Gibco™ Advanced DMEM

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ES cell maintenance and passaging. TIMING 30 min. In the biosafety cabinet, coat a 60 mm culture dish with 2 mL of 0.1% gelatin. Leave for 20 minutes and in the mean time proceed to step 2. Warm at least 1 mL of 0.25% Trypsin-EDTA and 20 mL of LIF-2i medium in a 37°C water bath. To begin dissociation of the 80% confluent ES cells, aspirate the spent LIF-2i medium and wash once with PBS. Add 400 μL of 0.25% Trypsin-EDTA and incubate at RT for ~1–2 min. After 30 seconds, shake the plate horizontally to break up the cells. Under a microscope, confirm that the cells have rounded and are detached from the surface of the plate. Collect the dissociated cells into 1 mL of LIF-2i medium and transfer the cells to a 2 mL microcentrifuge tube. Triturate the cells using a P1000 tip to break up clumps into single-cells. CRITICAL STEP Care should be taken to not introduce air bubbles into the medium. Centrifuge for 2–5 minutes at 200 g. While the cell suspension is in the centrifuge, aspirate the 0.1% gelatin from the 60 mm plate from step 1. Leave the plate for ~5 minutes to dry. Discard the supernatant and resuspend the cells from step 7 in 1 mL LIF-2i Medium. Plate the cells on the dried gelation coated plate (from step 8). Select plating density based on when you next wish to split the cells. Density should be between 1:10 (for next passage in ~2 days) and 1:50 (for next passage in ~4 days). Incubate cells at 37°C in 5.0% CO2 until ready for passage. Differentiation day 1: Addition of Matrigel. TIMING 30 min 21. Mix 10.56 mL of Ectodermal differentiation medium with 440 μL of Matrigel to make 11 mL of complete medium. CRITICAL STEP Keep the medium and Matrigel on ice or in a 4°C refrigerator immediately prior to mixing. Matrigel will become gelatinous at temperatures above 15°C. It is important to work quickly and hold the medium up to a light to see that the Matrigel is properly mixed. 22. Warm Ectodermal differentiation medium containing Matrigel in 37°C water bath. 23. Holding the multichannel pipette at an angle, carefully remove 50 μL of media from each well of the plate from step 20 so that the cell aggregate at the bottom of the well remains undisturbed. 24. Add 50 μL of the Ectodermal differentiation medium containing Matrigel to each well. Note that the final concentration of Matrigel is 2% v/v. 25. Mix by pipetting 4–6 times. Return plates to the incubator for 48 hours. Differentiation day 3: Addition of BMP4 and SB-431542 (BMP/SB). TIMING 30 min. 26. Prepare an appropriate amount of Ectodermal differentiation medium for each experimental condition. Typically 6 mL is sufficient for two 96-well plates. 27. Add BMP4 and SB-431542 to the Ectodermal differentiation medium at a 5X concentration. For 6 mL, add 3 μL of BMP4 and 3 μL of SB-431542 (i.e. 0.5 μL per 1 mL). 28. Add 25 μL of Ectodermal differentiation medium containing BMP/SB to each well of the plate from step 26. Each well now contains 125 μL of medium with a final concentration of 10 ng/mL BMP4 and 1 μM SB-431542. Incubate cells for a further 24–48 hours. Differentiation day 4–5: Addition of FGF-2 and LDN-193189 (FGF/LDN). TIMING 30 min. 29. Prepare an appropriate amount of Ectodermal differentiation medium for each experimental condition. Typically 6 mL is sufficient for two 96-well plates. 30. Add FGF-2 and LDN-193189 to the Ectodermal differentiation medium at a 6X concentration. For 6 mL, add 4.5 μL of FGF-2 (i.e. 0.75 μL per 1 mL) and 3.6 μL of LDN-193189 (i.e. 0.6 μL per 1 mL). 31. Add 25 μL of Ectodermal differentiation medium containing FGF/LDN to each well of the plates from step 28. Each well now contains 150 μL of medium with a final concentration of 25 ng/mL FGF-2 and 1 μM LDN-193189. Continue to incubate cells for 3–4 days. Differentiation day 8: Transition to long-term culture TIMING 45 min. 32. Using a P1000 pipette tip, transfer the aggregates from each well into a 15 mL conical tube. Use different 15 mL tubes for different conditions if necessary. 33. Allow the aggregates to settle at the bottom of the tube for 1 minute and then carefully aspirate the media. 34. Add at least 5 mL pre-warmed DMEM/F12 to wash the aggregates. 35. Repeat steps 33 and 34 at least 3 times. 36. Remove excess DMEM/F12. 37. Re-suspend the aggregates in complete Maturation medium containing 1% Matrigel. Transfer the aggregates to a 100 mm bacterial dish using a wide-mouth P1000. Add an appropriate volume of additional Maturation medium to allow 1 mL containing 1–3 aggregates to be distributed into individual wells of a 24-well plate. CRITICAL STEP Use an additional 1–2 mL of Maturation medium to account for medium spreading out across the surface of the plate. 38
Using a wide-mouth P1000 tip, pipet 1–3 aggregates in 1 mL into wells of a 24-well plate. Aggregates in the bacterial plate should be visible without a microscope. Alternatively, single aggregates can be transferred to a new 96-well plate in 150–200 μL of Maturation medium containing Matrigel. 39 Incubate cells for a further 22 days (24 well plates) or 12 days (96 well plates). For 24-well plates, beginning on day 10, replace half of the medium with Maturation medium that does not contain Matrigel every other day (i.e. day 12, 14, 16, etc.). Half of the medium should be replaced in 96-well plates every day beginning on day 9. 40
Under these conditions, organoids can be cultured for up to 30 days (24 well plates) or 20 days (96 well plates). Longer culture durations will likely require different media compositions or culture formats


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