Improved method for separating liver parenchymal cells and non-parenchymal cells of NAFLD mice induced by high fat diet

Abstract

The invention provides an improved method for separating liver parenchymal cells from non-parenchymal cells of a mouse with a non-alcoholic fatty liver disease induced by a high fat diet. According to the method, the NAFLD mouse model is successfully constructed by feeding a high-fat feed containing 60% of fat for 12-16 weeks. An improved two-step collagenase perfusion technology is adopted, a low-concentration enzyme solution is combined, and digestion time is accurately controlled, so that cell damage is effectively reduced. The efficient separation and purification of parenchymal hepatic cells and non-parenchymal cells are realized through a Percoll solution with a specific proportion by utilizing a density gradient centrifugation method. Flow cytometry and molecular biological technical verification show that the separated cells are high in survival rate and excellent in purity. The method provides a high-quality primary cell material for research on NAFLD pathogenesis and drug target discovery, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of experimental methods, specifically to an improved method for inducing the separation of parenchymal and non-parenchymal cells in NAFLD mice using a high-fat diet. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD), a chronic liver disease that is increasingly prevalent worldwide, has a complex and incompletely elucidated pathogenesis. Traditional research has largely focused on the role of hepatic parenchymal cells (HPCs) in lipid metabolism abnormalities. However, recent studies have revealed that the interaction between non-parenchymal cells (NPCs, including Kupffer cells, sinusoidal endothelial cells, and stellate cells) and HPCs plays a crucial regulatory role in NAFLD progression. Current cell isolation techniques primarily rely on the classic two-step collagenase perfusion method. However, this method faces challenges in high-fat diet-induced obese mouse models: lipid accumulation increases hepatocyte fragility, traditional high-concentration enzymatic digestion conditions easily induce cell damage, and altered NPC expression and function further reduce isolation efficiency, resulting in insufficient viable cell rate and purity, failing to meet the needs of pathogenesis research and drug target screening. Therefore, developing an efficient, mild isolation method for NAFLD mouse models that simultaneously maintains the activity and functional integrity of HPCs and NPCs has become an urgent need in the field of liver disease research.

[0003] To address the aforementioned issues, the applicant proposes an improved method for inducing the separation of parenchymal and non-parenchymal cells in NAFLD mice using a high-fat diet. Summary of the Invention

[0004] The purpose of this invention is to provide an improved method for inducing the separation of parenchymal and non-parenchymal cells in the liver of NAFLD mice by a high-fat diet, so as to solve the problems in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an improved method for separating hepatic parenchymal cells and non-parenchymal cells in mice with non-alcoholic fatty liver disease induced by a high-fat diet, comprising the following steps:

[0006] Mouse model construction:

[0007] Male C57BL / 6J mice aged six to eight weeks were selected and divided into a normal diet group and a high-fat diet group after acclimatization feeding.

[0008] A mouse model of non-alcoholic fatty liver disease induced by a high-fat diet was established by feeding the high-fat diet group with a high-fat diet containing 60% fat for 12 to 16 weeks.

[0009] The successful construction of the model was verified by detecting a significant increase in mouse body weight, elevated serum alanine aminotransferase and aspartate aminotransferase levels, and lipid accumulation in hepatocytes as shown by liver histological analysis.

[0010] Optional steps for separating hepatic parenchymal cells and non-parenchymal cells include:

[0011] Liver perfusion and digestion:

[0012] A two-step collagenase perfusion technique was employed.

[0013] First, the liver is perfused with an EGTA-containing perfusion solution to induce swelling. Then, the portal vein is clamped, the perfusion rate is slowed down for three minutes, and then accelerated until the liver turns yellowish-white.

[0014] Then, the patient was perfused with a perfusion solution containing CaCl2 and a low concentration of type IV collagenase (0.15 mg / mL) for five to seven minutes.

[0015] During perfusion, the mice were kept at a normal body temperature to maintain cell viability.

[0016] Optional further separation and purification steps for hepatocytes and non-parenchymal cells include:

[0017] Cell isolation and purification:

[0018] Hepatocytes were obtained by low-speed centrifugation at 50g, 4 degrees Celsius, for 3 minutes, and were washed 4 times to reduce non-plasmic cell contamination.

[0019] Non-plasmic cells were separated by density gradient centrifugation using a 25% / 60% Percoll density gradient. The centrifugation conditions were 1,800 g centrifugation force, 4 degrees Celsius, and 20 minutes. Non-plasmic cells were located at the interface between the two Percoll layers.

[0020] The collected non-plasmic cells were further purified by a double centrifugation process.

[0021] Optionally, the procedure may also include a quality assessment of the isolated hepatic parenchymal and non-parenchymal cells, specifically including:

[0022] Cell quality assessment:

[0023] Cell viability and cytotoxicity were analyzed using flow cytometry. Cell survival rate was detected by double staining with calcein AM and propidium iodide. The average survival rates of hepatocytes and non-parenchymal cells were 94.3% and 91.4%, respectively.

[0024] Western blotting and reverse transcription quantitative polymerase chain reaction (RT-PCR) techniques were used to detect the expression of specific markers in hepatocytes and non-parenchymal cells, and to verify cell purity and characteristics.

[0025] Optionally, this method is applicable to studying the interaction and pathogenesis between hepatic parenchymal cells and non-parenchymal cells in a mouse model of non-alcoholic fatty liver disease induced by a high-fat diet, providing high-quality primary cell material for drug target research.

[0026] Beneficial effects: Improved cell survival rate and purity: By optimizing collagenase concentration, perfusion time and density gradient centrifugation conditions, the vulnerability damage of hepatocytes in obese mice induced by a high-fat diet was significantly reduced, resulting in a survival rate of 94.3% for hepatocyte parenchymal cells (HPCs) and 91.4% for non-parenchymal cells (NPCs), while the purity was greatly improved, providing high-quality cell materials for subsequent functional studies.

[0027] Breaking through the limitations of traditional techniques: Addressing the issues of low separation efficiency and poor cell viability in high-fat models using traditional methods, this method effectively separates fragile hepatocytes that are susceptible to lipid accumulation by combining precise temperature control, low-speed centrifugation with Percoll gradient centrifugation, thus overcoming the technical bottleneck of liver tissue separation in obese mice.

[0028] Facilitating disease mechanisms and drug development: Efficiently isolated HPCs and NPCs can be used to analyze their interaction in the progression of NAFLD, reveal the cellular and molecular mechanisms of lipid metabolism disorders, inflammation and fibrosis, provide key technical support for the discovery of novel therapeutic targets, and accelerate the development of anti-NAFLD drugs.

[0029] Simplified operation procedures and costs: Manual perfusion is used instead of peristaltic pumps, and the perfusion rate is strictly controlled according to the changes in the state of mouse liver, reducing equipment dependence; the separation time is shortened to within half an hour, reducing the impact of in vitro operations on cell viability, while reducing the amount of collagenase used, significantly reducing experimental costs and improving technology accessibility. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the model construction and verification process in an embodiment of the present invention.

[0031] Figure 2 This is a flowchart illustrating the cell isolation and purification process according to an embodiment of the present invention.

[0032] Figure 3 This is a flowchart illustrating the cell viability and purity verification process in an embodiment of the present invention.

[0033] Figure 4 This is a flowchart illustrating the verification process of cell functional characteristics in an embodiment of the present invention.

[0034] Figure 5An overview of the improved HPC and NPC isolation procedures according to embodiments of the present invention;

[0035] (A) Mice fed a high-fat diet (12-16 weeks) were restrained and anesthetized. Then the anesthesia was lifted;

[0036] In the abdominal cavity, expose the inferior vena cava and insert an indwelling needle. Inject perfusion fluid P1 (containing EGTA). After the liver swells, proceed with the portal vein.

[0037] The catheter is clamped, and perfusion is slowed down for 3 minutes, then accelerated after 3 minutes until perfusion fluid P1 is pushed through. Simultaneously, the liver turns yellowish-white. This process should be controlled within 5-7 minutes; the indwelling needle is closed, and 30 ml of perfusion fluid P2 (containing CaCl2) is aspirated. Perfusion continues slowly, maintaining a slow rate for 3 minutes, then the rate is increased until digestion is complete. This process should be controlled within 5-7 minutes. Finally, the liver cell suspension is filtered through a 70 μm filter into a sterile 50 ml centrifuge tube. Throughout the process, the liver is kept on ice, which is then used to maintain the viability temperature of hepatocytes and non-parenchymal cells through low-dose perfusion.

[0038] (B) Centrifugation: 4°C, 50g, 3 minutes. The precipitate is hepatocytes; collect the precipitate and supernatant.

[0039] (C) Resuspend the precipitate in DMEM-1.0 g / L glucose. Centrifuge: 4°C, 50 g, 3 min, and collect the precipitate and supernatant;

[0040] (D) The precipitate was resuspended in DMEM-1.0 g / L glucose. Centrifugation: 4°C, 50 g, 3 min. The precipitate was derived from hepatocytes of high-fat fed mice.

[0041] (E) Resuscitate HPCs using DMEM containing 10% FBS and 1% penicillin-streptomycin. A small amount of cells is pipetted into a 3.5 cm cell culture dish, and trypan blue and complete culture medium are added to the 3.5 cm cells. After mixing, cell viability is observed under an electron microscope. If cell viability is high, primary hepatocytes can be seeded onto cell culture plates. Incubate in a cell culture incubator (37°C / 5% CO2) for 24 hours.

[0042] (F) Collect the supernatant from the BC process. Centrifuge: 4℃, 200g, 3min. The precipitate consists of residual hepatocytes; collect the supernatant.

[0043] (G) Collect the supernatant from process F. Centrifuge: 4°C, 900g, 8 min; the precipitate is NPC.

[0044] (H) Redissolve the granules with 6 ml of 25% Percoll and place 5 ml of 60% Percoll at the bottom of a 15 mL test tube. Centrifuge: 4°C, 900 g, 8 minutes. The upper layer is primary hepatocytes, the middle layer is non-parenchymal cells, and the lower layer is hepatocytes with residual red blood cells.

[0045] (I) Resuspend the intermediate layer suspension with PBS. Centrifuge: 4°C, 900g, 10 minutes, to precipitate NPC;

[0046] (J) Liver of mice fed a high-fat diet;

[0047] (K) Liver perfusion;

[0048] (L) Liver digestion;

[0049] (M) Hepatocyte dissociation;

[0050] (N) Filter the hepatocyte suspension using a 70 μm filter;

[0051] (O) Hepatocytes of mice fed a high-fat diet;

[0052] (P) Non-parenchymal cells of mice fed a high-fat diet;

[0053] Figure 6 The survival rates of liver HPCs and NPCs isolated from mice in this embodiment of the invention are shown. (A) Number of cells: HPCs and NPCs isolated from the livers of HFD-treated mice were detected by cell counter. (B) Percentage of viable cells in HPCs. (C) Percentage of viable cells in NPCs.

[0054] Figure 7 The expression and purity of HPCs and NPCs in mouse livers in this embodiment of the invention; the Western Blot method for proteins expressed before (A) and after (B) isolation of HPCs and NPCs was modified; RT-PCR detection of genes expressed in HPCs and NPCs after the isolation method was modified (C). Detailed Implementation

[0055] The preferred embodiments of the present invention are described below with reference to the accompanying drawings to make the technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0056] This patented technology focuses on the fields of biomedicine and cell separation, proposing an innovative method for efficiently separating hepatic parenchymal cells (HPCs) and non-parenchymal cells (NPCs) in a mouse model of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet. This method systematically solves the problems of low cell viability and insufficient purity in high-fat models using traditional techniques by optimizing perfusion conditions, density gradient centrifugation parameters, and cell viability maintenance strategies. This provides crucial technical support for research on the pathogenesis of NAFLD and the screening of drug targets.

[0057] Nonalcoholic fatty liver disease (NAFLD) has become a major type of chronic liver disease worldwide, with its incidence rising in both developed and developing countries, reaching a global prevalence of approximately 25%. This disease is characterized by excessive lipid accumulation in hepatocytes and can progress to nonalcoholic steatohepatitis (NASH), liver fibrosis, and even hepatocellular carcinoma (HCC). Traditional research has focused primarily on abnormal lipid metabolism in hepatocytes, but recent findings have revealed that the interaction between non-parenchymal cells (including Kupffer cells, sinusoidal endothelial cells, and stellate cells) and hepatocytes plays a crucial regulatory role in NAFLD progression. For example, Kupffer cells exacerbate liver damage by releasing inflammatory factors, stellate cell activation drives the fibrotic process, and dysfunction of sinusoidal endothelial cells affects lipid transport and the immune microenvironment. Therefore, simultaneously isolating HPCs and NPCs while maintaining their activity and functional integrity has become a core requirement for elucidating the cellular and molecular mechanisms of NAFLD.

[0058] Currently, hepatocyte isolation mainly relies on the classic "two-step collagenase perfusion method," but this method faces multiple challenges in high-fat diet-induced obese mouse models. A high-fat diet leads to lipid accumulation within hepatocytes, decreasing cell membrane stability. Traditional high-concentration collagenase (1-2 mg / mL) and prolonged digestion (10-20 minutes) easily induce cell rupture, significantly reducing viable cell rate. Simultaneously, the expression and function of NPCs in the liver tissue of obese mice are altered, with increased adhesion between Kupffer cells, stellate cells, and HPCs, making effective separation difficult under traditional centrifugation conditions (such as high-speed centrifugation), resulting in insufficient NPC purity. Furthermore, the classic method requires a peristaltic pump to control the perfusion rate, making it highly equipment-dependent, and requires a large amount of collagenase (75-100 mg per mouse), resulting in high experimental costs and preventing adjustments to the perfusion rate based on liver condition. The traditional procedure requires multiple centrifugations and washes, prolonging cell exposure to a non-physiological environment, further reducing cell viability. Although existing studies have attempted to improve cell viability by reducing collagenase concentration or shortening digestion time, a systematic optimization strategy for high-fat models is lacking. For example, low-concentration enzymatic digestion may lead to incomplete digestion, affecting cell yield, and the optimization of density gradient centrifugation parameters has not yet been validated in high-fat mice. Therefore, developing a separation method that balances cell viability, purity, and ease of operation has become an urgent need in the field of NAFLD research.

[0059] The improved method proposed in this patent achieves efficient separation through the following technical features: C57BL / 6J mice were fed a high-fat diet with 60% fat content for 12-16 weeks, and the NAFLD model was successfully constructed by combining serum biochemical indicators and histological staining; a low concentration of type IV collagenase (0.15 mg / mL) and precisely controlled perfusion time (5-7 minutes) were used, combined with manual perfusion to replace the peristaltic pump to reduce mechanical damage; a 20% / 60% double-layer Percoll gradient was introduced, and high-efficiency separation of HPCs and NPCs was achieved by low-speed centrifugation (1800g, 20 minutes), with an NPCs survival rate of 91.4%; after perfusion, hepatocytes were maintained in a 4°C ice bath environment throughout the process, and the centrifugation step adopted low temperature (4°C) and short operation to maximize the preservation of cell function.

[0060] The specific implementation process is as follows: First, a high-fat diet-induced NAFLD mouse model was constructed. Male C57BL / 6J mice aged 6-8 weeks were selected and, after one week of acclimatization, divided into a normal diet group (ND) and a high-fat diet group (HFD). The HFD group's diet contained 60% fat and was continued for 12-16 weeks. Body weight was measured weekly, and serum samples were collected at week 16 to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. The HFD group showed a significant increase in body weight and a 2-3 fold increase in ALT and AST levels. Liver tissue was collected for hematoxylin-eosin (H&E) staining and Oil Red O staining to observe hepatocyte lipid vacuolation and lipid droplet accumulation. The hepatocytes of the HFD group mice showed significant lipid degeneration.

[0061] In the separation of parenchymal and non-parenchymal cells, liver perfusion and digestion were performed first. Mice were anesthetized with isoflurane and fixed on the operating table, and the abdominal cavity was opened to expose the inferior vena cava and portal vein. Perfusion solution I contained sodium bicarbonate, magnesium sulfate, potassium dihydrogen phosphate, D-glucose, and EGTA. Perfusion solution II was perfusion solution I with the addition of calcium chloride and 0.15 mg / mL type IV collagenase. In the first perfusion step, 30 mL of perfusion solution I was infused at a rate of 5 mL / min for 6 minutes until the liver swelled and turned yellowish-white. In the second perfusion step, perfusion solution II was switched to 40 mL at a rate of 3 mL / min for 5-7 minutes until the liver softened and became brittle. Manual perfusion was used instead of a peristaltic pump, and the flow rate was precisely controlled by syringe pressure to avoid mechanical damage. Perfusion solution II was preheated to 37°C to maintain enzyme activity. After perfusion, the liver was removed and placed in a sterile culture dish, rinsed with DMEM medium, and minced. The cell suspension was collected by filtering through a 70 μm filter. The cell suspension was centrifuged at 4°C and 50g for 3 minutes. The supernatant was discarded, and the precipitate was liver parenchymal cells (HPCs). The supernatant was transferred to a new tube for NPCs separation.

[0062] In the non-plasmic cell density gradient centrifugation step, 5 mL of 60% Percoll and 6 mL of 25% Percoll were added sequentially to a 15 mL centrifuge tube to form a density gradient interface. The NPCs suspension was slowly spread on the top layer of the Percoll gradient and centrifuged at 1800g for 20 minutes at 4°C. After centrifugation, the tube separated into three layers: the upper layer consisted of dead cells and debris, the middle layer consisted of NPCs, and the lower layer consisted of red blood cells. The middle layer of cells was transferred to a new tube using a pipette. The NPCs were washed twice with PBS (centrifuged at 900g for 8 minutes at 4°C), and the supernatant was discarded to obtain purified NPCs; the HPCs precipitate was resuspended in DMEM, centrifuged at 50g for 3 minutes, washed twice, and the supernatant was discarded to obtain purified HPCs.

[0063] Cell quality assessment included viability testing, purity verification, and functional characterization. Flow cytometry analysis showed that HPCs had a viability of 94.3%, and NPCs had a viability of 91.4%. Western blot analysis revealed high expression of specific markers: hepatocyte nuclear factor 4α (HNF4α) in HPCs, and significant expression of vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA), and EMR1 in NPCs. Reverse transcription quantitative PCR was used to detect the mRNA levels of HNF4α, CD68, and α-SMA. HNF4α mRNA expression was significantly lower in NPCs than in HPCs, while CD68 and α-SMA mRNA expression were significantly increased. HPCs were cultured in low-glucose DMEM medium for 24 hours, and triglyceride (TAG) content and Plin2 protein expression were measured. The TAG content in the HFD group was 3 times higher than that in the ND group, and Plin2 protein expression was significantly upregulated, confirming the lipid degeneration phenotype.

[0064] In the implementation case, hepatocytes were isolated from three HFD mice using the method described above, and the cell yield and survival rate were statistically analyzed. Approximately 3.3 × 10⁻⁶ HPCs were obtained from each mouse. 9 There are approximately 2.2 × 10⁸ NPCs. 8 The survival rate of HPCs was 95.2% ± 1.1%, and that of NPCs was 92.7% ± 0.8%. Isolated HPCs and NPCs were co-cultured, and lipopolysaccharide (LPS, 100 ng / mL) was added to induce an inflammatory response. The secretion levels of inflammatory factors (TNF-α, IL-6) were measured. The concentrations of TNF-α and IL-6 in the supernatant of the co-culture system were significantly higher than those in HPCs cultured alone, suggesting that NPCs exacerbate inflammatory damage in HPCs through paracrine effects.

[0065] Compared with existing technologies, this patent offers significant advantages. By reducing collagenase concentration and shortening digestion time, combined with low-temperature operation, the survival rate of HPCs is increased from 80%-85% using traditional methods to 94.3%, and the survival rate of NPCs is increased from 75%-80% to 91.4%. Double-layer Percoll gradient centrifugation achieves NPC purity exceeding 90%, and the expression of specific markers remains consistent with traditional methods, ensuring the reliability of subsequent mechanistic studies. Manual perfusion replaces peristaltic pumps, reducing equipment dependence and decreasing collagenase usage by 80%, significantly lowering experimental costs. The overall separation process is shortened to within 30 minutes, minimizing the impact of in vitro operations on cell viability.

[0066] like Figure 5 As shown: Overview of the improved HPC and NPC isolation procedures

[0067] (A) Mice fed a high-fat diet (12-16 weeks) were restrained and anesthetized. Then the...

[0068] In the abdominal cavity, expose the inferior vena cava and insert an indwelling catheter. Inject perfusion solution P1 (containing EGTA). After the liver swells, infuse the portal vein...

[0069] The catheter is clamped, and perfusion is slowed for 3 minutes, then accelerated after 3 minutes until perfusion fluid P1 is pushed through. Simultaneously, the liver turns yellowish-white. This process should be controlled within 5-7 minutes. The indwelling needle is closed, and 30 ml of perfusion fluid P2 (containing CaCl2) is aspirated. Perfusion continues slowly, maintaining a slow rate for 3 minutes, then increasing the rate until digestion is complete. This process should be controlled within 5-7 minutes. Finally, the liver cell suspension is filtered through a 70 μm filter into a sterile 50 ml centrifuge tube. Throughout the process, the liver is kept on ice, which is then used to maintain the viability temperature of hepatocytes and non-parenchymal cells through low-dose perfusion.

[0070] (B) Centrifugation: 4°C, 50g, 3 minutes. The precipitate is liver cells, and the precipitate and supernatant are collected.

[0071] (C) Resuspend the precipitate in DMEM-1.0 g / L glucose. Centrifuge: 4°C, 50 g, 3 min, and collect the precipitate and supernatant.

[0072] (D) The precipitate was resuspended in DMEM-1.0 g / L glucose. Centrifugation: 4°C, 50 g, 3 min. The precipitate was derived from hepatocytes of high-fat fed mice.

[0073] (E) Resuscitate HPCs using DMEM containing 10% FBS and 1% penicillin-streptomycin. A small amount of cells is pipetted into a 3.5 cm cell culture dish, and trypan blue and complete culture medium are added to the 3.5 cm cells. After mixing, cell viability is observed under an electron microscope. If cell viability is high, primary hepatocytes can be seeded onto cell culture plates. Incubate in a cell culture incubator (37°C / 5% CO2) for 24 hours.

[0074] (F) Collect the supernatant from the BC process. Centrifuge: 4℃, 200g, 3min. The precipitate consists of residual hepatocytes. Collect the supernatant.

[0075] (G) Collect the supernatant from process F. Centrifuge: 4°C, 900g, 8min. The precipitate is NPC.

[0076] (H) Redissolve the granules with 6 ml of 25% Percoll and place 5 ml of 60% Percoll at the bottom of a 15 ml test tube. Centrifuge: 4°C, 900 g, for 8 minutes. The upper layer consists of primary hepatocytes, the middle layer consists of non-parenchymal cells, and the lower layer consists of hepatocytes.

[0077] The layer consists of residual red blood cells.

[0078] (I) Resuspend the intermediate layer suspension with PBS. Centrifuge: 4°C, 900g, 10 minutes to precipitate NPC.

[0079] (J) Liver of mice fed a high-fat diet.

[0080] (K) Liver perfusion.

[0081] (L) Liver digestion.

[0082] (M) Hepatocyte dissociation.

[0083] (N) The hepatocyte suspension was filtered using a 70 μm filter.

[0084] (O) Mouse hepatocytes fed a high-fat diet.

[0085] (P) Non-plasmic cells of mice fed a high-fat diet.

[0086] Survival rates of isolated liver HPC and NPC in DIO mice

[0087] Lipid-rich cells exhibit greater fragility, leading to lower post-isolation survival rates; further assessment of the feasibility of isolation is needed.

[0088] Hepatocytes (HPCs) and non-parenchymal cells (NPCs) were counted using both automated and manual hematology counters. The total number of HPCs was found to be 3.3 × 10⁹, while the total number of NPCs was 22 × 10⁸. Figure 5 A). Cytometric analysis of the flow cell components showed that the average viable percentages of HPCs and NPCs were 94.3% and 91.4%, respectively.

[0089] like Figure 6 As shown: Survival rates of liver HPCs and NPCs isolated from DIO mice. (A) Number of cells: HPCs and NPCs isolated from the livers of HFD-treated mice detected by cell counter. (B) Percentage of viable HPCs. (C) Percentage of viable NPCs. Experiment

[0090] HPCs and NPCs were isolated 5 times, and the cell count and percentage of viable cells were also measured 5 times.

[0091] Expression and purity of HPC and NPC in isolated livers of obese mice

[0092] To evaluate the purity of HPCs and NPCs in our improved isolation method, we assessed the expression and purity of these isolated cell types before and after the purity modification. Initially, we employed a literature that did not use Percoll in the isolation of NPCs. Western blot analysis showed that specific markers of HPCs and NPCs, including vascular endothelial cadherin (VE-cadherin, a liver sinusoidal marker), α-smooth muscle actin α-SMA (a liver marker in stellate cells), epidermal growth factor-like module containing mucin-like hormone receptor-like 1 (ERM1, a marker of Kupffer cells), and hepatocyte nuclear factor 4α (HNF4-α, a marker of HPCs), were expressed in their respective cell populations. However, it has been noted that HNF4-α is generally a marker of HPCs and is also present in NPCs, as well as EMR1 detected in HPCs. α-SMA is a marker of fibrosis, and we observed elevated levels in HPCs. Figure 6 A). Using our improved isolation method combined with Percoll for NPC isolation and an improved washing step for HPC isolation, Western blot (WB) analysis showed that HNF4a was expressed only in HPCs. In contrast, VE-cadherin, EMR1, and α-SMA were more prevalent in NPCs. Figure 7 B). These genes in NPCs at the mRNA level were also quantitatively quantified via reverse transcription using quantitative polymerase chain reaction (RT-qPCR). Figure 7 C). The results showed that HNF4a mRNA levels in NPCs decreased significantly under the following conditions, while CD68 and α-SMA mRNA levels increased significantly.

[0093] These results indicate that the improved method produces NPCs with higher purity.

[0094] like Figure 7 As shown: Expression and purity of HPCs and NPCs in the liver of DIO mice. Western blotting of proteins expressed before (A) and after (B) isolation of HPCs and NPCs. RT-PCR detection of genes expressed in HPCs and NPCs after isolation method modification (C).

[0095] HPCs Characterization: Duration of HPCs Cultured in Low-Glucose Dulbecco's Culture Modified Eagle Medium (DMEM) for Only 48 Hours After Electroplating. To Maintain Functional Properties and Phenotypic Characteristics, Hepatocytes Exhibited Typical Characteristics of Lipid-Rich HPCs Through Cell Morphology. Figure 7 A) and triglyceride (TAG) content ( Figure 7 B) HPCs cultured for 24 hours. In addition, plin2 protein expression was significantly increased ( Figure 7 C). In summary, these findings confirm the successful isolation of lipid-rich HPCs.

[0096] The implementation of this patent provides an efficient and stable cell isolation tool for NAFLD research, possessing significant scientific value and promising clinical applications. By simultaneously isolating HPCs and NPCs, their interactions in lipid metabolism, inflammation, and fibrosis can be systematically analyzed, deepening the understanding of NAFLD pathogenesis. High-quality primary cells provide an ideal model for in vitro screening of anti-NAFLD drugs (such as PPARα agonists and FGF21 analogs), accelerating the new drug development process. This method can be extended to hepatocyte research for other metabolic diseases (such as diabetes and obesity), improving the efficiency of basic medical research translation into clinical applications.

[0097] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0098] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An improved method of isolation of hepatocytes and non-parenchymal cells from mice with high-fat diet-induced non-alcoholic fatty liver disease, characterized in that, The method comprises the following steps: Mouse model construction: Six to eight-week-old male C57BL / 6J mice were selected and divided into a normal diet group and a high-fat diet group after adaptive feeding. The high-fat diet group was fed with high-fat feed containing 60% fat for 12 to 16 weeks to construct a high-fat diet-induced non-alcoholic fatty liver disease mouse model. The model construction was verified to be successful by detecting significant weight gain of the mice, elevated levels of serum alanine aminotransferase and aspartate aminotransferase, and histological analysis of liver tissue showing lipid accumulation in hepatocytes.

2. The improved method of claim 1 wherein, The separation steps of liver parenchymal cells and non-parenchymal cells include: Liver perfusion and digestion: A two-step collagenase perfusion technique was used. First, perfusion with a perfusion solution containing EGTA was performed to swell the liver, and then the portal vein was clamped, the perfusion speed was slowed down for three minutes, and then accelerated until the liver turned yellow-white. Then, perfusion was performed with a perfusion solution containing CaCl2 and low-concentration type IV collagenase at 0.15 mg / ml, and the digestion time was 5 to 7 minutes. During the perfusion process, the body temperature of the mouse was kept normal to maintain cell activity.

3. The improved method of claim 2 wherein, The further separation and purification steps of liver parenchymal cells and non-parenchymal cells include: Cell separation and purification: Hepatocytes were obtained by low-speed centrifugation, and the centrifugation conditions were 50 g, 4°C, and 3 minutes, and repeated 4 times of washing to reduce non-parenchymal cell contamination. Non-parenchymal cells were separated by density gradient centrifugation using a 25% / 60% Percoll density gradient, and the centrifugation conditions were 1,800 g, 4°C, and 20 minutes, and the non-parenchymal cells were located at the interface between the two layers of Percoll. The collected non-parenchymal cells were further purified by a double centrifugation step.

4. The improved method of claim 3 wherein, The method also includes the steps of quality assessment of the separated liver parenchymal cells and non-parenchymal cells, which specifically include: Cell quality assessment: Flow cytometry was used to analyze cell viability and cytotoxicity, and calcein AM and propidium iodide double staining were used to detect cell survival rate, and the average survival rates of liver parenchymal cells and non-parenchymal cells reached 94.3% and 91.4%, respectively. Western blotting and reverse transcription quantitative polymerase chain reaction techniques were used to detect the expression of specific markers of liver parenchymal cells and non-parenchymal cells to verify the purity and characteristics of the cells.

5. The improved method according to any one of claims 1 to 4, characterized in that, The method is suitable for studying the interaction between liver parenchymal cells and non-parenchymal cells in a high-fat diet-induced non-alcoholic fatty liver disease mouse model and the pathogenesis, and provides high-quality primary cell materials for drug target research.

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