Isolation and Culture of Mouse Kupffer Cells

Published: July 31, 2023

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 …

Açıklamalar

The authors have nothing to disclose.

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.

Etiketler

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Bu Makaleden Alıntı Yapın
Isolation and Culture of Mouse Kupffer Cells. J. Vis. Exp. (Pending Publication), e21515, doi: (2023).

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