A method for culturing rat primary hepatocytes in vitro

Abstract

The application relates to a method for culturing rat primary hepatocytes in vitro, which comprises the following steps: counting rat primary hepatocyte suspension, inoculating the counted rat primary hepatocyte suspension on a coated plate, and culturing to obtain purified rat primary hepatocytes by using a hepatocyte culture medium; the hepatocyte culture medium comprises small molecule additives, DMEM high-sugar culture medium, antibiotics, fetal bovine serum, dexamethasone and insulin, and the small molecule additives comprise FH1 and / or FPH1. The hepatocyte culture medium containing FH1 and / or FPH1 is used for culturing rat primary hepatocyte suspension, which can significantly enhance the function of rat primary hepatocytes, and improve the mRNA and protein expression levels of albumin (ALB) and hepatocyte nuclear factor 4 alpha (HNF4 alpha).

Description

Technical Field

[0001] This invention relates to the fields of cell biology and in vitro hepatocyte culture technology, specifically to a method for in vitro culture of primary rat hepatocytes. Background Technology

[0002] In fields such as drug metabolism and toxicology evaluation, analysis of liver physiological mechanisms, and bioartificial liver support systems, primary hepatocyte in vitro culture models, which can realistically reflect liver metabolic characteristics and drug responses, have irreplaceable research value. Primary hepatocytes are the main functional units of the liver parenchyma, responsible for the synthesis and secretion of plasma proteins such as albumin, drug oxidation metabolism centered on cytochrome P450 enzyme systems (such as CYP1A2, CYP2B1, and CYP3A1), as well as urea synthesis, bile acid production, and many other key physiological functions. Establishing an in vitro hepatocyte culture system capable of maintaining these functional activities long-term is a core prerequisite for conducting drug hepatotoxicity screening, drug-drug interaction assessment, and constructing bioartificial hepatocyte reactors.

[0003] Based on their species origin, primary hepatocyte culture can be broadly categorized into human primary hepatocyte culture and animal primary hepatocyte culture. Human primary hepatocytes best reflect the metabolic characteristics of the human liver; however, their availability is extremely limited due to scarce sources such as surgically discarded liver tissue or donor livers that do not meet transplantation standards. Furthermore, significant differences exist among donors in genetic background, underlying diseases, and medication history. They cannot proliferate under in vitro conditions, and liver function markers are rapidly lost during culture. These factors severely restrict their large-scale application. Rat primary hepatocytes are one of the most widely used in vitro models in liver physiology, toxicology, and drug metabolism studies, and are considered the gold standard model. However, primary hepatocytes rapidly dedifferentiate during isolation and in vitro culture, leading to a sharp loss of expression of liver-specific genes and proteins. How to effectively maintain the functional activity of rat primary hepatocytes under in vitro conditions remains a pressing technical problem in this field.

[0004] To slow down the dedifferentiation process, researchers have tried various strategies. At the culture system level, the combined addition of dexamethasone and insulin is the most classic approach. Studies have shown that the synergistic effect of dexamethasone and insulin can significantly improve the adhesion efficiency of rat primary hepatocytes, and the polygonal epithelial morphology of mature hepatocytes can be maintained for a certain period. However, this maintenance effect is very limited and short-lived: even under the continuous presence of hormones, rat primary hepatocytes still undergo irreversible, multi-level functional collapse during culture. Glucagon has also been used in combination with dexamethasone and insulin to regulate cAMP metabolism and glycogen synthesis in hepatocytes, but this multi-hormone approach is still not ideal in inhibiting dedifferentiation, and maintaining the activity of cAMP-related enzymes in the long term is equally difficult. Nicotinamide has been reported to support the proliferation of primary hepatocytes and promote the formation of "small hepatocyte" colonies under high concentrations, but the effect of nicotinamide is mainly focused on promoting proliferation rather than maintaining maturation and differentiation functions. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) have also been used to promote the long-term survival of hepatocytes in vitro. However, EGF mainly promotes proliferation rather than maintaining differentiation, and its ability to induce DNA synthesis is significantly reduced in aged rat hepatocytes. More importantly, the effects of the above-mentioned additives are one-dimensional—dexamethasone and insulin mainly improve adhesion and synthesis functions, nicotinamide tends to promote proliferation, and HGF / EGF tends to promote survival and proliferation—none of which can achieve multi-dimensional synergistic maintenance of hepatocyte synthesis function, metabolic enzyme activity, and differentiation regulation.

[0005] In multi-factor combination strategies, a 5C small molecule mixture consisting of SB431542 (TGF-β inhibitor), forskolin, DAPT (Notch inhibitor), IWP2 (Wnt inhibitor), and LDN193189 has been reported to inhibit hepatocyte dedifferentiation and maintain albumin secretion, urea synthesis, and drug metabolism during culture periods lasting over a month. Furthermore, small molecules such as IWR1 (Wnt signaling inhibitor), A83-01 (TGF-β signaling inhibitor), and Dihexa (HGF receptor agonist) have also been incorporated into various function maintenance regimens for combination trials. However, these regimens are complex (typically involving more than five signaling pathway modulators), costly, and the synergistic and antagonistic effects between factors are difficult to precisely control. There are also significant differences in activity between different batches, and all are primarily optimized based on human primary hepatocyte systems; their efficacy and applicability in rat primary hepatocytes have not yet been fully validated.

[0006] In summary, primary rat hepatocytes face a critical challenge of simultaneous and rapid decline in synthetic function, metabolic enzyme activity, and differentiation regulation capacity under conventional culture systems. Existing improved culture strategies are either singular in function or involve cumbersome components, significant batch-to-batch variations, and difficulties in standardization. Therefore, there is an urgent need to develop a culture protocol with relatively well-defined components, convenient operation, and the ability to enhance the expression levels of multiple key transcription factors to effectively address the problem of rapid dedifferentiation and functional decline of primary rat hepatocytes in vitro. Summary of the Invention

[0007] Based on the above description, the present invention provides a method for in vitro culture of primary rat hepatocytes, aiming to solve the problems of rapid dedifferentiation and functional decline of primary rat hepatocytes in vitro.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a method for in vitro culture of primary rat hepatocytes, comprising the following steps: S10. Obtain liver from rats; S20. The hepatocytes in the liver are dissociated and crudely extracted to obtain a single-cell suspension; S30. Centrifuge the single-cell suspension and wash and purify it multiple times to obtain purified rat primary hepatocyte precipitate. S40. The rat primary hepatocyte precipitate was resuspended in complete culture medium to obtain a rat primary hepatocyte suspension. S50. Count the rat primary hepatocyte suspension, inoculate the counted rat primary hepatocyte suspension onto a coated plate, and culture it in hepatocyte culture medium to obtain purified rat primary hepatocytes. In step S50, the hepatocyte culture medium includes small molecule additives, DMEM high glucose medium, antibiotics, fetal bovine serum, dexamethasone, and insulin, and the small molecule additives include FH1 and / or FPH1.

[0009] In some embodiments, the small molecule additive includes FH1, wherein the molar concentration of FH1 is 5 μmol / L to 30 μmol / L; and / or, The small molecule additive includes FPH1, and the molar concentration of FPH1 is 5 μmol / L to 30 μmol / L.

[0010] In some embodiments, the hepatocyte culture medium contains: The volume of the DMEM high-glucose culture medium is 500 ml; The volume percentage of the antibiotic is 1%-2%; The volume percentage of the fetal bovine serum is 10%-15%; The amount of dexamethasone added is 0.5 μM-2 μM; The insulin concentration is 0.5 μg / mL to 2 μg / mL.

[0011] In some embodiments, step S20 includes: placing the liver in a sterile culture dish at 4°C, removing the liver capsule, scraping the liver tissue, filtering, and obtaining a single-cell suspension; The sterile culture dish contains a cell washing solution, which includes DMEM high-glucose culture medium.

[0012] In some embodiments, step S30 includes: centrifuging the single-cell suspension, removing the supernatant, adding cell washing buffer to the precipitate, resuspending the cells, centrifuging, removing the supernatant, and repeating the washing 2-3 times until the supernatant is clear and bloodless, obtaining purified rat primary hepatocyte precipitate; wherein: In the centrifugation operation, the centrifugal force is 50g, the centrifugation time is 3-5 minutes, and the centrifugation temperature is 4℃; and / or, The cell washing solution includes DMEM high-glucose medium.

[0013] In some implementations, in step S40: The temperature of the complete culture medium is 37°C; and / or, The complete culture medium includes DMEM high glucose medium, antibiotics, fetal bovine serum, dexamethasone, and insulin.

[0014] In some embodiments, prior to step S50, the preparation of a coating plate is further included, wherein the method for preparing the coating plate includes the following steps: Prepare a 0.0006 mol / L acetic acid aqueous solution, filter to sterilize, and dilute with a 5 mg / ml rat tail collagen solution to obtain the coating solution; The culture plate was coated with coating solution. After coating, the excess coating solution was aspirated. The coated culture plate was washed 1-2 times with phosphate buffer to obtain the coated plate. The volume ratio of the aqueous acetic acid solution to the rat tail collagen solution is (100~250):1.

[0015] In some implementations, step S10 includes: After anesthetizing the rats, the first and second perfusion fluids were sequentially instilled through the portal vein. After the liver was digested and softened, it was cut off.

[0016] In some embodiments, the first perfusion solution comprises, by mass concentration, the following substances: Sodium chloride 8.3 g / L, potassium chloride 0.5 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 2.4 g / L, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid 0.95 g / L, balance water.

[0017] In some embodiments, each 300 ml of the second perfusion fluid comprises: 1.17g sodium chloride, 1g potassium chloride, 4.8g 4-hydroxyethylpiperazine ethanesulfonic acid, 1.5g bovine serum albumin, 0.158g anhydrous calcium chloride, and 0.3g collagenase IV.

[0018] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: (1) Adding FH1 and / or FPH1 to the hepatocyte culture medium can enhance the liver function of mature hepatocytes, increase the expression levels of albumin (ALB) and hepatocyte nuclear factor 4α (HNF4α) mRNA and protein, and achieve synergistic enhancement of the synthetic function and differentiation regulation ability of mature hepatocytes, effectively overcoming the problem of rapid dedifferentiation and functional decline in the in vitro culture process of rat primary hepatocytes.

[0019] (2) Rat primary hepatocyte suspension was cultured in hepatocyte culture medium containing FH1 and / or FPH1. The components are simple, the materials are stable, the batch-to-batch stability of the materials is high, and the inactivation is slower after being added to the culture medium and used for cell culture, thus making the culture effect more stable.

[0020] (3) This invention provides a new and effective means for maintaining the function of primary rat hepatocytes in vitro, which significantly enhances the application value of primary rat hepatocyte models in liver physiology, toxicology and drug metabolism research. Attached Figure Description

[0021] Figure 1 This is a bright-field microscopic image of purified primary rat hepatocytes from Comparative Example 1 of this invention.

[0022] Figure 2 These are bright-field microscopic images of primary rat hepatocytes purified in Examples 1-3 and Comparative Example 1 of this invention.

[0023] Figure 3 This is a microscopic image of purified rat primary hepatocytes stained with Oil Red O in Comparative Example 1.

[0024] Figure 4 This is a microscopic image of purified rat primary hepatocytes stained with PAS, as shown in Comparative Example 1.

[0025] Figure 5 The images show the Western Blot results of purified rat primary hepatocytes from Examples 1-3 and Comparative Example 1.

[0026] Figure 6 The images show the RT-qPCR results of purified rat primary hepatocytes from Examples 1-3 and Comparative Example 1. Detailed Implementation

[0027] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0029] Rat primary hepatocytes face a critical challenge under conventional culture systems: a simultaneous and rapid decline in synthetic function, metabolic enzyme activity, and differentiation regulation. Existing modified culture strategies are either singular in function or involve cumbersome components, significant batch-to-batch variations, and difficulties in standardization. Therefore, there is an urgent need to develop a culture protocol with relatively well-defined components, convenient operation, and the ability to synergistically enhance albumin secretion, CYP enzyme activity, and the expression levels of key transcription factors such as HNF4α, to effectively address the problem of rapid dedifferentiation and functional decline of rat primary hepatocytes in vitro.

[0030] In view of this, the present invention provides a method for in vitro culture of primary rat hepatocytes, comprising the following steps: S10. Obtain liver from rats; S20. The hepatocytes in the liver are dissociated and crudely extracted to obtain a single-cell suspension; S30. Centrifuge the single-cell suspension and wash and purify it multiple times to obtain purified rat primary hepatocyte precipitate. S40. The rat primary hepatocyte precipitate was resuspended in complete culture medium to obtain a rat primary hepatocyte suspension. S50. Count the rat primary hepatocyte suspension, inoculate the counted rat primary hepatocyte suspension onto a coated plate, and culture it in hepatocyte culture medium to obtain purified rat primary hepatocytes. In step S50, the hepatocyte culture medium includes small molecule additives, DMEM high glucose medium, antibiotics, fetal bovine serum, dexamethasone, and insulin, and the small molecule additives include FH1 and / or FPH1.

[0031] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: (1) Adding FH1 and / or FPH1 to the hepatocyte culture medium can enhance the liver function of mature hepatocytes, increase the expression levels of albumin (ALB) and hepatocyte nuclear factor 4α (HNF4α) mRNA and protein, and achieve synergistic enhancement of the synthetic function and differentiation regulation ability of mature hepatocytes, effectively overcoming the problem of rapid dedifferentiation and functional decline in the in vitro culture process of rat primary hepatocytes.

[0032] (2) Rat primary hepatocyte suspension was cultured in hepatocyte culture medium containing FH1 and / or FPH1. The components are simple, the materials are stable, the batch-to-batch stability of the materials is high, and the inactivation is slower after being added to the culture medium and used for cell culture, thus making the culture effect more stable.

[0033] (3) This invention provides a new and effective means for maintaining the function of primary rat hepatocytes in vitro, which significantly enhances the application value of primary rat hepatocyte models in liver physiology, toxicology and drug metabolism research.

[0034] Furthermore, the small molecule additive includes FH1, with a molar concentration of 5 μmol / L to 30 μmol / L; and / or, the small molecule additive includes FPH1, with a molar concentration of 5 μmol / L to 30 μmol / L. Using the above-mentioned molar concentrations of small molecule additives has a better promoting effect on the differentiation and maintenance of rat primary hepatocytes during in vitro culture, can more effectively inhibit cell dedifferentiation, and simultaneously enhance hepatocyte-specific functions such as synthesis and metabolism.

[0035] It should be noted that FH1 and FPH1 can be added simultaneously or selectively, and this application does not impose any restrictions. In one embodiment, the small molecule additive includes FH1, and when the molar concentration of FPH1 is 15 μmol / L, it has a further effect on promoting the maintenance and enhancement of hepatocyte function during the in vitro culture of rat primary hepatocytes. Using hepatocyte culture medium containing FH1 to culture rat primary hepatocyte suspension can significantly enhance the function of rat primary hepatocytes, while increasing the expression levels of mRNA and protein of albumin (ALB), cytochrome P450 1A2 (CYP1A2) and hepatocyte nuclear factor 4α (HNF4α), thereby achieving a multi-dimensional synergistic enhancement of the synthetic function, metabolic function and differentiation regulation of mature hepatocytes, effectively overcoming the problem of rapid dedifferentiation and functional decline during the in vitro culture of rat primary hepatocytes.

[0036] Furthermore, in the hepatocyte culture medium: the volume of the DMEM high-glucose medium is 500 ml; the volume percentage of the antibiotic is 1%-2%; the volume percentage of the fetal bovine serum is 10%-15%; the amount of dexamethasone added is 0.5 μM-2 μM; and the mass concentration of the insulin is 0.5 μg / mL-2 μg / mL.

[0037] In this hepatocyte culture medium, DMEM high glucose provides hepatocytes with a high concentration of glucose to support vigorous energy metabolism and basic nutritional needs; antibiotics (usually penicillin / streptomycin) inhibit bacterial contamination and ensure the stability of the culture system; fetal bovine serum provides adhesion factors, growth factors, and essential nutrients to promote cell survival and spread; dexamethasone, as a synthetic glucocorticoid, can induce hepatocyte-specific gene expression, stabilize the cytoskeleton, and inhibit inflammation-like activation; insulin promotes glycogen synthesis, protein synthesis, and cell survival by activating insulin receptors, and synergistically maintains some differentiation characteristics with dexamethasone. However, the above components are unlikely to enhance hepatocyte cellular function. When the small molecule FH1 is introduced on this basis, FH1 becomes the core driving force as a potent inducer of HNF4α: the hormone response environment pre-constructed by dexamethasone and insulin enables hepatocytes to maintain their ability to respond to differentiation signals, while the high glucose levels in serum and DMEM ensure cell viability and energy supply, and antibiotics maintain a sterile environment; on this platform, FH1 efficiently upregulates the expression of hepatocyte nuclear factor HNF4α, thereby strongly driving the transcription of its downstream albumin gene ALB and drug-metabolizing enzyme gene CYP1A2, and synergistically with the anti-dedifferentiation effect of dexamethasone to stabilize and block the mesenchymal pathway, thereby achieving synergistic inhibition of rapid dedifferentiation of primary hepatocytes in vitro and significantly improving synthetic and metabolic functions.

[0038] Furthermore, step S10 includes: after anesthetizing the rat, the first perfusion fluid and the second perfusion fluid are sequentially infused through the portal vein, and the liver is cut off after it has been digested and softened.

[0039] Specifically, the following steps can be taken: Clamp the proximal end of the portal vein (closer to the liver) with an arterial clamp. Make a small oblique incision (no more than 1 / 3 of the tube diameter) at the distal end of the portal vein with ophthalmic scissors. Quickly insert a 22G intravenous catheter filled with pre-perfusion fluid into the blood vessel, advance the cannula, and remove the needle core. Double-ligate and fix it with pre-threaded sutures to ensure no dislodgement or leakage. Immediately turn on the peristaltic pump and introduce the first perfusion fluid preheated to 37°C at a flow rate of 10-15 mL / min. Simultaneously, quickly cut open the inferior vena cava below the liver as the perfusion fluid outlet to drain waste fluid. During perfusion, continuously heat the first perfusion fluid. The liver will rapidly change from dark red to a uniform yellowish-brown color, indicating effective perfusion. Pre-perfusion continues for 10-15 minutes until the outflow is completely clear and there is no visible blood residue in the liver. Before the end of pre-perfusion, ligate the superior vena cava above the liver to prevent fluid shunting during subsequent enzyme perfusion and ensure that the perfusion fluid flows fully through the hepatic sinusoids. After pre-perfusion, quickly switch the perfusion fluid to a second perfusion fluid at 37°C and adjust the flow rate to 5-8 mL / min. Perform open perfusion for the first 2 minutes, discarding the outflow. Then, collect the outflow from the inferior vena cava below the liver into a sterile container and establish circulating perfusion using a peristaltic pump. Circulating perfusion lasts 8-12 minutes, closely monitoring the liver throughout. The digestion endpoint is determined when the liver capsule shrinks, softens, and indentation occurs when forceps gently touch the liver capsule; the liver tissue exhibits a slight "tofu-like" appearance. Immediately stop perfusion and quickly remove the entire liver.

[0040] Furthermore, by mass concentration, the first perfusion solution comprises the following substances: sodium chloride 8.3 g / L, potassium chloride 0.5 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 2.4 g / L, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid 0.95 g / L, and the balance being water. The aforementioned first perfusion solution is a calcium-free chelating buffer. Ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA) effectively chelates calcium ions in the interstitial fluid, forcing calcium-dependent hepatocyte junction complexes (such as cadherin-mediated junctions) to dissociate, thus softening the liver tissue and laying the foundation for subsequent digestion. Sodium chloride and potassium chloride together maintain appropriate osmotic pressure and ion balance, protecting cell morphology. 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) provides a stable, physiologically-range pH buffer system, preventing acidic substances produced by cell metabolism from causing acid-base imbalance. After overall perfusion, residual blood can be removed and the liver tissue structure can be loosened, which facilitates the subsequent entry of collagenase perfusion solution and efficient digestion of the extracellular matrix.

[0041] Furthermore, each 300ml of the second perfusion fluid includes: 1.17g of sodium chloride, 1g of potassium chloride, 4.8g of 4-hydroxyethylpiperazine ethanesulfonic acid, 1.5g of bovine serum albumin, 0.158g of anhydrous calcium chloride, and 0.3g of collagenase IV. This second perfusion fluid is a calcium-containing collagenase digestion solution. Sodium chloride and potassium chloride work together to maintain the osmotic pressure and ion balance of the extracellular fluid, protecting hepatocyte morphology; 4-hydroxyethylpiperazine ethanesulfonic acid provides a physiological pH buffer, preventing acid-base fluctuations during digestion; bovine serum albumin, as a protective colloid, can bind cytotoxic substances such as free fatty acids and maintain colloid osmotic pressure, reducing mechanical damage; anhydrous calcium chloride provides calcium ions, satisfying the enzyme activity requirements of collagenase IV and participating in cell membrane stability; collagenase IV specifically hydrolyzes collagen components in the extracellular matrix of liver cells, gently dissociating the connections between hepatocytes and digesting the tissue into single cells. Based on the softening of liver tissue by the initial perfusion, the perfusion fluid can efficiently release highly viable hepatocytes while maintaining cell integrity and function to the maximum extent.

[0042] Further, step S20 includes: placing the liver in a sterile culture dish at 4°C, removing the liver capsule, scraping the liver tissue, and filtering to obtain a single-cell suspension; wherein the sterile culture dish contains a cell washing solution, which includes DMEM high-glucose medium. Operating at 4°C rapidly inhibits the metabolic activity of isolated liver tissue and the activity of intracellular lysosomal enzymes and other hydrolytic enzymes, effectively slowing down cell autolysis and damage; using DMEM high-glucose medium as the cell washing solution provides an osmotic pressure, glucose energy substrate, and pH buffer system similar to that in vivo, maintaining the viability and stability of hepatocytes during the separation process. The sterile environment and removal of the liver capsule avoid contamination and exposure of the liver parenchyma; the scraping operation gently releases hepatocytes from the connective tissue matrix; combined with subsequent filtration to remove undispersed tissue fragments, cell clusters, and impurities, a high-purity, highly active single-cell suspension is finally obtained, laying a good foundation for subsequent inoculation and maintaining differentiation function.

[0043] Specifically, the liver can be placed in a sterile culture dish, and the liver capsule can be gently removed with ophthalmic forceps. The liver tissue can be gently scraped with micro-forceps to fully release the hepatocytes into the washing solution, forming a single-cell suspension. Avoid forceful tearing to reduce tissue fragmentation. The cell suspension is then filtered sequentially through 100-mesh and 200-mesh sterile cell sieves to remove undigested tissue fragments and connective tissue. The filtrate is collected in a 50mL sterile centrifuge tube to obtain the single-cell suspension.

[0044] Further, step S30 includes: centrifuging the single-cell suspension, removing the supernatant, adding cell washing buffer to the precipitate, resuspending the cells, centrifuging, removing the supernatant, and repeating the washing 2-3 times until the supernatant is clear and bloodless, obtaining purified rat primary hepatocyte precipitate; wherein: in the centrifugation operation, the centrifugation force is 50g, the centrifugation time is 3min, and the centrifugation temperature is 4℃; the cell washing buffer includes DMEM high-glucose culture medium. Using the conditions of 4℃ low temperature, 50g low centrifugation force, and short centrifugation time of 3 minutes, taking advantage of the large volume and rapid sedimentation of hepatocytes, selective and rapid precipitation can be achieved, while smaller red blood cells, cell debris, and non-parenchymal cells are suspended in the supernatant and removed; repeated gentle resuspension with cell washing buffer and centrifugation 2-3 times until the supernatant is clear and bloodless gradually washes away residual blood cells, cell debris, collagenase, and EGTA, etc. The entire process was conducted under low temperature and mild mechanical conditions to minimize cellular metabolic stress and mechanical damage, ultimately yielding high-purity, high-viability rat primary hepatocyte precipitates, laying the foundation for subsequent culture and functional maintenance.

[0045] It should be noted that the centrifugal force of 50g means that the relative centrifugal force is 50 times the acceleration due to gravity of the Earth.

[0046] Furthermore, in step S40: the temperature of the complete culture medium is 37°C; the complete culture medium includes DMEM high glucose medium, antibiotics, fetal bovine serum, dexamethasone, and insulin.

[0047] Furthermore, prior to step S50, the preparation of a coated plate is also included. The preparation method of the coated plate includes the following steps: preparing a 0.0006 mol / L acetic acid aqueous solution, filtering for sterilization, adding 200 μl to 500 μl of a 5 mg / ml rat tail collagen solution for dilution to obtain a coating solution; coating the culture plate with the coating solution, aspirating excess coating solution after coating, and washing the coated culture plate 1 to 2 times with phosphate buffer to obtain a coated plate; wherein the volume ratio of the acetic acid aqueous solution to the rat tail collagen solution is (100 to 250): 1.

[0048] In the above technical solution, rat tail collagen is dissolved in a 0.0006 mol / L dilute acetic acid aqueous solution, which keeps the collagen molecules in a stable, non-aggregated state. After filtration and sterilization, it is used to coat culture plates, forming a uniform extracellular matrix protein coating on the bottom of the plate. After coating, excess liquid is aspirated and the plate is washed with phosphate buffer to remove residual acidic solvents and unattached excess collagen, preventing local acidity from affecting subsequent cell activity. The resulting coated plate provides a substrate similar to the in vivo microenvironment for primary hepatocytes, effectively promoting hepatocyte adhesion, spreading, and survival.

[0049] In step S50, cell counting is performed on the purified rat primary hepatocyte suspension to precisely control the seeding density and ensure batch-to-batch consistency. The counted hepatocytes are seeded onto a pre-prepared collagen-coated plate and cultured using the aforementioned hepatocyte culture medium. Under the synergistic effect of the physical adhesion signals provided by the collagen matrix and the biochemical maintenance signals provided by factors such as dexamethasone, insulin, and FH1 in the culture medium, the hepatocytes rapidly adhere to the plate and form a typical epithelial-like polygonal morphology. This allows for the reconstruction of a stable differentiation phenotype in vitro, effectively inhibiting the dedifferentiation process and maintaining core liver functions such as albumin secretion and drug metabolism in the long term.

[0050] Furthermore, after the counted rat primary hepatocyte suspension is inoculated onto a coated plate, it can be cultured in complete culture medium before being cultured in hepatocyte culture medium.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The main reagents are shown in Table 1.

[0052] Table 1 Main Reagents

[0053] Example 1 This embodiment provides a method for in vitro culture of primary rat hepatocytes, including the following steps: S10. After weighing, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg). Once the corneal reflex disappeared and the muscles relaxed, the rats were fixed supine on a dissecting board. The abdomen was shaved, and the skin was disinfected three times with 75% alcohol. Heparin sodium 1000 U / kg was injected intraperitoneally and allowed to stand for 5 minutes to ensure systemic heparinization and prevent vascular clotting during perfusion. A 3-4 cm incision was made along the midline of the abdomen, extending from the pubic symphysis to below the xiphoid process, and the abdominal cavity was opened layer by layer. The abdominal wall was pulled apart laterally with hemostatic forceps to fully expose the surgical field. The intestines were wrapped with gauze soaked in 37°C warm saline and gently pushed towards the left side of the abdominal cavity, avoiding traction on the liver. The main portal vein and the inferior vena cava below the liver were fully exposed, and the perivascular connective tissue was bluntly dissected. Two sterile sutures were inserted below the main portal vein (one to fix the cannula, and one to ligate the distal end); one suture was inserted below the inferior vena cava below the liver for later use.

[0054] Clamp the proximal end of the portal vein (closest to the liver) with an arterial clamp. Make a small oblique incision (no more than 1 / 3 of the tube diameter) at the distal end of the portal vein using ophthalmic scissors. Quickly insert a 22G intravenous catheter filled with the first perfusion fluid into the vessel. After advancing the cannula, remove the needle core and double-ligate it with pre-threaded sutures to ensure no dislodgement or leakage. Immediately turn on the peristaltic pump and flow in the 37°C preheated first perfusion fluid at a flow rate of 15 mL / min. Simultaneously, quickly cut open the inferior vena cava below the liver as the perfusion fluid outlet to drain waste fluid. Continuously add the first perfusion fluid during perfusion. The liver will rapidly change from dark red to a uniform yellowish-brown color, indicating effective perfusion. Continue pre-perfusion for 15 minutes until the outflow is completely clear and no visible blood residue remains in the liver. Before ending the pre-perfusion, ligate the superior vena cava above the liver to prevent fluid shunting during subsequent enzyme perfusion and ensure that the perfusion fluid flows fully through the hepatic sinusoids.

[0055] After pre-perfusion, the perfusion fluid was quickly switched to a second perfusion fluid at 37°C, and the flow rate was adjusted to 8 mL / min. Open perfusion was performed for the first 2 minutes, and the outflow was discarded. Then, the outflow from the inferior vena cava was collected into a sterile container, and a peristaltic pump was used to establish circulating perfusion. Circulating perfusion lasted 10 minutes, with close monitoring of the liver throughout. The digestion endpoint was determined when the liver capsule became wrinkled and softened; gentle touch of the liver capsule with forceps caused indentation; and the liver tissue exhibited a slight "tofu-chew" appearance. Perfusion was immediately stopped at this point. After stopping perfusion, the entire liver was quickly removed.

[0056] S20. Place the liver in a sterile culture dish containing 4°C cell washing medium (DMEM high-glucose medium) and transfer it to a laminar flow hood for further processing. Gently remove the liver capsule with ophthalmic forceps and gently scrape the liver tissue with microforceps to form a cell suspension. Filter the cell suspension sequentially through 100-mesh and 200-mesh sterile cell sieves, and collect the filtrate in a 50mL sterile centrifuge tube.

[0057] S30. Centrifuge the cell suspension at 4℃ and 50g for 3 min, remove the supernatant, add pre-cooled cell washing buffer (DMEM high glucose medium) at 4℃ to the precipitate, gently pipette to resuspend the cells, centrifuge again at 4℃ and 50g for 3 min, remove the supernatant; repeat washing 3 times until the supernatant is clear and bloodless, to obtain the purified rat primary hepatocyte precipitate.

[0058] S40. The rat primary hepatocyte precipitate was resuspended in a complete culture medium preheated at 37°C to obtain a purified rat primary hepatocyte suspension.

[0059] S50. Prepare a 0.0006 mol / L acetic acid aqueous solution, filter to sterilize, and dilute with 5 mg / ml rat tail collagen solution (the volume ratio of acetic acid aqueous solution to rat tail collagen solution is 250:1) to obtain a coating solution; coat the culture plate with the coating solution, remove excess coating liquid after coating, and wash the coated culture plate twice with phosphate buffer to obtain a coated plate; count the rat primary hepatocyte suspension, inoculate the counted rat primary hepatocyte suspension onto the coated plate, culture in complete culture medium for 48 h, and then culture in hepatocyte culture medium for 96 h to obtain purified rat primary hepatocytes.

[0060] The preparation of the first perfusion solution includes the following steps: dissolving 8.3 g of NaCl, 0.5 g of KCl, 2.4 g of Hepes, and 0.95 g of EGTA in 950 mL of pure water, adjusting the pH to 7.4, bringing the volume to 1000 mL, and autoclaving to obtain the first perfusion solution. The preparation of the second perfusion solution includes the following steps: dissolve 1.17 g of NaCl, 1 g of KCl, 4.8 g of Hepes, 1.5 g of albumin, and 0.158 g of anhydrous calcium chloride in 250 mL of pure water, adjust the pH to 7.6, add distilled water to 300 mL, filter to remove bacteria, and then add 0.3 g of collagenase IV. The complete culture medium consisted of 500 mL DMEM high glucose medium, 5 mL antibiotics, 50 mL FBS, 1 μmol dexamethasone, and 0.5 μg / mL insulin.

[0061] The hepatocyte culture medium consisted of 500 mL DMEM high glucose medium, 5 mL antibiotics, 50 mL FBS, 1 μmol dexamethasone, 0.5 μg / mL insulin, and FH1 with a molar concentration of 15 μmol / L.

[0062] Example 2 Except for the different hepatocyte culture medium, the other conditions and steps were the same as in the previous example. The hepatocyte culture medium used in Example 2 included 500 mL of DMEM high glucose medium, 5 mL of antibiotics, 50 mL of FBS, 1 μmol of dexamethasone, 0.5 μg / mL of insulin, and FPH1 with a molar concentration of 15 μmol / L.

[0063] Example 3 Except for the different hepatocyte culture medium, the other conditions and steps were the same as in the previous example. The hepatocyte culture medium used in Example 3 included 500 mL of DMEM high glucose medium, 5 mL of antibiotics, 50 mL of FBS, 1 μmol of dexamethasone, 0.5 μg / mL of insulin, FH1 with a molar concentration of 15 μmol / L, and FPH1 with a molar concentration of 15 μmol / L.

[0064] Comparative Example 1 Except for not using hepatocyte culture medium, the other conditions and steps were the same as in Example 1.

[0065] Cell identification The purified primary rat hepatocytes obtained in Examples 1-3 and Comparative Example 1 were observed under a microscope and identified using Oil Red O staining and PAS staining. Bright-field images obtained under a microscope using direct transmission or reflection of white light are shown below. Figure 1 and Figure 2 As shown, Figure 1 The images show bright-field microscopic images of purified primary rat hepatocytes from Comparative Example 1. The left image (10X) is a bright-field image under a 10x objective lens, and the right image (20X) is a bright-field image under a 20x objective lens. In the left image, hepatocytes are seen adhering to the cell wall in an island-like pattern, with clear outlines, exhibiting a typical polygonal epithelial morphology and uniform distribution. The right image more clearly shows cell boundaries, granular texture within the cytoplasm, and the binuclear structure of some cells; the cells are plump and not shrunken or floating. Figure 1 It can be seen that the separated hepatocytes adhere well to the wall, have intact morphology, and strong three-dimensionality, proving that the hepatocyte separation was successful. Figure 2 These are bright-field microscopic images of purified rat primary hepatocytes from Examples 1-3 and Comparative Example 1. The top left image is from Example 1, the top right image is from Example 2, the bottom left image is from Example 3, and the bottom right image is from Comparative Example 1. Figure 2 It can be seen that compared with control example 1, the addition of FH1 and FPH1 in examples 1-3 resulted in higher cell density and stronger viability.

[0066] (1) Oil Red O staining (kit, Beijing Solarbio Science & Technology Co., Ltd., catalog number G1262) Remove the hepatocyte culture medium, wash twice with PBS, and fix with Oil Red O fixative for 20-30 min; Discard the fixative, wash twice with distilled water, and then soak in 60% isopropanol for 20-30 seconds. After discarding 60% isopropanol, add freshly prepared Oil Red O staining solution and immerse for 10-20 minutes. Discard the staining solution, rinse with 60% isopropanol for 10-20 seconds until the stroma is clear. Wash with distilled water 2-5 times until no excess staining solution is removed; Add Mayer's hematoxylin staining solution and counterstain the nuclei for 1-2 minutes. Discard the staining solution, wash with water 2-5 times, incubate with Oil Red O buffer for 1 minute to restore blue color, and then discard the solution. Add distilled water to cover the cells and observe them under a microscope.

[0067] Observation results as follows Figure 3 As shown, Figure 3 This is a microscopic image of purified rat primary hepatocytes stained with Oil Red O, as shown in Comparative Example 1.

[0068] Depend on Figure 3 As can be seen, all cells were stained, indicating that they have lipid metabolism function, meaning that the tested cells are hepatocytes.

[0069] (2) PAS staining (kit, Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0142S) Remove the hepatocyte culture medium, wash twice with PBS, and fix with 70% ethanol for 10 minutes.

[0070] Oxidation with periodic acid solution: Remove the periodic acid solution and equilibrate to room temperature. Add 100 μl of periodic acid solution to each sample and react in a humidified chamber in the dark for 10 minutes. After that, remove the periodic acid solution, soak in distilled water, and wash on a shaker for 5 minutes.

[0071] Schiff reagent staining: Add 100 μl of Schiff reagent to each sample, place in a humidified chamber, and stain in a 37ºC oven in the dark for 30 minutes to 1 hour. Remove the staining solution, soak in distilled water, and wash on a shaker for 5 minutes.

[0072] Hematoxylin staining: Add 100 μl of hematoxylin staining solution to each sample and stain for 30 seconds. Remove the staining solution and rinse with distilled water at least twice, 3 seconds each time, until the excess stain is removed. Wash away any excess color with distilled water and observe under a microscope. The results are as follows. Figure 4 As shown.

[0073] Figure 4 This is a microscopic image of purified primary rat hepatocytes from Comparative Example 1 after PAS staining.

[0074] Depend on Figure 4 As can be seen, all cells were stained, indicating that they have glucose metabolism function, meaning that the tested cells are liver cells.

[0075] Immunoblotting and RT-qPCR detection Proteins were extracted from the purified rat primary hepatocytes in Examples 1-3 and Comparative Example 1 and detected by Western blotting. RNA was extracted and detected by RT-qPCR.

[0076] (1) Immunoblotting detection Protein extraction: Discard the culture medium from the coated plate, wash cells twice with pre-cooled PBS, add 200 μL of RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to each well, and lyse on ice for 30 min; centrifuge at 12000g for 15 min at 4℃, discard the upper lipid layer and the lower precipitate, and collect the clear protein supernatant in the middle layer; determine the protein concentration using the BCA method, add 5×SDS loading buffer, and boil at 100℃ for 5 min to denature the protein.

[0077] Western Blot Detection: Equal volumes of denatured protein samples were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the proteins were transferred to an NC membrane using a wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then incubated overnight at 4°C with the corresponding primary antibody. After washing the membrane three times with TBST, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour. The membrane was washed three more times with TBST, and development was performed using ECL chemiluminescence. Figure 5 . Figure 5 The images show the Western Blot results of purified rat primary hepatocytes from Examples 1-3 and Comparative Example 1.

[0078] Depend on Figure 5 It can be seen that, compared with Comparative Example 1, the purified rat primary hepatocytes cultured in hepatocyte culture medium containing FH1 and / or FPH1 in Examples 1-3 showed clearer specific bands for ALB and HNF4α, indicating that the hepatocyte culture medium containing FH1 and / or FPH1 can enhance gene expression in SD rat primary hepatocytes. Among them, the purified rat primary hepatocytes cultured in hepatocyte culture medium containing FH1 in Example 1 showed clearer specific bands for ALB, CYP1A2 and HNF4α, indicating that adding 15 μM FH1 alone has a better effect on enhancing the function of rat primary hepatocytes.

[0079] (2) RT-qPCR detection: Total RNA extraction: Discard the culture medium in the six-well plate, wash the cells twice with PBS, and perform the RNA extraction according to the instructions of the RNA extraction kit (Hunan Aike Rui Biotechnology Co., Ltd., catalog number AG21024-50). After extraction, use Nanodrop to determine the RNA concentration and purity.

[0080] cDNA reverse transcription: Take an equal amount of total RNA and prepare the reaction system according to the reverse transcription kit instructions (Suzhou Nearshore Protein Technology Co., Ltd., catalog number E047-01B). The reaction program is: 42℃ for 15 min, 85℃ for 5 s. The obtained cDNA is stored at -20℃ for later use.

[0081] Real-time quantitative PCR (qPCR) detection: The qPCR reaction system was prepared using the SYBR Green fluorescent dye method. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for a total of 40 cycles. Melting curve analysis was performed after the reaction. GAPDH was used as an internal reference gene, and the relative expression level of the target gene was calculated using the 2^-ΔΔCt method. The test results are as follows: Figure 6 As shown.

[0082] Figure 6 The images show the RT-qPCR results of purified rat primary hepatocytes from Examples 1-3 and Comparative Example 1.

[0083] Depend on Figure 6 It can be seen that, compared with Comparative Example 1, the purified rat primary hepatocytes cultured in Examples 1-3 using hepatocyte culture medium containing FH1 and / or FPH1 had higher relative mRNA expression levels of ALB and HNF4α, indicating that the hepatocyte culture medium containing FH1 and / or FPH1 can enhance gene expression in SD rat primary hepatocytes. Among them, the purified rat primary hepatocytes cultured in Example 1 using hepatocyte culture medium containing FH1 had higher relative mRNA expression levels of ALB, CYP1A2 and HNF4α, indicating that adding 15 μM FH1 alone had a better effect on enhancing the function of rat primary hepatocytes.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro culture of primary rat hepatocytes, characterized in that, Includes the following steps: S10. Obtain liver from rats; S20. The hepatocytes in the liver are dissociated and crudely extracted to obtain a single-cell suspension; S30. Centrifuge the single-cell suspension and wash and purify it multiple times to obtain purified rat primary hepatocyte precipitate. S40. The rat primary hepatocyte precipitate was resuspended in complete culture medium to obtain a rat primary hepatocyte suspension. S50. Count the rat primary hepatocyte suspension, inoculate the counted rat primary hepatocyte suspension onto a coated plate, and culture it in hepatocyte culture medium to obtain purified rat primary hepatocytes. In step S50, the hepatocyte culture medium includes small molecule additives, DMEM high glucose medium, antibiotics, fetal bovine serum, dexamethasone, and insulin, and the small molecule additives include FH1 and / or FPH1.

2. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, The small molecule additive includes FH1, wherein the molar concentration of FH1 is 5 μmol / L to 30 μmol / L; and / or, The small molecule additive includes FPH1, and the molar concentration of FPH1 is 5 μmol / L to 30 μmol / L.

3. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, In the hepatocyte culture medium: The volume of the DMEM high-glucose culture medium is 500 ml; The volume percentage of the antibiotic is 1%-2%; The volume percentage of the fetal bovine serum is 10%-15%; The amount of dexamethasone added is 0.5 μM-2 μM; The insulin concentration is 0.5 μg / mL to 2 μg / mL.

4. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, Step S20 includes: placing the liver in a sterile culture dish at 4°C, removing the liver capsule, scraping the liver tissue, filtering, and obtaining a single-cell suspension; The sterile culture dish contains a cell washing solution, which includes DMEM high-glucose culture medium.

5. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, Step S30 includes: centrifuging the single-cell suspension, removing the supernatant, adding cell washing buffer to the precipitate, resuspending the cells, centrifuging, removing the supernatant, and repeating the washing process 2-3 times until the supernatant is clear and bloodless, thus obtaining purified rat primary hepatocyte precipitate; wherein: In the centrifugation operation, the centrifugal force is 50g, the centrifugation time is 3min, and the centrifugation temperature is 4℃; and / or, The cell washing solution includes DMEM high-glucose medium.

6. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, In step S40: The temperature of the complete culture medium is 37°C; and / or, The complete culture medium includes DMEM high glucose medium, antibiotics, fetal bovine serum, dexamethasone, and insulin.

7. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, Before step S50, the preparation of a coating plate is also included, and the method for preparing the coating plate includes the following steps: Prepare a 0.0006 mol / L acetic acid aqueous solution, filter to remove bacteria, and add 200 μl to 500 μl of rat tail collagen solution with a concentration of 5 mg / ml to obtain the coating solution; The culture plate was coated with coating solution. After coating, the excess coating solution was aspirated. The coated culture plate was washed 1-2 times with phosphate buffer to obtain the coated plate. The volume ratio of the acetic acid aqueous solution to the rat tail collagen solution is (100~250):

1.

8. The method for in vitro culture of primary rat hepatocytes as described in claim 1, characterized in that, Step S10 includes: After anesthetizing the rats, the first and second perfusion fluids were sequentially instilled through the portal vein. After the liver was digested and softened, it was cut off.

9. The method for in vitro culture of primary rat hepatocytes as described in claim 8, characterized in that, The first perfusion solution comprises the following substances by mass concentration: Sodium chloride 8.3 g / L, potassium chloride 0.5 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 2.4 g / L, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid 0.95 g / L, balance water.

10. The method for in vitro culture of primary rat hepatocytes as described in claim 8, characterized in that, Each 300ml of the second perfusion fluid includes: 1.17g sodium chloride, 1g potassium chloride, 4.8g 4-hydroxyethylpiperazine ethanesulfonic acid, 1.5g bovine serum albumin, 0.158g anhydrous calcium chloride, and 0.3g collagenase IV.

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