Dissection of human and mouse embryonic lungs and set-up of organoid culture

Human fetal lung dissection

• Collect lungs in Hibernate A medium & keep it at 4^oC
• On the dissection day need to:
  • Thaw Matrigel in the back of the tissue culture room fridge (takes 3 hr to thaw)
  • Place a 48-well plate (greiner bio-one, Cellstar, Cat No 677102) in the big incubator to warm up the plates, so that the Matrigel will set quickly and form a mound instead of spreading all over the well. 
• Wash tissues with T/C PBS.
• Use forceps to remove esophagus and trachea.
• Treat lung with dispase (8 U/ml PBS) at room temperature for 2 min. (Cut lungs into smaller pieces if big.) 
• Wash off dispase with PBS & keep the lungs in Advanced+++ DMEM/F12 on ice.
• Dissect a small piece of lung under microscope in a 5 cm petri-dish containing some Advanced+++ DMEM/F12; remove mesenchyme to reveal tips with tungsten needles.
• Cut tips with a sharp tungsten needle on the bud necks.
• Transfer tips with a 50 ul pipette to another petri-dish containing some Advanced+++ DMEM/F12.
• At the same time, prepare cold Matrigel in Eppendorf tubes in a T/C hood.
  • Cool Matrigel and 1.5ml Eppendorf tubes in an ice box.
  • 30ul Matrigel per Eppendorf tube.
•  Group 5 tips together and pipette them to a Matrigel Eppendorf prepared above with a 50 ul pipette under microscope. Keep on ice. 
• After all the tips have been transferred to the Matrigel eppendorfs, pipette the tips containing Matrigel above to a 48-well plate. 
  • Avoid bubbles
  • ~ 25 ul each well
  • ~ 5 tips per well
• Place the 48-well plate in a 37^oC incubator for 5 min to solidify the Matrigel
• Add self renewal medium along the side of the well (300 ul per well).
• Fill empty wells with PBS to minimise evaporation from Matrigel wells.
• Incubate in a 37oC CO2 incubator.
• Change medium on Tuesday and Friday; prepare fresh medium weekly
• Passage:
  • 1:4 dilution for the first passage, and then 1:5 dilution for further passages

Category