DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Refresh medium was refreshed every 2 days Passage of cultures every 7-10 days at a ratio of 1:4 to 1:8 (depending on the medium used, determining the growth efficiency)

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Refresh medium was refreshed every 2 days Passage of cultures every 7-10 days at a ratio of 1:4 to 1:8 (depending on the medium used, determining the growth efficiency)

Publication protocol

Organoid culture from mouse endometrium: Mice were euthanized by CO2 asphyxiation. Unless otherwise stated, female mice in estrous (as determined using vaginal smears; Caligioni, 2009) (see Fig. S1E) were utilized (2-3 animals per endometrial fragment dissociation). The protocol used to isolate the endometrial fragments was adapted from the method to isolate colonic crypts (Sato et al., 2009). Uterine horns were dissected and longitudinally opened to expose the endometrium, subsequently minced into small pieces and dissociated in a 30 mM EDTA solution in Ca2+/Mg2+-free PBS (PBS0; Life technologies) for 60 min at 37°C (see Kim et al., 2005). After addition of 10% fetal bovine serum (FBS; Sigma Aldrich), the endometrial tissue pieces were allowed to settle and mechanically dissociated by pipetting up and down at least 10 times in ice-cold PBS0. The supernatant was filtered through a 70 μm cell strainer (BD Biosciences), and the remaining tissue pieces were again mechanically triturated. The successive filtered fractions (from 1 to 4; see Fig. S1A) were pooled and centrifuged at 190 g for 5 min at 4°C. The pellet was resuspended in ice-cold DMEM/F12 (Life technologies) and centrifuged again. Finally, the pellet was gently resuspended in a mix of 70% Matrigel (growth factor- reduced; Corning) in DMEM/F12. Thirty μl of the homogeneous suspension was plated in pre-warmed 48- well plates and after solidification, culture medium containing a cocktail of growth and signaling factors (see below) was added. Cultures were kept at 37°C in a 5% CO2 incubator and medium was refreshed every 2 days. Outgrowing organoid cultures were passaged every 7-10 days at a ratio of 1:4 to 1:8 (depending on the medium used, determining the growth efficiency). Organoids were collected and incubated with TrypLE (Life Technologies) for 10 min at 37°C. The suspension was centrifuged, the pellet resuspended in DMEM/F12 and the organoids dispersed by vigorous pipetting yielding mostly single cells. After centrifugation, the cell pellet was resuspended in 70% Matrigel and deposited for organoid growth as described above. Different batches of Matrigel were used throughout the study which did not affect organoid growth characteristics and behavior (data not shown).
After testing several signaling and growth factors (see Results), the optimal medium composition for mouse EMOs was defined as: DMEM/F12 supplemented with penicillin/streptomycin (1%; Life Technologies), Glutamax (2 mM; Life Technologies), B27 (2%; Life Technologies), N2 (1%; Life Technologies), insulin-transferrin-selenium (ITS, 1%; Life Technologies), nicotinamide (1 mM; Sigma Aldrich), EGF (50 ng ml-1; R&D Systems), FGF10 (50 ng ml-1; Peprotech), Noggin (100 ng ml-1; R&D Systems), the TGFβ/Alk inhibitor A83-01 (0.5 μM; Tocris) and finally the WNT activators WNT3A and RSPO1, added as conditioned medium (CM) at the proportions mentioned. The WNT3A- and RSPO1-CM are widely used to grow organoids from other tissues (Barker et al., 2010; Karthaus et al., 2014; Kessler et al., 2015; Sato et al., 2011), and were prepared as described in Willert et al. (2003) and Drost et al. (2016). The cell lines were originally developed by Dr. Nusse’s group (Willert et al., 2003) and Dr. Kuo’ group (Ootani et al., 2009), repectively, and provided to us by Dr. Clevers’ group (Hubrecht Institute, Utrecht, The Netherlands). The Development • Supplementary information Development 144: doi:10.1242/dev.148478: Supplementary information cell lines are also available from ATCC (CRL-2647) and Amsbio (Cultrex R-spondin1 Cells). Every new batch of WNT3A- and RSPO1-CM was quantified using the TOP/FOP assay as described (van de Wetering et al., 2001; Barker and Clevers, 2006; Barker et al., 2010). Biological activity was comparable in different batches (data not shown). In some experiments, recombinant WNT3A (200 ng ml-1; R&D Systems) and recombinant RPSO1 (200 ng ml-1; R&D Systems) were used instead of the CM. ROCK inhibitor (Y27632, 9 μM; Millipore) was added during seeding after organoid dispersion and the first subsequent medium refreshment. Unless otherwise stated, organoids of low passage number (P3-P5) were used for the experiments described. To investigate whether EMOs could develop from individual cells, organoids derived from TdTomato+ endometrium were trypsinized into single cells which were mixed in a 1:1 ratio with single cells derived from wildtype EMOs. The mixture was seeded in Matrigel at a density of 10 cells per well. Pictures were taken using an Axiovert 40 CFL microscope (Zeiss).


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