Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging

Published: April 30, 2023

Abstract

Source: Van den Bijgaart R.J.E. et al., Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment, J. Vis. Exp. (2016)

This video describes a method of preparing lungs for ex vivo live imaging to study interactions between cancer cells and stromal cells, using agarose for inflation. In the sample protocol, agarose inflation procedure is described before observing live cellular interactions.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Lungs for Ex vivo Live Imaging

NOTE: Try to work as sterile and careful as possible to avoid unnecessary challenges of the immune cells within the lungs.

  1. Inject the mouse intraperitoneal (i.p.) with a lethal overdose of an anesthetic    permitted by the animal protocol approved by IACUC, e.g., 1 ml of 2.5% Avertin. Wait for the mouse to stop breathing and be completely non-responsive to noxious stimuli (hind paw pinch).
    NOTE: Cervical dislocation and carbon dioxide euthanasia should be avoided as it can detrimentally affect lung cell viability.
  2. Immobilize the mouse on a dissection board and sterilize the mouse with 70% ethanol.
  3. Use surgical scissors to first make a transverse epigastric incision through the skin, followed by a similar incision through the peritoneum. Hold the dissection board in a vertical position and cut the descending aorta, so that blood pools down in the abdomen and not in the chest cavity.
  4. Snip a small opening in the diaphragm to release vacuum. Cut along the 10th and 12th rib to excise the diaphragm and get visual access to the lungs.
  5. Use surgical scissors to cut the skin up to the trachea over the ribcage but leave the ribcage intact. Separate the skin from the ribcage. Expose the trachea by removing the surrounding connective tissue, being careful not to damage the trachea itself (Figure 1A).
  6. Snip a small opening approximately 1 mm in diameter in the exposed trachea parallel to the cartilaginous rings, as close to the larynx as possible (Figure 1B). Be careful not to cut completely through the trachea.
  7. Take a 20 G needle and gently insert the needle 4-5 mm into the trachea without any counter force (Figure 1D). The end of the needle should be visible through the trachea (Figure 1C). Use forceps to stabilize the needle in the trachea. Alternatively, a suture may be tied around the trachea to hold the needle in place.
    NOTE: By inserting too deep, the carina may be traumatized or only one side of the lungs might be inflated.
  8. Fill a syringe with 400 µl of 37 °C 2% low-melting-temperature agarose (taken directly from a constant temperature bath). Make sure the dissection board is standing up and slowly instill the warm agarose through the needle into the lungs, use ~400 µl to inflate the lungs.
    NOTE: Watch the lungs inflating inside the ribcage. Do not over-inflate the lung as it will rupture.
  9. Once the lungs are inflated, filling ~⅔ of the rib cage, detach the syringe and keep the needle inside the trachea to prevent any agarose from leaking.
  10. Pour approximately 50 ml of 20 °C PBS over the inflated lungs to allow the agarose inside the lungs to set and solidify. Slowly remove the needle and close the trachea with forceps to prevent any non-solidified agarose from leaking.
  11. Expose the lungs by performing a sternotomy and subsequently excise the lungs. For excision of the lungs, hold on to the trachea while cutting through the trachea completely. Gently pull the trachea up, cut away the connective tissue and esophagus while pulling the lungs out of the chest cavity until the lungs is separated from the mouse (Figure 1E).
  12. Immerse the lungs in warm RPMI-1640 to wash off excessive blood and gently separate the lobes by using scissors and forceps to cut the lobes’ main stem bronchus at hilum (Figure 1F).
  13. Place the lobes, with the flat surface down to maximize imaging surface, in a well of a 24-well imaging plate (Figure 1G). Add 100 µl of 37 °C RPMI-1640 on top of the lobes. Place several 15 mm circular microscope cover slides on top of the lobes to prevent it from floating.
  14. Pour warm PBS into the surrounding wells to prevent the RPMI-1640 media from evaporating. Insert the 24-well plate into the equilibrated climate chamber and maintain the lung lobes at 37 °C with air and 5% CO2. Insert the climate chamber on the stage of the confocal microscope.
    NOTE: Other gas mixtures (e.g., 5% O2, 5% CO2 in N2 to examine cell behavior under conditions of hypoxia/lower oxygen) could also be considered.

Representative Results

Figure 1
Figure 1. Protocol for preparation of lungs for live imaging. (A) Exposure of the trachea after preparation of the mouse. (B) Small snip made in the exposed trachea parallel to the cartilaginous rings. (C) 20 G needle inserted 4-5 mm into the trachea. (D) Instillation of 400 µl 2% low-melting-temperature agarose into the lungs. (E) Inflated lungs separated from the mouse. (F) Lobes separated after inflation. (G) Lobes placed in a well of a 24-well imaging plate. 

Disclosures

The authors have nothing to disclose.

Materials

Anesthetic NA NA Anesthesia approved by IACUC, used for anesthesia and/or euthanesia
PBS, USP sterile  Amresco INC  K813-500ML  Ultra pure grade for i.v. injection
1X PBS  UCSF cell culture facility NA
Styrofoam platform NA NA Will be used as dissection board
Fine scissors sharp   Fine Science Tools 14060-11
20 G x 1 needle, short bevel  BD  305178
Forceps   Roboz Surgical Store RS-5135
1 mL syringe without needle  BD  309659
Low-melting-temperature agarose  Lonza  50111 To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 °C water bath until used for inflation.
RPMI-1640 medium without phenol red  Life Technologies  11835-030
24 well Imaging plate  E&K scientific  EK-42892
Glass cover slides, 15 mm  Fisher Scientific  22-031-144
Digital CO2 and temperature controller  Okolab  DGTCO2BX  http://www.oko-lab.com
Climate chamber  Okolab  NA http://www.oko-lab.com
Cell Observer spinning disk confocal microscope  Zeiss NA
Air  UCSF  NA
Oxygen UCSF  NA
Carbon dioxide UCSF  NA

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Cite This Article
Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging. J. Vis. Exp. (Pending Publication), e20232, doi: (2023).

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