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Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Published: August 13, 2020 doi: 10.3791/60847
* These authors contributed equally


A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.


Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.


Isolated organ perfusion was originally developed to study organ physiology for transplantation1,2,3, and enabled understanding of functions of the organs without interference from other body systems. For example, isolated kidney and heart perfusion was immensely useful in understanding basic principles of hemodynamics and effects of vasoactive agents, whereas liver perfusion was important to understanding the metabolic function, including drug metabolism in healthy and diseased tissue4,5,6,7. In addition, perfusion studies were critical in understanding viability and function of organs intended for transplantation. In Cancer Researchearch, isolated tumor perfusion has been described by several groups using mouse, rat, and freshly resected human tissues8,9. In some isolated tumor perfusion, the tumor was implanted in the ovary fat pad to force the growth of tumor supplying blood vessels from the mesentery artery10. The Jain group performed pioneering studies using isolated perfusion of colon adenocarcinomas to understand tumor hemodynamics and metastasis8,11,12,13. Other innovative engineered ex vivo setups include a 96-well plate-based perfusion device to culture the primary human multiple myeloma cells14 and a modular flow chamber for engineering bone marrow architecture and function research15.

In addition to physiology and pathology studies, organ perfusion has been used to study the basic principles of drug delivery. Thus, one group described isolated rat limb perfusion and studied accumulation of liposomes in implanted sarcomas16, whereas another group performed dissected human kidney perfusion to study the endothelial targeting of nanoparticles17. Ternullo et al. used an isolated perfused human skin flap as a close-to-in vivo skin drug penetration model18.

Despite these advancements in perfusion of large organs and tissues, there have been no reports on in situ perfusion models in mice that: a) bypass clearance organs such as liver, spleen and kidneys; b) include pelvic organs, skin, muscle, reproductive organs (in male), bladder, prostate and bone marrow. Due to the small size of these organs and the supplying vasculature, ex vivo cannulation and establishment of a perfusion circuit has not been feasible. The mouse is the most important animal model in cancer and immunology research, and drug delivery. The ability to perfuse small mouse organs would allow interesting questions regarding drug delivery to these organs, including to tumors implanted in the pelvis (bladder, prostate, ovary, bone marrow), to be answered, as well as studies of basic physiology and immunology of diseases of these organs. To address this deficiency, we developed an in situ perfusion circuit in mice that can potentially avoid tissue injury and is much better suited for functional research than isolated organ perfusion.

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All methods described here have been approved by the University of Colorado’s Institutional Animal Care and Use Committee (IACUC).

1. Pre-heat the perfusion system

  1. Prepare the perfusion system before surgery by starting a 37 °C circulating water bath for all water-jacketed components (perfusate reservoir, moist chamber, and lid) as shown in a customized configuration in Figure 1A. Make sure the tubing is clean and replace if necessary. To limit perfusate volume, use a bubble trap within a moist chamber as the perfusate reservoir (Figure 1B-6).

2. Vascular catheterization

  1. Induce anesthesia in an 8-10 week old BALB/c mouse using an isoflurane veterinary anesthesia machine with 3-5% isoflurane and oxygen flow rate at 0.3 L/min. As an alternative, use ketamine/xylazine or any other type of intraperitoneal anesthesia. Evaluate the depth of anesthesia by 2 methods: toe-pinch and corneal reflex.
  2. Prewet a 4-0 silk suture with needle in double distilled water.
  3. Place the anesthetized mouse in a supine position on a Styrofoam board with the head facing the surgeon and immobilize forelimbs and hind limbs with tape. Wipe the abdomen with isopropyl alcohol and cut the abdomen along the midline in a "T" shape with scissors. Stop bleeding around the edge of the incision by electrocoagulation (cauterizing).
  4. Push the stomach, jejunum and colon to the right side of the abdomen to reveal the abdominal aorta, vena cava, and common iliac and iliolumbar arteries and veins.
  5. Under a dissection microscope, find and ligate the iliolumbar artery/vein in the male, and ovarian artery/vein and the iliolumbar artery/vein in the female using 4-0 silk sutures (Figure 2 yellow lines).
  6. Under a dissection microscope, loop two 4-0 silk sutures underneath the abdominal aorta and inferior vena cava (about 1 cm above iliac artery and vein, 1 mm apart, Figure 2), and make a loose knot in the suture closest to the iliac vessels (Figure 2, white dotted line). Alternatively, a 6-0 silk suture can be used for this knot.
  7. Under a dissection microscope, horizontally align and stretch both the inferior vena cava and the abdominal aorta with porte-aiguille. Use a 24 G winged shielded I.V. catheter to puncture the abdominal aorta, push the button to retract the needle core and insert the catheter about 5 mm into the vessel.
    1. Repeat the same procedure with the inferior vena cava and tie up knots of both sutures around the catheterized vessels.
      NOTE: The needle can easily puncture through the blood vessel; therefore, keep the vessels stretched and needle parallel with the vessel. Retract the needle core as soon as the needle penetrates about 1 mm into the vessel. The abdominal aorta is underneath the inferior vena cava and much thinner and more elastic due to being encased in connective tissue. Therefore, the aorta can “hold on” to the catheter, and should thus be catheterized before the vena cava to reduce the likelihood of the catheter slipping out.
  8. Apply instant glue to immobilize the catheters to the erector spinae, replace the abdominal organs, and end the surgery while maintaining anesthesia.
    NOTE: Organs that cannot be completely replaced into the abdominal cavity will need to be periodically moistened with perfusion medium during the perfusion process.

3. Set up the perfusion system

  1. Transfer the mouse into the water-jacketed moist chamber prewarmed to 37 °C on a silicon pad and immobilize the catheter wings to the pad with 19 G needles.
  2. Fill the arterial catheter’s end (inlet) with prewarmed perfusion buffer (Ringer’s lactate solution supplemented with 5% BSA), and then connect the catheter end with the inlet perfusion tubing using a screw-on connector (Figure 1B, red arrow).
    NOTE: Hold the connector with hemostatic forceps and immobilize the tubing with tape to avoid moving the catheters.
  3. Adjust the peristaltic flow rate to 0.6 mL/min and keep the perfusion outflow (Figure 1B, blue arrow) open-ended for 5-10 min to wash out the blood through the venous catheter. There will be some clots in the outlet catheter; flush out the clots with perfusate buffer before closing the perfusion circuit.
  4. Connect the venous catheter’s end with the outlet tubing using a screw-on connector to close the circuit (Figure 1B). At this point, perform CO2 gas euthanasia and verify by chest puncture or any other method.
  5. Cover the moist chamber with the warmed lid. Check the level of perfusate periodically and add more if needed. Perfusion can be performed for up to 2 hours.
    NOTE: 5 mL of perfusate will be needed to set up the closed perfusion system. If there is no leaking or edema, the volume of perfusate will decrease by less than 1 mL and additional buffer will not be needed. To avoid edema induced by peripheral circulating thrombus, perfusion buffer containing 0.002% heparin can be used in the first 10 minutes of perfusion, but should be changed to buffer without heparin to avoid the leaking at the edge of the incisions.
  6. If needed, add a reagent of choice into the perfusion reservoir or to the injection port at any time (Figure 1B-5). For example, 10 µL of 10 mg/mL Hoechst33342 can be added into the perfusate to stain the cell nuclei 2 h before the end of perfusion, or 50 µL of 1 mg/mL DyLight 649-lectin to stain the vascular endothelial cells 30 min before the end of perfusion.
    NOTE: If attempting to stain bone marrow with Hoechst solution, mice will need to be pre-injected 30 minutes before surgery.
  7. After perfusion with fluorescent reagents, wash out with perfusion buffer for another 10 minutes to minimize the background fluorescence.

4. Analysis of perfused organs

  1. Collect organs including testis, prostate, bladder, femur, muscle, and skin (e.g., feet). Excise a piece of organ about 1 mm3 and flatten between two glass slides.
    1. Study under inverted fluorescent confocal microscope using DAPI/Cy5 excitation and emission filters (excitation lasers : DAPI, 405 nm; Cy5,640 nm). Use at least 200x magnification objective with a 0.45 numerical aperture.
    2. Alternatively, fix the organs with 4% formaldehyde solution for 24 h and perform hematoxylin-eosin staining19.
  2. To create a bone window for intact bone marrow observation, immobilize both ends of the femur or tibia and scrape away the cortical bone with the lateral edge of a 19 G needle to expose the periosteum; take care to keep a thin layer of residual bone. Place bone on a cover slip with the window facing the glass and image with inverted fluorescent confocal microscope using DAPI and Cy5 channels as described above. The cells and vascular network in the bone marrow cavity can be readily observed.

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Representative Results

We set up a closed circuit perfusion system through cannulation of the abdominal aorta and the inferior vena cava of 8-10 week old mice while keeping the volume of perfusion buffer less than 10 mL. Figure 3A shows confocal images after perfusing tissues with Ringer’s solution containing Hoechst 33342 and DyLight 649-lectin. Muscle, bone marrow, testis, bladder, prostate, and foot skin show efficient nuclear and vascular staining. Figure 3B shows hematoxylin-eosin staining of organs after 3 hours of normothermic perfusion.

Figure 1
Figure 1. Simplified perfusion setup. (A) The system includes (1) a pressure/flow rate controller, (2) a circulating heated water bath, (3) a heated moist chamber with heated cover and custom Styrofoam spacer, and (4) a peristaltic pump. (B) The perfusion circuit shows inlet (red lines) and outlet (blue lines), (5) the injection port and (6) the customized perfusion reservoir. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Location of ligated and cannulated blood vessels in male and female mice. In order to outline the blood vessels, 5 µL/kg of 1% Evans Blue dye was added to perfusion medium 30 min before the end of the perfusion. In female mice, both iliolumbar and ovarian arteries and veins are ligated. In male mice, iliolumbar artery and vein are ligated. Yellow and white lines show approximate position of ligation knots; yellow arrows show actual sutures and knots. Both venous and arterial catheters are inserted. Intestines were removed for demonstration purposes. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Confocal and H&E images show successful perfusion of organs for 3 h with no apparent tissue injury. Confocal scale bar: 20 µm for bone marrow and 100 µm for other organs. H&E scale bar: 100 µm. Please click here to view a larger version of this figure.

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The described circuit can be used to probe various research questions, for example the role of different serum components and tissue barriers in drug delivery, or immune and stem cell trafficking. Different drug delivery systems (e.g., liposomes and nanoparticles) can be added to the perfusate in order to understand the role of physiological and biochemical factors in delivery. The duration of perfusion can vary, depending on the tissue studied, scientific goals, and the composition of perfusate. We present here the results of perfusion up to 2 h using a basic perfusion media consisting of Ringer’s solution with lactate and albumin. It must be noted that the goal of the present work was to establish the perfusion circuit rather than developing and optimizing perfusion medium and conditions to support optimal organ/tissue function and oxygenation. The addition of nutrients, vitamins, hormones, and blood cells have been extensively investigated in the previous literature, and numerous perfusion media and oxygenation protocols have been described in dozens of research publications in human and animal tissues7,9,10,11,12,13,16,17,20,21. Refinements of the perfusate composition as well as oxygenation can enable long-term maintenance of tissue metabolism at body temperature. Thus, some groups used red blood cells and blood oxygenation, which greatly improves viability of sensitive tissues17. Perfusion controls are also highly customizable; if necessary, a gas manifold to control oxygenation and pressure manometer can be added. In addition, a larger perfusate chamber can be used, and the physiological parameters such as pressure, temperature, and flow rate can be controlled by a computer.

There are several limitations in the perfusion circuit. The uterus and ovaries in female mice could not be perfused due to the ligation of ovarian artery and vein. Also, we observed that perfusion of testis was incomplete, possibly due to alternative blood supply not included in the perfusion circuit.

With sufficient practice, the cannulation procedure can be performed within 20-30 min with a success rate of over 80%. The success rate highly depends on the ability to cannulate the aorta and vena cava without puncturing the vessel as discussed in Step 2.7. It is important in step 2 to minimize injury to tissues and small blood vessels in the surgical area because of potential leaks and loss of perfusate. In step 2.5, one must always puncture the artery first, and take precaution so that the catheter will not be pulled out. In step 3, when connecting the catheters’ end to the tubing, make sure to hold the catheter tightly using porte-aiguille to avoid moving the needle. Blood pressure is directly proportional to flow rate; therefore, the flow rate of perfusate must be lower than 0.6 mL/min to maintain physiological pressure. Lastly, it is preferred to maintain the heartbeat until the perfusion circuit is closed, as this will improve the perfusion of microvasculature.

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The authors have nothing to disclose.


The study was supported by the NIH grant CA194058 to DS, Skaggs School of Pharmacy ADR seed grant program (DS); National Natural Science Foundation of China (Grant No. 31771093), the Project of International Collaboration of Jilin Province (No.201180414085GH), the Fundamental Research Funds for the Central Universities, the Program for JLU Science and Technology Innovative Research Team (2017TD-27, 2019TD-36).


Name Company Catalog Number Comments
3.5x-90x stereo zoom microscope on boom stand with LED light Amscope SKU: SM-3BZ-80S
Carbon dioxide, USP Airgas healthcare 19087-5283
Confocal microscope NIKON ECLIPSE Ti2
Disposable Sterile Cautery Pen with High Temp FIAB F7244
Moist chamber bubble trap (part 6 in Figure 1) Harvard Apparatus 733692 Customized as the perfisate container; also enabled constant pressure perfusion
Moist chamber cover with quartz window (part 3 in Figure 1) Harvard Apparatus 733524 keep the chamber's temperature
Moist chamber with metal tube heat exchanger Harvard Apparatus 732901 Water-jacketed moist chamber with lid to maintain perfusate and mouse temperature
Olsen-Hegar needle holders with suture cutters Fine Science Tools (FST) 125014
Oxygen compressed, USP Airgas healthcare C2649150AE06
Roller pump (part 4 in Figure 1) Harvard Apparatus 730113 deliver perfusate to cannula in the moist chamber
SCP plugsys servo control F/Perfusion (part 1 in Figure 1) Harvard Apparatus 732806 control the purfusion speed
Silicone pad Harvard Apparatus
Silicone tubing set (arrows in Figure 1) Harvard Apparatus (TYGON) 733456
Student standard pattern forceps Fine Science Tools (FST) 91100-12
Surgical Scissors Fine Science Tools (FST) 14001-14
Table for moist chamber Harvard Apparatus 734198
Thermocirculator (part 2 in Figure 1) Harvard Apparatus 724927 circulating water bath for all water-jacketed components
Three-way stopcock (part 5 in Figure 1) Cole-Palmer 30600-02
Veterinary anesthesia machine Highland HME109
19-G BD PrecisionGlide needle BD 305186 For immobilizing the Insyte Autoguard Winged needle and scratching the cortical bone
4-0 silk sutures Keebomed-Hopemedical 427411
6-0 silk sutures Keebomed-Hopemedical 427401
Filter (0.2 µm) ThermoFisher 42225-CA Filter for 5% BSA-RINGER’S
Permanent marker Staedtler 342-9
Syringe (10 mL) Fisher Scientific 14-823-2E
Syringe (60 mL) BD 309653 Filter for 5% BSA-RINGER’S
1% Evans blue ( w/v ) in phosphate-buffered saline (PBS, pH 7.5) Sigma 314-13-6
10% buffered formalin velleyvet 36692
BALB/c mice ( 8-10 weeks old ) Charles River
Baxter Viaflex lactate Ringer's solution EMRN Medical Supplies Inc. JB2324
Bovine serum albumin Thermo Fisher 11021-037
Cyanoacrylate glue Krazy Glue
DyLight-649-lectin Vector Laboratories,Inc. ZB1214
Ethanol (70% (vol/vol)) Pharmco 111000190
Hoechst33342 Life Technologies H3570
Isoflurane Piramal Enterprises Limited 66794-017-25
Phosphate buffered saline Gibco 10010023



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Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice
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Cite this Article

Ren, P., Yang, C., Lofchy, L. A., Wang, G., Chen, F., Simberg, D. Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice. J. Vis. Exp. (162), e60847, doi:10.3791/60847 (2020).More

Ren, P., Yang, C., Lofchy, L. A., Wang, G., Chen, F., Simberg, D. Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice. J. Vis. Exp. (162), e60847, doi:10.3791/60847 (2020).

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