Care Committees (CETEA), the Ministry of Agriculture of the Czech Republic, and the Expert Committee for Laboratory Animal Welfare at the Faculty of Medicine, Masaryk University. The study was performed by certified individuals (AC, JS, EC, and ZK) and carried out in accordance with the principles of the Basel Declaration.
Briefly, the mice were euthanized by cervical dislocation, the thoracic and inguinal mammary glands were collected, visible lymph nodes were excised, and the pooled mammary glands were finely chopped to approximately 1-mm3 pieces and digested in a solution of collagenase and trypsin [2 mg/mL collagenase (Roche, Switzerland or Sigma, United States), 2 mg/mL trypsin (∗Dutscher Dominique, France or Sigma, United States), 5 μg/mL insulin (Sigma, United States), 50 μg/mL gentamicin (Sigma, United States), 5% fetal bovine serum (Hyclone/GE Healthcare, United States) Dulbecco’s in modified Eagle medium (DMEM)/F12 (Thermo Fisher Scientific, United States)] for 30 min at 37°C with shaking at 100 rpm. Next, the tissue suspension was treated with 20 U/mL DNase I (Sigma, United States) and 0.5 mg/mL dispase II (Roche, Switzerland) and exposed to five rounds of differential centrifugation at 450 × g for 10 s, which resulted in separation of epithelial (organoid) and stromal fractions (Supplementary Figure 1A). The organoids were resuspended in basal organoid medium [BOM; 1× insulin–transferrin–selenium supplement, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, in DMEM/F12 (all from Thermo Fisher Scientific, United States)] and kept on ice up to 2 h before seeding for 3D culture.
3D Culture of Mammary Organoids: Freshly isolated primary mammary organoids were mixed with growth factor reduced Matrigel (Corning, United States) and plated in domes in 24-well culture plate (one dome per well, 70 μL of Matrigel per dome). 200, 400, or 1000 organoids per dome were seeded for histology, gene expression, and Western blot analysis, respectively. After setting the Matrigel for 45–60 min at 37°C, the 3D organoid cultures were overlaid with cell culture medium according to the experiment and incubated at 37°C in a humidified atmosphere with 5% CO2 (Supplementary Figure 1B). The media used were as follows: growth factor medium [BOM supplemented with different growth factors: 2.5 nM FGF2 (Peprotech, United States or Thermo Fisher Scientific, United States), 2.5 nM FGF7, 2.5 nM FGF10, 50 ng/mL EGF (all from Peprotech, United States), 5 nM TGFα (Sigma, United States), or a combination of 10 ng/mL WNT3A and 50 ng/mL R-spondin 1 (W3/R1, both from Peprotech, United States)] and lactation medium {LM; 1 μg/mL prolactin [mouse recombinant prolactin for quantitative polymerase chain reaction (qPCR), Western blot, immunohistochemistry and contraction experiments (Sigma, United States or Peprotech, United States), and sheep pituitary prolactin for confocal and time-lapse imaging, including contraction experiments (Sigma, United States)], and 1 μg/mL hydrocortisone (Sigma, United States) in BOM}. Media containing growth factors were changed every 3 days; LM was changed every 2 days. To induce contraction of lactation organoids grown with mouse recombinant prolactin, 40 μg/mL recombinant oxytocin (Sigma, United States) was used. For time-lapse imaging experiments, organoid cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C on Olympus IX81 microscope equipped with Hamamatsu camera and CellR system for time-lapse imaging. For morphological analysis of organoid development, the organoids were photographed from days 8 to 17 of culture; one image per organoid was taken every hour. The images were exported and analyzed using ImageJ (NIH, United States). For analysis of organoid contraction, the organoids were photographed from days 6 to 20 of culture. On each imaging day, the photographs were taken every second for 120 s. The images were exported to video at 10 frames per second using xCellence software (Olympus, Japan).
Replating of Organoids: To replate organoids, 3D cultures were rinsed with phosphate-buffered saline (PBS) and disintegrated by pipetting up and down in ice-cold PBS with a 1000 μL pipette. Successful disintegration of Matrigel was checked under a microscope. Organoid suspensions were centrifuged at 450 × g for 3 min. Organoid pellets were resuspended in fresh Matrigel and plated as described above. Organoids were maintained in BOM or in BOM supplemented with 2.5 nM FGF2; the medium was changed every 3 days. Organoid area was measured in ImageJ.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media Mouse primary mammary ephitelial cells- organoids using DMEM/F-12 from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for DMEM/F-12 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Mouse primary mammary ephitelial cells- organoids using DMEM/F-12 from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.