Protocol for isolation of lipid-laden hepatocytes from high-fat diet-fed and metabolically challenged mice

Summary

We present a protocol to isolate hepatocytes from mice fed a high-fat diet (HFD), modeling features of non-alcoholic fatty liver disease (NAFLD) and metabolically associated fatty liver disease (MAFLD). We perfuse and enzymatically digest the liver and then purify hepatocytes using a modified Percoll gradient (30%, 25%, and 10%) to retain lipid-laden cells. This protocol enables the isolation of both normal and lipid-filled hepatocytes for culture and downstream studies of drug metabolism, hepatotoxicity, and glucose or lipid metabolism in vitro.

Graphical abstract

Highlights

• •Steps for isolating hepatocytes from HFD-fed mice while preserving lipid-laden cells
• •Instructions to adjust Percoll gradients for recovery across age and diet conditions
• •Guidance on flow rates for cell viability and minimal vascular damage in obese models
• •Isolation of normal and lipid-filled hepatocytes for metabolic and imaging analyses

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

We present a protocol to isolate hepatocytes from mice fed a high-fat diet (HFD), modeling features of non-alcoholic fatty liver disease (NAFLD) and metabolically associated fatty liver disease (MAFLD). We perfuse and enzymatically digest the liver and then purify hepatocytes using a modified Percoll gradient (30%, 25%, and 10%) to retain lipid-laden cells. This protocol enables the isolation of both normal and lipid-filled hepatocytes for culture and downstream studies of drug metabolism, hepatotoxicity, and glucose or lipid metabolism in vitro.

Before you begin

Hepatocytes derived from HFD-fed or metabolically compromised mice exhibit substantial intracellular lipid accumulation,¹ which alters their buoyant density and complicates isolation. The isolation of lipid-laden hepatocytes is essential for metabolic research, as lipid droplet sizes vary with disease progression and animal age.² Lipid droplet size and distribution vary with disease progression, age, and liver zonation,³ necessitating careful optimization of perfusion and purification parameters. Compared with hepatocytes from young, healthy mice, those from steatotic livers are more fragile and require lower perfusion flow rates to maintain viability. Standard hepatocyte isolation methods often fail to recover lipid-rich hepatocytes because these cells are less dense and are lost during high-density Percoll purification. To address this limitation, we describe a protocol in which the Percoll gradients (≤20%–30%) were adjusted based on diet duration and metabolic status to retain both normal and lipid-laden hepatocytes. This approach improves recovery and yields a more representative hepatocyte population for downstream metabolic, imaging, and gene expression analyses.⁴ We adapted this protocol from published methods with modifications to improve the recovery of lipid-laden hepatocytes.^5,6 Filter-sterilize all buffers (0.2 μm) before use. Prepare a 90% Percoll stock solution by mixing 90 mL Percoll with 10 mL 10× PBS.

We used C57BL/6 J mice for hepatocyte isolation. C57BL/6 J mice were housed under standard conditions and fed a high-fat diet (HFD) or a chow diet, as specified in the experimental design. Mice were initially fed a standard chow diet for 3 months. High-fat diet (HFD) feeding was then initiated based on the total age of the experimental group.

 6-months group: 3 months chow + 3 months HFD.

 9-months group: 3 months chow + 6 months HFD.

 12-months group: 3 months chow + 9 months HFD.

Innovation

Conventional hepatocyte isolation protocols using fixed Percoll gradients are optimized for lean, healthy livers and often fail to recover lipid-laden hepatocytes from metabolically compromised models. In HFD-fed mice, hepatocytes accumulate intracellular lipid droplets, reducing their buoyant density and leading to their loss during standard purification steps. This protocol introduces an optimized approach that systematically adjusts Percoll gradient concentrations (10%–30%) based on age, diet, and metabolic status to improve recovery of both normal and lipid-laden hepatocytes. By integrating controlled perfusion flow rates tailored for obese or steatotic livers, the protocol minimizes vascular damage while preserving cell viability. In addition, it incorporates defined criteria for perfusion efficiency and enzymatic digestion to ensure consistent tissue dissociation. A key advancement of this protocol is its ability to retain and isolate hepatocytes with altered density resulting from lipid accumulation, thereby enabling the simultaneous recovery of heterogeneous hepatocyte populations from the same liver. This is particularly important for studying metabolic dysfunction, as conventional methods selectively bias against pathological cells. Overall, this protocol provides a reproducible and adaptable strategy for isolating functionally relevant hepatocytes from HFD-fed mice and can be applied to models of fatty liver disease. It enables downstream applications, including lipid imaging, metabolic assays, and gene expression analyses, thereby improving the physiological relevance of in vitro studies.

Institutional permissions

The isolation of hepatocytes from mice on standard and high-fat diets, and from mice with metabolic disorders, should be performed in accordance with local and institutional regulations and be approved by the Institutional Animal Care and Use Committee (IACUC). Mice should be kept under standard housing conditions with controlled temperature, humidity, and a 12-h light/dark cycle. Animals fed a high-fat diet require careful monitoring of body weight, liver size, and overall health before hepatocyte isolation. For the experiments described here, only diet-induced obese mice were used; although this protocol can be adapted for NAFLD or NASH models, such models were not employed in the current study.

Preparation of buffers and materials

Timing: 30–45 min

• 1.Pre-warm perfusion and digestion buffers to 37°C before liver perfusion to maintain enzymatic activity and cell viability.
• 2.Prepare Liberase solution fresh and keep it on ice until immediately before use.
• 3.Coat culture plates or coverslips with 1% collagen in PBS at least 2 h before plating hepatocytes.
• 4.
  Adjust Percoll gradient solutions (10%–30%) based on the animal’s age, diet, and metabolic condition to account for variations in hepatocyte density due to lipid accumulation. Suggested starting concentrations for optimization are:

  • a.
    30% Percoll: 6-, 9-, and 12-months-old mice on standard chow; 6- and 9-months-old mice on high-fat diet.
  • b.
    20% Percoll: 12-months-old chow-fed mice; 9- and 12-months-old high-fat diet mice.
  • c.
    10% Percoll: 12-months-old high-fat diet mice.
• 5.These adjustments allow improved recovery and viability of hepatocytes across different ages, diet durations, and disease models characterized by varying lipid droplet accumulation.

Note: Liberase concentration should be optimized according to user requirements, and experiments should be conducted to assess cell recovery and yield.

CRITICAL: Ensure proper catheterization of the inferior vena cava with minimal disruption to avoid inferior vena cava rupture and air bubbles, which can damage the liver and reduce cell yield.

CRITICAL: The hepatic portal vein is cut once the liver is fully perfused, indicated by uniform blanching of all lobes and free outflow of perfusion solution through the inferior vena cava. Perfusion can be standardized using a controlled flow rate (5–10 mL/min) to ensure reproducibility.

CRITICAL: Flow rate must be carefully adjusted: 5 mL/min is suitable for normal mice, while reduced rates (less than 4 mL/min or less as per requirement) are required for high-fat diet or metabolically challenged mice to prevent perfusion-related liver injury.

CRITICAL: For purification, Percoll density should be reduced from 30% to 20% or 10% in lipid-laden hepatocytes, as excess lipid accumulation decreases cell density.

CRITICAL: Sterility must be maintained throughout the procedure to ensure thriving downstream hepatocyte culture.

This preparation guarantees consistent hepatocyte isolation across diverse dietary and disease models, maximizes cell yield, and supports downstream applications, including culture, imaging, and functional assays.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Calcium chloride (CaCl2)	Sigma-Aldrich	C5670
Potassium chloride (KCl)	Sigma-Aldrich	P9333
Glucose (D-Glucose)	Sigma-Aldrich	BCCM3181
0.5 M EDTA	Invitrogen™	15575020
HEPES (1 M)	Gibco™	15630080
Sodium Pyruvate (100 mM)	Gibco™	11360070
10× PBS	Gibco™	70011044
DPBS (no calcium, no magnesium)	Gibco™	14190136
BODIPY 493/503	Invitrogen™	D3922
Collagen Type I, rat tail	Sigma-Aldrich	C3867
Ethanol (100%)	Fisher Scientific	BP2818-500
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881
Percoll®	Sigma-Aldrich	P4937
Liberase™ TM Research Grade	Roche	5401119001
Avertin (2,2,2-Tribromoethanol)	Sigma-Aldrich	T48402
Experimental models: Organisms/strains
C57/BL6 male mice with chow and a high-fat diet	Jackson	C57/BL6
Other
Gibco™ William’s E Medium, GlutaMAX™ Supplement	Gibco™	32551020
Penicillin-Streptomycin (100×) (10000 IU)	Gibco™	15140122
Fetal Bovine Serum (FBS)	HyClone	SH30396.03
Nonessential Amino Acids (100×)	Gibco™	11140–050
Sterile Deionized Water	Millipore	TMS-006-C
50 mL Conical Tubes	Corning	352070
Glass Coverslips	Fisher Scientific	12-545-80
100 μm Cell Strainer	Falcon	352360
EXCEL IV Catheter, 22G × 1″	EXCEL	26746
Fisher Scientific Mini Pump Variable Flow	Fisher Scientific	JAA103974
Silicone tubing; inner diameter (ID) 1/8 inch, outer diameter (OD) 1/4 inch	Fisher Scientific	JAA103974
Sutures (6-0 silk)	AD Surgical	S-S618R13
27-Gauge Needles	BD Biosciences	305109
Surgical Instruments (scissors, forceps, clamps)	Fine Science Tools	Various

Materials and equipment

Perfusion buffer

Reagent	Final concentration	Volume
PBS	1×	987.9 mL
HEPES/KCL buffer (1 M and 5% (w/v))	10 mM and 0.05%	10 mL
1 M Glucose	5 Mm	5 mL
0.5 M EDTA	100μM	200 μL

Perfusion buffer (Storage 4°C for 4 weeks maximum).

Digestion buffer

Reagent	Final concentration	Volume
PBS	1×	987.9 mL
HEPES/KCL buffer (1 M and 5% (w/v))	10 mM and 0.05%	10 mL
HEPES 1 M	1 Mm	20 mL
1 M Glucose	5 Mm	5 mL
0.5 M EDTA	100 μM	200 μL
1 M CaCl2	1 mM final concentration added before Liberase	1 ml

Digestion buffer (Storage 4°C for 4 weeks maximum).

Note: Both perfusion and digestion buffers should be adjusted to pH 7.4 and sterile-filtered through a 0.22 μm filter under aseptic conditions, and stored in a sterile environment to prevent contamination. CaCl₂ should be added to the digestion buffer immediately before adding Liberase to ensure optimal enzyme activity and prevent premature degradation.

Plating medium

Reagent	Final concentration	Volume
William’s E medium (Sigma)	–	500 mL
Penicillin/streptomycin	100 IU	1% (v/v)
HEPES	10 mM	1% (v/v)
Sodium pyruvate	1 mM	1% (v/v)
Heat-inactivated fetal bovine serum	50 mL	10% (v/v)
Nonessential amino acids	100 μM	1% (v/v)
L-glutamine	2 mM	1%(v/v)

Plating medium (Storage 4°C for 4 weeks maximum).

Cell Culture medium

Reagent	Final concentration	Volume
William’s E medium (Sigma)	-	500 mL
Penicillin/streptomycin	100 IU	1% (v/v)
HEPES	10 mM	1% (v/v)
Sodium pyruvate	1 mM	1% (v/v)
Nonessential amino acids	100 μM	1% (v/v)
L-glutamine	2 mM	1%(v/v)

Cell Culture medium (Storage 4°C for 4 weeks maximum).

Step-by-step method details

Animal housing and preparation for liver perfusion and hepatocyte isolation

Timing: 20–45 min

This section describes the preparation of mice for liver perfusion and catheterization to isolate primary hepatocytes. All procedures must comply with Institutional Animal Care and Use Committee (IACUC) guidelines.

• 1.
  Mouse preparation.

  • a.
    12 weeks old at the start of the study, mice were housed under standard conditions and fed a high-fat diet (HFD) or chow diet according to the experimental design.
  • b.
    Anesthetize the mouse using an approved anesthetic agent (e.g., isoflurane or ketamine/xylazine). Confirm deep anesthesia by pinching the hind limb with blunt forceps to test for the absence of reflex.
  • c.
    Disinfect the abdominal fur with 70% ethanol, and position the mouse supine on a dissection tray. Pin the limbs gently to expose the abdomen.
  • d.
    Make a midline abdominal incision and pin back the skin flaps to expose the peritoneal cavity.
  • e.
    Carefully move the intestines aside using sterile cotton swabs to visualize the inferior vena cava (IVC). Ensure the IVC is straight for optimal catheter insertion.
  • f.
    Adjust and stretch all four limbs to maintain proper alignment, preventing accidental puncture during catheterization (Figures 1A and 1B).

Note: Proper positioning of the mouse is critical to ensure a straight inferior vena cava (IVC) and facilitate smooth catheterization. The mouse was placed supine on a surgical board, and its limbs were gently secured with tape to maintain stability (Figures 1A and 1B). If required, the abdomen can be fully extended by slightly elevating the thoracic region with a small support under the upper back, thereby straightening the IVC along its longitudinal axis. Position the head slightly upward to reduce tension on abdominal structures. Care was taken to avoid excessive stretching, which may compromise vascular integrity. This positioning minimizes vessel curvature, reduces the risk of IVC tearing, and facilitates controlled catheter insertion.

• 2.
  Catheterization of the inferior vena cava.

  • a.
    Place two sutures around the IVC, approximately 5 mm apart, just below the renal veins (Figures 1C and 1D).
  • b.
    Gently insert the catheter at least 5 mm below the distal suture.
  • c.
    Secure the catheter by tightening both sutures. This stabilizes the catheter during perfusion, particularly in older or obese mice with enlarged or fat-encased IVCs.

Note: Ensure no air bubbles enter the catheter. To prevent air bubbles, all catheters and tubing were prefilled with perfusion buffer and visually inspected during insertion. Care was taken to maintain continuous flow. Bubble traps can be used when necessary to ensure bubble-free perfusion, as air bubbles can rupture hepatic microvessels and reduce hepatocyte yield.

Figure 1.

Isolation of hepatocytes from normal and fatty liver of diet-fed mice using in situ perfusion and Percoll gradient separation

(A and B) Anatomical positioning of the mouse for liver perfusion. The inferior vena cava is exposed, limbs are gently stretched, and the portal vein is supported to stabilize catheter placement. Representative liver morphology before perfusion in normal–diet–fed mice (A) and high-fat-diet (HFD)-fed mice with fatty liver (B).

(A.1 and B.1) Liver appearance before perfusion.

(C and D) Schematic depiction showing perfusion of the inferior vena cava and the drainage from the portal vein.

(C.1) Liver appearance from normal-diet-fed mice immediately after perfusion, showing uniform blanching and preserved tissue integrity.

(D.1) Post-perfusion fatty liver showing persistent lipid content characteristic of NAFLD/MAFLD.

(E and F) Percoll gradient separation of hepatocytes isolated from normal mice using 30% Percoll (E), and 10% Percoll for HFD mice (F).

Hepatocytes form a clean pellet. Distinct top-layer accumulation of dead cells, lipid droplets, and debris is observed.

(E.1 and F.1) Representative confocal images of isolated hepatocytes. Normal-diet hepatocytes display clear cytoplasm with few lipid droplets.

(F.1) Lipid-laden hepatocytes from HFD mice show enlarged, refractile lipid droplets and reduced cytoplasmic clarity, consistent with steatotic injury.

Setting up perfusion and digestion buffers

Timing: 15–30 min

This section describes the preparation of temperature-controlled perfusion and digestion buffers and the assembly of the perfusion apparatus. It includes sterilizing the tubing, calibrating the peristaltic pump, and adjusting flow rates to ensure efficient perfusion and optimal hepatocyte recovery.

• 3.
  Buffer setup.

  • a.
    Use a water bath to warm both the perfusion and digestion buffer to 37°C and maintain this temperature throughout the perfusion.
  • b.
    Assemble the entire perfusion setup 15–30 min before anesthesia to ensure smooth operation.
  • c.
    Sterilize the pump tubing by running 70% ethanol, followed by distilled water, PBS, and finally perfusion buffer through the system.
  • d.
    Calibrate the peristaltic pump for a flow rate of 5 mL/min for a regular mouse.
  • e.
    For obese or HFD-fed mice, reduce the flow rate (4 mL/min or less) to prevent hepatic vessel rupture and improve cell recovery.

Note: Confirm the actual flow rate before perfusion, as the tubing diameter may affect the flow rate.

Liver perfusion and enzymatic digestion

Timing: 30–40 min

This section describes cannulation of the portal vein, controlled perfusion to clear blood, and enzymatic digestion of liver tissue using Liberase-containing buffer. Proper perfusion is confirmed by uniform liver blanching, followed by controlled digestion to dissociate hepatocytes while preserving viability and morphology.

• 4.
  Perfusion with buffer.

  • a.
    Connect the perfusion tubing to the IVC catheter after ensuring the tubing is filled with buffer to the tip (no air bubbles).
  • b.
    Start the pump at 5 mL/min, perfusing approximately 50 mL of perfusion buffer.
  • c.
    Once perfusion begins, cut the hepatic portal vein to allow drainage.
  • d.
    Observe the liver for color change and bulging, indicating adequate perfusion (Figures 1C, 1C.1, 1D, and 1D.1).
• 5.
  Enzymatic digestion.

  • a.
    Immediately before use, the digestion buffer was supplemented with CaCl₂ to a final concentration of 1 mM and Liberase to a final concentration of 10 μg/mL. Stock solutions were prepared separately and diluted into the buffer just prior to perfusion. Adding CaCl₂ in advance can lead to crystallization if the buffer is warmed, and pre-adding Liberase may reduce enzyme activity. Just before use, supplement the digestion buffer with CaCl₂ (1 mM) and Liberase (10 μg/mL) freshly prepared on ice.
  • b.
    Do not pre-mix Liberase and CaCl₂ far in advance, as this can reduce enzyme activity.
  • c.
    For normal mice, perfuse the liver with digestion buffer at 5 mL/min. Adjust the flow rate (4 mL/min or less) for fatty or enlarged livers.
  • d.
    Continue perfusion until the liver softens and becomes pale (Figures 1C and 1D).
  • e.
    Gently probe the liver with cotton swabs; adequately digested tissue will feel soft and pliable.
• 6.
  Liver removal and cell dissociation.

  • a.
    Excise the perfused liver and transfer it into a dish containing 10 mL of digestion buffer without Liberase.
  • b.
    Gently tap or tease the tissue against the dish to release hepatocytes into suspension.
  • c.
    Add an equal volume (10 mL) of plating medium to stop enzymatic digestion immediately.
  • d.
    Filter the cell suspension through a 100 μm cell strainer to remove undigested tissue fragments.
  • e.
    Centrifuge the suspension at 50 × g for 2 min at room temperature.
  • f.
    Wash the cell pellet twice with plating medium.

Note: Gentle handling during tapping and centrifugation preserves hepatocyte viability and yield.

Hepatocyte purification using a Percoll gradient

Timing: 45–60 min

This section describes the separation of viable hepatocytes from debris, non-parenchymal cells, and damaged cells using a density-based Percoll gradient (10%–30%). Gradient concentrations are adjusted based on age, diet, and lipid accumulation to improve the recovery of both normal and lipid-laden hepatocytes while maintaining cell viability.

• 7.
  Density-based purification.

  • a.
    Prepare 90% Percoll by diluting with 10× PBS.
  • b.
    Prepare working Percoll solutions (30%, 20%, or 10%) by diluting the 90% Percoll solution with DMEM to the desired final concentration.
  • c.
    Select the appropriate Percoll concentration based on lipid content and mouse condition: Normal diet: 30%; HFD-fed mice: 20%; Severe steatosis (NAFLD/NASH models): 10%.
  • d.
    Resuspend hepatocytes in the corresponding Percoll solution.
  • e.
    Centrifuge at 200 × g for 20 min at 4°C with slow acceleration and deceleration.
  • f.
    Carefully remove the supernatant and collect the hepatocyte pellet (Figures 1E and 1F).
  • g.
    Wash cells twice with plating medium.

Note: Percoll purification effectively removes dead cells and digestion debris, improving cell viability for culture.

Plate preparation and cell culture

Timing: 2–3 h (including coating and cell attachment)

This section describes the preparation of collagen-coated culture plates or coverslips and the plating of freshly isolated hepatocytes at an appropriate density. It outlines attachment conditions, medium composition, and initial culture handling to preserve hepatocyte viability, morphology, and metabolic function for downstream assays.

• 8.
  Plate coating and cell seeding.

  • a.
    Coat culture plates or cover glass (25mm for 6-well plates) with 1% collagen solution and incubate at 37°C for 2 h.
  • b.
    Wash twice with sterile PBS before plating.
  • c.
    Adjust the plating density according to hepatocyte size and lipid content. Lipid-laden hepatocytes are larger, so fewer cells should be seeded per well to ensure proper attachment.
  • d.
    After 4–6 h, check for cell attachment under a microscope.
  • e.
    Replace the plating medium with culture medium once cells are attached.

Note: Rat tail collagen is diluted 1:10 from the stock solution using 1× PBS, followed by a further dilution to 1:100 in sterile 1× PBS for coating. If cells fail to attach, optimize perfusion flow rate and digestion time (see troubleshooting).

Measuring intracellular lipid accumulation using BODIPY staining

Timing: ∼30–45 min

This section describes staining of cultured hepatocytes with BODIPY to visualize and quantify intracellular neutral lipid droplets. It outlines dye incubation, washing steps, and fluorescence imaging parameters (Ex/Em) to assess lipid accumulation under different experimental conditions.

• 9.
  Staining lipids with BODIPY.

  • a.
    Make a 1 mM BODIPY stock by dissolving BODIPY in DMSO; store at −20°C.
  • b.
    Briefly rinse cells with 1× PBS.
  • c.
    Add BODIPY at a working concentration of 1 μM to cells (The BODIPY stock solution was dissolved in DMSO and diluted into culture medium immediately before use).
  • d.
    Incubate at 37°C for 20 mins, protecting from light.
  • e.
    Wash 2 and 3 times with PBS to remove unbound dye.
  • f.
    Mount the coverslips in an open perfusion microincubator (PDMI-2; Harvard Apparatus) and image them using 488 nm excitation and 510–540 nm emission (Ex: 488 nm/Em: 510–540 nm) (Figures 1E.1, 1F.1, and 2).

Note: Handle pipetting and washing steps gently to prevent detachment or damage to the cover glass.

Figure 2.

Differential enrichment of lipid-laden hepatocytes across Percoll gradients in chow- and high-fat–diet-fed mice

(A) Distribution of lipid-laden cells within the supernatant following centrifugation across Percoll gradients (30%, 20%, and 10%) using hepatocytes isolated from normal and HFD-fed mice. Increased lipid accumulation in HFD hepatocytes results in decreased cellular density and greater retention of lipid-laden cells in upper layers of the gradient.

(B) Representative confocal images of pelleted hepatocytes collected from each Percoll concentration. Some concentrations (e.g., very low or very high) yielded no hepatocytes; corresponding images are omitted. Pellets from normal diet mice show dense, uniform hepatocytes with minimal lipid content, whereas HFD hepatocytes exhibit enlarged, refractile cells with substantial lipid accumulation, consistent with steatotic remodeling.

Expected outcomes

This protocol yields highly viable, morphologically intact hepatocytes suitable for downstream applications, including lipid accumulation assays (BODIPY staining), immunofluorescence, and metabolic profiling or gene expression studies. Optimizing the perfusion flow rate and Percoll gradient concentration improves hepatocyte yield and viability in high-fat diet (HFD)-fed mice. Successful perfusion is indicated by uniform liver blanching and smooth tissue digestion.

Estimated total time for the procedure

Step	Approximate time
Animal preparation and catheterization	20–45 min
Perfusion setup and buffer preparation	15–30 min
Liver perfusion and digestion	30–40 min
Cell dissociation and washing	20–30 min
Percoll purification	45–60 min
Plate coating and hepatocyte culture	2–3 h
BODIPY staining and imaging	30-45 mins
Total duration (excluding culture time)	5–6 h

Limitations

Hepatocyte isolation efficiency varies with mouse strain, age, and diet, with high-fat diet (HFD) models presenting enlarged, fragile livers that require careful adjustment of perfusion flow rates. The procedure is sensitive to technical variability, as minor differences in catheterization, flow rate, or perfusion pressure can affect digestion efficiency and cell viability. Enzyme activity, particularly Liberase, may vary between batches, necessitating routine validation. Lipid-laden hepatocytes are more fragile and prone to damage during isolation, resulting in reduced viability compared to cells from lean controls. Strict temperature control (37°C) and rapid processing (within 60–90 min) are essential but limit throughput. Percoll gradient optimization is required for each condition to balance yield and purity, as lower densities may increase contamination with non-parenchymal cells. The protocol is susceptible to microbial contamination and is difficult to scale. Primary hepatocytes also have a limited culture lifespan (3–5 days), and residual Percoll may interfere with downstream assays. Finally, isolation from severely steatotic or fibrotic livers remains challenging due to impaired perfusion. While this protocol improves recovery of lipid-laden hepatocytes from HFD-fed mice, we did not perform systematic quantitative comparisons of cell yield, viability, or metabolic function relative to conventional Percoll gradients. Consequently, the advantages of the modified approach are supported primarily by qualitative assessments, including cell morphology, attachment, and lipid droplet preservation. Future studies incorporating quantitative yield measurements and functional assays will be necessary to rigorously evaluate hepatocyte recovery and metabolic competence.

Troubleshooting

Problem 1

Catheter puncturing the inferior vena cava during catheterization and while perfusing.

Potential solution

As mentioned earlier, in the Catherization of the inferior vena cava subheading, try to place two sutures around the inferior vena cava, spaced apart, to keep the catheter straight.

Problem 2

Blood remains in the liver after perfusion.

Potential solution

This typically occurs due to incorrect catheter placement or low perfusion flow rate. Ensure the catheter is correctly inserted into the inferior vena cava (Catherization of the inferior vena cava) and that a small amount of blood backflows before starting perfusion. Confirm the portal vein is cut to allow drainage, and maintain a flow rate of 5 mL/min for normal mice. Remove any air bubbles in the tubing to prevent obstruction.

Problem 3

Liver does not blanch or swell during perfusion.

Potential solution

Lack of liver blanching indicates incomplete perfusion, often due to low pressure, blocked tubing, or improper catheter alignment. Check that the perfusion pump is calibrated, the tubing is free of kinks, and the perfusion buffer is prewarmed to 37°C (Perfusion with buffer). Gently reinsert the catheter if needed to reestablish flow.

Problem 4

Liver ruptures or swells excessively during perfusion.

Potential solution

A ruptured or overly swollen liver suggests the perfusion rate is too high, leading to vascular rupture. For older or high-fat diet (HFD) mice, reduce the flow rate to 3 to 4 mL/min (Enzymatic Digestion). Avoid over-tightening sutures and monitor the liver visually for gentle expansion during perfusion.

Problem 5

Reduced single cells in suspension.

Potential solution

Tap the liver tissue gently against the petridish to induce hepatocyte dissociation (Liver removal and cell dissociation) at the single-cell level. Increase the liberase concentration and reduce the flow rate of the digestion buffer to increase the digestion.

Problem 6

Liver remains firm after digestion due to poor enzymatic digestion.

Potential solution

Insufficient digestion results from reduced enzyme activity (Enzymatic digestion). Always prepare the digestion buffer fresh, adding CaCl₂ to a final concentration of 1 mM and Liberase to a final concentration of 10 μg/mL immediately before perfusion. Keep Liberase on ice, avoid repeated freeze-thaw cycles, and confirm that the perfusion temperature remains at 37°C to optimize enzyme efficiency.

Problem 7

Excessive tissue damage or over-digestion of liver tissue.

Potential solution

Prolonged enzyme perfusion can damage hepatocytes, reducing viability (Liver removal and cell dissociation). Stop perfusion once the liver turns pale and softens. Immediately transfer the liver into the digestion buffer without Liberase, and add plating medium to neutralize residual enzyme activity.

Problem 8

Low hepatocyte yield after centrifugation.

Potential solution

Low yield may result from cell loss during centrifugation or aspiration (Liver removal and cell dissociation). Use gentle centrifugation at 50 × g for 2 min, ensuring the rotor is appropriately balanced. Do not aspirate too close to the pellet. If yield remains low, check digestion efficiency and perfusion completeness.

Problem 9

High proportion of dead or damaged hepatocytes

Potential solution

Cell death often results from the introduction of air bubbles, overdigestion, or temperature fluctuations (setting up perfusion and digestion buffers for perfusion). Prime the perfusion line to remove all air bubbles, maintain buffers at 37°C, and minimize handling time. If viability is below 80%, reduce enzyme concentration or perfusion duration.

Problem 10

Cells floating in the Percoll gradient.

Potential solution

When fewer cells are in the pellet, and more cells are in the supernatant or floating in the Percoll, the Percoll concentration is reduced (Density-based purification). If the cells in the supernatant contain larger or smaller droplets, reduce the Percoll concentration, as collecting from the supernatant may include non-viable cells, which can decrease attachment and affect assay efficiency.

Problem 11

Cell clumping or debris in suspension.

Potential solution

Clumping can occur due to free DNA from lysed cells or incomplete filtration (Liver removal and Cell dissociation). Pass the suspension through a 100 μm cell strainer twice, and include DNase I (10 μg/mL) in the digestion buffer to degrade free DNA. Avoid vigorous pipetting to minimize shear stress.

Problem 12

Poor hepatocyte attachment to collagen-coated plates.

Potential solution

Weak attachment may result from insufficient collagen coating or plating density (Plate preparation and cell culture). Coat plates with 1% collagen for 2 h at 37°C and wash twice with PBS. Adjust plating density for lipid-laden hepatocytes, as they are larger and attach less efficiently. Verify cell viability before plating.

Problem 13

Abnormal hepatocyte morphology after plating.

Potential solution

Cells that remain rounded or fail to spread have likely experienced temperature shock or mechanical stress. Prewarm all media and solutions to 37°C, and handle cells gently during transfer (Buffer set up). Avoid repeated pipetting.

Problem 14

Low recovery of lipid-laden hepatocytes from HFD mice.

Potential solution

Lipid-rich hepatocytes are less dense and can be lost during Percoll centrifugation if the density is too high (Density-based purification). Reduce Percoll concentration to 10%–20% and centrifuge at 200 × g for 20 min with slow acceleration/deceleration at 4°C. Adjust density based on mouse age and lipid accumulation.

Problem 15

Contamination in cultures after plating.

Potential solution

Contamination commonly results from incomplete sterilization of perfusion tubing or instruments (Buffer setup). Clean the entire setup by flushing with 70% ethanol, followed by sterile PBS before perfusion. Perform all post-isolation procedures under a biosafety cabinet (Class II laminar flow hood) using sterile reagents.

Problem 16

Low reproducibility between isolations.

Potential solution

Variability in yield or viability may arise from differences in enzyme potency, mouse age, or metabolic status. Use mice of similar age, weight, and diet, and maintain consistent perfusion temperature and flow rate. Validate each new Liberase batch for activity before use. Consistency in handling, timing, and reagent preparation is key to reproducibility.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Santhanam Shanmughapriya (santhanam@rowan.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Thiruvelselvan Ponnusamy (ponnusamy@rowan.edu).

Materials availability

This study did not generate new, unique reagents.

Data and code availability

This study did not generate a dataset or code.

Acknowledgments

This study was supported by the National Institutes of Health (R00 HL138268 and NIH R01HL176747 to S.S.), the W.W. Smith Charitable Trust (H2103) to S.S., a Career Development Grant from the American Heart Association (935207) to S.S., NIH R21 DA051798, and Osteopathic Heritage Foundation (OHFE-F-2025-46) to K.N.

Author contributions

K.N. and S.S. conceptualized the study and supervised the project. T.P. performed animal maintenance, conducted animal surgeries, and performed hepatocyte isolation, BODIPY staining, and imaging. T.P. and K.N. wrote the manuscript with input and edits from S.S. All authors read and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this manuscript, the authors used Grammarly in order to improve the readability and language of the manuscript. After using Grammarly, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.