DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Change medium every second or third day Culture organoids for five to seven days	Organoids can be cryo conserved at −150 °C

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Change medium every second or third day Culture organoids for five to seven days
Downstream tips
Organoids can be cryo conserved at −150 °C

Publication protocol

Two to three duodenal biopsies per individual were taken with standard endoscopic forceps during routine gastroduodenoscopy and placed in ice-cold 5 ml cell basal medium (DMEM/F12, 15 mM HEPES (Gibco, Germany), 2mM L-glutamine (Gibco, Germany), 100 u/ml penicillin, and 0,1 mg/ml streptomycin (Gibco, Germany) immediately. Crypt units were isolated according the protocol of VanDussen et al.1 with minor variations. The biopsy samples were washed twice with 5 ml cold basal medium (BM) before they were minced with a scalpel and enzymatically digested in 1 ml BM with collagenase (2 mg/ml, Sigma-Aldrich C9407) at 37 °C. The digest was pipetted every 5 minutes and microscopically controlled. When the tissue structure was mostly dispersed, usually after 20–25 minutes, the digest was filtered through a 100 µm strainer (Falcon, Germany) and strainer rinsed with additional 10 ml of BM. Crypt units were collected by low-threshold centrifugation (150–200 × g) for 5 minutes to separate from single cells and cell debris. In case of high impurity with cell debris the crypt cells were resuspended once more in 10 ml BM and pelleted at 150–200 g for 3 min. Cell pellets were resuspended in 1 ml BM, transferred to 2 ml vials and sedimented at 2000 × g for 5 min. The supernatant was discarded and crypts were carefully resuspended in 120 µl of ice-cold Matrigel matrix (Corning 35623) to enable three dimensional growths. Aliquots à 30 µl were settled in 24 well plates and plates were incubated in cell culture incubator at 37 °C, 5% carbondioxid for 10 minutes to allow the Matrigel to solidify. Afterwards, 250 µl cell culture medium enriched with supplements (CM-S) was added to each well and replaced every second day. Organoids were used for assays or cryo conserved at −150 °C. Therefore, organoids were washed with ice cold BM to remove Matrigel and collected by centrifugation at 2000 × g for 5 min. Organoid pellets were suspended in 1 ml BM, 10% fecal calf serum (FCS, Biochrom, Germany), 10% dimethyl sulfoxide, slowly frozen to −80 °C in cryo freezing container (Nalgene, Germany), and transferred to −150 °C for long-term storage. For further research the cryo conserved organoids were quickly thawed at 37 °C, transferred to10 ml BM, centrifuged at 2000 × g for 5 min, plated with Matrigel and cultured in CM-S medium.
Culture medium to maintain organoids (CM-S): Mouse L-cells that express Wnt3a, R-spondin, and noggin, were commercially purchased (ATCC CRL-3276, Bio Tech Standards, Germany) and conditioned medium (L-WRN) was prepared according instructions and protocol of company and Miyoshi et al.3. Culture medium with supplements (CM-S) was prepared using 50% BM and 50% conditioned L-WRN medium, and further supplemented with 15% FCS, 1 mM N-acetyl-L-cysteine (Sigma, Germany), 1x N-2 supplements (Gibco, Germany), 1 × B-27®supplements (Gibco, Germany), 50 ng/ml epidermal growth factor, 10 mM nicotinamide (Sigma, Germany), 10 nM Leu15-gastrin I (Sigma, Germany), 500 nM A-83–01 (inhibitor for ALK4/5/7; Sigma, Germany), 10 µM SB202190 (p38 MAP kinase inhibitor; Sigma, Germany), and 10 µM Y-27632 (p160 ROCK inhibitor; Tocris, Germany) in accordance with the protocols of Sato et al., VanDussen et al., and Miyoshi et al.1–3. Organoids were cultured with 300 µl culture medium and medium changed every second or third day. After five to seven days, when organoids have formed big-circled structures, they were isolated from Matrigel matrix and splitted. Therefore, organoids were washed once with ice-cold Dulbecco’s phosphate buffered saline (PBS; Gibco, Germany) and sheared by pipetting several times in 500 µl ice-cold PBS. Crushed organoids were transferred into 12 ml vials (Falcon, Germany), diluted with 10 ml PBS, pelleted by centrifugation at 150–200 × g and plated with Matrigel matrix as described above into 6–8 wells.

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