Isolation and Culture of Mouse Kupffer Cells

Abstract

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.

Protocol

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) Prepare freshly all reagents described in the material table. Warm the EGTA (Ethylene Glycol Tetra-acetic Acid)/HBSS (Hank's Balanced Salt Solution) Solution (50 ml per mouse) and the Collagenase Solution (100 ml per mouse) for 30 min at 40 °C. Rinse …

Representative Results

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 …

Materials

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.