A Simple Protocol for the Isolation and Culture of Hepatocytes from MASLD Mice

Summary

This study establishes a standardized protocol for isolating primary hepatocytes from metabolic dysfunction-associated steatotic liver disease (MASLD) mice, enabling disease-relevant investigations of steatosis and metabolic dysfunction without artificial lipid loading. The method yields high-yield and high-viability hepatocytes suitable for mechanistic and therapeutic studies.

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by pathological lipid accumulation (steatosis) within hepatocytes, represents a significant and growing global health burden. Primary mouse hepatocyte cultures serve as indispensable ex vivo models for elucidating MASLD pathogenesis and therapeutic interventions. However, reliable isolation of high-quality hepatocytes from steatotic livers remains technically challenging. Here, we present a simple protocol for the efficient isolation of primary hepatocytes from MASLD mice using the collagenase perfusion technique. This protocol generates hepatocytes exhibiting inherent steatosis without requiring artificial induction via palmitate and oleic acid supplementation, thereby preserving a more physiologically relevant phenotype. The protocol yields highly viable and high-purity hepatocytes and is universally applicable for isolating high quantities of hepatocytes from MASLD mice. Furthermore, it is suitable for pharmacological evaluation or genetic intervention studies. In summary, we provide a reproducible protocol to isolate high-yield, high-purity, and highly viable hepatocytes for downstream cell biological studies to facilitate the discovery of novel therapeutic targets and drugs for MASLD.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by pathological lipid accumulation in hepatocytes and represents the most prevalent chronic liver disorder globally¹. MASLD is estimated to affect 38% of the world's adult population and has become a significant threat to human health². Mechanisms of MASLD have not been fully elucidated and treatment options are very limited. Discovering new therapeutic targets and developing targeted agents for MASLD has become a critical clinical need. Hepatocytes, as the predominant cell type composing the liver, are the primary site of hepatic glucose-lipid-drug metabolism. Primary hepatocytes retain a more complete function of hepatocytes than hepatocyte cell lines and are a useful tool for studying liver disease mechanisms and drug development³. Isolation and culture of primary hepatocytes from mice with fatty liver is a fundamental technique for studying liver pathophysiology, including lipid metabolism, insulin resistance, and inflammatory responses. A prior protocol established for hepatocyte isolation from murine models of fatty liver disease employed gradient centrifugation to specifically enable the separation of both normal and lipid-accumulating hepatocyte populations. These isolated lipid-laden hepatocytes thus constitute a critical in vitro model system for investigating fatty liver disease pathobiology³. The primary goal of this approach is to easily and economically obtain highly viable and high-yield hepatocytes that retain disease-specific phenotypic characteristics and thus are able to mimic MASLD in vitro.

The general procedure for hepatocyte isolation in mice consists of a two-step process that begins with flushing the blood from the liver with an isotonic perfusion solution, such as Hank's Balanced Salt Solution (HBSS), and then digesting the liver using a collagenase-containing digestion solution. Recent advances in liver perfusion and enzymatic digestion techniques have improved the yield and viability of primary hepatocytes in female mice⁴. Improvements to traditional two-step collagenase perfusion methods, such as optimizing buffer composition and shortening digestion times, have been critical in adapting experimental protocols to fatty liver samples. Primary hepatocytes serve as indispensable models for elucidating the pathophysiological mechanisms underlying MASLD and its cardiometabolic complications, particularly through their role in mediating heart-liver crosstalk and liver-vascular axis interactions through endothelial mediators and angiogenic factors^5,6. These cells provide critical insights into the multi-organ communication networks driving disease progression, as evidenced by clinical associations among MASLD, chronic kidney disease, and ischemic heart disease⁷, while offering a physiologically relevant platform for developing targeted therapies addressing both hepatic and cardiovascular manifestations of metabolic syndrome. Current in vitro research of MASLD predominantly relies on palmitic/oleic acid (PA/OA)-stimulated normal hepatocytes or human hepatocyte cell lines (e.g., HepG2 and Huh7), which inadequately replicate the pathophysiological features of steatotic hepatocytes under diseased conditions, limiting their translational relevance for studying heart-liver-vascular interactions. This approach fails to capture the chronic metabolic dysfunction, inflammatory microenvironment, and disease-specific interorgan crosstalk characteristic of MASLD progression.

This protocol can be used to isolate hepatocytes from MASLD mice, without the need to artificially add palmitic and oleic acid treatments. In conclusion, we present a simple and reproducible method for obtaining high-yield hepatocytes with excellent viability that is suitable for downstream cell biology research to support the discovery of new therapeutic targets and drug development for MASLD.

Protocol

All experimental steps were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Science and Technology of China. Male C57BL/6J mice were fed a high-fat diet (HFD, #12492, Research Diet) for more than 12 weeks in this study.

1. Preparation of a collagen-coated culture plate or dish

1. Add 15 mL 100% EtOH to 35 mL of double-distilled H₂O (ddH₂O). Prepare a solution of collagen type I 50 μg/mL in 30% EtOH. Filter with a sterile 0.22 µm filter tip.
2. Coat a cell culture plate or dish with 30 µL of collagen solution per cm². Incubate at room temperature (RT) for 1 h.
3. Remove excess liquid with a pipette and dry the culture plate or dish. Irradiate the coated dishes with ultraviolet light for 0.5 h for sterilization. Store at 4 °C for less than 1 month.
   NOTE: The wavelength of the UV lamp is 254 nm, and the UV intensity is not less than 400 mw/m². The distance of the UV lamp source from the collagen-coated dishes is 1 m.

2. Preparation of perfusion solution

1. A few days before isolating the hepatocytes, prepare hepatocyte isolation reagents and culture media specified in Table 1. Filter perfusion solution A and B with a sterile 0.22 µm filter tip. Store at 4 °C for less than 1 month. Prepare a water bath and set the temperature to 37 °C.
2. Preheat perfusion solutions A and B in a water bath at 37 °C. Add 40 mg of collagenase type IV to 80 mL of perfusion solution B before surgery.
3. Pump 75% EtOH at a rate of 4 mL/min for 5 min using a peristaltic pump at RT. Switch the pump tubing from 75% EtOH to preheated perfusion solution A.
   NOTE: Assemble the heated circulation filling unit according to the configuration illustrated in Figure 1A.

3. Surgical procedure

1. Anesthetize the mouse using an approved method. Inject sodium pentobarbital (50 mg/kg) intraperitoneally. Verify the loss of turning reflex before proceeding.
2. Secure the mouse's upper and lower limbs on a dissection board. Sterilize the surgical area with 75% alcohol.
3. Cut through the inner and outer abdominal membranes using dissection instruments. Pivot the visceral organs to the mouse's left side with a cotton swab to expose the inferior vena cava (IVC) and hepatic portal vein (PV).
4. Press the inferior vena cava with a cotton swab to bulge it out. Insert a 22-G indwelling needle parallel to the inferior vena cava.
5. Withdraw the iron part of the indwelling needle. Leave the catheter in the inferior vena cava. Connect the needle to a perfusion line with a 0.22 µm filter tip.
   NOTE: An indwelling needle consists of an iron needle part and a plastic catheter part. Ensure accurate insertion into the inferior vena cava and wait until the indwelling needle fills with blood. Get rid of all air bubbles from the indwelling needle. Complete the entire procedure rapidly to avoid thrombosis.
6. Set the perfusion rate to 5 mL/min of perfusion fluid A. Begin perfusion and observe the liver until it appears swollen. Cut off the portal vein immediately.
7. Gently massage the liver using a cotton swab to ensure thorough blood flushing. Continue perfusion until the liver turns earthy yellow.
8. Switch to perfusion fluid B containing 0.05% collagenase type IV. Continue perfusion until the liver is fully digested.
   NOTE: It takes at least 10 min per mouse for the liver to be digested. A volume of 80 mL of perfusion solution B containing collagenase is enough for one mouse to digest its liver. The hepatic tissue undergoes complete enzymatic digestion until achieving maximal softening, with the capsular-enclosed parenchyma reaching a state of extreme fragility susceptible to mechanical disruption.
9. Remove the liver from the abdomen of the mouse. Place the liver in a 10 cm dish containing DMEM.
10. Proceed in a biosafety cabinet. Shred the liver into powder using tweezers (well-digested liver releases hepatocytes easily).
11. Filter the suspension across a 70 µm mesh filter into a sterile 50 mL centrifuge tube. Adjust the volume to 45 mL (per mouse) with DMEM medium.
12. Centrifuge at 50 × g for 4 min. Pour off the supernatant completely.
13. Resuspend the pellet with DMEM medium to a final volume of 25 mL (per mouse). Centrifuge again at 50 × g for 2 min.
14. Pour off the supernatant completely. Resuspend the pellet in 25 mL of culture medium supplemented with 10% FBS.
15. Mix the suspension thoroughly. Stain the cells with Trypan blue for counting. Proceed to inoculate for culture immediately.
    NOTE: Place 2 mL of medium containing 2 × 10⁵ cells per well into collagen-coated 6-well dishes. Replace the DMEM medium after 2 h of cell inoculation.

Results

The current protocol was implemented on male C57BL/6J mice fed HFD for more than 12 weeks. The isolated primary hepatocytes were examined for morphology and function. We obtained an average yield of 2 × 10⁷ cells per isolation from approximately 40 g of mouse liver. Cell viability was determined to be between 85%-86% by Calcein AM staining of live cells. The yield from each diet-induced obese (DIO) mouse isolation was sufficient to seed 15 standard 6-well plates.

Discussion

The isolation of viable mouse hepatocytes involves a critical two-step procedure beginning with thorough vascular perfusion using isotonic solution to remove blood components, followed by controlled enzymatic digestion with active collagenase solution^9,10. Successful isolation depends on maintaining optimal physiological conditions throughout the process, particularly stable temperature (37 °C) and pH (7.2-7.4) of perfusion solutions, as these parameters dir...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL sterile centrifuge tubes	Nest	602052
anti-HNF4α antibody	Immunoway	YM8517
anti-IgG antibody	Proteintech	30000-0-AP
BODIPY	Thermo Fisher Scientific	D3922
CaCl2	Merck (SigmaAldrich)	C5670
Centrifuge	Thermo	Sorvall ST16R
Collagen Type I	Corning	354236
Collagenase, Type 4	Sangon Biotech	A004186-0001
Detaining needle	BRAUN	4254074B
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	C11995500BT
EGTA	Yeasen	60339ES10
Ethanol	Merck (SigmaAldrich)	493511
Fetal Bovine Serum	Sigma	F2442
Glucose	Merck (SigmaAldrich)	G7021
HEPES	Gibco	15630080
Isoginkgetin	MedChemExpress	HY-N2117
K2HPO4	Merck (Supelco)	PHR2900
KCl	Merck (SigmaAldrich)	P3911
Na2HPO4 ·12H2O	Merck (SigmaAldrich)	04273
NaCl	Merck (SigmaAldrich)	S9888
NaHCO3	Merck (SigmaAldrich)	S6014
Needle filter 0.22µM	Millex-GP	SLGV033RB
Nile Red	Invitrogen	N1142
Penicillin/streptomycin (100X)	Gibco	15140122
Recombinant adenovirus	Obio Technology Corp., ltd.		Recombinant adenoviruses encoding Flag-tagged mouse IER2 (Ad-Ier2, NM_010499.4) and control adenovirus (Ad-NC) were constructed by Obio Technology Corp., ltd.
Sodium pentobarbital	Merck (SigmaAldrich)	P3761
Sterile cell strainers, 100 μm	Fisher Scientific	22363549
Trypan Blue	Beyotime	ST2780-5g
Variable-flow peristaltic pump	Longer	BT100-1F