CN110724661A - Separation method of mouse primary hepatocytes, mouse primary hepatocytes prepared by separation method and application of mouse primary hepatocytes - Google Patents

Abstract

The invention provides a separation method of mouse primary hepatocytes, the prepared mouse primary hepatocytes and application thereof, and relates to the technical field of primary cell separation. The method for separating the primary hepatocytes of the mouse comprises the following steps of: sequentially exposing an abdominal cavity of an anesthetized mouse, ligating a perihepatic duct, perfusing in situ in three steps and separating cells to obtain primary hepatocytes of the mouse; the in-situ three-step perfusion comprises a first perfusion, a second perfusion and a third perfusion in sequence; first perfusion, removing red blood cells by using first perfusion liquid, and performing anticoagulation on blood; second perfusion, removing the anticoagulant by using second perfusion liquid, and performing primary digestion on the liver tissue; third perfusion, further digestion with third perfusion fluid. The separation method of the mouse primary hepatocytes solves the technical problems that the separation method of the mouse primary hepatocytes, which is high in activity of the cells after separation, rapid in adherence, high in yield of the surviving cells, low in separation cost and not easy to pollute in the separation process, is lacking in the prior art.

Description

Separation method of mouse primary hepatocytes, mouse primary hepatocytes prepared by separation method and application of mouse primary hepatocytes

Technical Field

The invention relates to the technical field of primary cell separation, in particular to a separation method of mouse primary hepatocytes, the mouse primary hepatocytes prepared by the same and application of the mouse primary hepatocytes.

Background

The primary mouse hepatocytes are widely applied in the technical field of life science, and the preparation technology of the primary mouse hepatocytes is a basic flow in many scientific experiments at present and is very widely applied.

At present, the isolation method of mouse primary hepatocytes includes the following steps: (1) liquid nitrogen grinding method: the method has the advantages that the cost of liquid nitrogen is high, part of tissues can be adhered to the mortar and the pestle during grinding to generate certain liver tissue waste, meanwhile, in the liquid nitrogen grinding process, the mortar is dried due to untimely liquid nitrogen supplement, cells are heated to reduce the activity and even die, the cell activity of the primary hepatocytes prepared by separation is further reduced, the yield of the viable hepatocytes is reduced, and the method is not suitable for large-scale preparation of the primary hepatocytes. (2) A trypsin digestion method: the method has high cost by using pancreatin, and the phenomenon that external cells are over digested and internal cells are difficult to digest often occurs in the process of pancreatin digestion, so that the cell activity of the primary hepatocytes prepared by separation and the yield of the surviving hepatocytes are reduced. (3) Extracorporeal perfusion: the method is easy to cause fungus pollution when separating liver, and has inconvenient sterile operation, and often causes rupture of liver capsule to cause failure of perfusion. In addition, the posterior vena cava is adopted for perfusion during perfusion, the posterior vena cava is thick, in the process that perfusion liquid enters thin blood vessels from thick blood vessels, the damage of liver cells can be caused due to the change of the speed and power of the perfusion liquid, once the blood vessels are damaged, excessive bleeding can be caused due to overlarge blood pressure in the perfusion process, the perfusion needle is not easy to be punctured and ligated, the experiment efficiency is reduced, the experiment time is prolonged, the activity of the obtained primary liver cells is reduced due to the prolonging of the time, the yield of the survival liver cells is reduced, the existing perfusion liquid often causes incomplete digestion, and the yield of the survival liver cells prepared by separation is further reduced.

Therefore, the method for separating the primary mouse hepatocytes, which can effectively improve the cell activity of the primary hepatocytes prepared by separation, enable the separated cells to adhere to the walls more quickly, effectively improve the yield of the surviving hepatocytes, reduce the preparation cost of the mouse hepatocytes, is suitable for preparation of a large number of mouse primary hepatocytes, and is not easy to cause bacterial contamination, has important scientific research value and practical application value.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a separation method of primary mouse hepatocytes, which is characterized in that a anesthetized mouse is subjected to abdominal cavity exposure, perihepatic duct ligation, in-situ three-step perfusion and cell separation in sequence to obtain primary mouse hepatocytes; the in-situ three-step perfusion comprises a first perfusion, a second perfusion and a third perfusion in sequence; first perfusion, removing red blood cells by using first perfusion liquid, and performing anticoagulation on blood; second perfusion, removing the anticoagulant by using second perfusion liquid, and performing primary digestion on the liver tissue; and (4) performing third perfusion and further digesting by using third perfusion liquid. The separation method of the mouse primary hepatocytes can effectively improve the activity of the separated mouse primary hepatocytes, so that the adhesion of the mouse primary hepatocytes to the walls is quicker, the yield of the primary hepatocytes is high, the method is suitable for larger experiment dosage requirements, and fungus pollution is not easily generated in the separation process; in addition, the method has the advantages of simple program and low cost.

The second purpose of the invention is to provide the mouse primary hepatocyte prepared by the mouse primary hepatocyte separation method.

The invention also aims to provide the application of the mouse primary hepatocytes in cell biology tests or virology tests.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

in a first aspect, a method for isolating mouse primary hepatocytes is provided, which comprises the following steps: sequentially exposing an abdominal cavity of an anesthetized mouse, ligating a perihepatic duct, perfusing in situ in three steps and separating cells to obtain primary mouse hepatocytes;

the in-situ three-step perfusion comprises a first perfusion, a second perfusion and a third perfusion in sequence;

first perfusion, removing red blood cells by using first perfusion liquid, and performing anticoagulation on blood;

second perfusion, removing the anticoagulant by using second perfusion liquid, and performing primary digestion on the liver tissue;

third perfusion, further digestion with third perfusion fluid.

Preferably, on the basis of the scheme of the invention, the perfusion blood vessel for in-situ perfusion is a hepatic portal vein.

Preferably, on the basis of the scheme of the invention, the third perfusion step further comprises a step of perfusing and cleaning the mouse liver by PBS after perfusing the mouse liver by the third perfusion solution;

preferably, the temperature of PBS is 36-37 ℃;

preferably, the isolation method of mouse primary hepatocytes further comprises the step of pre-heating mouse liver with 37 ℃ PBS after the second perfusion;

preferably, the third perfusion fluid is a collagenase IV solution, the temperature is 37 ℃, and the concentration of the collagenase IV solution is 0.03-0.05%;

preferably, the concentration of the collagenase type IV solution is 0.048%;

preferably, the first, second and third perfusates perfusate 150-.

Preferably, on the basis of the present invention, the perihepatic duct is ligated, and the ligated perihepatic duct comprises: common bile duct, gastroduodenal artery, splenic artery, left gastric artery and posterior cavity artery;

preferably, the perihepatic duct ligation ligates common bile duct, gastroduodenal artery, splenic artery, left gastric artery and posterior cavity artery in sequence;

preferably, the perihepatic duct ligation comprises the following steps:

(1) the knot cuts the ligament of the duodenum;

(2) ligating common bile duct;

(3) ligating gastroduodenal artery, tracking blood flowing direction of hepatic common artery until spleen artery and left gastric artery are exposed;

(4) ligating and cutting the spleen artery and the left stomach artery, and exposing the abdominal artery trunk;

(5) turning down the liver, exposing the liver and the diaphragm at the back side, and separating the sickle ligament of the liver and the left and right tricuspid ligaments;

(6) ligating the left gastric vein and double ligating at the pancreas neck, and cutting the pancreas (7) ligated posterior vena cava between ligations;

(8) liver was turned up, perihepatic portal lymphatic tissue was isolated, hepatic portal vein was exposed, and heparin was injected at 100U.

Preferably, on the basis of the scheme of the invention, the cell separation sequentially comprises liver tissue separation and cell screening separation;

the liver tissue separation comprises the following steps: separating the liver after digestion and perfusion to the outside of an anesthetized mouse, immersing the anesthetized mouse in a PBS (phosphate buffer solution) solution at 37 ℃, removing the liver envelope, connective tissues and blood vessels, and repeatedly blowing and beating by using a liquid-transfering device to obtain mouse liver homogenate;

the cell screen separation comprises the following steps: sequentially screening the homogenate through cell screens of 100 meshes, 200 meshes and 400 meshes to obtain cell suspension, and carrying out solid-liquid separation on the cell suspension to obtain primary mouse hepatocytes;

preferably, the solid-liquid separation method is centrifugation, the centrifugation rotating speed is 450-;

preferably, the centrifugal speed is 500 g;

preferably, the centrifugation is for a period of 5 min.

Preferably, on the basis of the scheme of the invention, a micro-injection pump is adopted for in-situ perfusion;

preferably, the perfusion rate of in situ perfusion is 1.2-1.5 mL/min.

Preferably, on the basis of the scheme of the invention, the first perfusion liquid is perfused until the liver begins to swell, the posterior vena cava is punctured, and the first perfusion liquid is released;

preferably, during the first perfusion, the liver surface is tapped.

Preferably, on the basis of the scheme of the invention, the placement of the indwelling needle is further carried out after the perihepatic duct ligation, and the placement of the indwelling needle comprises the following steps:

surrounding the preset suture on the back side of the hepatic portal vein, puncturing the hepatic portal vein by using an indwelling needle, removing a hard needle of the indwelling needle, and fixing the indwelling needle by using the preset suture; then placing a heparin cap and connecting a scalp needle;

preferably, the abdominal cavity is exposed, comprising the step of making a Mercedes surgical incision in the abdomen of the anesthetized mouse;

preferably, the first perfusion fluid comprises, in mass percent: NaCl0.7-0.9%, KCl0.03-0.05%, NaH₂PO₄·2H₂O0.007-0.008％、Na₂HPO₄·12H₂O0.014-0.016％、EGTA0 .018-0.02％、NaHCO₃0.03-0.04%, HEPES 0.023-0.024%, glucose 0.08-0.1%, penicillin-streptomycin 0.8-1.5%, and the balance of water.

Preferably, the first perfusion fluid comprises, in mass percent: NaCl0.8%, KCl0.04%, NaH₂P O₄·2H₂O0.0078％、Na₂HPO₄·12H₂O0.0151％、EGTA0.019％NaHCO₃0.035%, HEPES 0.238%, Glucose 0.09%, penicillin-streptomycin 1%, and water in balance;

preferably, the second perfusion fluid comprises, in mass percent: NaCl0.7-0.9%, KCl 0.03-0.05%, CaCl₂0.05-0.06％、NaH₂PO₄·2H₂O0.007-0.008％、Na₂HPO₄·12H₂O0.014- 0.016％、HEPES0.23-0.24％、NaHCO₃0.03-0.04%, 0.8-1.5% of cyan-streptomycin and the balance of water;

preferably, the second perfusion fluid comprises, in mass percent: NaCl0.8%, KCl0.04%, CaCl₂0.056％、NaH₂PO₄·2H₂O0.0078％、Na₂HPO₄·12H₂O 0.0151％、HEPES0 .238％、NaHCO₃0.035g, 1% of cyan-streptomycin and the balance of water.

In a second aspect, a mouse primary hepatocyte prepared by the method for isolating a mouse primary hepatocyte is provided.

In a third aspect, the application of mouse primary hepatocytes in cell biology tests or virology tests is provided;

preferably, the cell biology experiment comprises: a cytotoxicity test, an apoptosis test, an oxidative stress test, an autophagy test, or a viral infection test.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the method for separating the primary hepatocytes of the mouse, the liver of the mouse is perfused by in-situ three-step perfusion, and compared with in-vitro perfusion in the prior art, the in-situ perfusion is not easy to cause bacterial pollution and damage to a liver capsule, so that the perfusion success rate is high, and the cost is effectively reduced; in addition, the mouse liver perfusion method adopts a three-step perfusion mode to perfuse the mouse liver, and sequentially comprises a first perfusion, a second perfusion and a third perfusion. Compared with the prior art, the method has the advantages that perfusion is divided into three steps, anticoagulant is removed in the second perfusion process and is primarily digested, and further digested in the third perfusion process, the cell digestion effect is better in the perfusion mode, the phenomena of excessive digestion and insufficient digestion of cells are effectively relieved, the cell activity of the primary hepatocytes prepared by separation is improved, the separated hepatocytes can be more quickly attached to the wall, and the yield of the surviving hepatocytes is more effectively improved by matching with an in-situ perfusion method. In addition, the method can obtain a large amount of primary hepatocytes at one time, is suitable for large-scale experimental application, and has the advantages of simple procedure and low cost.

(2) The mouse primary hepatocytes prepared by the method for separating the mouse primary hepatocytes are fast in adherence and high in cell activity; the mouse primary hepatocytes of the present invention can be used in cell biology tests or virology tests.

Drawings

FIG. 1 is a graph of exposed portal vein of example 1 of the present invention;

FIG. 2 is a hepatic portal vein perfusion diagram of example 1 of the present invention;

FIG. 3 is a 12h adherence map of mouse primary hepatocytes prepared in example 1 of the present invention;

FIG. 4 is a 12h adherence map of mouse primary hepatocytes prepared in comparative example 1 of the present invention;

FIG. 5 is a 12h adherence map of mouse primary hepatocytes prepared in comparative example 2 of the present invention;

FIG. 6 shows the cell activity (%) of primary cells obtained in example 1 of the present invention cultured in control group and 1, 10, 100, 1000nM T-2 dose group for 24h in experimental example;

FIG. 7 shows the cell activities (%) of primary cells prepared in comparative example 1 of the present invention cultured for 24 hours in control group and 1, 10, 100, 1000nM T-2 dose group in experimental example;

FIG. 8 shows the cell activities (%) of the primary cells prepared in comparative example 2 of the present invention cultured for 24 hours in the control group and the T-2 dose groups of 1, 10, 100, and 1000nM in the experimental examples.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

According to a first aspect of the present invention, there is provided a method for isolating mouse primary hepatocytes, comprising the steps of: sequentially exposing an abdominal cavity of an anesthetized mouse, ligating a perihepatic duct, perfusing in situ in three steps and separating cells to obtain primary hepatocytes of the mouse;

the in-situ three-step perfusion comprises a first perfusion, a second perfusion and a third perfusion in sequence;

first perfusion, removing red blood cells by using first perfusion liquid, and performing anticoagulation on blood;

second perfusion, removing the anticoagulant by using second perfusion liquid, and performing primary digestion on the liver tissue;

third perfusion, further digestion with third perfusion fluid.

Exposing the abdominal cavity, namely exposing the abdominal cavity of the mouse, and further exposing the liver of the mouse; the perihepatic vessel ligation refers to ligation of main vessels around the liver which are not used for perfusion, so as to control the flow direction of perfusion liquid in the subsequent perfusion process to a certain extent; the three-step perfusion is a perfusion process that the perfusion process is divided into three steps, different perfusion liquids are respectively used for perfusion, namely, the first perfusion liquid is a perfusion process that the anticoagulant agent is contained in the first perfusion liquid to prevent blood from coagulating and remove red blood cells at the same time of anticoagulation, and the first perfusion liquid flows through blood vessels of the mouse liver to remove the red blood cells, so that the red blood cells are effectively prevented from being mixed in cells obtained by subsequent separation; the second perfusion liquid contains an anticoagulant and digestive enzyme, can prevent blood in the liver from coagulating, avoids blood coagulation from blocking blood vessels, further prevents subsequent digestive perfusion process from being blocked, and is an anticoagulant and primary digestive process; the third perfusion liquid mainly contains digestive enzymes, can act on the liver cells more fully through some capillary vessels, is a further digestion process, is anticoagulated and digested simultaneously compared with the traditional process, can act on the liver cells more fully, is digested more quickly and fully, has better digestion effect, and can obtain better activity and more rapid adherence of cells; typical but non-limiting digestive fluids are for example: collagenase type IV, collagenase type II, trypsin, etc. The three processes are carried out in sequence, firstly, the erythrocyte is removed while anticoagulation is carried out, then, the residual anticoagulant is removed, preliminary digestion is carried out, and then, further digestion is carried out.

In a preferred embodiment, the perfused vessel perfused in situ is the portal vein of the liver.

In the prior art, the posterior vena cava is usually adopted for perfusion, but the posterior vena cava is thick, excessive bleeding can be caused due to overlarge blood pressure when a blood vessel is punctured during operation, and the perfusion needle is not easy to puncture and ligate, so that the technical requirement on an operator is high, the operation needs to be careful, and the consumed time is long. The hepatic portal vein is easy to find, convenient to fix and not easy to slip, the bleeding amount in the puncture process is less, the puncture visual field is not influenced, the perfusion efficiency is improved, the hepatic portal vein is easy to prick a perfusion needle relatively easily, ligation is easy, operation is convenient, operation time is further saved, the activity of subsequently obtained hepatic cells is facilitated, and the prepared hepatic cells are adhered to the wall more rapidly.

In a preferred embodiment, the third perfusion step further comprises a step of perfusing and washing the mouse liver with PBS after perfusing the mouse liver with the third perfusion solution. Washing with PBS (phosphate Buffered saline), residual digestive enzymes contained in the third perfusion solution can be removed to prevent over-digestion of the cells.

Preferably, the temperature of PBS is 36-37 ℃; the temperature of PBS is 36-37 ℃ which is close to the temperature of the liver tissue of the mouse, and the activity of the liver tissue cells can be effectively maintained.

Preferably, the isolation method of mouse primary hepatocytes further comprises the step of pre-heating mouse liver with 37 ℃ PBS after the second perfusion; after the second perfusion, the mouse liver is preheated by PBS at 37 ℃, so that the liver tissue is prevented from being cooled gradually in the operation process, on one hand, the activity of the cells is maintained, more importantly, the mouse liver is preheated at 37 ℃, the activity of digestive enzymes is the best under the condition of 37 ℃, the liver tissue cells can be quickly digested, the activity of the digestive enzymes is fully exerted, and the digestion efficiency is improved.

Preferably, the third perfusion fluid is a collagenase type IV solution at 37 ℃ and the concentration of the collagenase type IV solution is 0.03-0.05%. The commercially available common type IV collagenase usually contains at least 7 protease components, has good digestion effect on mouse liver tissues, causes less cell death in the digestion process, has high yield of the living cells harvested after digestion, is mild in the digestion process, has good digestion effect, and has good activity and quick wall adhesion of the harvested cells. The temperature is 37 ℃, the temperature is closer to the body temperature of a mouse, the stimulation to liver tissues is small, the activity of liver cells is favorably kept, the concentration of the IV collagenase solution is controlled to be 0.03-0.05%, the digestion is more sufficient, the cell death is not easily caused, the digestion effect is good, the over digestion is not easily caused, and the cell activity after perfusion is better. Typical but non-limiting concentrations of collagenase type IV are for example: 0.03%, 0.04%, 0.05%, etc.

Preferably, the concentration of the collagenase type IV solution is 0.048%. When the concentration of the type IV collagenase is 0.048%, the digestion effect is better, the digestion is not required for too long time, the activity of subsequently obtained cells is better maintained, and the adherence is quicker.

Preferably, the first, second and third perfusates perfusate 150-. According to practical experience, the first perfusion solution is anticoagulated and red blood cells are removed, and according to the liver blood volume and liver volume of the mouse, the red blood cells can be sufficiently removed by perfusion of 150-. In order to avoid blockage of blood vessels in the liver due to coagulation, particularly blockage of some small blood vessels, which can cause insufficient subsequent digestion, influence the yield of living cells and waste liver tissues, an anticoagulant is also added into the second perfusion solution, the anticoagulation effect is achieved while primary digestion is carried out, and according to actual experience, the perfusion is carried out by 150-200 mL. And the third perfusion liquid is essentially perfused by digestive liquid which plays a role in digestion, and the perfusion of 150-200mL can generally digest the liver tissues of the mice more fully. Typical but non-limiting separate perfusions of the first, second and third perfusates are for example: 150mL, 160mL, 170mL, 180mL, 190mL, 200mL, or the like.

In a preferred embodiment, the perihepatic duct is ligated, the ligated perihepatic duct comprising: common bile duct, gastroduodenal artery, splenic artery, left gastric artery and posterior cavity artery; the perihepatic duct includes a main blood vessel and a bile duct around the liver, and ligation of the perihepatic duct can control the flow direction of perfusion fluid better, so that perfusion is more rapid and sufficient.

Preferably, the perihepatic duct ligation ligates common bile duct, gastroduodenal artery, splenic artery, left gastric artery and posterior cavity artery in sequence;

preferably, the perihepatic duct ligation comprises the following steps:

(1) the knot cuts the ligament of the duodenum;

(2) ligating common bile duct;

(3) ligating gastroduodenal artery, tracking blood flowing direction of hepatic common artery until spleen artery and left gastric artery are exposed;

(4) ligating and cutting the spleen artery and the left stomach artery, and exposing the abdominal artery trunk;

(5) turning down the liver, exposing the liver and the diaphragm at the back side, and separating the sickle ligament of the liver and the left and right tricuspid ligaments;

(6) ligating the left gastric vein and double ligating at the pancreas neck, and cutting the pancreas (7) ligated posterior vena cava between ligations;

(8) liver was turned up, perihepatic portal lymphatic tissue was isolated, hepatic portal vein was exposed, and heparin was injected at 100U.

The ligation is carried out according to the steps, the ligation is carried out from the outside to the inside, the liver dissociation is increased in the ligation process, and the rupture of a liver capsule is not easy to cause. The perfusion success rate is high, and liver tissue waste is not easy to cause.

In a preferred embodiment, cell separation comprises liver tissue separation and cell sieve separation in this order;

the liver tissue separation comprises the following steps: separating the liver after digestion and perfusion to the outside of an anesthetized mouse, immersing the anesthetized mouse in a PBS (phosphate buffer solution) solution at 37 ℃, removing the liver envelope, connective tissues and blood vessels, and repeatedly blowing and beating by using a liquid-transfering device to obtain mouse liver homogenate;

the cell screen separation comprises the following steps: sequentially screening the homogenate through cell screens of 100 meshes, 200 meshes and 400 meshes to obtain cell suspension, and carrying out solid-liquid separation on the cell suspension to obtain primary mouse hepatocytes;

the separation of liver tissue is the process of separating the whole liver tissue, and the cell screening is the process of obtaining the dispersed cells. In the process, the liver is separated firstly, then the liver is put into PBS solution with the temperature of 37 ℃ to better keep the activity of liver cells, the envelope, connective tissue and vascular tissue of the liver are removed, the digested tissue is easy to break in the repeated blowing process, and then the liver homogenate of the mouse is obtained.

Preferably, the solid-liquid separation method is centrifugation, the centrifugation rotating speed is 450-; the centrifugation can be relatively rapid cell separation, is favorable for maintaining the activity of the primary liver cells, rapidly obtains the high-activity primary liver cells of the mouse, and in addition, the cell damage is easily caused by overlarge centrifugal force, the sedimentation of the undersized centrifugal force cells is not easy to occur, and the cell sedimentation is more complete after the PBS is repeatedly centrifuged for 2-3 times after the resuspension.

Preferably, the centrifugal speed is 500 g; the sedimentation speed of the cells is higher and the cells are not easy to be damaged at the rotating speed of 500 g.

Preferably, centrifugation is for a period of 5 min; the centrifugation time is not suitable for being too long or too short, the too long centrifugation time easily causes damage to cells, the too short centrifugation time easily causes incomplete sedimentation, and the centrifugation time of 5min relatively ensures the completeness of sedimentation without causing cell damage.

In a preferred embodiment, the in situ perfusion uses a micro-syringe pump. The micro-injection pump is adopted, so that the flow rate can be better controlled, the perfusion effect is better, and the cost of the micro-injection pump is lower.

Preferably, the perfusion rate of in situ perfusion is 1.2-1.5 mL/min. The perfusion speed is between 1.2 and 1.5mL/min, blood vessels in the liver tissue are easily damaged too fast, various perfusion liquids used for perfusion cannot sufficiently act on the liver tissue, particularly the digestion process, the control of the perfusion speed is very important, when the perfusion speed is between 1.2 and 1.5mL/min, the digestion is sufficient, and the damage to cells is small, and the typical but non-limiting perfusion speed is as follows: 1.2mL/min, 1.3mL/min, 1.4mL/min, 1.5mL/min, etc. In a preferred embodiment, the first perfusion solution is perfused until the liver begins to swell, the posterior vena cava is punctured, and the first perfusion solution is released; this process is the process of removing red blood cells from blood vessels within the liver tissue.

Preferably, during the first perfusion, the liver surface is tapped. The surface of the liver is tapped to promote the liquid flow in the liver tissue, so that the bad red blood cell removing effect caused by the unsmooth flow of the red blood cell removing liquid in the perfusion process is effectively avoided, and the discharge of blood cells in the liver is accelerated.

In a preferred embodiment, the perihepatic duct ligation further comprises placing an indwelling needle, wherein the placing of the indwelling needle comprises the following steps:

surrounding the preset suture on the back side of the hepatic portal vein, puncturing the hepatic portal vein by using an indwelling needle, removing a hard needle of the indwelling needle, and fixing the indwelling needle by using the preset suture;

surrounding the preset suture on the back side of the hepatic portal vein, puncturing the hepatic portal vein by using an indwelling needle, removing a hard needle of the indwelling needle, and fixing the indwelling needle by using the preset suture; the indwelling needle can effectively avoid the phenomenon that the blood vessel is punctured due to mechanical collision or movement in the perfusion process of the common hard needle, so that the perfusion fails, can effectively improve the probability of successful perfusion, avoids unnecessary waste and saves liver tissues.

Then placing a heparin cap and connecting a scalp needle; the heparin cap is a device connected with the indwelling needle, can prevent liquid from flowing back and has certain anticoagulation effect.

Preferably, the abdominal cavity is exposed, comprising the step of making a Mercedes surgical incision in the abdomen of the anesthetized mouse; the incision can expose abdominal viscera more fully, and is convenient for operation and perfusion.

Preferably, the first perfusion fluid comprises, in mass percent: NaCl0.7-0.9%, KCl0.03-0.05%, NaH₂PO₄·2H₂O0.007-0.008％、Na₂HPO₄·12H₂O0.014-0.016 ％、EGTA0.018-0.02％、NaHCO₃0.03-0.04%, HEPES 0.023-0.024%, glucose 0.08-0.1%, penicillin-streptomycin 0.8-1.5%, and the balance of water.

The addition of 0.8-1.5% of penicillin-streptomycin into the first perfusion liquid further reduces the probability of fungus contamination in the perfusion process, EGTA has an anticoagulation effect, and the rest salts form a neutral perfusion liquid together, and simultaneously have a certain buffering effect and a certain protection effect on cells.

Preferably, the first perfusion fluid comprises, in mass percent: NaCl0.8%, KCl0.04%, NaH₂P O₄·2H₂O0.0078％、Na₂HPO₄·12H₂O0.0151％、EGTA0.019％NaHCO₃0.035%, HEPES 0.238%, Glucose 0.09%, penicillin-streptomycin 1%, and water in balance. The first perfusion solution with a specific formula is preferably selected, so that the anticoagulation effect can be better exerted, the antibiotic concentration and the salt solution concentration are more reasonable, and the cell protection effect is better.

Preferably, the second perfusion fluid comprises, in mass percent: NaCl0.7-0.9%, KCl 0.03-0.05%, CaCl₂0.05-0.06％、NaH₂PO₄·2H₂O0.007-0.008％、Na₂HPO₄·12H₂O0.014- 0.016％、HEPES0.23-0.24％、NaHCO₃0.03-0.04%, 0.8-1.5% of penicillin-streptomycin and the balance of water.

The addition of 0.8-1.5% of penicillin-streptomycin into the second perfusion liquid also reduces the probability of fungus contamination, CaCl2 has the effect of removing EGTA, and the rest salts jointly form a neutral perfusion liquid which has a certain buffering effect and a certain protection effect on cells.

Preferably, the second perfusion fluid comprises, in mass percent: NaCl0.8%, KCl0.04%, CaCl₂0.056％、NaH₂PO₄·2H₂O0.0078％、Na₂HPO₄·12H₂O 0.0151％、HEPES0 .238％、NaHCO₃0.035g, 1% of cyan-streptomycin and the balance of water.

The second perfusion liquid with a specific formula is preferably selected, so that the anticoagulant in the first perfusion liquid remained after the first perfusion can be better exerted, the antibiotic concentration and the saline solution concentration are optimized, and the cell protection effect is better.

According to a second aspect of the present invention, there is provided a mouse primary hepatocyte obtained by a method for isolating a mouse primary hepatocyte.

The mouse primary hepatocytes prepared by the method for separating the mouse primary hepatocytes provided by the invention are fast in adherence and high in cell activity.

According to a third aspect of the invention, there is provided the use of mouse primary hepatocytes in a cell biology assay or a virology assay.

The mouse primary hepatocyte provided by the invention can be applied to cell biology tests or virology tests.

Preferably, the cell biology experiment comprises: a cytotoxicity test, an apoptosis test, an oxidative stress test, an autophagy test, or a viral infection test.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.

Example 1

Example 1 the reagents used were formulated as follows:

first perfusion liquid: weighing 1.6g NaCl, 0.08g KCl and 0.0156g NaH₂PO₄·2H₂O、 0.0302gNa₂HPO₄·12H₂O、0.038g EGTA、0.07g NaHCO₃0.476g of HEPES, 0.18g of glucose and 0.5g of penicillin-streptomycin, and ultrapure water is added to the mixture to reach the constant volume of 200 mL.

Second perfusion liquid: weighing 1.6g NaCl, 0.08g KCl and 0.112g CaCl₂、0.0156g NaH₂PO₄·2H₂O、0.0302g Na₂HPO₄·12H₂O、0.07g NaHCO₃0.476g of HEPES, 0.5g of penicillin-streptomycin, and ultrapure water was added to the solution to a volume of 200 mL.

Digestion solution: 0.048% collagenase type IV.

A method for separating primary hepatocytes of a mouse comprises the following steps:

exposure of the abdominal cavity: injecting ICR mouse into abdominal cavity at 0.3mg/g lomannnin, and fixing on operating table after completely anaesthetizing. The whole body was sterilized with 75% alcohol, and then sterile gauze was placed around the mice to expose only the operative site, Mercedes surgical incision (herringbone incision), followed by cutting the muscle and peritoneal membrane to expose the abdominal cavity.

Ligation of perihepatic duct: (1) the knot cuts the ligament of the duodenum; (2) ligating common bile duct; (3) ligaturing gastroduodenal artery, tracking blood flowing direction of hepatic common artery until spleen artery and left gastric artery are exposed; (4) ligating and cutting the spleen artery and the left stomach artery, and exposing the abdominal artery trunk; (5) turning down the liver to expose the liver and diaphragm at the back side, and separating the sickle ligament and the left and right triangular ligaments of the liver; (6) ligating the left gastric vein and double ligating at the pancreas neck, and cutting the pancreas (7) ligated posterior vena cava between ligations; (8) turning up the liver, isolating the peripheral lymphoid tissue of the hepatic portal vein, exposing the hepatic portal vein, see fig. 1, fig. 1 is a view of exposing the hepatic portal vein, and then injecting 100U of heparin.

In-situ three-step perfusion: the preset suture is wound around the back side of the portal vein, a 26 # indwelling needle is used for puncturing the portal vein, a hepatic portal vein perfusion diagram is shown in figure 2, a hard needle in the indwelling needle is removed, and then the indwelling needle is fixed by the preset suture. Place the heparin cap and attach the scalp needle. Perfusing 200mL of first perfusion liquid at the speed of 1.5mL/min by using a micro-injection pump, puncturing a posterior vena cava by using a needle when the liver begins to swell, and releasing the first perfusion liquid, wherein in the first perfusion process, the surface of the liver is lightly tapped by a hand until blood cells in the liver are completely perfused out of the liver; then perfusing 200mL of second perfusion liquid at the speed of 1.5mL/min by using a micro-injection pump; 0.048% type IV collagenase and PBS prepared in advance are placed in a water bath kettle at 37 ℃ for preheating, the preheated PBS is used for soaking the in-situ liver, a microinjection pump is used for carrying out third perfusion by using 0.048% type IV collagenase at the speed of 1.5mL/min, and the same perfusion is carried out for 200 mL. PBS was pumped at 1.5mL/min using a micro syringe pump to remove type IV collagenase.

Cell separation: separating the digested liver to the outside of the body, soaking the digested liver in PBS at 37 ℃, transferring the separated mouse liver to cells, placing the cells on a sterilized flat dish, taking out the liver capsule by using forceps of the ophthalmology department, separating connective tissues and blood vessels, then adding the PBS into the separated liver tissue, and repeatedly blowing the mouse liver tissue by using a liquid transfer gun until no obvious agglomeration exists, thus obtaining the mouse liver homogenate. Then, screening the homogenate of the mouse liver through 100-mesh, 200-mesh and 400-mesh cell sieves respectively; collecting the cell suspension, transferring the cell suspension to a 10mL centrifuge tube, centrifuging for 5min at 500g, centrifuging after resuspending by PBS, repeating for 3 times, and discarding the supernatant.

The prepared mouse primary hepatocytes are cultured for 12h in a DMEM culture medium at 37 ℃ and 5% CO2, and the adherence map of the prepared mouse primary hepatocytes is 12h as shown in figure 3.

Comparative example 1

Comparative example 1 differs from example 1 only in that perfusion was performed in an ex vivo perfusion mode.

The prepared mouse primary hepatocytes are attached to the wall after being cultured for 12h, and are shown in figure 4.

Comparative example 2

Comparative example 2 differs from example 1 only in that perfusion is divided into two steps, anticoagulant and digestive fluids are mixed, and anticoagulation perfusion and digestive perfusion are performed in one step.

The prepared mouse primary liver cells were cultured for 12h, as shown in figure 5.

Test examples

Mouse primary hepatocytes obtained in example 1 and comparative examples 1 to 2 were each added at a concentration of 5X 10⁵each.mL^-1The cells were seeded in 96-well cell culture plates (200. mu.l per well), cultured in a 5% CO2 incubator at 37 ℃ and then observed for cell status under a microscope at 0h, 24h, 48h and 72h, respectively.

The mouse primary hepatocytes obtained in example 1 and comparative examples 1 to 2 were used at a concentration of 5X 10⁵each.mL^-1Cells were seeded into 96-well cell culture plates, 200. mu.l per well, in 5% CO₂After culturing at 37 ℃ for 12 hours in a constant temperature incubator, a T-2 toxicity test was performed. The primary mouse cells obtained in each example were inoculated into 10 wells, and the experiment was divided into five groups of 2 wells, each of which was: treating adherent cells with T-2 toxin of different concentrations in control group, 1, 10, 100 and 1000nM T-2 toxin dose groups for 24h, performing toxicity test, and determining the activity of hepatocytes of different toxin groups by MTT method, wherein the activity determination results are shown in FIGS. 6-8; FIG. 6 shows the cell activity (%) of primary cells obtained in example 1 of the present invention cultured in control group and 1, 10, 100, 1000nM T-2 dose group for 24h in experimental example; FIG. 7 shows the cell activities (%) of primary cells prepared in comparative example 1 of the present invention cultured for 24 hours in control group and 1, 10, 100, 1000nM T-2 dose group in experimental example; FIG. 8 shows the cell activities (%) of the primary cells prepared in comparative example 2 of the present invention cultured for 24 hours in the control group and the T-2 dose groups of 1, 10, 100, and 1000 nM.

As can be seen from the comparison of the detection results of example 1 and comparative example 1, the adherent effect of the prepared mouse primary cells is relatively poor, adherent cells in a visual field are fewer, the activity of the prepared primary cells is poor, the adherent is slower, and the cell survival rate is lower by adopting an isolated perfusion mode.

As can be seen from comparison of the detection results of example 1 and comparative example 2, a two-step perfusion mode is adopted, that is, anticoagulation perfusion and digestion perfusion are performed simultaneously, so that after the prepared mouse primary hepatocytes are cultured, adherent cells in a visual field are relatively few, the prepared primary hepatocytes have poor activity, are slow in adherence, and have a lower cell survival rate.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (10)

1. A method for separating primary hepatocytes of a mouse is characterized by comprising the following steps: sequentially exposing an abdominal cavity of an anesthetized mouse, ligating a perihepatic duct, perfusing in situ in three steps and separating cells to obtain primary hepatocytes of the mouse;

the in-situ three-step perfusion comprises a first perfusion, a second perfusion and a third perfusion in sequence;

the first perfusion, removing red blood cells by using a first perfusion solution, and performing anticoagulation on blood;

the second perfusion, the anticoagulant is removed by using a second perfusion liquid, and the liver tissue is subjected to primary digestion;

the third perfusion, further digested with a third perfusion fluid.

2. The method of isolating mouse primary hepatocytes according to claim 1, wherein the perfused vessels perfused in situ are hepatic portal veins.

3. The method of isolating mouse primary hepatocytes according to claim 2, wherein the third perfusion step further comprises a perfusion wash step of the mouse liver with PBS after perfusing the mouse liver with the third perfusion solution;

preferably, the temperature of the PBS is 36-37 ℃;

preferably, the isolation method of the mouse primary hepatocytes further comprises a step of pre-heating mouse livers with 37 ℃ PBS after the second perfusion;

preferably, the third perfusion fluid is a collagenase IV solution, the temperature is 37 ℃, and the concentration of the collagenase IV solution is 0.03-0.05%;

preferably, the concentration of the collagenase type IV solution is 0.048%;

preferably, the first perfusion liquid, the second perfusion liquid and the third perfusion liquid are respectively perfused by 150-.

4. A method of isolating mouse primary hepatocytes according to any one of claims 1 to 3, wherein the perihepatic ducts are ligated, the ligated perihepatic ducts comprising: common bile duct, gastroduodenal artery, splenic artery, left gastric artery and posterior cavity artery;

preferably, the perihepatic duct ligation ligates common bile duct, gastroduodenal artery, splenic artery, left gastric artery and posterior cavity artery in sequence;

preferably, the perihepatic duct ligation comprises the following steps:

(1) the knot cuts the ligament of the duodenum;

(2) ligating common bile duct;

(3) ligating gastroduodenal artery, tracking blood flowing direction of hepatic common artery until spleen artery and left gastric artery are exposed;

(4) ligating and cutting the spleen artery and the left stomach artery, and exposing the abdominal artery trunk;

(5) turning down the liver, exposing the liver and the diaphragm at the back side, and separating the sickle ligament and the left and right triangular ligaments of the liver;

(6) ligating the left gastric vein and double ligating at the pancreas neck and cutting the pancreas between ligations

(7) Ligating the posterior vena cava;

(8) liver was turned up, perihepatic portal lymphoid tissue was isolated, hepatic portal vein was exposed, and heparin was injected at 100U.

5. The method of isolating mouse primary hepatocytes according to claim 4, wherein the cell isolation comprises in sequence a liver tissue isolation and a cell sieving;

the liver tissue isolation comprises the following steps: separating the liver after digestion and perfusion to the outside of an anesthetized mouse, immersing the anesthetized mouse in a PBS (phosphate buffer solution) solution at 37 ℃, removing a liver envelope, connective tissues and blood vessels, and repeatedly blowing and beating by using a pipette to obtain mouse liver homogenate;

the cell screen separation comprises the following steps: sequentially sieving the homogenate by using 100-mesh, 200-mesh and 400-mesh cell sieves to obtain cell suspensions, and performing solid-liquid separation on the cell suspensions to obtain primary mouse hepatocytes;

preferably, the solid-liquid separation method is centrifugation, the centrifugation rotating speed is 450-;

preferably, the centrifugal rotation speed is 500 g;

preferably, the centrifugation is for a period of 5 min.

6. The method for isolating mouse primary hepatocytes according to claim 5, wherein the in situ perfusion uses a micro-syringe pump;

preferably, the perfusion rate of the in-situ perfusion is 1.2-1.5 mL/min.

7. The method for isolating mouse primary hepatocytes according to claim 6, wherein the first perfusion is performed by puncturing the posterior vena cava when the liver begins to swell and releasing the first perfusion;

preferably, during the first perfusion, the liver surface is tapped.

8. The method of isolating mouse primary hepatocytes according to claim 7, wherein the perihepatic duct ligation further comprises indwelling needle placement, the indwelling needle placement comprising the steps of:

surrounding the preset suture on the back side of the hepatic portal vein, puncturing the hepatic portal vein by using an indwelling needle, removing a hard needle of the indwelling needle, and fixing the indwelling needle by using the preset suture; then placing a heparin cap and connecting a scalp needle;

preferably, the abdominal cavity exposure comprises the step of making a Mercedes surgical incision in the abdomen of the anesthetized mouse;

preferably, the first perfusionThe liquid comprises the following components in percentage by mass: NaCl0.7-0.9%, KCl0.03-0.05%, NaH₂PO₄∙2H₂O0.007-0.008％、Na₂HPO₄∙12H₂O0.014-0.016％、EGTA0.018-0.02％、NaHCO₃0.03-0.04%, HEPES 0.023-0.024%, glucose0.08-0.1%, penicillin-streptomycin 0.8-1.5%, and the balance of water;

preferably, the first perfusion fluid comprises, in mass percent: NaCl0.8%, KCl0.04%, NaH₂PO₄∙2H₂O0.0078％、Na₂HPO₄∙12H₂O0.0151％、EGTA0.019％NaHCO₃0.035%, HEPES 0.238%, Glucose 0.09%, penicillin-streptomycin 1%, and water in balance;

preferably, the second perfusion fluid comprises, in mass percent: NaCl0.7-0.9%, KCl 0.03-0.05%, CaCl₂0.05-0.06％、NaH₂PO₄∙2H₂O0.007-0.008％、Na₂HPO₄∙12H₂O0.014-0.016％、HEPES0.23-0.24％、NaHCO₃0.03-0.04%, 0.8-1.5% of penicillin-streptomycin and the balance of water;

preferably, the second perfusion fluid comprises, in mass percent: NaCl0.8%, KCl0.04%, CaCl₂0.056％、NaH₂PO₄∙2H₂O0.0078％、Na₂HPO₄∙12H₂O0.0151％、HEPES0.238％、NaHCO₃0.035g, 1% of cyan-streptomycin and the balance of water.

9. Mouse primary hepatocytes produced by the method of isolating mouse primary hepatocytes of any one of claims 1 to 8.

10. Use of the mouse primary hepatocyte of claim 9 in a cell biology assay or a virology assay;

preferably, the cell biology experiment comprises: a cytotoxicity test, an apoptosis test, an oxidative stress test, an autophagy test, or a viral infection test.

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