Hepatocyte cryopreservation liquid and cryopreservation method and recovery method of primary mouse hepatocytes

Abstract

The invention discloses a hepatocyte cryopreservation solution which comprises a first cryopreservation mixed solution, a second cryopreservation mixed solution and fetal calf serum, the first cryopreservation mixed solution is prepared from a Leibovitz's L-15 culture medium, lactobionic acid, raffinose, reduced glutathione, ademetionine, curcumin, bicyclol, ursodesoxycholic acid, silymarin and glycyrrhizic acid; the second cryopreservation mixed solution comprises cane sugar, trehalose, hydroxyethyl starch and polyene phosphatidylcholine. On the other hand, the invention discloses a cryopreservation method and a recovery method of primary mouse hepatocytes based on the hepatocyte cryopreservation liquid. The cryopreservation method comprises the following steps: pre-culturing, re-suspending, slowly dropwise adding DMSO (dimethylsulfoxide) and cooling and cryopreserving step by step. The resuscitation method comprises the steps of rapid unfreezing, centrifugal purification and resuscitation culture. The liver cell cryopreservation liquid and the cryopreservation and resuscitation method can play roles in regulating osmotic pressure, inhibiting ice crystal formation, stabilizing cell membrane structure, resisting oxidation, promoting protein synthesis and the like, and the resuscitated cells are high in survival rate and strong in adherence capability and functional activity.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation technology, and in particular to a hepatocyte cryopreservation solution, a method for cryopreserving primary mouse hepatocytes, and a method for thawing. Background Technology

[0002] Hepatocytes are the main cells of the liver, accounting for 80% of the liver's volume and number. They are highly differentiated cells. Hepatocytes are metabolically active, synthesizing essential clotting factors, fatty acids, cholesterol, phospholipids, etc., and storing glycogen, proteins, lipids, and vitamins to meet the body's needs.

[0003] Hepatocytes serve as seed cells for cell transplantation therapy of chronic liver failure and as target cells for drug screening. The preparation and culture of large-scale, highly viable hepatocytes is a fundamental step in these studies. Therefore, the isolation, culture, and cryopreservation techniques of hepatocytes are receiving increasing attention and have become a current research hotspot.

[0004] Hepatocytes are highly sensitive to external conditions and have poor tolerance to cryopreservation. Various chemical and physical damages caused during cryopreservation and thawing can affect hepatocyte viability and function. While cryopreservation technology for hepatocytes has been studied for many years, an ideal method for cryopreservation has yet to be found. A suitable cryopreservation solution will effectively help hepatocytes resist changes in osmotic pressure during cryopreservation and reduce damage during the process. Current technology allows for the cryopreservation of hepatocytes using UW solution, but UW solution can only preserve hepatocytes at low temperatures for short periods; long-term cryopreservation results are unsatisfactory. Therefore, there is an urgent need to establish a simple and efficient technique for long-term cryopreservation of primary mouse hepatocytes. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a simple and efficient cryopreservation and resuscitation method for obtaining mouse primary hepatocytes with good growth status and high purity after long-term cryopreservation, and to establish a complete and reliable in vitro isolation and cryopreservation technology for mouse primary hepatocytes.

[0006] To achieve the above objectives, the present invention provides a hepatocyte cryopreservation solution, comprising a first cryopreservation mixture, a second cryopreservation mixture, and fetal bovine serum;

[0007] The first cryopreservation mixture includes Leibovitz's L-15 medium, lactobionic acid, raffinose, reduced glutathione, S-adenosylmethionine, curcumin, bicyclol, ursodeoxycholic acid, silymarin, and glycyrrhizic acid.

[0008] The second cryopreservation mixture includes sucrose, trehalose, hydroxyethyl starch and polyene phosphatidylcholine, with a pH of 7.2-7.4.

[0009] Preferably, the first cryopreservation mixture and the second cryopreservation mixture are prepared in advance, and the hepatocyte cryopreservation solution is prepared and used immediately. The hepatocyte cryopreservation solution is prepared by mixing the first cryopreservation mixture and the second cryopreservation mixture, adjusting the pH to 7.2-7.4, and then adding fetal bovine serum.

[0010] Preferably, the contents of each component in the hepatocyte cryopreservation solution are as follows:

[0011] Lactobionic acid 35.84 g / L;

[0012] Raffinose 17.83 g / L;

[0013] Reduced glutathione 0.922 g / L;

[0014] S-adenosylmethionine 5-10 μM;

[0015] Curcumin 5-10 μM;

[0016] Bicyclic alcohol 10-20 μM;

[0017] Ursodeoxycholic acid 40-60 μM;

[0018] Silymarin 500-800 μg / mL;

[0019] Glycyrrhizic acid 1-5mM;

[0020] Sucrose 10-20mM;

[0021] Trehalose 10-20 mM;

[0022] Hydroxyethyl starch 0.05-0.2 mM;

[0023] Polyene phosphatidylcholine 10-20 μM;

[0024] And, fetal bovine serum 10-20%.

[0025] Another aspect of the present invention provides a method for cryopreserving primary mouse hepatocytes, comprising the following steps:

[0026] The primary mouse hepatocytes were precultured in pre-culture medium, then centrifuged and the cell pellet was obtained.

[0027] The cell pellet was resuspended in the aforementioned hepatocyte cryopreservation solution;

[0028] Additionally, slowly add 2-6% DMSO dropwise onto ice, mix well, and then gradually cool down before freezing.

[0029] The pre-culture medium includes Leibovitz's L-15 medium, insulin, penicillin-streptomycin antibiotics, lactulose, N-acetyl-L-cysteine, glucose, alpha-lipoic acid, hepatocyte growth factor, interleukin-6, and ω-3 fatty acids.

[0030] Preferably, before pre-culturing the primary mouse hepatocytes, the following steps are also included:

[0031] Mice underwent surgery to expose the abdominal cavity, ligate the portal vein, and perfuse to remove red blood cells and digestive enzymes.

[0032] The liver was removed, further digested with digestive enzymes, and the mouse primary hepatocytes were obtained.

[0033] Furthermore, the mouse primary hepatocytes were purified by continuous density gradient centrifugation;

[0034] The digestive enzyme solution comprises 0.05-0.2% type IV collagenase and 0.01-0.05% DNase;

[0035] The continuous density gradient centrifugation purification process includes first performing density gradient centrifugation with 70-90% Percoll solution, aspirating the hepatocyte layer and resuspending the cells, and then performing density gradient centrifugation with 40-60% Percoll solution and 20%-30% Percoll solution.

[0036] Preferably, the contents of each component in the pre-culture solution are as follows:

[0037] Insulin 0-100mM;

[0038] Penicillin-streptomycin bispecific antibody 1×;

[0039] Lactulose 100-200mM;

[0040] N-acetyl-L-cysteine ​​1-5mM;

[0041] Glucose 2-6mM;

[0042] α-Lipoic acid 5-10 mM;

[0043] Hepatocyte growth factor 100-200 ng / mL;

[0044] Interleukin-6 30-50 μg / mL

[0045] In addition, 10-20 μM of ω-3 fatty acids.

[0046] Another aspect of the present invention provides a method for the resuscitation of primary mouse hepatocytes, wherein the primary mouse hepatocytes are cryopreserved using the aforementioned cryopreservation method; the resuscitation method includes the following steps:

[0047] The frozen primary mouse hepatocytes were rapidly transferred to a 37°C water bath for rapid thawing.

[0048] The rapidly thawed primary mouse hepatocytes were transferred to a preheated density gradient centrifugation solution and purified by density gradient centrifugation.

[0049] Furthermore, the primary mouse hepatocytes were revived and cultured using a resuscitation medium;

[0050] The resuscitation medium includes Leibovitz's L-15 medium, fetal bovine serum, disodium adenosine triphosphate, and silymarin.

[0051] Preferably, the resuscitation method further includes the step of: continuing to culture the resuscitated cells using a complete culture medium;

[0052] The complete culture medium includes Leibovitz's L-15 medium, fetal bovine serum, insulin, dexamethasone, glutamine, penicillin-streptomycin bispecific antibody, CaCl2, hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, interleukin-6, and hepatocyte growth factor.

[0053] Preferably, the components in the resuscitation medium are present in the following amounts:

[0054] Fetal bovine serum 10-20%;

[0055] Adenosine triphosphate disodium 10-20 μg / mL;

[0056] And silymarin 500-800 μg / mL.

[0057] Preferably, the components in the complete culture medium are present in the following amounts:

[0058] FBS 10-20%;

[0059] Insulin 50-100mM;

[0060] Dexamethasone 50-100mM;

[0061] Glutamine 1-5mM;

[0062] Penicillin-streptomycin bispecific antibody 1×;

[0063] CaCl2 1-5mM;

[0064] Hepatocyte growth factor 100-200 ng / mL;

[0065] Epidermal growth factor 100-200 ng / mL;

[0066] Fibroblast growth factor 100-200 ng / mL;

[0067] Interleukin-6 30-50 μg / mL;

[0068] In addition, hepatocyte growth factor 100-200 ng / mL.

[0069] In summary, the present invention has the following beneficial effects:

[0070] (1) The components in the hepatocyte cryopreservation solution of the present invention can play a role in regulating osmotic pressure, inhibiting ice crystal formation, stabilizing cell membrane structure, and anti-oxidation through synergistic effects. Using the hepatocyte cryopreservation solution of the present invention can reduce the amount of DMSO used in the cryopreservation process, thereby achieving more efficient and less toxic protection.

[0071] (2) The hepatocyte cryopreservation solution and resuscitation culture medium of the present invention can further stabilize the cell membrane structure and promote protein synthesis during resuscitation culture, thereby promoting cell repair;

[0072] (3) The cryopreservation method of the present invention uses a cryopreservation solution specifically for hepatocytes, which is prepared and used immediately. The cells are cryopreserved using a step-down cooling method, reducing the cytotoxic damage caused by the cryoprotectant during freezing. This results in a larger number of cells obtained after thawing, with less cell damage and higher cell purity. The cryopreservation and thawing method of the present invention is simple and efficient. After 30 days of cryopreservation, the cell viability and purity after thawing reach over 95%. Compared with traditional cryopreservation methods, the method of the present invention can significantly improve the survival rate, adhesion ability, and functional activity of cryopreserved hepatocytes.

[0073] (4) The cryopreservation and thawing method of the present invention is economical, practical, simple and easy to implement, which is conducive to establishing in vitro experimental model cells, studying the characteristics of hepatocytes, and providing high-quality hepatocytes for cell transplantation and drug screening research. Attached Figure Description

[0074] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the 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.

[0075] Figure 1 This is a flowchart of a hepatocyte isolation and cryopreservation method according to an embodiment of the present invention;

[0076] Figure 2 This is a statistical chart showing the survival rate of freshly isolated hepatocytes according to an embodiment of the present invention.

[0077] Figure 3 This is a flowchart of a hepatocyte resuscitation and culture method according to an embodiment of the present invention;

[0078] Figures 4 to 6 This is a statistical chart showing the survival rate of hepatocytes at 0h, 4h, and 6h after resuscitation, according to an embodiment of the present invention.

[0079] Figure 7 and Figure 8 This is a microscopic observation of hepatocytes after resuscitation and adherence culture, according to an embodiment of the present invention. Detailed Implementation

[0080] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0081] The instruments, consumables, and reagents used in the embodiments of this invention include:

[0082] A set of surgical instruments, a cell counter (Thermo, USA), an inverted microscope (XDS-1A, Shanghai), a fluorescence microscope (Leica, USA), a low-temperature centrifuge (Eppendorf, USA), an ultra-low temperature freezer (Zhongke Meiling), a pipette (Eppendorf, USA), an electronic analytical balance (Sartorius, USA), a clean bench (HJ-CJ-1D, Shanghai), an LC-MS (Agilent, USA), and a CO2 cell incubator (Boxun, Shanghai).

[0083] L15 medium (Leibovitz's L-15, purchased from Gibco), penicillin-streptomycin antibiotics (100× solution, purchased from Gibco), fetal bovine serum (FBS, purchased from Bioind), rat tail collagen (self-made), type IV collagenase (purchased from Thermo Fisher Scientific), cell culture plates, centrifuge tubes (purchased from Corning Scientific), and trypan blue (purchased from Thermo Fisher Scientific).

[0084] First buffer solution: Dissolve 8.0g NaCl, 0.2g KCl, 1.56g Na2HPO4, 0.06g KH2PO4, 0.35g NaHCO3, 0.9g glucose, 2.38g Hepes and 0.19g EGTA in water, adjust the pH to 7.2-7.4, and bring the volume to 1L.

[0085] Second buffer solution: Dissolve 8.0g NaCl, 0.2g KCl, 1.56g Na2HPO4, 0.2g KH2PO4, 0.35g NaHCO3, 0.9g glucose, 2.38g Hepes, 0.098g MgSO4 and 0.14g CaCl2 in water, adjust the pH to 7.2-7.4, and bring the volume to 1L.

[0086] Digestive enzyme solution: Add 0.05-0.2% type IV collagenase and 0.01-0.05% DNase to the second buffer solution;

[0087] Pre-culture medium: Add 50-100mM insulin, 1% penicillin-streptomycin (i.e., 1×), 100-200mM lactulose, 1-5mM N-acetyl-L-cysteine, 2-6mM glucose, 5-10mM α-lipoic acid, 100-200ng / mL hepatocyte growth factor (HGPF), 30-50μg / mL interleukin-6 (IL-6), and 10-20μM ω-3 fatty acids to L15 medium;

[0088] First cryopreservation mixture: According to the preset total volume of hepatocyte cryopreservation solution, add 35.84 g / L lactobionic acid, 17.83 g / L raffinose, 0.922 g / L reduced glutathione, 5-10 μM S-adenosylmethionine, 5-10 μM curcumin, 10-20 μM bicyclol, 40-60 μM ursodeoxycholic acid, 500-800 μg / mL silymarin, and 1-5 mM glycyrrhizic acid to L15 medium. The above concentrations are based on the final concentration.

[0089] Second cryopreservation mixture: Prepare an aqueous solution of 10-20 mM sucrose, 10-20 mM trehalose, 0.05-0.2 mM hydroxyethyl starch and 10-20 μM polyene phosphatidylcholine according to the preset total volume of hepatocyte cryopreservation solution, and adjust the pH to 7.2-7.4. The above concentrations are based on the final concentration.

[0090] Resuscitation medium: Add 10-20% FBS, 10-20 μg / mL adenosine triphosphate disodium and 500-800 μg / mL silymarin to L15 medium;

[0091] Complete culture medium: Add 10-20% FBS, 50-100mM insulin, 50-100mM dexamethasone, 1-5mM glutamine, 1% penicillin-streptomycin antibiotics, 1-5mM CaCl2, 100-200ng / mL hepatocyte growth factor (HGF), 100-200ng / mL epidermal growth factor (EGF), 100-200ng / mL fibroblast growth factor (FGF), 30-50μg / mL interleukin-6 (IL-6), and 100-200ng / mL hepatocyte growth factor (HGPF) to L15 medium.

[0092] Example 1

[0093] like Figure 1 As shown, the isolation and cryopreservation of mouse hepatocytes includes the following steps:

[0094] S1: Preheating solution: Preheat 50mL of the first buffer solution and 50mL of digestive enzyme solution in a water bath at 42℃, so that they reach 37℃ in the liver.

[0095] S2: Surgical exposure of the peritoneum, ligation of the portal vein and removal of red blood cells: Heparin sodium (to prevent clotting) was injected into the peritoneum of mice, followed by sodium pentobarbital. The peritoneum was opened, and a ligation loop was loosely placed 5 mm outside the liver at the portal vein. The vascular cannula was inserted into the palmar surface of the liver and ligated. The subhepatic vessels were quickly cut open, and 50 mL of preheated first buffer solution was infused at a flow rate of 5-10 mL / min.

[0096] S3: Perfusion of digestive enzyme solution: Further perfuse 50 mL of digestive enzyme solution at a flow rate of 5-10 mL / min;

[0097] S4: Further digestion: The liver is cut off and placed in 50mL of digestive enzyme solution. Then, the liver tissue is torn apart with tweezers to detach the liver cells.

[0098] S5: Obtaining hepatocytes: Shake the cell suspension at 150 rpm for 10-15 min at 37℃; then filter the cell suspension through a 100 μm cell sieve, and centrifuge at 40-80 g / min for 5 min; resuspend the precipitated cells in a second buffer solution, and centrifuge at 40-80 g / min for 5 min, repeating the washing once;

[0099] S6: Continuous density gradient centrifugation purification: Resuspend hepatocytes in 10 mL of L15 basal medium, add 10 mL of 70-90% Percoll solution to the bottom layer of the test tube, gently add cell suspension to the top layer, and centrifuge at 400-600g for 10 min; after centrifugation, the mixture in the test tube will separate into four layers: the first layer is dead cells, the second layer is other contaminating cells, and the third layer is hepatocytes. Take the cells in the third layer with a pipette, resuspend them in the second buffer solution, and then add the bottom layer of 40-60% Percoll solution, the middle layer of 20%-30% Percoll solution, and the top layer of cell suspension to the centrifuge tube in a 1:1:1 volume ratio, centrifuge at 400-600g for 10 min, and purify again. The bottom precipitate in the centrifuge tube after centrifugation is hepatocytes;

[0100] S7: Cell pre-culture: Pour out the supernatant from the centrifuge tube, loosen the hepatocytes in the bottom sediment, and resuspend the hepatocytes in the pre-culture medium; pre-culture in a cell culture incubator at 37℃ and 5% CO2 for 30-60 min, transfer the cells to a 50 mL cryovial, and centrifuge at 50-100 g for 5 min at room temperature.

[0101] The pre-culture medium was supplemented with IL-6 and HGPF, which synergistically enhance the in vitro proliferation efficiency and cell viability of hepatocytes. To assess the cell viability after this pre-culture, a portion of the hepatocyte pellet was taken, stained with trypan blue, and the cell number and viability were calculated using an automated cell counter. Figure 2 As shown, the survival rate of freshly isolated cells is close to 100%, and the cell viability in repeated experiments is ≥96%, which is higher than that of conventional methods (usually ≥90%). This shows that the additives in the pre-culture can significantly improve cell activity, which is more conducive to the subsequent cryopreservation steps.

[0102] S8: Suspension cryopreservation: Mix the first and second cryopreservation mixtures, adjust the pH to 7.2-7.4, and add 10%-20% FBS to obtain hepatocyte cryopreservation solution. Prepare and use immediately. Resuspend the hepatocyte pellet that has been pre-cultured and centrifuged in step S7 in the hepatocyte cryopreservation solution. Place the hepatocyte suspension on ice and slowly add 2-6% DMSO at a rate of 1-5 mL / min. The slow addition method can maintain osmotic balance between the intracellular matrix and the culture medium, reducing cell damage. After the addition is complete, gently invert the cryopreservation tube to mix the cell suspension. Then place the cryopreservation tube in a -20°C freezer for 10-20 min, then transfer it to a -80°C freezer for 1-2 hours, and finally transfer it to a liquid nitrogen tank for cryopreservation.

[0103] Using the hepatocyte cryopreservation solution of this embodiment reduces the amount of DMSO used in the cryopreservation process, and further achieves highly efficient and low-toxicity protection by using a stepwise cooling method. Simultaneously, the components in the cryopreservation solution, such as S-adenosylmethionine, curcumin, glycyrrhizic acid, and silymarin, synergistically exert antioxidant effects; sugars and their derivatives regulate osmotic pressure and inhibit ice crystal formation; and components such as bicyclol, ursodeoxycholic acid, silymarin, and polyene phosphatidylcholine stabilize cell membranes and promote cell repair. Silymarin also promotes protein synthesis during the thawing process, further enhancing cell repair. Compared to traditional cryopreservation methods, the method of this embodiment significantly improves the survival rate, adhesion ability, and functional activity of cryopreserved hepatocytes.

[0104] Example 2

[0105] like Figure 3 As shown, the resuscitation and culture of hepatocytes in suspension cryopreservation includes the following steps. The hepatocytes in this embodiment were cryopreserved for 30 days according to the cryopreservation method of Example 1:

[0106] S9: Preheat density gradient centrifugation solution: Mix 20-30 mL of second buffer solution and 10-20 mL of 70-90% Percoll solution to obtain density gradient centrifugation solution. Add the density gradient centrifugation solution to a 15 mL centrifuge tube and preheat in a 37℃ constant temperature water bath for 20 min.

[0107] S10: Cell thawing: Transfer the cryovials containing hepatocytes from the liquid nitrogen tank to a constant temperature water bath maintained at 37°C; the transfer from the liquid nitrogen tank to the water bath should be completed within 10 seconds to facilitate rapid thawing.

[0108] S11: Centrifugal purification: Transfer the hepatocytes from the cryopreservation tube to a preheated 15mL centrifuge tube containing density gradient centrifugation solution, and then centrifuge at 50-100g for 5min.

[0109] S12: Resuscitation culture: Discard the supernatant after centrifugation in step S11, resuspend the cell pellet with 6 mL of resuscitation culture medium, and culture in a cell culture incubator at 37℃ and 5% CO2 for 30 min. The time point when the resuscitation culture is completed is recorded as 0 h after resuscitation.

[0110] To assess the resuscitation effect, a small amount of cell suspension can be taken, stained with trypan blue, and the total cell count and cell viability can be calculated. For example... Figure 4 As shown, the cell viability was 96% after 0 hours of thawing, and the cell viability was ≥95% in multiple repeated experiments, which is significantly higher than the level of conventional cryopreservation methods (usually around 80%).

[0111] S13: Plate culture: Take an appropriate amount of cell suspension, add complete culture medium, inoculate into culture plates, and then place them in a cell culture incubator at 37℃ and 5% CO2 for further culture; the culture plates include untreated plates and rat tail collagen-coated plates; the hepatocytes in the untreated plates are in suspension culture and are used to continue to observe the activity of the resuscitated hepatocytes in the further culture process; the hepatocytes in the rat tail collagen-coated plates are in adherent culture and are used to observe the adhesion rate of the resuscitated hepatocytes.

[0112] like Figure 5 and Figure 6 As shown, the viability of suspended cells in untreated plates 4 hours after resuscitation was 91% (the viability was ≥90% in multiple repeated experiments), and the viability of suspended cells 6 hours after resuscitation was 88% (the viability was ≥85% in multiple repeated experiments). Figure 7 and Figure 8 The cell morphology of hepatocytes after resuscitation and adherence culture was shown. The adhesion rate of hepatocytes was ≥90% after 2 hours of resuscitation. Further enzyme activity assays of hepatocytes showed that 7-ethoxycoumarin O-deethylation (ECOD) activity was ≥400 pmol / min / million cells; 7-hydroxycoumarin glucuronidation (UGT) activity was ≥100 pmol / min / million cells; and 7-hydroxycoumarin sulfation (ST) activity was ≥30 pmol / min / million cells.

[0113] Compared with existing cryopreservation technologies, this invention establishes a simple and efficient method for the cryopreservation and resuscitation of primary mouse hepatocytes. After resuscitation, the primary cells, observed by cell morphology and enzyme activity assays, exhibit high quantity, high purity, and good activity, with a cell viability and purity exceeding 95%. Furthermore, this method is economical, practical, and easy to implement, facilitating the establishment of in vitro experimental cell models, the study of hepatocyte characteristics, and the provision of high-quality hepatocytes for cell transplantation and drug screening research.

[0114] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A hepatocyte cryopreservation solution, characterized in that, Includes the first cryopreservation mixture, the second cryopreservation mixture, and fetal bovine serum; The first cryopreservation mixture includes Leibovitz's L-15 medium, lactobionic acid, raffinose, reduced glutathione, S-adenosylmethionine, curcumin, bicyclol, ursodeoxycholic acid, silymarin, and glycyrrhizic acid. The second cryopreservation mixture includes sucrose, trehalose, hydroxyethyl starch and polyene phosphatidylcholine, with a pH of 7.2-7.

4.

2. The hepatocyte cryopreservation solution according to claim 1, characterized in that, The first cryopreservation mixture and the second cryopreservation mixture are prepared in advance, while the hepatocyte cryopreservation solution is prepared and used immediately. The hepatocyte cryopreservation solution is prepared by mixing the first cryopreservation mixture and the second cryopreservation mixture, adjusting the pH to 7.2-7.4, and then adding fetal bovine serum.

3. The hepatocyte cryopreservation solution according to claim 1, characterized in that, The contents of each component in the hepatocyte cryopreservation solution are as follows: Lactobionic acid 35.84 g / L; Raffinose 17.83 g / L; Reduced glutathione 0.922 g / L; S-adenosylmethionine 5-10 μM; Curcumin 5-10 μM; Bicyclic alcohol 10-20 μM; Ursodeoxycholic acid 40-60 μM; Silymarin 500-800 μg / mL; Glycyrrhizic acid 1-5mM; Sucrose 10-20mM; Trehalose 10-20 mM; Hydroxyethyl starch 0.05-0.2 mM; Polyene phosphatidylcholine 10-20 μM; And, fetal bovine serum 10-20%.

4. A method for cryopreserving primary mouse hepatocytes, characterized in that, Includes the following steps: The primary mouse hepatocytes were precultured in pre-culture medium, then centrifuged and the cell pellet was obtained. The cell pellet was resuspended in the hepatocyte cryopreservation solution as described in any one of claims 1 to 3; Additionally, slowly add 2-6% DMSO dropwise onto ice, mix well, and then gradually cool down before freezing. The pre-culture medium includes Leibovitz's L-15 medium, insulin, penicillin-streptomycin antibiotics, lactulose, N-acetyl-L-cysteine, glucose, alpha-lipoic acid, hepatocyte growth factor, interleukin-6, and ω-3 fatty acids.

5. The cryopreservation method according to claim 4, characterized in that, Before preculturing the primary mouse hepatocytes, the following steps are also included: Mice underwent surgery to expose the abdominal cavity, ligate the portal vein, and perfuse to remove red blood cells and digestive enzymes. The liver was removed, further digested with digestive enzymes, and the mouse primary hepatocytes were obtained. Furthermore, the mouse primary hepatocytes were purified by continuous density gradient centrifugation; The digestive enzyme solution comprises 0.05-0.2% type IV collagenase and 0.01-0.05% DNase; The continuous density gradient centrifugation purification process includes first performing density gradient centrifugation with 70-90% Percoll solution, then resuspending the hepatocyte layer, and finally performing density gradient centrifugation with 40-60% Percoll solution and 20%-30% Percoll solution.

6. The cryopreservation method according to claim 4, characterized in that, The contents of each component in the pre-culture solution are as follows: Insulin 0-100mM; Penicillin-streptomycin bispecific antibody 1×; Lactulose 100-200mM; N-acetyl-L-cysteine ​​1-5mM; Glucose 2-6mM; α-Lipoic acid 5-10 mM; Hepatocyte growth factor 100-200 ng / mL; Interleukin-6 30-50 μg / mL In addition, 10-20 μM of ω-3 fatty acids.

7. A method for resuscitating primary mouse hepatocytes, characterized in that, The primary mouse hepatocytes were cryopreserved using the cryopreservation method as described in any one of claims 4 to 6; the revival method includes the following steps: The frozen primary mouse hepatocytes were rapidly transferred to a 37°C water bath for rapid thawing. The rapidly thawed primary mouse hepatocytes were transferred to a preheated density gradient centrifugation solution and purified by density gradient centrifugation. Furthermore, the primary mouse hepatocytes were revived and cultured using a resuscitation medium; The resuscitation medium includes Leibovitz's L-15 medium, fetal bovine serum, disodium adenosine triphosphate, and silymarin.

8. The resuscitation method according to claim 7, characterized in that, It also includes the step of: continuing to culture the revived cells using complete culture medium; The complete culture medium includes Leibovitz's L-15 medium, fetal bovine serum, insulin, dexamethasone, glutamine, penicillin-streptomycin bispecific antibody, CaCl2, hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, interleukin-6, and hepatocyte growth factor.

9. The resuscitation method according to claim 7, characterized in that, The components in the resuscitation medium are as follows: Fetal bovine serum 10-20%; Adenosine triphosphate disodium 10-20 μg / mL; And silymarin 500-800 μg / mL.

10. The resuscitation method according to claim 8, characterized in that, The components in the complete culture medium are as follows: FBS 10-20%; Insulin 50-100mM; Dexamethasone 50-100mM; Glutamine 1-5mM; Penicillin-streptomycin bispecific antibody 1×; CaCl2 1-5mM; Hepatocyte growth factor 100-200 ng / mL; Epidermal growth factor 100-200 ng / mL; Fibroblast growth factor 100-200 ng / mL; Interleukin-6 30-50 μg / mL; In addition, hepatocyte growth factor 100-200 ng / mL.

Images