Gibco™DMEM/F-12, no glutamine

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Generation of bud tip progenitor organoids using serial needle passaging or bulk passaging

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Generation of bud tip progenitor organoids using serial needle passaging or bulk passaging

Publication protocol

Generation of human lung organoids or bud tip progenitor organoids. Generate either human lung organoids (option A), which contain airway-like structures surrounded by lung mesenchyme and a population of cells with alveolar markers, or bud tip progenitor organoids (options B and C). Use option B if doing needle passaging. Use option C if using bulk passaging.
A. Generation and maintenance of human lung organoids. Timing Organoids with airway-like structures and surrounding mesenchyme will take ~50 d to grow and are optimal for use on days 50–85, as this is when the organoids have the maximum amount of airway-like structures. They need to be re-embedded in fresh Matrigel every 2–3 weeks. Add 1% (vol/vol) FBS and 500 ng/mL of FGF10 to foregut basal medium. Add 500 µL of media to each well of spheroids, ensuring that the media completely covers the Matrigel droplets. Change media every 3–5 d. See Table 2 for a summary of the media components. Re-embedding of organoids. Every 2–3 weeks, or sooner if organoids appear to be accumulating cellular debris within lumens or sinking to the bottom of the Matrigel droplet, cut the tip off a P1000 pipette tip and gently scrape the bottom of the well to dislodge the Matrigel droplet(s) containing organoids. Pick up the entire droplet and surrounding media with the pipette tip and move to a Petri dish. Under a dissecting scope in a sterile environment, use a scalpel and 27-gauge needle to gently cut and remove the old Matrigel away from the organoids. Be cautious not to cut the tissue. Once finished, move the now-cleaned organoids to a 1.5-mL snap-cap tube and remove any media. Cut the tip off a p200 pipette tip and transfer 200 µL of fresh Matrigel (kept on ice) to the tube with the organoids; mix and draw up all Matrigel into the p200 tip. Serially pipette ~50 µL of the Matrigel–organoid mixture into single wells in a fresh 24-well plate. Aim to put one to three organoids in each droplet. Place plate in the incubator for ~10 min, or until Matrigel droplets have solidified. Once droplets have solidified, add 500 µL of media.Maintain organoids for the desired time period, repeating Step 20A(ii–v) to re-embed organoids every 2–3 weeks. Once foregut spheroids are placed in Matrigel, it takes roughly 50 d to obtain pseudostratified epithelial structures that contain basal cells, mucus-producing cells and ciliated cells, as well as surrounding lung mesenchymal cell types and primitive alveolar cell types. The organoids can be maintained for more than 100 d; however, we find that the epithelium is gradually lost over time, and utilization between 60 and 85 d is optimal.
B. Generation and maintenance of bud tip progenitor organoids using serial needle passaging ● Timing Epithelium-only cysts will form after ~2 weeks and will contain a nearly homogeneous population of bud tip progenitor cells. If culture is not homogeneous, organoids with clear epithelial cystic structures can be isolated by hand at this time point (2 weeks). This population of epithelial organoids can be maintained and expanded by serial needle passaging for over 120 d in culture. On the day of use, add 50 µg/mL l-ascorbic acid, 0.04 µL/mL monothioglycerol, 10 ng/mL FGF7, 50 nM ATRA and 3 µM CHIR-99021 to basal medium composed of DMEM/F12 + N-2 supplement + B27 supplement + 1× l-glutamine + 1× pen–strep to make bud tip progenitor organoid complete medium (as also detailed in Table 2). Feed foregut spheroids with 500 µL of bud tip progenitor organoid complete medium. Replace media every 3–5 d. Needle passage. After 2–3 weeks, or sooner if organoids appear to be accumulating cellular debris within lumens or sinking to the bottom of the Matrigel droplet, use a P1000 pipette tip to dislodge Matrigel droplets containing organoids. Place the dislodged droplets together into a 1.5-mL snap-cap tube. Using a 1-mL syringe and a 27-gauge needle, draw the Matrigel and cells into the syringe and forcefully expel the contents back into the tube. Repeat two to three times.Spin down the contents of the tube in a benchtop minicentrifuge (e.g., Thermo Fisher Scientific mySPIN 6 Mini Centrifuge, cat. no. 75004061) at full speed (6,300g) for ~3–5 s. It is also acceptable to spin cells down at 300g for 3 min at 4 °C. Epithelial fragments will settle to the bottom, and Matrigel will remain suspended in the media. Under a dissecting scope in a sterile hood, use the needle and syringe to remove the media and Matrigel from the tube, leaving behind the cell pellet. Add ~200 µL of fresh ice-cold Matrigel to the tube using a p200 pipette with the tip cut off. Mix the cells and Matrigel, avoiding bubbles. Draw up all 200 µL of the Matrigel mixture and serially pipette ~25 µL of the mixture into a single well of a 24-well plate. Place the plate in the incubator for ~10 min or until Matrigel droplets have solidified. Add 750 µL of bud tip progenitor organoid complete medium. Repeat the needle passage (Step 20B(iii–viii)) every 2–3 weeks, or at any time to select for the bud tip progenitor population.
C. Generation and maintenance of bud tip progenitor organoids using bulk passaging ● Timing Epithelium-only cysts will form after ~2 weeks and will contain a nearly homogeneous population of bud tip progenitor cells. To generate patterned lung organoids with discrete bud tip and airway-like regions, keep their structure intact during passaging. Bud tip progenitor organoids that are not needle passaged will develop budded epithelial structures and elements of proximal–distal patterning, as well as functional mucus-producing cells on the interior of the structure, after roughly 6 weeks in culture. ▲ CRITICAL Bud tip progenitor organoids that are not needle passaged will secrete mucus into the lumen of the organoid, causing a dense appearance and leading to increased cell death; therefore, these organoids must be cleaned out regularly.On the day of use, add 50 µg/mL l-ascorbic acid, 0.04 µL/mL monothioglycerol, 10 ng/mL FGF7, 50 nM ATRA and 3 µM CHIR-99021 to basal medium composed of DMEM/F12 + N-2 supplement + B27 supplement + 1× l-glutamine + 1× pen–strep to make bud tip progenitor organoid complete medium (as also detailed in Table 2). Feed foregut spheroids with 500 µL of bud tip progenitor organoid complete medium. Replace media every 3–5 d.Passage of bud tip progenitor organoids without a needle. After 2–3 weeks, or sooner if organoids appear to be accumulating cellular debris within lumens or sinking to the bottom of the Matrigel droplet, scrape Matrigel droplets containing bud tip progenitor organoids using a cut P1000 pipette and transfer both the media within the well and the Matrigel droplet to a Petri dish. Use a scalpel to cut away old Matrigel without disturbing the tissue structure, and reembed in fresh Matrigel droplets as described in Step 20A(ii–v) above. These organoids contain bud tip progenitor cells in budded structures at the periphery of the organoid. Interior regions will predominantly show SOX2+ airway-like structures with differentiated mucus-producing cells. They can be needle passaged as described in Step 20B(iii–viii) at any time to select for the bud tip progenitor population. Clearing mucus from non-needle-passaged organoids. Mucus or cellular debris will often accumulate within the lumens of organoids and will appear as an optically dense, dark and granulated substance within the lumen after ~2 weeks in culture without passage. To clear this debris, get a sterile scalpel, a clean 27-gauge needle, a 1-mL syringe and a fresh aliquot of prewarmed DMEM/F12. Transfer organoids plus Matrigel to a Petri dish with prewarmed DMEM/F12 and transfer to a sterile hood with a stereomicroscope. Under the stereomicroscope, manually cut organoids in half with the sterile scalpel. Using the 1-mL syringe with the 27-gauge needle, draw up clean prewarmed DMEM/F12 into the syringe. Gently expel the media through the needle to wash the mucus away from the organoids. Timing See Fig. 1 for an overview of the timeline of the protocol. Splitting hPSCs to establish the cell cultures required takes 0.5–1 h (Steps 1–10). The culture reaches appropriate confluency after 1–2 d. The generation of foregut spheroids from hPSCs takes 9 d (Steps 11–14). It takes roughly 1 h to collect foregut spheroids and culture them in a Matrigel droplet (Steps 15–20). From there, human lung organoids with airway-like structures and multiple immature epithelial and mesenchymal cell types can be generated after roughly 50 d in culture (41 d from foregut spheroid stage; Step 20A(i–v)), whereas bud tip progenitor organoids can be generated within 22 d (14 d from foregut spheroids stage; Step 20B(i,ii)) and can be maintained as proliferating progenitors (Step 20B (iii–ix)) or as budded patterned lung organoids with proximal–distal patterning (Step 20C(i–vi)).


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 hiPSC-derived lung organoids using Gibco™DMEM/F-12, no glutamine from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™DMEM/F-12, no glutamine below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media hiPSC-derived lung organoids using Gibco™DMEM/F-12, no glutamine 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.