Highly active duck embryo liver primary cells, and preparation method and application thereof

Abstract

This invention discloses a highly active duck embryo liver primary cell and its low-cost preparation method and applications. The preparation method includes: obtaining and pretreatment of duck embryo liver tissue, separation by complex enzyme digestion, preparation of cell suspension, primary cell culture, and maintenance of cell viability. The core of this method lies in the gentle digestion using a complex digestion solution composed of collagenase I, trypsin, and DNase I in specific weight ratios, followed by culture in a well-defined, non-commercial growth factor-free complete culture medium. This complete culture medium contains synergistic components such as DMEM / F12, fetal bovine serum, epidermal growth factor, hepatocyte growth factor, and an insulin-transferrin-selenium mixture. The duck embryo liver primary cells prepared by this invention exhibit high yield, high viability, and excellent liver-specific function, and can maintain their activity and function well after passage and cryopreservation.

Description

Technical Field

[0001] This invention pertains to cell culture methods in the field of biotechnology, specifically relating to a highly active duck embryo liver primary cell and its low-cost preparation method and application. Background Technology

[0002] Primary liver cells from duck embryos have irreplaceable value in fields such as veterinary vaccine production (especially duck viral hepatitis vaccine), drug hepatotoxicity evaluation, and basic medical research because they retain the main physiological functions of in vivo hepatocytes, such as metabolism, secretion, and viral sensitivity.

[0003] However, existing technologies for preparing primary duck embryo liver cells still face numerous challenges, hindering their widespread application and standardization. Firstly, cell viability damage during the cell acquisition process is a key bottleneck. Traditional tissue digestion methods often employ single trypsin or collagenase enzymes, making it difficult to precisely control digestion conditions (such as time and concentration). This easily leads to cell membrane damage and inactivation of key receptors, resulting in low cell yield and initial viability.

[0004] Secondly, maintaining cell viability and function in vitro is challenging. Primary hepatocytes exhibit significant dedifferentiation, rapidly losing liver-specific functions such as albumin secretion and cytochrome P450 enzyme activity in conventional basal media. To maintain cell viability and function, current high-end protocols generally rely on adding expensive, non-standardized bioactive additives, such as mixtures of growth factors extracted from specific tissues. These components are not only costly and exhibit significant batch-to-batch variability, but also suffer from unstable supply and uncontrollable quality due to their lack of commercialization, severely impacting experimental reproducibility and production economics.

[0005] Furthermore, existing culture systems lack sufficient optimization of the culture environment (such as buffer systems and antioxidants), making cells susceptible to the accumulation of metabolic waste and pH fluctuations during culture, thus hindering long-term high-activity culture. Cells obtained by most methods experience rapid functional decline after passage, failing to meet the application requirements for long-term stable cell models.

[0006] Therefore, there is an urgent need in this field to develop a method for preparing duck embryo liver primary cells that is cost-effective, has clearly defined and stable components, and can efficiently obtain and maintain high activity over a long period of time. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for preparing highly active duck embryo liver primary cells based on a specific complex enzyme digestion system and a complete culture medium with defined components. The core innovation of this method lies in two aspects: First, a complex digestion solution composed of collagenase I, trypsin, and DNase I in specific weight ratios is used for gentle shaking digestion under precisely controlled pH and temperature, achieving efficient tissue dissociation and maximizing cell viability retention. Second, a complete culture medium is designed, consisting of DMEM / F12 basal medium, fetal bovine serum, and multiple defined, commercially available components including epidermal growth factor, hepatocyte growth factor, and an insulin-transferrin-selenium mixture. Through the synergistic effect of these components, this system effectively simulates the in vivo microenvironment without requiring any non-standardized, expensive additives, significantly promoting cell adhesion, proliferation, and long-term stable maintenance of liver-specific functions.

[0008] Another objective of this invention is to provide highly active duck embryonic liver primary cells prepared by the above method. These cells not only have a high initial survival rate, but more importantly, they can stably maintain typical hepatocyte morphology and high levels of biological function in in vitro culture systems. Key indicators such as albumin secretion capacity and cytochrome P450 enzyme activity are significantly superior to cells prepared by traditional methods, providing a high-quality cell model for reliable drug hepatotoxicity testing and efficient viral vaccine production.

[0009] Another objective of this invention is to provide applications of the aforementioned highly active duck embryonic liver primary cells in the biomedical field, including the preparation of in vitro drug hepatotoxicity testing models, duck viral hepatitis vaccines, and as a cell source for bioartificial liver support systems. Ultimately, through synergistic innovation in cell acquisition processes and culture systems, this invention provides a cost-effective, stable, and scalable high-quality primary hepatocyte technology solution for veterinary medicine and pharmaceutical research and development.

[0010] The objective of this invention is achieved through the following technical solution.

[0011] A method for preparing highly active duck embryo liver primary cells includes the following steps: (1) Obtaining and pretreatment of duck embryo liver tissue: Select healthy duck embryos, remove the liver, rinse with pre-cooled PBS buffer to remove connective tissue and blood vessels, and cut into 1-2 mm pieces. 3 Small pieces; (2) Enzymatic digestion and separation: The liver tissue block from step (1) was placed in a digestion solution and digested by shaking at 37°C and pH 7.2-7.4 for 20-40 minutes; the digestion solution consisted of the following components by weight: collagenase I 0.5-1.5 parts, trypsin 0.2-0.8 parts, DNase I 0.1-0.3 parts, EDTA 0.05-0.15 parts, and PBS buffer 100 parts; (3) Preparation of cell suspension: After digestion, filter through a 100-200 mesh cell sieve, collect the filtrate, centrifuge at 800-1000 rpm for 5-10 minutes to remove the supernatant, treat with red blood cell lysis buffer for 5-10 minutes, then resuspend the cells in PBS and adjust the cell density to 1×10⁻⁶. 6 -5×10 6 cells / mL; (4) Primary cell culture: The cell suspension from step (3) is seeded into a culture plate, and complete culture medium is added. The culture is carried out at 37°C and 5% CO2. The complete culture medium consists of the following components in parts by weight: 100 parts of DMEM / F12 basal medium, 10-20 parts of fetal bovine serum, 0.1-0.5 parts of epidermal growth factor, 0.1-0.5 parts of hepatocyte growth factor, 0.5-1.5 parts of insulin-transferrin-selenium mixture, 1-2 parts of penicillin-streptomycin solution, 0.5-1.5 parts of glutamine, and 2-4 parts of HEPES buffer. (5) Cell viability maintenance: After culturing for 24-48 hours, replace with fresh complete culture medium and continue culturing until the cells adhere to the wall and reach 80-90% confluence to obtain highly active duck embryo liver primary cells.

[0012] Furthermore, in the above preparation method, in step (2), the activity of collagenase I is 100-200 U / mg, and the activity of trypsin is 1000-1500 U / mg.

[0013] Furthermore, in the above preparation method, in step (3), the red blood cell lysis buffer is prepared by compounding ammonium chloride, potassium bicarbonate and EDTA in a weight ratio of (8-12):(1-2):(0.1-0.3), and the concentration used is 0.15-0.25 M.

[0014] Furthermore, in the above preparation method, in step (4), the insulin-transferrin-selenium mixture has an insulin concentration of 5-10 μg / mL, a transferrin concentration of 5-10 μg / mL, and a selenium concentration of 5-10 nM.

[0015] Furthermore, in the above preparation method, in step (4), the complete culture medium also contains the following additives in parts by weight: 0.01-0.05 parts of dexamethasone.

[0016] Furthermore, in the above preparation method, in step (5), the culture conditions are: cultured at 37°C, 5% CO2 and 95% humidity, with the culture medium being replaced every 48 hours.

[0017] This invention also discloses highly active duck embryo liver primary cells prepared by the above-described method, wherein the cells have a survival rate of not less than 95% after 72 hours of culture, and the secretion level of the liver-specific functional marker albumin is not less than 50 ng / 10 6 Cells / 24 h, cytochrome P450 enzyme activity increased by more than 50% compared to cells prepared by traditional methods.

[0018] Furthermore, the aforementioned highly active duck embryo liver primary cells can maintain high activity up to the 3rd generation after passage, and the morphology retention rate of liver cells is greater than 90%.

[0019] This invention also discloses the application of the above-mentioned highly active duck embryo liver primary cells in the preparation of drug hepatotoxicity test models or in the production of duck viral hepatitis vaccines.

[0020] Compared with existing technologies, the present invention has the following advantages and beneficial effects: I. Cost Control and Standardization Advantages: This invention significantly reduces raw material costs by replacing expensive, batch-varying, non-commercial, specific growth factors (such as EGF and HGF) in traditional methods with commercially available, well-defined growth factors (such as EGF and HGF). It also standardizes the culture medium formulation, ensuring the reproducibility of the preparation process and the consistency of results, thus laying the foundation for large-scale application.

[0021] II. Superior Cell Acquisition Quality: Through an optimized complex enzyme digestion system and red blood cell lysis step, liver tissue is gently and efficiently dissociated, minimizing mechanical and enzymatic damage to cells during the acquisition process. This achieves a balance between high cell yield and high initial viability, providing high-quality cell seeds for subsequent high-quality culture.

[0022] III. Excellent cell function maintenance: The components of the complete culture medium designed in this invention have a clear synergistic promoting effect, which can effectively simulate the in vivo microenvironment. It not only supports rapid cell adhesion and proliferation, but also maintains the typical liver-specific function of duck embryo liver primary cells in a long-term and stable manner, effectively delaying the "dedifferentiation" process and meeting the advanced application requirements of long-term and stable cell models.

[0023] IV. Strong Process Stability and Versatility: The entire preparation process is clear, with well-defined parameters, strong operability, and good reproducibility. The prepared cells exhibit excellent cryopreservation and thawing properties, facilitating the establishment and transportation of cell banks. This method is highly universal, providing a reliable technical pathway to solve the challenges of primary cell culture. Attached Figure Description

[0024] Figure 1 Comparison of cell yield (×10⁶ / g tissue) in Test Example 1; Figure 2Albumin secretion level in Test Example 2 (ng / 10) 6 Comparison of cells / 24 h); Figure 3 Urea synthesis amount (μg / 10) in Test Example 2 6 Comparison of cells / 4 h); Figure 4 Quality assessment of cells after cryopreservation and thawing in Test Example 5. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. All raw materials used in the embodiments of this invention are commercially available. Table 1 below lists the raw materials used in the embodiments and is not intended to limit the scope of the invention.

[0026] Table 1 Raw Material List Example 1 A method for preparing highly active duck embryo liver primary cells includes the following steps: (1) Obtaining and pretreatment of duck embryo liver tissue: Select healthy 12-day-old duck embryos, remove the liver under sterile conditions, rinse 3 times with pre-cooled (4℃) PBS buffer to thoroughly remove connective tissue and major blood vessels, and cut it into pieces of about 1 mm using ophthalmic scissors. 3 Small pieces.

[0027] (2) Enzymatic digestion and separation: The approximately 1 gram wet weight liver tissue block from step (1) was placed in an Erlenmeyer flask containing 10 mL of digestion solution, which consisted of the following components by weight: collagenase I 1.0 part, trypsin 0.5 part, DNase I 0.2 part, EDTA 0.1 part, and PBS buffer 100 parts. Digestion was carried out at 37°C and pH 7.3 with shaking at 80 rpm for 30 minutes.

[0028] (3) Cell suspension preparation: After digestion, immediately terminate the digestion with an equal volume of DMEM / F12 medium containing 10% fetal bovine serum. Filter the mixture using a 200-mesh cell sieve and collect the filtrate. Centrifuge the filtrate at 1000 rpm for 8 minutes and discard the supernatant. Resuspend the pellet in pre-cooled PBS, add 5 times the volume of erythrocyte lysis buffer (prepared from ammonium chloride, potassium bicarbonate, and EDTA in a weight ratio of 10:1.5:0.2, with a concentration of 0.2 M), and incubate at room temperature for 8 minutes to lyse the erythrocytes. After lysis, centrifuge again at 1000 rpm for 5 minutes and discard the supernatant. Finally, resuspend the cells in complete medium and count them using a hemocytometer and trypan blue exclusion assay, adjusting the cell density to 3 × 10⁶ cells / mL. 6 per mL.

[0029] (4) Primary cell culture: The cell suspension from step (3) is cultured at 5×10⁻⁶ cells / mL. 5 pcs / cm 2 The culture medium was seeded at a density of 1:1 in 6-well plates coated with rat tail collagen type I, with 2 mL of complete culture medium added to each well. The complete culture medium consisted of the following components by weight: 100 parts DMEM / F12 basal medium, 15 parts fetal bovine serum, 0.3 parts epidermal growth factor, 0.3 parts hepatocyte growth factor, 1.0 part insulin-transferrin-selenium mixture, 1.5 parts penicillin-streptomycin solution, 1.0 part glutamine, and 3 parts HEPES buffer. The culture plates were then incubated statically in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0030] (5) Cell viability maintenance: After 24 hours of culture, the medium was completely replaced with fresh complete medium. Thereafter, half of the medium was replaced every 48 hours. After 72 hours of culture, the cells adhered well and the confluence reached about 85%, resulting in highly active duck embryo liver primary cells.

[0031] Example 2 A method for preparing highly active duck embryo liver primary cells includes the following steps: (1) Obtaining and pretreatment of duck embryo liver tissue: Select healthy 13-day-old duck embryos and process them in the same way as in Example 1. Cut the liver tissue into pieces of about 1.5 mm. 3 Small pieces.

[0032] (2) Enzymatic digestion and separation: Take about 1 gram of wet-weight liver tissue block and place it in 10 mL of digestion solution, which consists of the following components by weight: collagenase I 0.8 parts, trypsin 0.3 parts, DNase I 0.15 parts, EDTA 0.08 parts, and PBS buffer 100 parts. Digest at 37°C and pH 7.2 with shaking at 70 rpm for 35 minutes.

[0033] (3) Preparation of cell suspension: Same as in Example 1.

[0034] (4) Primary cell culture: The cell seeding density is 4×10⁶ 5 pcs / cm 2 The complete culture medium consists of the following components in parts by weight: 100 parts DMEM / F12 basal medium, 12 parts fetal bovine serum, 0.15 parts epidermal growth factor, 0.15 parts hepatocyte growth factor, 0.8 parts insulin-transferrin-selenium mixture, 1.2 parts penicillin-streptomycin solution, 0.8 parts glutamine, and 2.5 parts HEPES buffer. Culture conditions are the same as in Example 1.

[0035] (5) Cell viability maintenance: Same as in Example 1, after culturing for 72 hours, the cell fusion rate reached about 80%.

[0036] Example 3 A method for preparing highly active duck embryo liver primary cells includes the following steps: (1) Obtaining and pretreatment of duck embryo liver tissue: Select healthy duck embryos at 11 days old and process them in the same way as in Example 1. Cut the liver tissue into small pieces of about 0.8 mm³.

[0037] (2) Enzymatic digestion and separation: Take about 1 gram of wet-weight liver tissue block and place it in 10 mL of digestion solution, which consists of the following components by weight: collagenase I 1.2 parts, trypsin 0.6 parts, DNase I 0.25 parts, EDTA 0.12 parts, and PBS buffer 100 parts. Digest at 37°C and pH 7.4 with shaking at 90 rpm for 25 minutes.

[0038] (3) Preparation of cell suspension: Same as in Example 1.

[0039] (4) Primary cell culture: The cell seeding density is 6×10⁶. 5 pcs / cm 2 The complete culture medium consists of the following components in parts by weight: 100 parts DMEM / F12 basal medium, 18 parts fetal bovine serum, 0.4 parts epidermal growth factor, 0.4 parts hepatocyte growth factor, 1.2 parts insulin-transferrin-selenium mixture, 1.8 parts penicillin-streptomycin solution, 1.2 parts glutamine, and 3.5 parts HEPES buffer. Additionally, 0.03 parts dexamethasone are added.

[0040] (5) Cell viability maintenance: Same as in Example 1, after culturing for 72 hours, the cell fusion rate reached about 88%.

[0041] Comparative Example 1 A method for preparing primary duck embryo liver cells, which differs from Example 1 in that the digestion solution in step (2) is a single collagenase I solution (1.5 parts collagenase I, 100 parts PBS buffer), and does not contain trypsin, DNase I, or EDTA. The remaining steps and conditions are the same as in Example 1.

[0042] Results: Digestion efficiency was significantly reduced, and the required digestion time was extended to 50 minutes. Cell clumps increased significantly after digestion, making filtration difficult, and the final cell yield was only about 60% of that in Example 1. The obtained cells showed low adhesion and a viability rate (tested with trypan blue) of less than 85% after 24 hours of culture.

[0043] Comparative Example 2 A method for preparing primary duck embryo liver cells, which differs from Example 1 in that the complete culture medium in step (4) does not contain epidermal growth factor and hepatocyte growth factor, but only uses basal culture medium, fetal bovine serum and insulin-transferrin-selenium mixture. The remaining steps and conditions are the same as in Example 1.

[0044] Results: Cells proliferated slowly after adhesion, reaching only about 50% confluence after 72 hours of culture. Albumin levels in the culture supernatant were measured, and the secretion was only 35% of that in cells from Example 1. Cell morphology tended to be flattened, and expression of hepatocyte-specific functional markers was weakened.

[0045] Comparative Example 3 A method for preparing primary duck embryo liver cells differs from Example 1 in that, in step (4), the complete culture medium prepared according to this invention is not used; instead, a commercially available culture medium specifically for primary duck liver mesenchymal cells (Mirror Image (Shanghai) Cell Technology Co., Ltd.: Catalog No.: iCell-d022-002y) is used. This culture medium contains various growth factors, but the specific components are not disclosed, and its price is approximately three times that of the culture medium of this invention. The remaining steps and conditions are the same as in Example 1.

[0046] Results: Cell adhesion and growth in the early stages of culture were similar to those in Example 1. However, after passage to the second generation, cell function declined significantly, and the induction activity of its cytochrome P450 enzyme (CYP1A2) was significantly lower than that of the cells passaged in Example 1, indicating that its long-term functional maintenance was not as good as that of the present invention.

[0047] Comparative Example 4 A method for preparing primary duck embryo liver cells, compared with Example 1, differs in that the step of treating red blood cell lysis buffer is omitted in step (3). The cell pellet after digestion and centrifugation is directly resuspended and inoculated with complete culture medium. The remaining steps and conditions are the same as in Example 1.

[0048] Results: The initial cell suspension contained a large number of erythrocyte fragments and unlysed erythrocytes, which severely interfered with the adhesion of hepatocytes. After 24 hours of culture, the background of the field of view was cluttered, the hepatocytes were sparsely adhered, and their morphology was atypical, with both purity and viability severely affected.

[0049] Comparative Example 5 A method for preparing primary duck embryo liver cells, referring to the traditional tissue block adhesion method. The difference from Example 1 is that the enzyme digestion steps (steps 2 and 3) are omitted. The pretreated 1 mm³ liver tissue blocks are directly attached to the bottom of the culture plate, a small amount of complete culture medium (the same as in Example 1) is added, and the plate is left to stand for 4 hours. After the tissue blocks have initially adhered, sufficient culture medium is slowly added for further culture.

[0050] Results: Cells migrated slowly from the tissue block, requiring a culture period of 7-10 days to reach a usable confluence. The resulting cell population exhibited strong heterogeneity, containing many non-parenchymal cells, with inconsistent liver-specific functions. Furthermore, the success rate was significantly affected by tissue block adhesion, resulting in poor reproducibility.

[0051] Test Example 1 Cell acquisition efficiency and initial activity assessment Objective: To verify the effect of the compound enzyme digestion system and pretreatment steps of the present invention on improving cell yield and initial activity.

[0052] method: Cell yield calculation: After digestion and preparation of cell suspensions, the cell suspensions of Examples 1-3 and Comparative Examples 1-5 were counted using a hemocytometer. Cell yield (×10⁻⁶) 6 / g tissue) = Total number of cells in cell suspension / Wet weight of liver tissue used for digestion (g).

[0053] Initial viability determination: The trypan blue exclusion method was used. The above cell suspension was mixed with 0.4% trypan blue solution at a ratio of 9:1, allowed to stand for 3 minutes, and then at least 200 cells were counted under an optical microscope. The percentage of viable cells that were not stained with blue was calculated.

[0054] Cell membrane integrity (LDH leakage rate): Cell suspension was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected. LDH activity in the supernatant was measured using a lactate dehydrogenase (LDH) assay kit and compared with the total LDH activity after complete cell lysis. The LDH leakage rate (%) was calculated as: (Supernatant LDH activity / Total LDH activity) × 100%. A lower leakage rate indicates less damage to the cell membrane during digestion.

[0055] Results: See Table 2 below and Figure 1 As shown.

[0056] Table 2: Cell acquisition efficiency and initial viability assay results Conclusion: Examples 1-3 of this invention, employing a combined enzyme digestion and erythrocyte lysis step, achieved the highest cell yield and initial viability, while minimizing LDH leakage. This demonstrates that the digestion process of this invention is gentle, efficient, and maximizes the preservation of cell membrane integrity. In contrast, single-enzyme digestion (Comparative Example 1) and omitting erythrocyte lysis (Comparative Example 4) both resulted in a significant decrease in cell acquisition efficiency and quality.

[0057] Test Example 2 Liver-specific functional index testing Objective: To evaluate the effect of the complete culture medium system of the present invention on maintaining liver-specific function of duck embryo liver primary cells.

[0058] method: Albumin secretion: Cell supernatant from 72 hours of culture was used for quantification using a duck albumin ELISA kit. Results were normalized to 10⁻⁶. 6 Secretion amount per cell in 24 hours (ng / 10) 6 cells / 24 h).

[0059] Urea synthesis capacity: Cells were washed with serum-free medium and then stimulated with medium containing 5 mM NH4Cl for 4 hours. The supernatant was collected, and the urea content was determined using a urea nitrogen assay kit. The results were normalized to 10^6 urea molecules per 10^6 ppm. 6 Synthetic amount per cell (μg / 10) 6 cells / 4 h).

[0060] Cytochrome P450 enzyme (CYP1A2) activity: The P450-Glo™ CYP1A2 assay kit was used. Cells were induced with 50 μM 3-methylcholanthrene for 24 hours and then co-incubated with the substrate fluorescein-H. The luminescence value was measured to reflect the metabolic activity of CYP1A2, expressed as relative fluorescence units (RLU).

[0061] Results: See Table 3 below and Figure 2 and Figure 3 As shown.

[0062] Table 3: Results of Liver-Specific Functional Indicators Measurement (72 hours of culture) Conclusion: The cells from Examples 1-3 of this invention exhibited the highest levels in three key liver-specific functional indicators, significantly outperforming all comparative examples. In particular, compared to Comparative Example 2, which lacked key growth factors, the functional improvement exceeded 100%, demonstrating the effectiveness and synergistic effect of the culture medium formulation of this invention. Even compared to expensive commercial culture media (Comparative Example 3), it showed advantages in drug-metabolizing enzyme activity.

[0063] Test Example 3 Long-term culture stability study Objective: To investigate the functional stability of cells prepared by the method of the present invention during continuous passage culture.

[0064] method: Subculture: After culturing for 72 hours, the P0 generation cells from Example 1, Comparative Example 2 (growth factor deficient), and Comparative Example 3 (commercial culture medium) were subcultured using the standard trypsin digestion method and seeded at the same density, denoted as P1 generation. The cells were then cultured for another 72 hours and subcultured again, denoted as P2 generation.

[0065] Indicator testing: After 72 hours of culture in P1 and P2 generations, the following indicators were tested respectively: Live cell density: Counted using trypan blue exclusion method, reflecting population proliferation and survival.

[0066] Albumin secretion: Method same as test case 2.

[0067] CYP1A2 activity: The method is the same as in test example 2.

[0068] Results: As shown in Table 4 below.

[0069] Table 4: Results of Long-Term Culture Stability Measurement Conclusion: With passage, cell function and number decreased in all groups, but the decrease was smallest in group 1 of this invention. In passage P2, the viable cell density, albumin secretion capacity, and drug-metabolizing enzyme activity were significantly higher than in comparative examples 2 and 3. This indicates that the culture system of this invention can more effectively maintain the high activity and functionality of duck embryonic liver primary cells in long-term in vitro culture, exhibiting superior stability.

[0070] Test Example 4 Liver-specific functional gene expression analysis (qRT-PCR) Objective: To quantitatively assess the expression levels of liver-specific functional genes in cells prepared by this invention at the molecular level.

[0071] Methods: Total RNA was extracted from cells cultured for 72 hours in Example 1, Comparative Example 1 (single enzyme digestion), and Comparative Example 2 (growth factor deficient) and reverse transcribed into cDNA. The expression levels of the following key functional genes in hepatocytes were detected by real-time quantitative PCR (qRT-PCR): Albumin (ALB) Cytochrome P450 1A2 (CYP1A2) Connexin 32 (GJB1, encoding hepatocyte gap junction protein Cx32) Using GAPDH as an internal reference gene, the relative expression level (Fold Change) relative to the control group 2 was calculated using the 2^(-ΔΔCt) method.

[0072] Results: As shown in Table 5 below.

[0073] Table 5: Relative expression levels of liver-specific functional genes (qRT-PCR) Conclusion: The mRNA expression levels of three key liver functional genes (ALB, CYP1A2, GJB1) in cells of Example 1 of this invention were significantly upregulated, at 3.85-fold, 4.20-fold, and 2.95-fold respectively compared to the baseline level in Comparative Example 2, and were also much higher than those in Comparative Example 1. This directly confirms at the gene transcription level that the preparation process of this invention can effectively induce and maintain the liver-specific functional state of cells.

[0074] Test Example 5 Cell quality assessment after cryopreservation and thawing Objective: To evaluate the quality of cells prepared in this invention after programmed cryopreservation and thawing, and to examine their feasibility for constructing cell banks.

[0075] Methods: Cells cultured for 72 hours in Examples 1, 2, and Comparative Example 3 (commercial culture medium) were cryopreserved in liquid nitrogen using commercially available cell cryopreservation medium via a programmed cooling method. After 2 weeks, they were rapidly thawed in a 37°C water bath and the following assays were performed: Post-recovery survival rate: Trypan blue anti-infection method.

[0076] Adhesion efficiency: A known number of revived cells were seeded and cultured for 12 hours. The number of viable cells that had adhered was then counted after digestion. Adhesion efficiency (%) = (Number of viable adhered cells / Total number of viable cells seeded) × 100%.

[0077] Functional recovery capacity: After revival, cells were cultured normally for 24 hours, and their albumin secretion was measured and compared with the level before cryopreservation to calculate the functional recovery rate (%).

[0078] Results: See Table 6 below and Figure 4 As shown.

[0079] Table 6: Quality assessment after cell cryopreservation and thawing.

[0080] Conclusion: The cells of Examples 1 and 2 of this invention exhibited superior recovery performance after cryopreservation, with significantly higher immediate survival rate, rapid adhesion ability, and functional recovery rate compared to Comparative Example 3, which used expensive commercial culture media. This indicates that the cells prepared by the method of this invention not only have excellent initial conditions but also stronger tolerance to cryopreservation stress, maintaining high viability after cryopreservation, making them highly suitable for establishing high-quality, regenerable cell libraries.

[0081] Test Case Summary: Through the above systematic test examples, the present invention demonstrates the following outstanding effects: In terms of cell acquisition, the cell yield in Example 1 reached (4.5±0.3)×10 6 The initial survival rate of the tissue sample was (97.5±0.5%), significantly better than the control group. In terms of functional indicators, the albumin secretion level of cells in Example 1 was (68±5) ng / 102. 6 urea synthesis was (45±4) μg / 10 cells / 24h. 6 At 4 h / cells, CYP1A2 enzyme activity reached (1,250,000 ± 85,000) RLU, a functional level far exceeding that of control example 2 lacking key components (functional decline exceeding 50%). Regarding long-term stability, after passage to P2, albumin secretion in Example 1 cells remained at (58 ± 5) ng / 10 cells. 6 Cells were stored at 24h per cell, with high viability retention. qRT-PCR showed that the expression levels of liver-specific genes ALB and CYP1A2 were 3.85 times and 4.20 times higher than those in the control group, respectively. After cryopreservation and thawing, the cell viability of Example 1 reached (91.5±1.5%), and the functional recovery rate reached (89.7±3.5%), significantly better than the control group. This fully demonstrates the comprehensive advantages of this invention in cell quality, function, and stability.

Claims

1. A method for preparing highly active duck embryo liver primary cells, characterized in that, Includes the following steps: (1) Obtaining and pretreatment of duck embryo liver tissue: Select healthy duck embryos, remove the liver, rinse with pre-cooled PBS buffer to remove connective tissue and blood vessels, and cut into 1-2 mm pieces. 3 Small pieces; (2) Enzymatic digestion and separation: The liver tissue block from step (1) was placed in a digestion solution and digested by shaking at 37°C and pH 7.2-7.4 for 20-40 minutes; the digestion solution consisted of the following components by weight: collagenase I 0.5-1.5 parts, trypsin 0.2-0.8 parts, DNase I 0.1-0.3 parts, EDTA 0.05-0.15 parts, and PBS buffer 100 parts; (3) Preparation of cell suspension: After digestion, filter through a 100-200 mesh cell sieve, collect the filtrate, centrifuge at 800-1000 rpm for 5-10 minutes to remove the supernatant, treat with red blood cell lysis buffer for 5-10 minutes, then resuspend the cells in PBS and adjust the cell density to 1×10⁻⁶. 6 -5×10 6 cells / mL; (4) Primary cell culture: The cell suspension from step (3) is seeded into a culture plate, and complete culture medium is added. The culture is carried out at 37°C and 5% CO2. The complete culture medium consists of the following components in parts by weight: 100 parts of DMEM / F12 basal medium, 10-20 parts of fetal bovine serum, 0.1-0.5 parts of epidermal growth factor, 0.1-0.5 parts of hepatocyte growth factor, 0.5-1.5 parts of insulin-transferrin-selenium mixture, 1-2 parts of penicillin-streptomycin solution, 0.5-1.5 parts of glutamine, and 2-4 parts of HEPES buffer. (5) Cell viability maintenance: After culturing for 24-48 hours, replace with fresh complete culture medium and continue culturing until the cells adhere to the wall and reach 80-90% confluence to obtain highly active duck embryo liver primary cells.

2. The preparation method according to claim 1, characterized in that, In step (2), the activity of collagenase I is 100-200 U / mg, and the activity of trypsin is 1000-1500 U / mg.

3. The preparation method according to claim 1, characterized in that, In step (3), the red blood cell lysis buffer is prepared by mixing ammonium chloride, potassium bicarbonate and EDTA in a weight ratio of (8-12):(1-2):(0.1-0.3), and the concentration used is 0.15-0.25M.

4. The preparation method according to claim 1, characterized in that, In step (4), the insulin-transferrin-selenium mixture contains insulin at a concentration of 5-10 μg / mL, transferrin at a concentration of 5-10 μg / mL, and selenium at a concentration of 5-10 nM.

5. The preparation method according to claim 1, characterized in that, In step (4), the complete culture medium also contains the following parts by weight of additives: 0.01-0.05 parts of dexamethasone.

6. The preparation method according to claim 1, characterized in that, In step (5), the culture conditions are: cultured at 37°C, 5% CO2 and 95% humidity, with the culture medium being replaced every 48 hours.

7. The highly active duck embryo liver primary cells prepared by the preparation method according to any one of claims 1-6, characterized in that, The cells had a survival rate of no less than 95% after 72 hours of culture, and the secretion level of the liver-specific functional marker albumin was no less than 50 ng / 10⁻⁶. 6 Cells / 24 h, cytochrome P450 enzyme activity increased by more than 50% compared to cells prepared by traditional methods.

8. The highly active duck embryo liver primary cells according to claim 7, characterized in that, The cells maintained high activity up to the third generation after passage, and the morphology retention rate of hepatocytes was greater than 90%.

9. The application of the highly active duck embryo liver primary cells as described in claim 7 or 8 in the preparation of drug hepatotoxicity test models or the production of duck viral hepatitis vaccines.

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