Name: an improved time- and labor- efficient protocol for mouse primary hepatocyte isolation
Uploaded: 2021-10-25T13:00:00.000+00:00
Duration: 5 min 42 s
Description: Watch this Scientific Journal Video about An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation at JoVE.com

This work was supported by the National Institutes of Health (Grant 5R01HD095512-02 to S.W.).

All procedures were approved by the Johns Hopkins Animal Care and Use Committee. C57BL/6 female mice (8-week old) were used in this study.
1. Preparation:
<ol>
	<li>Mix William&#39;s E Medium (GlutaMAX Supplement) with 10% FBS and 1% antibiotic-antimycotic solution to make the culture medium.</li>
	<li>Filter 25 mL of collagenase-dispase medium (e.g., Liver Digest Medium) through a 0.45 &#181;m syringe filter to remove particle debris.</li>
	<li>Warm 50 mL of double-distilled H2O (ddH2O), 35 mL of perfusion Medium (e.g., Liver Perfusion Medium) (or 50 mL at the first time using this protocol), and 25 mL of filtered collagenase-dispase medium in a 45 &#176;C water bath for 30 min.</li>
	<li>Within a sterile tissue culture hood, mix 2 mL of 10x HBSS and 18 mL of Percoll in a 50 mL tube to make 20 mL 1x Percoll-HBSS and keep on ice or 4 &#176;C. 
	NOTE: 1x Percoll-HBSS can be kept at 4 &#176;C for at least 6 months.</li>
	<li>Within a sterile tissue culture hood, pour 30 mL of wash medium (e.g., Hepatocyte Wash Medium) into a clean Petri dish, and keep on ice.</li>
	<li>Submerge the pumping tube in the water of a 45 &#176;C water bath. Results are most reliable if the room temperature is at 25 &#176;C.</li>
	<li>Prepare 2 mL of 1x anesthetics by mixing 225 &#181;L of Ketamine HCL, 93.75 &#181;L of Xylazine, and 1681 &#181;L of 1x PBS.</li>
	<li>Anesthetize one mouse using an approved method. Here the mouse was intraperitoneally injected with 150 &#181;L of 1x anesthetics. Perform the tests for loss of reflexes such as reaction to toe pinching to ensure full anesthesia.</li>
	<li>Secure the mouse on its back onto the dissection pad by four limbs, by either pining or using water-proof tape or other methods approved by the institution&#39;s Animal Care and Use Committee (or equivalents).</li>
	<li>Prepare sterilized forceps and scissors for dissection. To avoid possible contamination, conduct all steps within a sterile hood.</li>
</ol>
2. Procedure:
<ol>
	<li>Using a peristaltic pump, start pumping warmed-up ddH2O at a speed of 4 mL/min for 5 min. Change the pumping tube from water to a warmed-up perfusion medium.</li>
	<li>Disinfect the anesthetized mouse&#39;s abdomen with 70% EtOH and cut open with a scissor to expose the liver, portal vein, and inferior vena cava (IVC).</li>
	<li>Stop the peristaltic pump. Insert a 24 G catheter (e.g., Closed IV Catheter, 24 G, 0.75 IN) into IVC. Start pumping and cut the portal vein open.</li>
	<li>Continue pumping until the flushed-out liquid is clear (around 3-5 min). Press the portal vein every minute to let liquid reach every corner of the liver. Be careful not to let air bubbles enter the IVC. 
	NOTE: This step is to flush out as much blood as possible from the liver.</li>
	<li>Change the pumping tube from the perfusion medium to the collagenase-dispase medium. Continue pumping until all 25 mL of the collagenase-dispase medium is depleted while doing step 2.6.</li>
	<li>Press the portal vein every minute to let liquid reach every corner of the liver. 
	NOTE: At this stage, the complete loss of blood is fatal to the mouse. The death of the mouse can be confirmed by a lack of heartbeat after the experiment. Dispose of the carcass as per facility policies.</li>
	<li>Isolate the whole liver out without gallbladder to the 30 mL wash medium in the Petri dish on ice.</li>
	<li>Tear it up into pieces with forceps to release primary hepatocytes into solution. This step would turn the wash medium into a cloudy solution full of released primary hepatocytes and small liver pieces.</li>
	<li>Filter the cloudy solution in step 2.8 through a 70 &#181;m cell strainer into the 20 mL 1x Percoll-HBSS in a 50 mL tube on ice. Mix by inverting the tube 20 times.</li>
	<li>Centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Within the tissue culture hood, aspirate the supernatant. Wash the pellet with cold 30 mL of wash medium.</li>
	<li>Centrifuge at 50 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the pellet in 25 mL of the culture medium (or appropriate other volumes) within the tissue culture hood.</li>
	<li>Count the cell number and plate the cells on desired culture plates according to the experimental design. 
	NOTE: Primary hepatocytes properly isolated from one 8-week-old mouse are usually sufficient to be plated on four 6-well plates or four 12-well plates.</li>
</ol>

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The authors have nothing to disclose.

Various primary hepatocyte isolation protocols have been developed. They also have been kept optimized and adapted based on different needs (Table 1). Isolation protocols are generally composed of two parts: perfusion (including enzyme digestion) and purification.
The perfusion can be performed with the entire liver in vivo2,20,21,22,23 or with dissected liver lobes24. The perfusion of dissected lobes was generally restricted to the left and medium lobes for technical ease. Theoretically, in vivo perfusion should yield more primary hepatocytes due to the usage of the entire liver with all lobes. Keeping the liver in place during perfusion may also reduce cell death since hepatocytes in vivo can be bathed in fluid, either blood or perfusion buffers, at all times during this step.
The maintenance of sterile conditions also plays an important role in this step. If the condition permits, it is better to conduct perfusion in a clean hood. All the tools with direct touch to tissues should be sterile, which can be achieved by autoclave. In order to prevent any contamination from bacteria and fungi, an antibiotic-antimycotic solution was added into the culture medium.
The IVC and portal vein are two main vessels connecting the liver with other organs. Most protocols utilize either of these veins to insert needles for perfusion: IVC2,20,21,23, portal vein22. The position of the portal vein is at a skewed angle, which leads to difficulty in positioning and stabilizing the inserted needle. A suture is thus usually needed to tie the needle in place with a surgical knot, which must be done very carefully as the vein is prone to damage. Generally, the diameter of the portal vein is also smaller than IVC, complicating needle insertion. Considering this, the use of IVC can be more convenient, although in some protocols, the needle is also surgically knotted to the IVC20. It has been reported that perfusion with IVC insertion resulted in less viable primary hepatocytes than portal vein25. However, in the present protocol, we successfully used IVC insertion, and the isolated primary hepatocyte viability (&#62;96% in optimized conditions, measured with Trypan Blue staining according to manufacturer&#39;s protocol) was as high, if not more, as other protocols (varies between 80% and 96% based on protocols and conditions2,22,23,24).
There are two aims for the perfusion step: to flush blood out of liver lobes and digest the liver to release primary hepatocytes. In order to achieve these objectives, at least two buffers should be used, one for blood flux (Flux Buffer), and one for digestion (Digestion Buffer). Most of the protocols prepare HBSS-based Flux Buffer and add collagenase within to make it capable for digestion, as Digestion Buffer. The preparation of these buffers is time-consuming, and the buffers could vary slightly in some delicate properties, like pH, from batch to batch, therefore introducing uninvited variables. Considering this, utilizing commercially available buffers saves valuable labor and time while minimizing these variables. Gibco developed a protocol for primary hepatocyte isolation from adult rats26, based on the use of commercially available Liver Perfusion Medium (as Flux Buffer), and Liver Digestion Medium (as Digestion Buffer). Mouse and rat, as rodents, share high similarities in body properties; one may integrate these two rat-optimized buffers into mouse primary hepatocyte isolation. Indeed, Gon&#231;alves et al.24, successfully carried out the perfusion of dissected liver lobes with these buffers, raising the promise of their use in liver perfusion in vivo. Here we successfully tested Liver Perfusion Medium and Liver Digestion Medium in the present mouse primary hepatocyte protocol, which yielded high-quality viable primary hepatocytes (Figure 3 and Figure 4).
The temperature of the perfusion buffer determines how well primary hepatocytes survive the isolation. If the temperature is too high, the collagenase within Digestion Buffer may have reduced activity; if too low, the primary hepatocytes may suffer cold shock, causing possible compromised isolation yield. The time that buffer takes to flow through perfusion tubing also determines the temperature of buffer when it reaches the liver. In previous protocols, 40 &#176;C2 or 42 &#176;C21 water bath was used. This temperature may be subject to change based on lab conditions, like the environment temperature, length of the perfusion tube, and the type of Peristaltic Pump. In the present protocol, after multiple times of testing, we optimized the water bath temperature to be 45 &#176;C to warm up the buffers.
The purity of isolated primary hepatocytes plays an important role in subsequent experiments, as the higher the purity ratio, the less possible interference. Although the liver is mainly composed of hepatocytes, accounting for 60%-80% by mass27, various types of other cells are also present, like immune cells, stellate cells, and endothelial cells, which are important for hepatic immunological activities. Each of these cells could post a potential interference in later-on experiments28. For instance, stellate cells in the liver also respond to pathogen invasions and liver injuries, involving in scar formation29. In numbers, 5%-8% of the total hepatic cell population is contributed by stellate cells30. Normally, stellate cells are in quiescence, but this status can be broken when stresses are present. Therefore, stellate cell activities may be triggered by stresses introduced during isolation or subsequent treatment if some of these cells remain in the final isolated primary hepatocyte pool. Other types of cells also contribute to a considerable portion of the hepatic cell population, like liver sinusoidal endothelial cells (LSECs), accounting for 20% of the total hepatic cell population31. How to eliminate the presence of these cells has been a puzzling part in primary hepatocyte isolation process. Therefore, purification is a key part in primary hepatocyte isolation, which usually takes advantages of the weight and size differences between different types of cells. By optimizing the centrifuge speed and/or purification buffer, primary hepatocytes can be pelleted down to the bottom of the tube.
Separating live cells from dead cells is also critical in obtaining healthy primary hepatocytes and accurate cell counting. Usually, counting cell number is a must after purification since the results of numerous experiments vary as cell confluence changes. Gradient centrifuge reagents can be used in this step to fulfill this mission, like Percoll. 36%-40% of Percoll in centrifuge pellets down live cells and keeps dead cells in supernatant. We successfully optimized centrifugation speed and time to reduce the non-primary hepatocyte cell population in the present protocol. Cell type-specific markers were used to test the purity of isolated primary hepatocytes here, like other protocols. The hepatocyte markers used here were TTR2, CD9524,32, ASGR133, and ASGR234. Markers for other types of cells include CD45 (immune cell marker), COL1A1 (Stellate cell marker), TIE2 (Endothelial cell marker)2,35. With these markers, the primary hepatocytes isolated with the present protocol showed a high level of purity (Figure 4), comparable to the previous protocols2,24.
During perfusion, collagenase in Digestion Buffer loosens attachment between cells by breaking down collagen within the extracellular matrix. This leads to difficulty in cell attainment during plating. Most of the previous protocols require/recommend pre-coating of plates with collagen2,22 or gelatine24 for better attachment of primary hepatocytes. To our knowledge, while Salem et al.21 and Li et al.20 did not discuss this step, other protocols assessed in this study clearly stated/recommended the usage of pre-coated plates2,22,23,24. In the present protocol, we found that the plate coating was not required for plates of certain types. While we are not sure whether this was because different types of plates, especially if they are from different manufacturers, have different surface smoothness, and whether this varied smoothness, if ever exists, plays an important role in primary hepatocyte attachment, it is beneficial to note so that another step (plate-coating) could be skipped for efficacy. It is also important to note that this protocol generates a significant proportion of terminally differentiated, e.g., diploid hepatocytes, along with mononuclear hepatocytes, similar to in vivo hepatic sinusoid.
In this study, the advantages of previous primary hepatocyte isolation protocols were combined, and the process was simplified as much as possible, using commercially available reagents and eliminating unnecessary steps. The centrifugation steps were successfully reduced to two, which is, to our knowledge, the fewest in published protocols. To confirm the purity and bioactivity of isolated primary hepatocytes, the level of various hepatic cell markers was assessed, confirming that the present protocol could greatly enhance primary hepatocyte purity and reduce other hepatic cell populations, such as immune cells, stellate cells, and endothelial cells. The activity of pharmaceutical biomarkers like ASGR1, ASGR2, CD81, and TLR4 was well-preserved in primary hepatocytes isolated with present protocol, which also had confirmed insulin sensitivity and glucose production activity. The main limitation of this protocol is the expense since all reagents were purchased commercially for efficiency. We did not specifically verify that glycogenosis was intact in primary hepatocytes isolated using the present protocol, and this may need further research for related studies. This protocol has similar perfusion steps to previous ones, like Salem et al.21, Severgnini et al.2 and Li et al.20, and Korelova et al.23, using IVC insertion. Their perfusion and digestion buffers, which may require extra labor to prepare, may also work with the present protocol with little modification of enzyme digestion time. Therefore, combining reagents of prior protocols with steps of the present protocol may also be beneficial, both time- and economically friendly.
In summary, an improved time- and labor-efficient protocol for primary hepatocyte isolation from mouse liver was developed. This protocol utilizes commercially available reagents entirely and can be completed in ~35 min, from dissecting mouse to plating primary hepatocytes, thus providing a useful technique to primary hepatocyte-related studies.

The liver serves as one of the most important organs in the vertebrate body due to the vital role it plays in numerous life-supporting functions like food digestion, blood circulation, and detoxification. Usage of mouse primary hepatocyte in vitro culture is increasingly popular in studies of carbohydrate metabolism and hepatic carcinoma. Therefore, it is important to develop a convenient method for mouse primary hepatocyte isolation while maintaining its innate physiological function. Due to its function as a hub of glucose metabolism, the liver is also central to glucose production and storage1. Experiments with primary hepatocytes in vitro are a must to most glucose metabolism studies. Therefore, for years, various research groups have developed protocols for mouse primary hepatocyte isolation.
The general procedure of mouse hepatocyte isolation is to first flush out blood in the liver with an isosmotic liquid such as phosphate-buffered saline (PBS) or Hanks&#39; Balanced Salt Solution (HBSS) and then use collagenase-containing solution to dissociate hepatocytes. These protocols share a general procedure but differ in reagents and steps based on different needs. However, preparing required reagents and performing isolation steps take time. In developing the present protocol, efficiency was set as a priority, with all reagents ready-to-use and available from the market, and as few steps as possible. The overall goal of this protocol is to provide a fast and labor-efficient method to isolate primary hepatocytes from mouse, without jeopardizing the isolated primary hepatocyte purity and viability.

<table><tbody><tr><td>1x PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>10x HBSS</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>12-well Plate</td><td>FALCON</td><td>353043</td><td>Coating not required</td></tr><tr><td>6-well Plate</td><td>FALCON</td><td>353046</td><td>Coating not required</td></tr><tr><td>anti-AKT</td><td>Cell Signaling</td><td>2920S</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100x), Stabilized</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>anti-FOXO1</td><td>Cell Signaling</td><td>97635S</td><td></td></tr><tr><td>anti-GAPDH</td><td>Cell Signaling</td><td>2118S</td><td></td></tr><tr><td>anti-p-AKT (S473)</td><td>Cell Signaling</td><td>9271L</td><td></td></tr><tr><td>anti-PEPCK</td><td>Santa Cruz</td><td>SC-166778</td><td></td></tr><tr><td>anti-p-FOXO1 (S256)</td><td>Cell Signaling</td><td>84192S</td><td></td></tr><tr><td>Cell Strainer, 70 &amp;micro;m</td><td>CELLTREAT</td><td>229483</td><td></td></tr><tr><td>Closed IV Catheter, 24 Gauge 0.75 IN</td><td>Becton Dickinson</td><td>383511</td><td></td></tr><tr><td>DMEM, no glucose, no glutamine, no phenol red</td><td>ThermoFisher Scientific</td><td>A1443001</td><td></td></tr><tr><td>EnzyChrom Glucose Assay Kit</td><td>BioAssay Systems</td><td>EBGL-100</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>Forskolin</td><td>MilliporeSigma</td><td>F3917-10MG</td><td></td></tr><tr><td>Glucagon</td><td>Sigma-Aldrich</td><td>G2044</td><td></td></tr><tr><td>Goat Anti-mouse IgG Secondary Antibody</td><td>LI-COR</td><td>926-68070</td><td></td></tr><tr><td>Goat Anti-rabbit IgG Secondary Antibody</td><td>LI-COR</td><td>926-32211</td><td></td></tr><tr><td>GraphPad Prism 8</td><td>GraphPad Software</td><td>NA</td><td></td></tr><tr><td>Hepatocyte Wash Medium</td><td>Gibco</td><td>17704-024</td><td></td></tr><tr><td>IBMX</td><td>Cell Signaling</td><td>13630S</td><td></td></tr><tr><td>Insulin</td><td>Lilly</td><td>NDC 0002-8215-01</td><td></td></tr><tr><td>Ketamine HCL (100 mg/mL)</td><td>Hospira Inc</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Liver Digest Medium</td><td>Gibco</td><td>17703-034</td><td>Aliquot within tissue culture hood to 25 mL each in 50 mL tube, and keep in -20 &amp;deg;C freezer</td></tr><tr><td>Liver Perfusion Medium</td><td>Gibco</td><td>17701-038</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Gilson</td><td>Minipuls 2</td><td>Capable of pumping at 4 mL/min</td></tr><tr><td>Petri Dish</td><td>Fisherbrand</td><td>08-757-12</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Sorvall</td><td>Legend RT</td><td>Capable to centrifuge 50 mL tube at 4 &amp;deg;C</td></tr><tr><td>Sodium L-Lactate</td><td>Sigma-Aldrich</td><td>L7022</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>Syringe Filter, PVDF 0.45 &amp;micro;m 30mm diameter</td><td>CELLTREAT</td><td>229745</td><td></td></tr><tr><td>Syringe, 0.5 mL</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>Syringe, 60 mL</td><td>Becton Dickinson</td><td>309653</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Tube, 15 mL</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Tube, 50 mL</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>Water Bath Tank</td><td>Corning</td><td>CLS6783</td><td>Or any water bath tank capable of heating up to 45 &amp;deg;C</td></tr><tr><td>William&amp;rsquo;s E Medium (GlutaMAX Supplement)</td><td>Gibco</td><td>32551020</td><td></td></tr><tr><td>Xylozine (100 mg/mL)</td><td>Vetone Anased LA</td><td>NDC13985-704-10</td><td></td></tr></tbody></table>

an improved time- and labor- efficient protocol for mouse primary hepatocyte isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Watch this Scientific Journal Video about An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation at JoVE.com

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

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Nanyang Technological University

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JoVE Journal

Biology

9.6K Views.  Johns Hopkins University School of Medicine. Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Video: An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Immunology and Infection

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Bioengineering

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Medicine

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 ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...