Extraction and cryopreservation method of mouse liver cells
By combining two-step perfusion and specific centrifugation with specialized cryopreservation solution and cooling method, the problem of cell damage during mouse hepatocyte isolation and cryopreservation was solved, and high-purity, high-activity, and high-resuscitation-rate hepatocytes were prepared.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BOKANG BIOENGINEERING (SHANDONG) CO LTD
- Filing Date
- 2026-02-24
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, mouse liver cells are easily damaged during isolation, and conventional cryopreservation methods result in low cell viability and function, as well as significant quality differences between different batches, making long-term preservation difficult.
A two-step perfusion design was employed, combining a specific concentration of collagenase digestion solution with low-speed centrifugation, followed by Percoll gradient centrifugation. A specialized cryopreservation solution formulation and gradient cooling program were used, including Williams' E medium, fetal bovine serum, DMSO, and trehalose, along with a specific cooling method.
It significantly improved the purity and activity of mouse hepatocytes, greatly increased the recovery rate after cryopreservation, and the operation was stable and reproducible with good intercellular consistency.
Smart Images
Figure CN122012377A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, specifically to methods for extracting and cryopreserving mouse liver cells. Background Technology
[0002] The liver is the most important detoxification organ in the body. The metabolic, synthetic, secretory, and storage capabilities of hepatocytes are directly related to the body's health. Primary hepatocytes are ideal tools for studying liver diseases, and mouse hepatocytes are particularly important in biomedical research, widely used in drug metabolism and toxicity testing, liver physiology and pathology studies, virology research, and cell transplantation therapy, demonstrating high application value in scientific research. However, the in vitro application of primary hepatocytes has been constrained by two major technical bottlenecks: First, cells are easily damaged during the separation process. The efficiency of obtaining liver cells using commonly used collagenase digestion and mechanical separation methods is affected by various factors, such as perfusion pressure and flow rate, digestion time, collagenase concentration and activity, leading to low cell viability and function, and significant differences in cell quality between different batches. Second, primary cells are difficult to culture and preserve for long periods. Conventional cryopreservation methods cause severe ice crystal damage and osmotic stress, making it impossible to guarantee cell viability and function after thawing. Therefore, obtaining suitable hepatocyte models is crucial for studying the pathogenesis of liver diseases, drug pharmacology, and molecular genetics. Developing a standardized, reproducible method for preparing mouse hepatocytes that yields high-activity, high-purity cells suitable for long-term cryopreservation has significant practical application value. Summary of the Invention
[0003] To address the aforementioned issues, this application provides a method for extracting and cryopreserving mouse liver cells. This method is stable, has a high cell yield, good cell activity, and a high recovery rate after cryopreservation.
[0004] To achieve the above objectives, this application provides a method for extracting and cryopreserving mouse liver cells, comprising the following steps: S1. Liver perfusion and digestion: Mouse carcasses were cannulated through the inferior vena cava, the portal vein was cut open, and the superior vena cava was clamped. First, the liver was perfused with perfusion fluid for 6-8 minutes, and then perfused with digestive fluid for 10-15 minutes. S2. Cell isolation and purification: After circulatory perfusion, the liver membrane is removed and torn open in the pre-cooled digestion solution at 3-5℃. The liver is filtered through a sieve and the filtrate is collected. The filtrate is centrifuged for the first time, and complete culture medium is added. Then, it is centrifuged a second time and the cell pellet is collected. The pellet is washed 2-3 times with complete culture medium and centrifuged again. The cell pellet at the bottom is retained to obtain purified liver parenchymal cells. S3. Resuspend the purified hepatocytes in pre-cooled cryopreservation solution to a final concentration of 1-2 × 10⁻⁶. 7 cells / mL, aliquoted and frozen.
[0005] Furthermore, the perfusion fluid is 1×Krebs-Ringer buffer, the perfusion fluid temperature is 36-38℃, and the perfusion fluid flow rate is 5-6ml / min.
[0006] Furthermore, the digestion solution consists of 100 CDU / ml of type IV collagenase and 1×Krebs-Ringer buffer, with a digestion solution temperature of 36-38℃ and a flow rate of 7-8 ml / min.
[0007] Furthermore, the terminating digestion solution is a Williams' E solution containing 1.5-2.5% fetal bovine serum and 4-6 mM CaCl2.
[0008] Furthermore, the sieve has an aperture size of 70 μm or 100 μm.
[0009] Furthermore, for the initial centrifugation, the centrifugal force is 45-55×g, the centrifugation time is 3-5min, and the centrifugation temperature is 3-5℃.
[0010] Furthermore, the complete culture medium is DMEM medium containing 8-12% fetal bovine serum.
[0011] Furthermore, the secondary centrifugation is carried out under the following conditions: a 35-40% Percoll gradient solution is prepared in advance in the centrifuge tube, the cell suspension is added to the upper layer of the gradient solution, the volume ratio of the gradient solution to the cell suspension is 2-3:1, the centrifugation temperature is 3-5℃, the centrifugation force is 750-850×g, and the centrifugation time is 10-15min.
[0012] Furthermore, the centrifugation is performed with a centrifugal force of 45-55 × g and a centrifugation time of 3-5 min.
[0013] Furthermore, the cryopreservation solution comprises the following materials in parts by weight: 40-50 parts Williams' E, 30-40 parts fetal bovine serum, 4-6 parts DMSO, 4-6 parts trehalose, and Trolox at a final concentration of 45-55 μM.
[0014] Furthermore, the cryopreservation method involves storing the product at 4°C for 30-40 minutes, cooling it down to -20°C at a rate of 1-2°C / min and storing it for 30-40 minutes, cooling it down to -80°C at a rate of 1-2°C / min and storing it for 8-9 hours, and then storing it in liquid nitrogen.
[0015] In summary, this application has the following beneficial effects: This application employs a two-step perfusion design. First, preheated perfusion fluid removes blood and loosens cell connections. Then, a digestive solution containing a specific concentration of collagenase IV is circulated for perfusion. This avoids cell damage caused by direct enzyme digestion and improves digestion efficiency through the combination of pre-relaxation and re-digestion. Collagenase IV is an enzyme used to isolate primary hepatocytes; it has high specificity for collagen in liver tissue and causes minimal damage to the hepatocyte membrane structure, maximizing cell viability. The purification stage uses a specific combination of low-speed centrifugation and Percoll centrifugation. Low-speed centrifugation removes most blood cells and non-parenchymal cells, such as erythrocytes and platelets. Percoll density gradient centrifugation further removes dead cells, cell debris, and residual non-parenchymal cells. This two-step purification effectively improves the purity of hepatocytes, avoiding cell loss caused by a single centrifugation step. The two-step perfusion system and purification methods significantly improve cell purity and viability. The cryopreservation solution formulation in this application balances nutrient supply, cryoprotection, and antioxidant properties. (Williams's...) E-med medium provides the specific nutrients required by hepatocytes. Fetal bovine serum contains various growth factors and adhesion factors, which can improve cell adhesion efficiency after resuscitation. Trolox, as an antioxidant, can effectively inhibit the generation of free radicals during cryopreservation, further improving cell viability. DMSO, a permeable cryoprotectant, and trehalose, as non-permeable cryoprotectants, work synergistically with a gradient cooling program to greatly improve cell viability. DMSO, as a permeable cryoprotectant, has a small molecular structure that can quickly penetrate the cell membrane of hepatocytes and form hydrogen bonds with intracellular water molecules, lowering the freezing point of the intracellular solution and delaying intracellular water freezing. During the cooling process to -20℃, the extracellular solution freezes first, leading to an increase in solute concentration. Slow freezing allows sufficient intracellular water to seep out of the cell, reducing the probability of intracellular ice crystal formation. By combining DMSO with a concentration gradient to promote intracellular water exudation, the formation of intracellular ice crystals is further reduced. Trehalose provides additional extracellular protection, forming a stable hydrogen bond network with extracellular water molecules, inhibiting ice crystal nucleation and growth, and reducing mechanical damage to the cell membrane. Incubation at -20℃ for 30-40 min stabilizes cells during dehydration, preventing cell damage from ice crystal recrystallization caused by temperature fluctuations. Cooling to -80℃ results in almost complete exudation of intracellular water, and the extracellular solution forms a vitrified state, preventing further ice crystal growth and recrystallization. An 8-9 h incubation period ensures cells fully adapt to the -80℃ environment, completely stabilizing the cell membrane and organelle structures, preparing them for subsequent liquid nitrogen preservation, further reducing cell cryopreservation damage, and significantly improving cell viability after thawing. The method described in this application has clear steps and well-defined key parameters, greatly improving the consistency and reproducibility of experimental results between different operators and batches, and significantly enhancing cell activity after extraction and cell viability after thawing. Attached Figure Description
[0016] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 This is a flowchart of the process for extracting and cryopreserving mouse hepatocytes according to this application. Figure 2 The image shows the morphology of mouse hepatocytes after separation using the method described in this application, as detected by the trypan blue rejection assay. Figure 3 This image shows the state of mouse hepatocytes after being isolated using the method described in this application and cultured adherently for 24 hours. Figure 4 The image shows the state of mouse hepatocytes after cryopreservation and thawing using the method described in this application. Figure 5 This is a diagram showing the state of mouse hepatocytes after isolation using the method described in Example 1. Figure 6 This image shows the state of mouse liver parenchymal cells after cryopreservation and resuscitation using the method described in Example 2. Detailed Implementation
[0018] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this invention.
[0019] The raw materials used in the specific embodiments of this application are of analytical grade. Additionally: Percoll was purchased from Santa Cruz Biotechnology Company, catalog number: sc-500790A; 1×Krebs-Ringer buffer was purchased from Sigma-Aldrich, catalog number: K4002; Williams' E was purchased from Gibco, catalog number: 12551-032; fetal bovine serum was purchased from Gibco, catalog number: A5669701; DMEM was purchased from Biological Industries, catalog number: 01-050-1A; type IV collagenase was purchased from Sigma-Aldrich, catalog number: C5138.
[0020] Example 1 A method for extracting and cryopreserving mouse liver cells includes the following steps: S1. Liver perfusion and digestion: Nine-week-old C57BL / 6 mice were cadaverized and fixed. The abdominal cavity was cut along the midline to expose the inferior vena cava, portal vein, and superior vena cava. A 0.55mm disposable intravenous infusion needle was inserted into the inferior vena cava and fixed with hemostatic forceps. First, the perfusion fluid (1×Krebs-Ringerbuffer) preheated at 37°C was perfused at a flow rate of 5 mL / min for 7 min. Then, the perfusion fluid (100 CDU / ml type IV collagenase and 1×Krebs-Ringerbuffer) preheated at 37°C was used for circulation perfusion at a flow rate of 7 mL / min for 12 min. S2. Cell Isolation and Purification: After perfusion, the entire liver was quickly removed and placed in a culture dish containing pre-cooled terminating digestion solution at 4°C (Williams' E solution containing 2% fetal bovine serum and 5mM CaCl2). The liver capsule was torn open with forceps to release hepatocytes and form cell suspension A. Cell suspension A was filtered through a 70μm cell sieve, and the filtrate was collected. The filtrate was centrifuged at 4°C and 50×g for 3 min, and the supernatant was discarded. Complete culture medium (DMEM containing 10% fetal bovine serum) was added to form cell suspension B. 37.5% Percoll gradient solution was prepared in advance in a centrifuge tube. Cell suspension B was then added to the upper layer of the gradient solution (gradient solution: cell suspension B volume ratio = 2:1). The mixture was centrifuged at 4°C and 800×g for 10 min. The upper cell layer and liquid were then carefully aspirated, and the cell pellet at the bottom was retained. The cell pellet was washed twice with complete culture medium and then centrifuged at 50×g for 3 min to obtain purified hepatocytes. S3. Resuspend the purified hepatocytes in pre-chilled cryopreservation buffer (containing 50 parts (w / v) Williams' E, 40 parts fetal bovine serum, 5 parts DMSO, 5 parts trehalose, and Trolox to a final concentration of 50 μM) to a final concentration of 1 × 10⁻⁶. 7 Cells / mL, aliquoted into 1mL / tube cryopreservation tubes, immediately placed in a programmed cooling box, set the program, store at 4℃ for 30min, cool to -20℃ at 1℃ / min and store for 30min, cool to -80℃ at 1℃ / min and store for 8h, then place in a liquid nitrogen tank for long-term storage.
[0021] Compare with Example 1 The difference between this comparative example and Example 1 lies in the method of extracting and cryopreserving mouse liver cells, which includes the following steps: S1. Liver perfusion and digestion: Nine-week-old C57BL / 6 mice were cadaverized and fixed. The abdominal cavity was cut along the midline to expose the inferior vena cava, portal vein, and superior vena cava. A 0.55mm disposable intravenous infusion needle was inserted into the inferior vena cava and fixed with hemostatic forceps. First, the perfusion fluid (1×Krebs-Ringerbuffer) preheated at 37°C was perfused at a flow rate of 5 mL / min for 7 min. Then, the perfusion fluid (100 CDU / ml type IV collagenase and 1×Krebs-Ringerbuffer) preheated at 37°C was used for circulation perfusion at a flow rate of 7 mL / min for 12 min. S2. Cell Isolation and Purification: After perfusion, the entire liver was quickly removed and placed in a culture dish containing pre-cooled terminating digestion solution at 4°C (Williams' E solution containing 2% fetal bovine serum and 5mM CaCl2). The liver capsule was torn open with forceps to release hepatocytes and form a cell suspension. The cell suspension was filtered through a 70μm cell sieve, and the filtrate was collected. A 37.5% Percoll gradient solution was prepared in advance in a centrifuge tube, and the filtrate was added to the upper layer of the gradient solution (gradient solution: filtrate volume ratio = 2:1). The mixture was centrifuged at 4°C and 800×g for 10 min. The upper cell layer and liquid were then carefully aspirated, and the cell pellet at the bottom was retained. The cell pellet was washed twice with complete culture medium and then centrifuged at 50×g for 3 min to obtain purified hepatocytes. S3. Resuspend the purified hepatocytes in pre-chilled cryopreservation buffer (containing 50 parts (w / v) Williams' E, 40 parts fetal bovine serum, 5 parts DMSO, 5 parts trehalose, and Trolox to a final concentration of 50 μM) to a final concentration of 1 × 10⁻⁶. 7 Cells / mL, aliquoted into 1mL / tube cryopreservation tubes, immediately placed in a programmed cooling box, set the program, store at 4℃ for 30min, cool to -20℃ at 1℃ / min and store for 30min, cool to -80℃ at 1℃ / min and store for 8h, then place in a liquid nitrogen tank for long-term storage.
[0022] Compare with Example 2 The difference between this comparative example and Example 1 is that the cryopreservation solution consists of 90 parts fetal bovine serum and 10 parts DMSO.
[0023] Compare with Example 3 The difference between this comparative example and Example 1 is that it was directly frozen at -80°C.
[0024] Performance testing Cells purified in Example 1 and Control Example 1 were counted and their viability was determined using the trypan blue exclusion assay. Mouse liver parenchymal cells cryopreserved in Example 1 and Control Examples 2-3 were removed from the cryopreservation tubes in liquid nitrogen and immediately placed in a 37°C water bath. The tubes were shaken rapidly until only a small ice crystal remained. The tube walls were wiped with 70% alcohol, and the cell suspension was transferred to a centrifuge tube containing 10 times the volume of preheated complete culture medium in a clean bench. The cells were mixed with a Pasteur pipette and centrifuged at 4°C and 50×g for 3 minutes. The supernatant was discarded, and the cells were resuspended in fresh complete culture medium for activity testing.
[0025] The results are shown in Table 1: Table 1
[0026] As shown in Table 1, the two-step perfusion method combined with a specific two-step centrifugation method used in this application can obtain a large number of hepatocytes with extremely high activity. Compared with the single low-speed centrifugation method used in Control Example 1, the method used in this application has better separation and purification effects, and the amount of hepatocytes obtained reaches 7.0 × 10⁻⁶. 7 The activity of liver cells reached 91.67%, far exceeding the method used in Control Example 1. This application greatly improved the survival rate of liver parenchymal cells after recovery by using a special cryopreservation solution and a specific cooling method. Compared with the conventional cryopreservation solution (90 parts fetal bovine serum and 10 parts DMSO) and the method of directly placing the cells at -80°C for cryopreservation and then thawing in Control Examples 2 and 3, the cryopreservation method used in this application resulted in a liver parenchymal cell activity of 88.28% after thawing, which is far higher than the method used in Control Examples 2 and 3. This indicates that the method used in this application can greatly improve the isolation and purification effect of liver parenchymal cells and the survival rate after cryopreservation and thawing.
[0027] The above description is merely an example and illustration of the concept of this application. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the inventive concept or exceed the scope defined in the claims, they should all fall within the protection scope of this application.
Claims
1. A method for extracting and cryopreserving mouse liver cells, characterized in that, Includes the following steps: S1. Liver perfusion and digestion: Mouse carcasses were cannulated through the inferior vena cava, the portal vein was cut open, and the superior vena cava was clamped. First, the liver was perfused with perfusion fluid for 6-8 minutes, and then perfused with digestive fluid for 10-15 minutes. S2. Cell isolation and purification: After circulatory perfusion, the liver membrane is removed and torn open in the pre-cooled digestion solution at 3-5℃. The liver is filtered through a sieve and the filtrate is collected. The filtrate is centrifuged for the first time, and complete culture medium is added. Then, it is centrifuged a second time and the cell pellet is collected. The pellet is washed 2-3 times with complete culture medium and centrifuged again. The cell pellet at the bottom is retained to obtain purified liver parenchymal cells. S3. Resuspend the purified hepatocytes in pre-cooled cryopreservation solution to a final concentration of 1-2 × 10⁻⁶. 7 cells / mL, aliquoted and frozen.
2. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The perfusion fluid is 1×Krebs-Ringer buffer, the temperature of the perfusion fluid is 36-38℃, and the flow rate of the perfusion fluid is 5-6ml / min.
3. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The digestion solution consists of 100 CDU / ml of type IV collagenase and 1×Krebs-Ringer buffer, with a temperature of 36-38℃ and a flow rate of 7-8 ml / min.
4. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The terminating digestion solution is a Williams' E solution containing 1.5-2.5% fetal bovine serum and 4-6 mM CaCl2.
5. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, For the initial centrifugation, the centrifugal force is 45-55×g, the centrifugation time is 3-5min, and the centrifugation temperature is 3-5℃.
6. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The complete culture medium is DMEM medium containing 8-12% fetal bovine serum.
7. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The secondary centrifugation is performed under the following conditions: a 35-40% Percoll gradient solution is prepared in a centrifuge tube beforehand; the cell suspension is added to the upper layer of the gradient solution; the volume ratio of the gradient solution to the cell suspension is 2-3:1; the centrifugation temperature is 3-5℃; the centrifugation force is 750-850×g; and the centrifugation time is 10-15min.
8. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The re-centrifugation is performed with a centrifugal force of 45-55×g and a centrifugation time of 3-5min.
9. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The cryopreservation solution comprises the following materials in parts by weight: 40-50 parts Williams' E, 30-40 parts fetal bovine serum, 4-6 parts DMSO, 4-6 parts trehalose, and Trolox at a final concentration of 45-55 μM.
10. The method for extracting and cryopreserving mouse liver cells according to claim 1, characterized in that, The cryopreservation method involves storing the product at 4°C for 30-40 minutes, cooling it down to -20°C at a rate of 1-2°C / min and storing it for 30-40 minutes, cooling it down to -80°C at a rate of 1-2°C / min and storing it for 8-9 hours, and then storing it in liquid nitrogen.