Source: Bourgognon, M., et al. Kupffer Cell Isolation for Nanoparticle Toxicity Testing. J. Vis. Exp. (2015)
In this video, we demonstrate the isolation of Kupffer cells or liver macrophages from the mouse liver. The liver is perfused with a digestion solution followed by density gradient separation. The isolated adherent Kupffer cells can be used for further analysis.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Perfusion and Cell Collection (Figure 1)
2. Preparation of the Density Gradient for Centrifugation (Figure 2)
3. Kupffer Cell Purification (Figure 3)
Figure 1: Liver Perfusion. After anesthesia of the mouse, the digestive tract is laterally moved to the left of the abdomen in order to make the portal vein (PV) accessible. The PV is cannulated using a slow flow rate (1-3 ml/min) of EGTA/HBSS Solution and the inferior vena cava (IVC) is immediately ruptured to avoid any excess pressure within the liver. Within the first minute of perfusion, the flow rate is gradually increased to 7 ml/min. The Collagenase Solution is then perfused at 10 ml/min until its full digestion is achieved.
Figure 2: Preparation of the density gradient. All procedures are carried out under sterile conditions. The SIP is prepared by mixing 15.3 ml of coated silica particle solution with 1.7 ml of 10 x PBS. A 5 ml of SIP is mixed with 15 ml of PBS to make 20 ml of 25% SIP solution. 10 ml of SIP is mixed with 10 ml of PBS to make 20 ml of 50% SIP solution. A centrifuge tube containing the 50% SIP solution (20 ml) is tilted and the 25% SIP solution (20 ml) is slowly added using a serological 25 ml pipette.
Figure 3: Purification of Kupffer Cells by density gradient centrifugation and cell adhesion. All procedures are carried out under sterile conditions. (A) After the rupture of the Glisson’s capsule, liver cells are filtered through a 100 µm strainer. The suspension is centrifuged at x 50 g to discard hepatocytes (pellets) and collect the non-parenchymal cell fraction (supernatant). This step is repeated 3 times. Non-parenchymal cells are added on top of a discontinuous isotonic gradient and centrifuged at 800 x g for 15 min. Kupffer cells collected from the 25% SIP cushion are purified further by cell adhesion selection. (B) Cells are plated in a 24-well plate and incubated at 37 °C and 5% CO2 for 30 min. Non-adherent cells are washed once with 500 µl of HBSS. Kupffer cells display their adherent morphology 4 hr after plating when f-CNTs can be added to the cells. The scale bar represents 25 µm.
The authors have nothing to disclose.
Euthatal (pentobarbital sodium) | Merial | ||
CD1 mice | Charles River | – | Mouse weight should vary from 35 to 45 g. We advise the use of male CD1 mice as their weight increase rapidly (e.g. a male CD1 mouse of 7 weeks old exceeds 35 g in weight). It is also advised to contact the animal supplier in advance to arrange for the delivery of older animals. Alternatively animals can be in-house to reach the desired weight. |
HBSS (Ca2+ and Mg2+ free, with bicarbonate) | Life Technologies | 14175-053 | HBSS must be Ca2+ free. |
Ethylene Glycol Tetraacetic Acid (EGTA) tetrasodium salt | Sigma-Aldrich | E8145 | EDTA can also be used but EGTA has the advantage of chelating Ca2+ selectively. |
HEPES (1M) | Life Technologies | 15630-056 | 100 ml |
Collagenase type-IV | Worthington | CSL-4 | It is advised to test different batches of collagenase or at least mention to the supplier the product has to be suitable for liver cell isolation |
Low glucose DMEM | Sigma-Aldrich | D5523 | 500 ml |
RPMI (with sodium pyruvate and Glutamax®) | Life Technologies | 12633-012 | 500 ml |
Penicillin/Steptomycin | Life Technologies | 15140-122 | 100 ml |
Fetal Bovine Serum | First-Link | 60-00-850 | 500 ml |
Trypan blue solution | Sigma-Aldrich | T8154 | 100 ml |
Coated silica particle solution (Percoll®) | GE Healtcare | 17-0891-02 | Percoll® is very stable and can be kept for several years |
1x Phosphate-Buffered Saline | Life Technologies | 10010-023 | 500 ml |
10x Phosphate-Buffered Saline | Life Technologies | 70011-036 | 500 ml |
Butterfly blood collection set (23G/305mm long tubing) | BD | 367288 | |
Syringe Filters (0.22 μm Blue Rim) | Minisart | 16534-K | |
Centrifuge Tubes (50 ml Blue Cap) | BD Biosciences, Falcon | 35 2070 | |
Petri dish (90 x 15 mm) | Thermo Fisher Scientific | BSN 101VR20 | |
100 μm cell strainer | BD | 352360 | |
Peristaltic pump | Watson Marlow | SciQ 300 | Rinse tubing before and after each usage with sterile PBS and 70% ethanol. |
24-well plates | Corning | 3526 | |
Centrifuge | Eppendorf | 5810R | |
Serrefine forceps | Hammacher GmbH | Art. Nr. HSE 004-35 / Cat. Nr. 221-0051 | The serrefine forceps allow to clamp the vessel cannulated with the 23G needle without the need of holding the forceps during the perfusion procedure. URL: (http://www.hammacher.de/Laboratory-Products/Clamps-forceps/Serrefines/HSE-004-35-Serrefine::25126.html) |
Cell scraper | BD Biosciences, Falcon | 353086 | Cut blade extremities with a pair of scissors to scrape cells in 24-well plates. |
EGTA (Ethylene Glycol Tetraacetic Acid)/HBSS (Hank's Balanced Salt Solution) Solution | HBSS containing 0.5 mM EGTA and 25 mM HEPES. Adjust pH to 7.4. Prepare 50 ml for each liver to perfuse. | ||
Collagenase Solution | DMEM low glucose containing collagenase type IV at 100 UI/ml, 15 mM HEPES and 1% Penicllin/Streptamycin (v/v). Adjust pH to 7.4. Prepare 100 ml for each liver to perfuse. After adding the collagenase, it is advised to warm up the solution for 30 min before use. This allows the collagenase activity to be optimum. | ||
Kupffer Cell Isolation Medium | RPMI containing, 1% Non-Essential Amino-Acids (v/v),1% glutamax® (v/v) and 1% Penicllin/Streptomycin (v/v). Prepare at least 100 ml for 1-3 livers. | ||
Kupffer Cell Culture Medium | RPMI containing 10% Fetal Bovine Serum, 1% Non-Essential Amino-Acids (v/v),1% Glutamax® (v/v) and 1% Penicllin/Streptamycin (v/v). Prepare at least 100 ml for 1-3 livers. | ||
SIP (solution of isotonic coated silica particles) | Mix 1.7 ml of 10x Phosphate Buffered Saline with 15.3 ml of Percoll® to obtain 17mL of SIP. | ||
25% SIP solution | Mix 5 ml of SIP with 15 ml of 1x Phosphate Buffered Saline | ||
50% SIP solution | Mix 10 ml of SIP with 10 ml of 1x Phosphate Buffered Saline | ||
Lysis Buffer | DMEM media with 0.9% Triton X-100 | ||
PBS/BSA Solution | Prepare fresh Phosphate-Buffered Saline pH 7.4 with 1% Bovine Serum Albumin. |