This work was supported by EU FP7-ITN Marie-Curie Network program RADDEL. MB and KAJ would like to acknowledge Joe Varguese and Rui Serra Maia for their help and suggestions along the optimization of the Kupffer cell isolation.

All animal experiments were executed in compliance with all relevant guidelines, regulations and regulatory agencies. The protocol being demonstrated was performed under the guidance and approval of the UK Home office regulation
1. Perfusion and Cell Collection (Figure 1)
<img alt="Figure 1" src="/files/ftp_upload/52989/52989fig1.jpg" /> 
Figure 1: Liver Perfusion. After anaesthesia 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 veina 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.
<ol>
	<li>Prepare freshly all reagents described in the material table.</li>
	<li>Warm the EGTA (Ethylene Glycol Tetra-acetic Acid)/HBSS (Hank&#39;s Balanced Salt Solution) Solution (50 ml per mouse) and the Collagenase Solution (100 ml per mouse) for 30 min at 40 &#176;C.</li>
	<li>Rinse the pump flexible tubing first with 70% ethanol. Pour 40 ml of EGTA/HBSS Solution into a centrifuge tube immersed in the water bath and rinse the pump flexible tubing with pre-warmed EGTA/HBSS Solution.</li>
	<li>Perform terminal anesthesia using a barbiturate to reliably produce unconsciousness before respiratory depression and death. Inject phenobarbitone at 1 mg/kg, i.p. into a female or male CD1 mouse (35-45 g). Confirm the anesthesia by toe pinching.</li>
	<li>Shave abdominal hairs and sterilize the abdominal surface using 70% ethanol solution.</li>
	<li>Cut through the abdominal cavity and expose the portal vein and inferior vena cava by moving the intestine laterally to the left of the abdomen.</li>
	<li>Start the pump at a speed of 1-3 ml/min with EGTA/HBSS Solution and cannulate the portal vein using a 23 g butterfly needle (wings cut). Clamp the section of the portal vein cannulated with the 23 G needle and then flip the serrefine forceps at the surface of the opened mouse abdomen. The liver should quickly pale&#160;within the first 30 sec of EGTA/HBSS Solution perfusion.</li>
	<li>Rapidly incise the lower part of the inferior vena cava to avoid excess pressure building in the liver, and then increase the flow rate gradually to 7 ml/min, over the first minute of perfusion.&#160;The animal dies due to bleeding secondary to vena caval venipuncture.&#160;</li>
	<li>When less than 5 ml of EGTA/HBSS Solution is remaining into the centrifuge tube, fill it up with Collagenase Solution (40-50 ml). Increase the flow rate gradually to 10 ml/min, over 30 sec.</li>
	<li>Make the liver swell by applying pressure to the inferior vena cava, using tweezers, for 5-10 sec intervals. This can be done periodically (5-10 times during digestion). This step will improve the liver cell dissociation and reduce the perfusion time with collagenase.</li>
	<li>When less than 10 ml of Collagenase solution remains within the centrifuge tube, pour 40-50 ml of pre-warmed Collagenase solution into the centrifuge tube.</li>
	<li>After 10-15 min of perfusion and about 70-80 ml of Collagenase Solution perfused, apply a small pressure on the surface of the liver with a forceps. A print of the pressure indicates that liver cells are dissociated.</li>
	<li>Remove the liver from the abdominal cavity as one piece and place it in a centrifuge tube containing 20-30 ml of Kupffer Cells Isolation Medium. Keep liver cells on ice or at 4 &#176;C for a maximum period of 3 hr to avoid affecting liver cell viability.</li>
	<li>Repeat the perfusion procedure on several animals if needed, while keeping the perfused liver on ice. Pool one to three livers for purification and proceed with stage 3.</li>
</ol>
2. Preparation of the Density Gradient for Centrifugation (Figure 2)
<img alt="Figure 2" src="/files/ftp_upload/52989/52989fig2.jpg" /> 
Figure 2: Preparation of the density gradient. All procedures are carried out under sterile condition. 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 are mixed with 15 ml of PBS to make 20 ml of 25% SIP solution. 10 ml of SIP are 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.
<ol>
	<li>Proceed with the following steps (section 2., 3. &#38; 5.) under sterile condition and keep cells on ice or at 4 &#176;C. Prepare the SIP (Solution of Isotonic coated silica Particles) by mixing 15.3 ml of coated silica particle solution with 1.7 ml of 10 x PBS.</li>
	<li>To make 20 ml of 25% SIP solution, mix 5 ml of SIP with 15 ml of PBS. To make 20 ml of 50% SIP solution, mix 10 ml of SIP with 10 ml of PBS.</li>
	<li>Fill one centrifuge tube with 20 ml of the 50% SIP solution. Tilt the centrifuge tube at an angle close to 90&#176; and add slowly the 25% SIP solution (20 ml) on the side wall of the tube using a 25 ml serological pipette, without touching the surface of the 50% SIP layer. When adding the 25% SIP solution, progressively reduce the angle of the tube. Keep the density gradient on ice until use.</li>
</ol>
3. Kupffer Cell Purification (Figure 3)
<img alt="Figure 3" src="/files/ftp_upload/52989/52989fig3.jpg" /> 
Figure 3: Purification of Kupffer Cells by density gradient centrifugation and cell adhesion. All procedures are carried out under sterile condition. (A) After rupture of the Glisson&#8217;s capsule, liver cells are filtered through a 100 &#181;m 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 24-well plate and incubated at 37 &#176;C and 5% CO2 for 30 min. Non-adherent cells are washed once with 500 &#181;l 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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52989/52989fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Put one perfused liver in a Petri dish with 15 ml of Kupffer Cell Isolation Medium. Rupture the Glisson&#8217;s capsule (membrane of the liver) using scissors and release all liver cells into the Kupffer cell isolation medium. Filter the solution though a 100 &#181;m cell strainer to be collected in a centrifuge tube. Repeat the perfusion procedure on additional perfused livers and pool the cells in the same centrifuge tube (15 ml from one mouse, up to 45 ml from three mice).</li>
	<li>Centrifuge the cell suspension at 50 x g for 2 min at 4 &#176;C. Parenchymal cells (hepatocytes) will be in the pellet and non-parenchymal cells (including Kupffer cells) will be in the supernatant.</li>
	<li>Collect the supernatant in a clean centrifuge tube and centrifuge at 50 x g for 2 min each. Repeat this step three more times.</li>
	<li>Centrifuge at 1,350 x g for 15&#160;min to pellet non-parenchymal cells. Discard the supernatant and resuspend the pellet in 10 ml of Kupffer Cell Isolation Medium.</li>
	<li>Add the non-parenchymal cell solution on the discontinuous isotonic gradient 25/50% as described in 2.3. Centrifuge at 850 x g for 15 min without acceleration or break.</li>
	<li>Localize the enriched Kupffer cell fraction appearing turbid within the 25% SIP fraction close to the 25/50% SIP interface (Figure 3). Using a 10 ml serological pipette, aspirate about 12 ml of the enriched Kupffer cell fraction. Transfer cells into a centrifuge tube containing 35-40 ml of Kupffer Cell Isolation Medium. Mix gently and centrifuge at 1,350 x g for 15 min at 4 &#176;C to pellet cells.</li>
	<li>Discard the supernatant and resuspend cells in 5-10 ml of pre-warmed Kupffer Cell Culture Medium. Count cells (haemocytometer) and measure the viability using trypan blue staining.</li>
	<li>Plate the purified non-parenchymal cells in 24-well plates at a density of 5 x 105 cells/well. Incubate the cells at 37 &#176;C and 5% CO2 (Figure 3). After 30 min, gently remove the medium, wash with pre-warmed Hank&#39;s Balanced Salt Solution (HBSS) once then replace it with 500 &#181;l of fresh and pre-warmed Kupffer Cell Culture Medium (per well).</li>
	<li>Leave Kupffer cells for at least 4 hr at 37 &#176;C and 5% CO2 before treatment with nanoparticles, to allow them to acquire their adherent morphology.</li>
</ol>
4. Characterization
<ol>
	<li>Kupffer Cells Purity
	<ol>
		<li>Prepare fresh PBS/BSA solution used to maintain Kupffer cell viability. Wash cells once with 500 &#181;l of PBS/BSA solution. Remove the PBS/BSA solution and detach Kupffer cells in 500 &#181;l of fresh PBS/BSA solution using gentle scraping. When cells are plated in 24-well plates, shorten the extremities of scraper blades with scissors to make the detachment easier to proceed. Transfer cells into flow cytometry tubes.</li>
		<li>Count Kupffer cells using a haemocytometer and centrifuge 1 x 105 cells at 1,350 x g. Resuspend Kupffer cells in 30 &#181;l of F4/80 antibody (neat). Mix well and incubate at room temperature for 30 min. Keep the unstained cells (not incubated with the antibody) on ice.</li>
		<li>Add 2 ml of PBS/BSA solution in stained cells, centrifuge at 1,350 g for 5 min and discard the resulting supernatant. Resuspend stained cells in 200 &#181;l of PBS/BSA.</li>
		<li>For flow cytometry analysis, gate 10,000 unstained Kupffer cells according to their size (forward scatter) and granularity (side scatter). Analyze fluorescence of gated cells in FL-1 channel.</li>
		<li>Repeat the procedure for stained Kupffer cells keeping the same settings used for the flow cytometry analysis of unstained Kupffer cells. Select and quantify the fluorescence of stained cells to assess the purity of Kupffer cell.</li>
	</ol>
	</li>
	<li>Kupffer Cell Phagocytic Activity
	<ol>
		<li>Replace media with 0.5% (v/v) fluorescent beads in Kupffer Cell Culture Medium and incubate for 4 hr at 37 &#176;C and 5% CO2.</li>
		<li>In order to remove non-internalized beads, centrifuge Kupffer cells at 1,350 x g, discard the supernatant and resuspend cells in 500 &#181;l PBS/BSA Solution. Repeat this step 2 more times.</li>
		<li>Scrape Kupffer cells in 500 &#181;l of PBS/BSA Solution and transfer cells into a flow cytometry tube.</li>
		<li>Centrifuge Kupffer cells at 1,350 x g, discard the supernatant and resuspend cells in 200 &#181;l of PBS/BSA Solution.</li>
		<li>For flow cytometry analysis, gate 10,000 unstained Kupffer cells according to their size (forward scatter) and granularity (side scatter). Analyze fluorescence of gated cells in FL-2 channel.</li>
	</ol>
	</li>
</ol>
5. Incubation of Chemically Functionalized Carbon Nanotubes (f -CNTs)
<ol>
	<li>Prepare a dispersion of f-CNTs (1 mg/ml) in water by sonication for 15 min before use. f-CNTs are prepared in-house22.</li>
	<li>Prepare 2x concentrated f-CNTs dispersion in Kupffer Cell Culture Medium. Sonicate f-CNTs dispersed in medium for 2 min before proceeding with step 5.3).</li>
	<li>For each well, replace 250 &#181;l of old media with 250 &#181;l of 2x f-CNTs dispersion. Untreated wells (250 &#181;l of conditioned medium + 250 &#181;l of fresh Kupffer Cell Culture Medium) and wells treated with 10% DMSO are used as negative and positive control, respectively.</li>
	<li>Incubate Kupffer cells at 37 &#176;C and 5% CO2 for 24 or 72 hr.</li>
</ol>
6. Toxicity Assessment of f -CNTs in Kupffer Cells by the Modified Lactate Dehydrogenase (LDH) Assay
<ol>
	<li>Discard the supernatant.</li>
	<li>Add 200 &#181;l (per well of 24-well plate) of Lysis Buffer and incubate at 37 &#176;C for 30 min-1 hr.</li>
	<li>After carrying out vigorous pipetting, collect lysed Kupffer cells into microcentrifuge tubes and centrifuge at 40,000 x g for 10 min to pellet f-CNTs that were taken up by cells and the cell debris.</li>
	<li>Collect the supernatants (&#402;-CNT free) into microcentrifuge tubes and store at -20 &#176;C or proceed to step 6.5).</li>
	<li>Transfer 50 &#181;l into a 96-well plate and add an equal volume of the substrate mix solution (LDH kit) to each well. Cover the plate and incubate at room temperature for 15 min before adding 50 &#181;l of the stop solution (LDH kit). Add triplicates of blank wells containing 50 &#181;l of lysis buffer, 50 &#181;l of substrate mix solution and 50 &#181;l of stop solution.</li>
	<li>Read the absorbance at 490 nm in a microplate reader and use the following formula to calculate the (%) cell viability.</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/52989/52989eq1.JPG" />

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The authors declare that they have no competing financial interests.

The following steps are critical to achieve high yield and high viability of Kupffer cells. Aseptic conditions should be used to limit the risk of bacterial and fungal contamination. All instruments need to be sterilized before use. Reagents should be freshly prepared before carrying out the isolation procedure.
The choice of collagenase IV, with low tryptic activity, is crucial. Different lots from the same supplier have different enzymatic activity and they may need to be compared initially ...

The field of nanotoxicology research aims to characterize the biological effect of nanoparticles. Toxicological studies based on in vivo investigations remain the most accurate methods. However, their use is limited by their cost, labor and time requirements1. As alternatives, in vitro assays have been used because of their simplicity and the possibility of developing high-throughput in vitro testing platforms2 - so extending the number of conditions tested. Most nanotoxicological studies are performed using in vitro assays with immortalized cell lines. However, there are concerns regarding the extrapolation of these experimental findings to in vivo toxicological effects3. Indeed, the properties of immortalized cell lines can be significantly different from tissues they were derived from, e.g. genetic transformation4, deterioration of key morphological features5, loss of cellular polarity6 and functional alterations such as the regulation of inflammatory mediators7.
Kupffer cells are the most abundant macrophage population in the body and are directly in contact with blood by lining the wall of liver sinusoids. As part of the reticulo-endothelial system (RES), these macrophages are responsible for the capture of circulating nanoparticles and therefore, constitute a highly suitable model to study nanoparticle toxicity. In vivo8 and in vitro9 studies of inflammatory responses associated with Kupffer cells exposed to nanoparticles have been published elsewhere. Kupffer cells have also been involved in the pathogenesis of liver diseases such as alcoholic liver disease10, liver fibrosis11 or viral hepatitis12. It was reported that isolated Kupffer cells provide useful insight to describe cellular mechanisms involved in liver disruption13,14.
Several methods have been reported to isolate and purify Kupffer cells. Cell isolation can result from mechanical or enzymatic dissociation15. Collagenase digestion shows the advantage of conserving the functional integrity of Kupffer cells, while leading to high Kupffer cell yield16. Many experimental approaches, varying in complexity and costs, have been used to separate Kupffer cells from other liver cell populations. For example, Kupffer cell purity can be achieved by immunoaffinity17, flow electrophoresis18, selective adhesion16 or by centrifugal techniques16, which select cells according to their size and density. A combination of these methods can be chosen to increase the purity of the population16. There is no consensus about the ideal method for Kupffer cell isolation, as it mostly depends on the application and available equipment. However, in the case of nanoparticle toxicity testing, the simplicity and high yield of the technique associated with the functional preservation of Kupffer cells appeared to be most suitable for this application.
The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method was modified from the original protocol developed by Smedsr&#248;d et al.16 designed for rat liver cell isolation. Most studies reported and described the isolation of Kupffer cells from rat livers. Herein, we describe a method to isolate Kupffer cells from mouse liver, at a high yield and purity. The use of mice reduces the experiment cost and allows the processing of several livers to obtain large amounts of Kupffer cells for nanoparticle toxicity testing.
In the following protocol, Kupffer cells were incubated with functionalized carbon nanotubes (f-CNTs). The unique physico-chemical properties of CNTs &#8211;i.e. high length to diameter ratio and large surface area, have made CNTs interesting candidates as vectors for therapy and diagnosis purposes. However, concerns have been raised regarding the toxicity of CNTs19, and the development of new in vitro tests aims at increasing the understanding of CNT biological effect. Toxicity in Kupffer cell is associated with a lack of structural integrity of the cell membrane. This is measured by the loss of the cytoplasmic enzyme LDH from the cell into the supernatant. The principle of this method, therefore, is to remove any released LDH and measure what is left in the cells20. This is done in preference to measuring the released LDH in the supernatant because the presence of CNTs in the supernatant interferes with the assay21.
We propose the use of this simple and cost effective Kupffer cell isolation method to isolate high number of functional Kupffer cells. This allows the screening of toxicity of a range of nanoparticles, in a relevant primary macrophage model.

<table><tbody><tr><td>Euthatal (pentobarbital sodium)</td><td>Merial</td><td></td><td></td></tr><tr><td>CD1 mice</td><td>Charles River&amp;nbsp;&amp;nbsp;</td><td>-</td><td>Mouse weight should vary from 35 to 45 g. We advise the use of male CD1 mice as their weight increase rapidly (&lt;em&gt;e.g.&lt;/em&gt; 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.&amp;nbsp;</td></tr><tr><td>HBSS (Ca&lt;sup&gt;2+ &lt;/sup&gt;and Mg&lt;sup&gt;2+ &lt;/sup&gt;free, with bicarbonate)&amp;nbsp;&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>14175-053</td><td>HBSS must be&amp;nbsp; Ca&lt;sup&gt;2+&lt;/sup&gt; free.&amp;nbsp;</td></tr><tr><td>Ethylene Glycol Tetraacetic Acid (EGTA) tetrasodium salt&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>E8145&amp;nbsp;&amp;nbsp;</td><td>EDTA can also be used but EGTA has the advantage of chelating Ca&lt;sup&gt;2+ &lt;/sup&gt;selectively.&amp;nbsp;</td></tr><tr><td>HEPES (1 M)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>15630-056&amp;nbsp;</td><td>100 ml&amp;nbsp;</td></tr><tr><td>Collagenase type IV&amp;nbsp;</td><td>Worthington&amp;nbsp;</td><td>CSL-4&amp;nbsp;</td><td>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&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Low glucose DMEM&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>D5523&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>RPMI (with sodium pyruvate and Glutamax)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>12633-012&amp;nbsp;</td><td>500 ml</td></tr><tr><td>Penicillin/Steptomycin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>15140-122</td><td>100 ml</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>First-Link&amp;nbsp;&amp;nbsp;</td><td>60-00-850&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>Trypan blue solution&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T8154&amp;nbsp;</td><td>100 ml&amp;nbsp;</td></tr><tr><td>Coated silica particle solution (Percoll)</td><td>GE Healtcare&amp;nbsp;&amp;nbsp;</td><td>17-0891-02&amp;nbsp;</td><td>Percoll&amp;reg; is very stable and can be kept for several years&amp;nbsp;</td></tr><tr><td>1x Phosphate Buffered Saline&amp;nbsp;&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>10010-023</td><td>500 ml&amp;nbsp;</td></tr><tr><td>10x Phosphate Buffered Saline&amp;nbsp;&amp;nbsp;</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>70011-036</td><td>500 ml</td></tr><tr><td>Dimethyl sulfoxide&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>D/4120/PB08&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>LDH kit (CytoTox 96)&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>G1781&amp;nbsp;</td><td>Keep protected from light&amp;nbsp;</td></tr><tr><td>DMEM phenol red free</td><td>Life Technologies&amp;nbsp;&amp;nbsp;</td><td>31053-028&amp;nbsp;</td><td>500 ml&amp;nbsp;</td></tr><tr><td>F4/80 antibody (Alexa 488)</td><td>AbD Serotec</td><td>MCA497A488</td><td>Do not dilute, used neat for flow cytometry</td></tr><tr><td>Fluorescent beads</td><td>Sigma</td><td>L2778</td><td>Latex beads, amine-modified polystyrene, fluorescent red. 1 ml</td></tr><tr><td>Name of the Material</td><td>Company&amp;nbsp;</td><td>Catalog number&amp;nbsp;</td><td>Comments/Description&amp;nbsp;</td></tr><tr><td>Butterfly blood collection set (23 G/305 mm long tubing)&amp;nbsp;&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>367288</td><td></td></tr><tr><td>Syringe Filters (0.22 &amp;mu;m Blue Rim)&amp;nbsp;</td><td>Minisart&amp;nbsp;</td><td>16534-K&amp;nbsp;</td><td></td></tr><tr><td>Centrifuge Tubes (50 ml Blue Cap)&amp;nbsp;</td><td>BD Biosciences, Falcon&amp;nbsp;</td><td>35 2070&amp;nbsp;</td><td></td></tr><tr><td>Petri dish (90 x 15 mm)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>BSN 101VR20&amp;nbsp;</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer&amp;nbsp;&amp;nbsp;</td><td>BD&amp;nbsp;&amp;nbsp;</td><td>352360</td><td></td></tr><tr><td>Peristaltic pump&amp;nbsp;</td><td>Watson Marlow&amp;nbsp;</td><td>SciQ 300&amp;nbsp;&amp;nbsp;</td><td>Rinse tubing before and after each usage with sterile PBS and 70% ethanol.</td></tr><tr><td>24-well plates</td><td>Corning&amp;nbsp;&amp;nbsp;</td><td>3526</td><td></td></tr><tr><td>96-well plates&amp;nbsp;</td><td>Corning&amp;nbsp;&amp;nbsp;</td><td>3595</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Plate reader</td><td>BMG Labtech</td><td>FLUOstar Omega</td><td></td></tr><tr><td>Serrefine forceps</td><td>Hammacher GmbH&amp;nbsp;</td><td>Art. Nr. HSE 004-35 / Cat. Nr. 221-0051</td><td>The serrefine forceps allow to clamp the vessel cannulated with the 23 G 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)&amp;nbsp;</td></tr><tr><td>Flow cytometry tubes</td><td>BD Biosciences, Falcon&amp;nbsp;</td><td>352052</td><td></td></tr><tr><td>Microcentrifuge tubes (1.5 ml)</td><td>Elkay</td><td>&amp;nbsp; 000-MICR-150</td><td></td></tr><tr><td>Cell scraper</td><td>BD Biosciences, Falcon</td><td>353086</td><td>Cut blade extremities with a pair of scissors to scrape cells in 24-well plates.</td></tr><tr><td>Name of the Reagent</td><td>Company&amp;nbsp;</td><td>Catalog number&amp;nbsp;</td><td>Comments/Description&amp;nbsp;</td></tr><tr><td>&amp;nbsp;EGTA (Ethylene Glycol Tetraacetic Acid)/HBSS (Hank&#039;s Balanced Salt Solution) Solution&amp;nbsp;</td><td></td><td></td><td>HBSS containing 0.5 mM EGTA and 25 mM HEPES. Adjust pH to 7.4. Prepare 50 ml for each liver to perfuse.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Collagenase Solution&amp;nbsp;</td><td></td><td></td><td>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.&amp;nbsp;</td></tr><tr><td>Kupffer Cell Isolation Medium&amp;nbsp;</td><td></td><td></td><td>RPMI containing, 1% Non-Essential Amino-Acids (v/v),1% glutamax&amp;reg; (v/v) and 1% Penicllin/Streptomycin (v/v). Prepare at least 100 ml for 1-3 livers.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Kupffer Cell Culture Medium&amp;nbsp;</td><td></td><td></td><td>RPMI containing 10% Fetal Bovine Serum, 1% Non-Essential Amino-Acids (v/v),1% Glutamax&amp;reg; (v/v) and 1% Penicllin/Streptamycin (v/v). Prepare at least 100 ml for 1-3 livers.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>SIP (solution of isotonic coated silica particles)</td><td></td><td></td><td>Mix 1.7 ml of 10x Phosphate Buffered Saline&amp;nbsp; with 15.3 ml of Percoll&amp;reg; to obtain 17mL of SIP.</td></tr><tr><td>25% SIP solution&amp;nbsp;</td><td></td><td></td><td>Mix 5 ml of SIP with 15 ml of 1x Phosphate Buffered Saline&amp;nbsp;&amp;nbsp;</td></tr><tr><td>50% SIP solution&amp;nbsp;</td><td></td><td></td><td>Mix 10 ml of SIP with 10 ml of 1x Phosphate Buffered Saline&amp;nbsp;</td></tr><tr><td>Lysis Buffer</td><td></td><td></td><td>DMEM media with 0.9% Triton X-100</td></tr><tr><td>PBS/BSA Solution</td><td></td><td></td><td>Prepare fresh Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin.&amp;nbsp;</td></tr></tbody></table>

kupffer cell isolation for nanoparticle toxicity testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

The number of non-purified parenchymal cells was consistent and ranged between 8 and 14 x 106 cells per mouse (17 isolations were performed). Each mouse isolation was sufficient to plate 16 to 28 wells. Cell viability by trypan blue staining showed cell viability ~95%. Kupffer cells showed a round shape within 30 min of incubation at 37 &#176;C, related to their incomplete adherent morphology (Figure 4A). At 4 hr of incubation and afterwards, cells were spread and cell clusters started to form...

Watch this Scientific Journal Video about Kupffer Cell Isolation for Nanoparticle Toxicity Testing at JoVE.com

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

kupffer-cell-isolation-for-nanoparticle-toxicity-testing

Research

JoVE Journal

Immunology and Infection

23.7K Views.  King's College London. Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells. 

Video: Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Environment

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Bioengineering

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Cancer Research

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Neuroscience

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Biology

Isolation and Culture of Mouse Kupffer Cells

Source: Bourgognon, M., et al. <a href="https://app.jove.com/v/52989">Kupffer Cell Isolation for Nanoparticle Toxicity Testing.</a> 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.

Source: Bourgognon, M., et al. Kupffer Cell Isolation for Nanoparticle Toxicity Testing. J. Vis. Exp. (2015)
In this video, we demonstrate the isolation ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

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