Material preparation TIMING 20 min
1- Sterilize a beaker containing a magnetic stirrer.
2- Heat the sterile stock agarose solution (1.5% type IV) in the microwave at full power (900 Watts) to liquefy and pour it in a 100 mm culture dish (25 ml/dish). Allow dish to gel at 4 °C for at least 3 hours. PAUSE POINT Gels can be left at 4 °C up to 2 weeks.
3- For assay using transgenic mouse, collect autologous mouse serum. Pay attention to make this experiment in a minimum of time in order to prevent blood clotting.
(A) Kill the mouse by cervical dislocation.
(B) Lay the mouse back down on the dissecting board and fix forelegs upwards and hind legs downwards with pins.
(C) Surface-sterilize the skin using Norvanol D or proprietary compounds.
(D) Remove the skin and sternum to allow a direct access to heart.
(E) Suck about 1 to 1.5 ml of blood with an insulin syringe inserted in the left ventricle of heart still beating.
(F) Transfer blood in a MiniCollect serum tubes and centrifuge at 12,000g for 10 minutes. Keep supernatant at 4 °C until use. TROUBLESHOOTING
Mouse lymphatic thoracic duct dissection TIMING 1h for 3 mice
4- Place an isofluran-soaked Tork paper in a bell-jar and keep it closed to saturate its atmosphere. Place a dish onto the paper to support the mouse in order to avoid direct contact with the isofluran.
5- Sterilize instruments by immersing in Norvanol D or proprietary compounds.
6- Kill the mouse by beheading after making animals unconscious in the saturated-Isofluran bell-jar. CRITICAL STEP Do not leave the mouse a long time in the Isofluran atmosphere, otherwise lymphatic cells would be fixed.
7- Lay the mouse back down on the dissecting board and fix forelegs upwards and hind legs downwards with pins.
8- Surface-sterilize the skin with Norvanol D or proprietary compounds.
9- Cut the skin with sterile scissors and forceps along the abdominal midline and along the left and right anterior sides of the rib cage. Open the sternal plate and cut ribs, leaving the diaphragm intact.
10- On the caudal side of the rib cage, cut the esophagus and the vena cava to remove all thoracic organs with sterile scissors and forceps (Fig. 1b).
11- Fill rib cage with sterile PBS; wash out the blood with sterile gauze pads if needed. CRITICAL STEP Take care of maintaining the rib cage filled with liquid during the dissection. TROUBLESHOOTING
12- Dissect carefully the periaortic fibroadipose tissue with very thin Norvanol-dry microdissection forceps under a binocular microscope (magnification 12X) (Fig. 1c). TROUBLESHOOTING
13- Break carefully the fat beneath the lymphatic duct. It appears as a thin white transparent strand surrounded by fat. For training, it may be useful to identify the lymphatic thoracic duct by preliminary intradermal injection of Evans blue (1.5% PBS) into the paws and ears of donor mice 15 minutes before sacrifice (Fig. 1a).
14- Remove the surrounding fat from the lymphatic duct (Fig. 1d). CRITICAL STEP Take care to clean the lymphatic dust as much as possible. TROUBLESHOOTING
15- When the duct is clean from fat, cut its edges with Vannas scissors and immediately transfer it to a culture dish containing penicillin/streptomycin supplemented ice-cold DMEM culture medium (Fig. 1e). Duct can be preserved for up to 3 hours at 4 °C during the preparation of collagen gels.
Culture of mouse lymphatic thoracic duct rings TIMING 1 h - 1 h 30
16- Punch agarose cylinders out of the previously prepared agarose gel with a 17-mm diameter puncher. Each agarose cylinder is then punch in its center with a 10-mm diameter puncher (24 agarose cylinders can be obtained from each 100 mm culture dish). Remove the inner agarose piece to get final cylinders.
17- Transfer four of the agarose cylinders with a sterile spatula to an 86x12 bacterial Petri dish.
18- Prepare the collagen solution on ice in a sterile beaker containing a magnetic stirrer by mixing 7.5 volumes of Collagen R (2 mg/ml) with 1 volume of 10x MEM, 1.5 volume of NaHCO3 (186 mM) and approximately 0.1 volume of NaOH (1 M) to adjust the pH to 7.4 (according to modification of MEM color from yellow to purple). Keep mixing collagen mixture on ice during all experiment with a moderate magnetic stirrer rotation speed (100 rpm). TROUBLESHOOTING
19- Add 150µl of the collagen solution in each agarose cylinder. Allow it to polymerize 10-20 minutes at 37 °C.
20- During collagen polymerisation, prepare the lymphatic thoracic duct pieces. Manipulator will choose between 2 procedures (option A and B).
(A) First option
In a vertical laminar airflow cabinet and with sterile microdissection forceps, transfer the lymphatic thoracic duct into an 86x12-mm Petri dish whose bottom is entirely recovered by 50% (vol/vol) DMEM/collagen mix. The collagen maintains the duct wet and DMEM avoids full adherence of the duct to collagen. Unfold the duct and cut it transversally with surgical blade into about 1mm-long pieces. According to the initial lymphatic thoracic duct length, it is possible to obtain 10 to 15 pieces from it. After cutting, immediately cover the pieces with 50% (vol/vol) DMEM/collagen mix and take care of maintaining pieces wet. CRITICAL STEP Do never allow the duct to dry.
(B) Second option
In a horizontal laminar airflow cabinet, with sterile microdissection forceps and Vannas scissor and under about 12X magnification binocular microscope, cut the duct in the Petri dish filled with DMEM. By pulling duct with the forceps on one end, cut it with Vannas scissors on the other end. According to the initial lymphatic thoracic duct length, it is possible to cut 10 to 15 pieces from it (Fig. 1f). CRITICAL STEP Try to have pieces of the same length.
21- With sterile microdissection forceps, transfer duct pieces to each previously polymerized collagen gels in agarose cylinder. CRITICAL STEP If manipulator choose option B in the step 20, first soak briefly duct pieces in a mix of 75% (vol/vol) DMEM/collagen before transferring pieces in the final collagen medium contained in the agarose cylinder. This step avoids the formation of a liquid layer surrounding the duct pieces. TROUBLESHOOTING
22- Allow the duct pieces to fix to collagen in agarose cylinder by incubating dishes at 37 °C for 10 minutes. This step can be skipped.
23- Cover the duct by adding 150µl of collagen solution. Allow it to polymerized 10 to 20 minutes at 37 °C.
24- Add 6 ml of supplemented MCDB131 medium. To perform drug response assay, compounds diluted in PBS or DMSO (with a maximum of 0.1%) may be added once at the beginning of experiment to the culture. Depending of used drugs, choose one of the three following culture conditions:
(A) For assay using stimulators of lymphangiogenesis: add 4% Ultroser G to culture medium.
(B) For assay using inhibitors of lymphangiogenesis: add 10% FCS to culture medium.
(C) For assay using transgenic mice: use 3% autologuous mouse serum isolated as explained at step 3.
25- Maintain cultures at 37 °C in a humidified water-jacketed incubator under reduced oxygen conditions (5% O2, 5% CO2 and 90% N2) for 7 to 14 days.
26- Observe rings with a phase contrast microscope after 7, 9 or 11 days of culture.
Immunostaining of lymphatic rings
26- Two methods for immunostaining are conceivable: whole mount fluorescence immunostaining (option A) or immunohistochemistry on paraffin-embedded sections (option B). Immunohistochemistry on frozen-sections is not possible because of the coarse-texture of collagen gel.
(A) First option: Whole mount fluorescence immunostaining
(i) At the end of the culture, harvest lymphatic rings with a little spoon from the agarose cylinder and transfer them in PBS for a wash of 1 hour.
(ii) Fix washed lymphatic rings with 70% (vol/vol) ethanol/H2O or 4% paraformaldehyd for 30 minutes depending on the antibody to be tested. Disperse the rings in a 24-well plate filled with 70% (vol/vol) ethanol/H2O or in NaN3/PBS (0.1%) respectively. PAUSE POINT Rings can be kept for long period at 4°C. Look out for evaporation.
(iii) Wash the lymphatic rings to be used for immunostaining with PBS for 3 rounds of 20 minutes at 50 rpm on an orbital shaker.
(iv) Block unspecific sites with 1.5% BSA-3% powder milk for 1 h at room temperature at 50 rpm on an orbital shaker.
(v) Incubate the lymphatic rings with primary antibody diluted in 0.15% BSA/0.3% powder milk/PBS. Antibody suitable for cryosection staining can be used in our system provided an adaptation of the antibody dilution and incubation time (e.g. rabbit anti-LYVE-1 1/600 overnight at room temperature). To identify the putative unspecific labeling of the gel, a negative control (incubation of a lymphatic ring without the primary antibody but 0.15% BSA/0.3% Powder Milk/PBS) is required.
(vi) Wash 4 times 15 minutes with PBS.
(vii) Incubate lymphatic rings with the appropriate fluorochrome conjugated-secondary antibody as for the first antibody incubation (e.g. swine anti-rabbit-FITC 1/40, 1h30 at room temperature).
(viii) Wash 4 times 15 minutes with P.B.S.
(ix) Mount the lymphatic rings on a microscope slide with Vectashield-propidium iodide or-dapi mounting medium. TROUBLESHOOTING
(B) Second option: Immunohistochemistry of paraffin-embedded lymphatic rings
(i) Fix the lymphatic rings with 4% formaldehyd for 1 hour before transferring them in 70% (vol/vol) ethanol/H2O overnight.
(ii) Transfer the specimens alternatively in a 95% (vol/vol) ethanol/H2O for 2 baths of 1 hour each, isopropanol for 2 baths of 1 hour each, xylene for 2 baths of 1 hour each and finally in paraffin overnight, for a first bath at 56 °C and 1 hour for second bath at 56 °C. CRITICAL STEP Pay attention to locate the piece of lymphatic thoracic duct otherwise gel will not be visible anymore.
(iii) Section paraffin blocks at 5µm on a microtome and let the slides dry on water drop on a Superfrost Plus slide layed on a 40 °C plate.
(iv) Perform usual immunohistochemistry. CRITICAL STEP Tag the specimen location because it becomes invisible.