CN117264876B

CN117264876B - Method for preparing multi-donor liver cells

Info

Publication number: CN117264876B
Application number: CN202311550264.XA
Authority: CN
Inventors: 陈双; 张彬; 宋楠
Original assignee: Miaoshun Shanghai Biotechnology Co ltd
Current assignee: Miaoshun Shanghai Biotechnology Co ltd
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2024-07-30
Anticipated expiration: 2043-11-21
Also published as: CN117264876A

Abstract

The invention discloses a method for preparing multi-donor liver cells. The invention designs a novel process for preparing multi-donor liver cells, and the whole process is accurately controlled, so that all steps are coordinated, the large-scale and high-efficiency preparation of liver cells with high cell activity and purity based on a plurality of donors can be realized, the stability among batches is high, the prepared cells can be stored for a long time, the prepared cells still have high activity rate and activity after recovery, and CYP450 metabolic enzyme still has higher activity.

Description

Method for preparing multi-donor liver cells

Technical Field

The invention belongs to the technical field of biology, relates to a method for preparing multi-donor liver cells, and particularly relates to a method for preparing primary liver cells with high cell activity and function on a large scale and application thereof.

Background

Liver is an important in vivo substance metabolism and detoxification organ, and hepatocytes are involved in regulating the metabolism of sugar, fat and protein, bioconversion and detoxification of various compounds, and have various functions of synthesizing and secreting plasma proteins and bile. The liver is the main place of drug metabolism, and most of drugs firstly reach the liver for metabolism and then enter the blood circulation after being orally taken or intramuscular injection through blood transmission, which is the first pass effect of the liver. Since the liver is an important metabolic site in vivo, the transformation of drugs is mostly completed in the liver, and thus the initial development of new drugs is necessary to study the effect of the liver on drug metabolism. Its main content includes the metabolic rate of the drug in the liver, the main metabolic pathways, the main metabolites and the enzyme systems involved in the metabolism of the compound in the main liver. Meanwhile, the toxic effect of the drug on the liver needs to be evaluated correspondingly, and all the information has important significance for early screening of the drug.

The primary liver cells have the following advantages: can obtain a large number of specimens with the same characters, keep the specimens consistent with the in-vivo conditions, and can study the metabolism of medicines under the condition of approaching physiological states, and the like. The long-term cultured single-layered primary liver cells subjected to sandwich culture treatment can form a biliary tubular-like structure and express a carrier peculiar to liver cells, and thus can be further used for evaluating the possibility of bile excretion of the compound. The primary cells are basically consistent with the conditions in organisms in terms of gene expression and cell behaviors, and have been considered as the best research object for in vitro experiments, however, the primary cells have complicated extraction process and low cell activity, and are key bottlenecks for restricting the application of the primary cells. In 1976 Seglen et al, optimized the extraction procedure of primary cells from the liver (Preparation of isolated RAT LIVER CELLS. Methods in Cell Biology, 1976), proposed a two-step method of liver infusion, which to date was still routine in large research institutions and laboratories. However, the number, viability and purity of the hepatic parenchymal cells obtained using this method are very limited.

In view of the above, it is important to develop a method capable of efficiently producing hepatocytes with high cell viability and purity on a large scale.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides a method for preparing multi-donor liver cells, which can be used for preparing liver cells with high cell activity and purity in a large scale and high efficiency.

In order to achieve the above purpose, the invention adopts the following technical scheme:

The present invention provides a method for preparing multi-donor liver cells, comprising the steps of:

(1) The method comprises the steps of anaesthetizing and fixing animals, picking an infusion needle of a perfusion system into a lower vena cava, cutting a hepatic portal vein, starting the perfusion system, and perfusing a first perfusion liquid with constant temperature of 42-44 ℃ at a perfusion flow rate of 10-50 mL/min for 4-5 min;

(2) Switching the first perfusion liquid into a second perfusion liquid with the temperature of 42-44 ℃ for constant-temperature perfusion, wherein the perfusion flow rate is 5-25 mL/min, and the time is 5-10 min;

(3) Taking the liver after perfusion in the step (2), removing the envelope, and preparing a cell suspension;

(4) Mixing the cell suspension obtained in the step (3) with a culture medium, sieving cells, centrifuging, discarding the supernatant, collecting cells, and mixing the cells with a cell preservation solution to obtain a cell suspension;

(5) Centrifuging the cell suspension obtained in the step (4), discarding the supernatant, collecting cells, mixing with a culture medium, adding the mixture into a percoll solution, centrifuging, and collecting cell sediment at the bottom to obtain the liver cells.

The invention designs a novel process for preparing multi-donor liver cells, and the whole process is accurately controlled, so that all steps are coordinated, and the large-scale and high-efficiency preparation of liver cells with high cell activity and purity based on a plurality of donors can be realized.

In the invention, the specific perfusion temperature is controlled, so that the cell activity can be better reserved on the basis of maintaining the cell activity rate, the perfusion temperature in the conventional method is usually set to 10-15 ℃, because the low temperature can reduce the metabolic demand of liver and increase the tolerance of liver cells to hypoxia, but the temperature and the in-vivo temperature are greatly different, even if the finally prepared cell activity rate (the living cell number ratio) is not low, the cell activity is difficult to maintain at a high level, the invention can achieve the effect of 'oxygen explosion' by continuously circulating the perfusion liquid in the perfusion system, and give enough oxygen to cells, so that the high activity rate is not needed to be replaced at the expense of the cell activity, and the perfusion temperature is finally set to 42-44 ℃ through deep analysis and test, meanwhile, the constant temperature is kept, and the cell activity and the activity rate can be influenced by unstable temperature; the specific perfusion speed and time are controlled, so that the activity and the activity rate of cells are ensured, and in a liquid phase environment, the perfusion speed is too high to cause massive cell death, and too slow to lengthen the operation time is also unfavorable for cell survival.

In the invention, in the step (5), the percoll solution is controlled to be added firstly, then the cell suspension is added into the percoll solution, so that the cell suspension can be fully centrifuged, if the cell suspension is added firstly and then the percoll separating liquid is added, the cells can sink to the bottom of the centrifuge tube quickly, then part of the cells cannot be fully centrifuged, and the yield of the centrifuged cells can be greatly reduced.

Preferably, the experimental animal of step (1) comprises a mouse, rat, dog, monkey or beagle.

Preferably, the first perfusion liquid in the step (1) is a calcium-free balanced salt solution, and contains 0.31% of sodium lactate, 0.6% of sodium chloride, 0.03% of potassium chloride, 0.02% of calcium chloride and 99.04% of sterile water.

Preferably, the first perfusion liquid contains 0.2% -0.4% of sodium lactate, 0.5% -0.7% of sodium chloride, 0.02% -0.04% of potassium chloride, 0.01% -0.03% of calcium chloride and 99% -99.06% of sterile water.

Preferably, the first perfusion fluid contains 0.31% sodium lactate, 0.6% sodium chloride, 0.03% potassium chloride, 0.02% calcium chloride and 99.04% sterile water.

Preferably, the second perfusion fluid of step (2) contains a type II collagenase.

Preferably, the medium of step (4) comprises Williams 'E medium, more preferably concentrated Williams' E medium.

Preferably, the hepatocyte preservation solution of step (4) contains a cell culture medium and additives including electrolytes, buffers, oxygen radical scavengers, impermeable substances adenosine and deferoxamine; the electrolyte includes metal ions and chloride ions; the oxygen radical scavenger comprises glutathione, allopurinol and alpha-lipoic acid; the impermeable substance comprises glucose, raffinose, mannitol, lactobionic acid, sucrose, fructose and dextran; the metal ions comprise any one or a combination of at least two of potassium ions, sodium ions, magnesium ions or calcium ions; the buffer comprises any one or a combination of at least two of dihydrogen phosphate, sulfate, bicarbonate or 4-hydroxyethyl piperazine ethane sulfonic acid.

In the invention, a specific hepatocyte preservation solution is designed, compared with a conventional basal medium, the cell viability and activity in the cold storage stage at 0-5 ℃ can be better maintained, and meanwhile, the cell preservation solution can be maintained for a protection period of not more than 48 hours, and the period of time is enough for preparing multi-donor mixed hepatocytes, so that efficient large-scale preparation is realized.

Preferably, the additive further comprises polyethylene glycol and/or an antibiotic.

Preferably, the hepatocyte preservation solution includes, in concentration: DMEM basal medium, 30-40 mM of potassium ion, 90-130 mM of sodium ion, 4-6 mM of magnesium ion, 0.15-0.25 mM of calcium ion, 15-22 mM of chloride ion, 14-20 mM of dihydrogen phosphate, 2-3 mM of sulfate, 2-3 mM of bicarbonate, 10-15 mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 2.5-3.5 mM of glutathione, 0.4-0.6 mM of allopurinol, 1.5-2.5 mM of alpha-lipoic acid, 2-3 mM of glucose, 12-18 mM of raffinose, 8-12 mM of mannitol, 120-140 mM of lactobionic acid, 8-12 mM of sucrose, 8-12 mM of fructose, 4-6 mM of glucan, 2.5-5 mM of adenosine, 0.8-1.2 mM of deferoxamine, 0.4-0.6 g/L of polyethylene glycol, 75-125 mM of penicillin and 75-125 mM of streptomycin.

Preferably, the pore diameter of the cell sieve in the step (4) is 30-40 μm.

Preferably, the concentration of the percoll solution in the step (5) is 25% -35%, including but not limited to 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%, etc.

Preferably, the step (1) further comprises the steps of preheating and loading the perfusion liquid, wherein the steps comprise placing the first perfusion liquid and the second perfusion liquid in a water bath with the temperature of 42-44 ℃ for at least 30 min, and the first perfusion liquid is poured into a conduit of a perfusion system and circulated.

According to the invention, the perfusion liquid circulates in the perfusion system, so that on one hand, oxygen in the air can be dissolved into the perfusion liquid as soon as possible; on the other hand, the temperature of the perfusion liquid can be prevented from being reduced, and constant-temperature perfusion is ensured.

Preferably, step (4) further comprises the step of cell counting the cell suspension, said cell counting method comprising trypan blue staining counting.

Preferably, step (5) further comprises a step of screening the cell suspension obtained in step (4) comprising selecting a cell suspension having a cell viability of 80% or more and a dead cell mass of less than 4.

Preferably, the method for preparing multi-donor hepatocytes comprises the steps of:

(1) Placing the first perfusion liquid and the second perfusion liquid in a water bath with the temperature of 42-44 ℃ for at least 30min, and pouring the first perfusion liquid into a conduit of a perfusion system and circulating;

(2) The method comprises the steps of anaesthetizing and fixing animals, picking an infusion needle of a perfusion system into a lower vena cava, cutting a hepatic portal vein, starting the perfusion system, cutting a diaphragm after the liver removes blood, clamping the lower vena cava at a centripetal end, and running for 4-5 min at a perfusion flow rate of 10-50 mL/min; switching the first perfusion liquid into the second perfusion liquid for constant-temperature perfusion at 42-44 ℃, wherein the perfusion flow rate is 5-25 mL/min, and the operation is 5-10 min;

(4) Mixing the cell suspension obtained in the step (3) with a culture medium, sieving with a 30-40 mu m cell sieve, centrifuging, discarding the supernatant, collecting cells, mixing the cells with a cell preservation solution to obtain a cell suspension, and performing trypan blue staining counting; the hepatocyte preservation solution comprises, in terms of concentration: DMEM basal medium, 30-40 mM of potassium ion, 90-130 mM of sodium ion, 4-6 mM of magnesium ion, 0.15-0.25 mM of calcium ion, 15-22 mM of chloride ion, 14-20 mM of dihydrogen phosphate, 2-3 mM of sulfate, 2-3 mM of bicarbonate, 10-15 mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 2.5-3.5 mM of glutathione, 0.4-0.6 mM of allopurinol, 1.5-2.5 mM of alpha-lipoic acid, 2-3 mM of glucose, 12-18 mM of raffinose, 8-12 mM of mannitol, 120-140 mM of lactobionic acid, 8-12 mM of sucrose, 8-12 mM of fructose, 4-6 mM of glucan, 2.5-5 mM of adenosine, 0.8-1.2 mM of deferoxamine, 0.4-0.6 g/L of polyethylene glycol and 150-250 mM of penicillin-streptomycin.

(5) And (3) taking the cell suspension obtained in the step (4), selecting the cell suspension with the activity ratio of more than or equal to 80% and the visual field dead cell mass of less than 4, centrifuging, discarding the supernatant, collecting cells, mixing with a culture medium, adding the cells into a percoll solution with the concentration of 25% -35%, centrifuging, and collecting cell sediment at the bottom to obtain the liver cells.

Preferably, the method for preparing multi-donor liver cells further comprises the steps of sub-packaging and cryopreserving the stem cells, wherein the steps comprise centrifuging the liver cells obtained in the step (5), discarding the supernatant, re-suspending cell sediment by using a culture medium, performing trypan blue staining counting, centrifuging after counting, discarding the supernatant, mixing the cell sediment with the cryopreservation solution, and sub-packaging; cooling and freezing the packaged cells.

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

The invention designs a novel process for preparing multi-donor liver cells, which is used for accurately controlling the whole process, including controlling specific perfusion temperature, so that the cell activity can be better reserved on the basis of maintaining the cell activity rate, the perfusion temperature in the conventional method is usually set to be 10-15 ℃, the metabolic demand of liver can be reduced due to low temperature, the tolerance of liver cells to hypoxia is increased, but the temperature and the in-vivo temperature are large in difference, even if the finally prepared cell activity rate (the ratio of the number of living cells) is not low, the cell activity is difficult to maintain at a high level, the invention has been proposed to achieve the effect of 'bursting oxygen' by continuously circulating perfusion liquid in the perfusion system, so that the cell sufficient oxygen is not required to be exchanged for high activity rate, and then the perfusion temperature is finally set to be 43+/-1 ℃ through deep analysis and test, and meanwhile, the temperature is kept constant, so that the cell activity and the activity rate are not influenced; the specific perfusion speed and time are controlled, so that the activity and the activity rate of cells are ensured, and in a liquid phase environment, a large number of cells die due to the too high perfusion speed, and the cell survival is not facilitated due to the too slow perfusion speed and the too long operation time; the specific hepatocyte preservation solution is designed, so that the activity and the activity of cells in the cold storage stage at 0-5 ℃ can be better maintained, and meanwhile, the cell preservation solution can be maintained for a protection period of not more than 48 hours, and the period of time is enough for preparing multi-donor mixed hepatocytes; the steps are coordinated, so that the liver cells with high cell activity and purity are prepared in a large scale and high efficiency based on a plurality of donors, the cells are easy to store for a long time, and the high activity can be maintained after recovery.

Drawings

FIG. 1 is a morphology of hepatocyte incubation 24 h cells from C57BL-6 mice;

FIG. 2 is a morphology of C57BL-6 mice incubated with hepatocytes 48 h cells;

FIG. 3 is a morphology of rat hepatocyte incubation 24 h cells;

FIG. 4 is a morphology of rat hepatocytes incubated 48 h cells;

FIG. 5 is a morphology of the liver cell incubation 24 h cells of beagle dogs;

FIG. 6 is a morphology of 72 h cells incubated with liver cells from beagle dogs;

FIG. 7A is a graph showing the cell viability of cryopreserved hepatocytes for 12 months;

FIG. 7B is a graph showing the results of cell number of cryopreserved hepatocytes for 12 months;

FIG. 7C is a graph showing the results of CYP1A PHENACETIN metabolic enzyme activity of cryopreserved hepatocytes for 12 months;

FIG. 7D is a graph showing the results of CYP2C S-Mephenytoin metabolizing enzyme activity of cryopreserved hepatocytes for 12 months;

FIG. 8A is a graph showing the cell viability of cryopreserved hepatocytes for 33 months;

FIG. 8B is a graph showing the cell number results of 33 months of cryopreserved hepatocytes;

FIG. 8C is a graph showing the results of metabolic enzyme activities of 33 months of cryopreserved hepatocytes;

FIG. 9A is a graph showing the results of cell viability within a batch;

FIG. 9B is a graph showing the results of cell numbers in a batch;

FIG. 10 is a schematic diagram comparing the preparation process of the present invention with the prior art.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.

The reagents used in the specific examples of the present invention are shown in Table 1.

TABLE 1

Example 1

This example was performed to prepare C57BL-6 mice and CD1 mice multi-donor hepatocytes.

1. The temperature of the water bath was set at 43 ℃, and the first perfusion liquid (containing sodium lactate 0.31%, sodium chloride 0.6%, potassium chloride 0.03%, calcium chloride 0.02% and sterile water 99.04%) and the second perfusion liquid (Sigma type II collagenase solution 100MG, cat# C2-BIOC, batch # C2-28-100 MG) were placed in a50 mL centrifuge tube in the water bath, and after preheating for at least 30min, the experiment was started.

2. Opening a peristaltic pump, replacing a needle head and a filter head, cleaning a perfusion system, performing alcohol once and then performing sterile water three times, pouring preheated first perfusion liquid into a conduit, avoiding bubbles in the conduit, paying attention to the continuous circulation of the first perfusion liquid in the conduit, and firstly, dissolving oxygen in air into the water liquid as soon as possible through an air inlet of the perfusion system for the purpose of oxygen explosion; secondly, the temperature of the reagent is prevented from decreasing, and the temperature is a key factor which affects the cell viability in the process.

3. Placing the anesthetized animals in a sterilized tray, fixing the animals by adhesive tape, cutting off abdominal cavity by 'U' -shaped, poking off viscera by a cotton swab, exposing the inferior vena cava, adjusting the peristaltic pump to 10 mL/min, picking up the inferior vena cava by an infusion needle, taking care of discharging air of a needle head, fixing the position of the infusion needle by a clamp, cutting off the portal vein of the liver, starting the peristaltic pump, cutting off diaphragm after preliminary blood removal of the liver, clamping the inferior vena cava at the centripetal end, and running 5min until the first perfusion liquid in the centrifuge tube runs empty; switching into a second perfusion liquid, adjusting the flow rate to 5 mL/min, running 6 min, cutting off the liver leaves as completely as possible, placing the liver leaves in the rest second perfusion liquid (the rest second perfusion liquid is larger than 7 mL), opening the envelope by using a Pasteur pipette, and stirring gently to prepare the cell suspension.

4. The subsequent steps were all operated on ice.

5. Adding the resuspension culture medium to 50mL to neutralize the digestive juice, sieving with a 40 μm cell sieve, centrifuging at 50×g for 2min, and discarding the supernatant; the cells obtained in the morning are resuspended and preserved by using 3mL hepatocyte preservation solution, and the cells obtained in the afternoon are preserved by using a resuspension culture medium until the volume reaches 20 mL; 50 The donor number is marked by the mL centrifuge tube, the centrifuge tube is preserved on ice, and the hepatocyte preservation solution comprises, in terms of concentration: DMEM basal medium, potassium 35 mM, sodium 110 mM, magnesium 5mM, calcium 0.16mM, chloride 30 mM, dihydrogen phosphate 17 mM, sulfate 2.5 mM, bicarbonate 2.5 mM, 4-hydroxyethylpiperazine ethanesulfonic acid 12 mM, glutathione 3mM, allopurinol 0.5 mM, alpha-lipoic acid 2mM, glucose 2.5 mM, raffinose 15 mM, mannitol 10 mM, lactobionic acid 130 mM, sucrose 10 mM, fructose 10 mM, dextran 5%, adenosine 3mM, deferoxamine 1mM, polyethylene glycol 0.5 g/L, and penicillin-streptomycin 200 mM.

6. After all donors are completed, trypan blue staining counts are performed.

7. Selecting donor with the activity ratio of more than or equal to 80% and the visual field dead cell mass of less than 4, centrifuging for 2min at 50 Xg, discarding the supernatant, and re-suspending the culture medium at 5 mL. Loading and purifying with 35% percoll and 15 mL centrifuge tube, adding 5 mL percoll, adding the suspended cells of 1 donor, centrifuging at 180×g, and centrifuging at 2 min; the Pasteur pipette sequentially sucks away the dead cell layer in the middle, the upper culture medium and the percoll at the lower layer; the bottom cell pellet was retained, gently resuspended in 2 mL resuspension medium, and the cell suspension was pooled into a 50mL centrifuge tube.

8. The collected cells were centrifuged at 50 Xg for 2min, the supernatant was discarded, the medium was resuspended to 100mL, and the mixture was resuspended and counted on a 0.1% trypan blue stain. Centrifuging at 50 Xg for 2min, discarding supernatant, adding the frozen stock solution to cell density of 10 mol/mL, and packaging, wherein each frozen stock tube is packaged with 1mL of cell suspension.

9. And (5) cooling the packed frozen cells by using a full-automatic cell freezing device, and storing the cooled cells in liquid nitrogen.

The product was prepared with reference to the above described multiple batches.

Example 2

This example was performed to prepare rat multi-donor hepatocytes.

1. The temperature of the water bath kettle is set to be 43 ℃, the water bath is filled with a first perfusion liquid and a second perfusion liquid of a 50 mL centrifuge tube, and the experiment is started after the water bath kettle is preheated for at least 30 min.

3. Placing the anesthetized animals in a sterilized tray, fixing the animals by adhesive tape, cutting off abdominal cavity by 'U' -shaped, poking off viscera by a cotton swab, exposing the inferior vena cava, adjusting the peristaltic pump to 50 mL/min, picking up the inferior vena cava by an infusion needle, taking care of discharging air of a needle head, fixing the position of the infusion needle by a clamp, cutting off the portal vein of the liver, starting the peristaltic pump, cutting off diaphragm after preliminary blood removal of the liver, clamping the inferior vena cava at the centripetal end, and running 4 min until the first perfusion liquid in the centrifuge tube runs empty; switching to a second perfusion liquid, adjusting the flow rate to 25 mL/min, running 5 min, cutting off the liver leaves as completely as possible, placing the liver leaves in the rest second perfusion liquid (the rest second perfusion liquid is larger than 7 mL), opening the envelope by using a Pasteur pipette, and stirring gently to prepare the cell suspension.

4. The subsequent steps were all operated on ice.

5. Adding the resuspension culture medium to 50 mL to neutralize the digestive juice, sieving with 30 μm cell sieve, centrifuging at 50×g for 2min, and discarding the supernatant; the cells obtained in the morning are resuspended and preserved by using 3 mL hepatocyte preservation solution, and the cells obtained in the afternoon are preserved by using a resuspension culture medium until the volume reaches 20 mL; 50 The donor number is marked by the mL centrifuge tube, the centrifuge tube is preserved on ice, and the hepatocyte preservation solution comprises, in terms of concentration: DMEM basal medium, potassium ion 30 mM, sodium ion 90 mM, magnesium ion 4 mM, calcium ion 0.15 mM, chloride ion 15 mM, dihydrogen phosphate 14 mM, sulfate 2mM, bicarbonate 2mM, 4-hydroxyethylpiperazine ethanesulfonic acid 10 mM, glutathione 2.5 mM, allopurinol 0.4 mM, alpha-lipoic acid 1.5 mM, glucose 2mM, raffinose 12 mM, mannitol 8 mM, lactobionic acid 120mM, sucrose 8 mM, fructose 8 mM, dextran 4, adenosine 2.5 mM, deferoxamine 0.8 mM, polyethylene glycol 0.4 g/L, and penicillin-streptomycin 150 mM.

6. After all donors are completed, trypan blue staining counts are performed.

7. Selecting donor with the activity ratio of more than or equal to 80% and the visual field dead cell mass of less than 4, centrifuging for 2min at 50 Xg, discarding the supernatant, and re-suspending the culture medium at 5 mL. Loading and purifying with 25% percoll and 15 mL centrifuge tube, adding 5 mL percoll, adding 1 donor cell, and centrifuging at 180×g for 2 min; the Pasteur pipette sequentially sucks away the dead cell layer in the middle, the upper culture medium and the percoll at the lower layer; the bottom cell pellet was retained, gently resuspended in 2 mL resuspension medium, and the cell suspension was pooled into a 50mL centrifuge tube.

Example 3

This example was performed to prepare beagle multi-donor hepatocytes.

1. The temperature of the water bath is set to be 43 ℃, the water bath is filled with a first perfusion liquid and a second perfusion liquid of a 50mL centrifuge tube, and the experiment is started after preheating for at least 30 min.

3. Placing the anesthetized animals in a sterilized tray, fixing the animals by adhesive tape, cutting off abdominal cavity by 'U' -shaped, poking off viscera by a cotton swab, exposing the inferior vena cava, adjusting the peristaltic pump to 25 mL/min, picking up the inferior vena cava by an infusion needle, taking care of discharging air of a needle head, fixing the position of the infusion needle by a clamp, cutting off the portal vein of the liver, starting the peristaltic pump, cutting off diaphragm after preliminary blood removal of the liver, clamping the inferior vena cava at the centripetal end, and running 4 min until the first perfusion liquid in the centrifuge tube runs empty; switching into a second perfusion liquid, regulating the flow rate to 10 mL/min, running 10 min, cutting off the liver leaves as completely as possible, placing the liver leaves in the rest second perfusion liquid (the rest second perfusion liquid is larger than 7 mL), opening the envelope by using a Pasteur pipette, and stirring gently to prepare the cell suspension.

4. The subsequent steps were all operated on ice.

5. Adding the resuspension culture medium to 50mL to neutralize the digestive juice, sieving with a 35 μm cell sieve, centrifuging at 50×g for 2min, and discarding the supernatant; the cells obtained in the morning are resuspended and preserved by using 3mL hepatocyte preservation solution, and the cells obtained in the afternoon are preserved by using a resuspension culture medium until the volume reaches 20 mL; 50 The donor number is marked by the mL centrifuge tube, the centrifuge tube is preserved on ice, and the hepatocyte preservation solution comprises, in terms of concentration: DMEM basal medium, potassium ion 40 mM, sodium ion 130 mM, magnesium ion 6mM, calcium ion 0.25 mM, chloride ion 22 mM, dihydrogen phosphate 20 mM, sulfate 3mM, bicarbonate 3mM, 4-hydroxyethylpiperazine ethanesulfonic acid 15 mM, glutathione 3.5 mM, allopurinol 0.6 mM, alpha-lipoic acid 2.5 mM, glucose 3mM, raffinose 18 mM, mannitol 12 mM, lactobionic acid 140 mM, sucrose 12 mM, fructose 12 mM, dextran 6, adenosine 5mM, deferoxamine 1.2 mM, polyethylene glycol 0.6 g/L, and penicillin-streptomycin 250 mM.

6. After all donors are completed, trypan blue staining counts are performed.

Test case

This test example resuscitates and tests for activity on hepatocytes prepared in each example.

The specific method for resuscitation and activity test comprises the following steps:

hepatocyte resuscitation and viability detection:

(1) Laboratory instrument preparation: opening the water bath kettle, irradiating the operation table with ultraviolet rays for 30min, and preparing materials;

(2) The cells were clamped from liquid nitrogen with forceps, placed in a 37 ℃ water bath, and immediately removed when shaking to defrost until a few ice crystals remained in the tube.

(3) Spraying alcohol on the outer wall of the cell cryopreservation tube, wiping the cell cryopreservation tube with sterile paper, and transferring the cell cryopreservation tube into a biological safety cabinet for operation;

(4) Resuspension the thawed cell suspension with pre-heated 1640 medium plus 10% fetal bovine serum, transferring to a 15 mL centrifuge tube, thoroughly mixing with a Pasteur pipette, centrifuging for 350g for 5min, and removing the supernatant;

(5) mu.L of cells were diluted in 180ul DPBS and mixed with 0.4% trypan blue 1:1, after dilution, counting and microscopic observation are carried out.

Hepatocyte activity test method:

measuring the mRNA expression level of the CYP450 enzyme subtypes (CYP 1A, CYP2B, CYP2C, CYP 3A) in the cells induced under each condition by a q-PCR instrument; and metabolic activity of related metabolic enzymes such as UGT, ST, UGT1 A1. The data of the mouse hepatocyte resuscitation phase I metabolic enzyme are shown in Table 2 (CB indicates the beginning of different batch numbers of C57BL/6 mice, CD indicates the beginning of different batch numbers of CD1 mice), and the data of the mouse hepatocyte resuscitation phase II metabolic enzyme are shown in Table 3, and the results indicate that three groups of cells still have metabolic activity after being resuscitated.

TABLE 2

TABLE 3 Table 3

The cell viability of C57BL-6 mice after hepatic cell resuscitation is 93%, the morphology of the incubated 24 h cells is shown in figure 1, and the morphology of the incubated 48 hours is shown in figure 2; the cell activity rate of the rat after hepatic cell recovery is 91%, the morphology of the incubated 24 h cells is shown in figure 3, and the morphology of the rat after 48 hours is shown in figure 4; the cell activity rate of the beagle dogs after hepatic cell recovery is 92%, the morphology of the incubated 24 h cells is shown in figure 5, and the morphology of the incubated 72 hours cells is shown in figure 6; the invention can prepare the liver cells with high cell activity and purity in a large scale and high efficiency, and has certain advantages compared with the existing liver cell products (Table 4).

TABLE 4 Table 4

Further, the prepared liver cells are tested for freezing stability, and the method specifically comprises the following steps: a certain amount of hepatocytes were spot checked in stock, and the methods of hepatocyte resuscitation, activity detection were referred to above.

The results of the 12-month freeze stability test are shown in fig. 7A-7D, and cell viability (fig. 7A) and cell number (fig. 7B) are measured by trypan blue exclusion, wherein 1 st and 2 nd represent 2 parallel test groups, CD1-1133 represents CD1 mouse hepatocyte results, SD-1055 represents rat hepatocyte test results, and C57-1838 represents C57BL/6 mouse hepatocyte test results, which indicate that the hepatocytes prepared by the invention can still maintain higher viable cell and active cell number after long-term freeze; the results of the cryopreservation change of the phase I metabolic enzyme activity are shown in FIG. 7C and FIG. 7D, and it can be seen that CYP1A PHENACETIN (phenacetin) and CYP2C S-Mephenytoin (S-merphenytoin) metabolic enzymes in primary hepatocytes remain high in activity after 12 months of cryopreservation.

The freezing stability test results of 33 months are shown in fig. 8A-8C, and the cell viability (fig. 8A) and the cell number (fig. 8B) are measured by trypan blue exclusion, wherein 1 st and 2 nd represent 2 parallel test groups, BE-1201 represents a beagle liver cell test result, CB-1202 represents a C57BL/6 mouse liver cell test result, and the results show that the liver cells prepared by the method can still keep higher viability and active cell number after long-term freezing; the results of the CYP450 enzyme subtype metabolic activity are shown in FIG. 8C, and can be seen that CYP2B Bupropion (bupropion), CYP3A Tsstosterone (testosterone) and CYP3A Midazolam (midazolam) metabolic enzymes still keep high activity in primary liver cells before and after 33 months of frozen storage.

Cell viability and cell number of the products between the different batches were analyzed, batch stability analysis was performed, and three test groups (tube 1, tube2 and tube 3) were repeated by C57BL/6 mouse hepatocytes (C57-1939, C57-1141), CD1 mouse hepatocytes (CD 1-1588, CD 1-1688), SD rat hepatocytes (SD-1199), respectively, and the results of cell viability and cell number within the batches are shown in FIGS. 9A and 9B.

In summary, the present invention develops a process flow capable of efficiently and massively preparing hepatic parenchymal cells for the first time, and compared with the existing process, the schematic diagram is shown in fig. 10 (for example, mice), and the existing low-efficiency large-scale preparation process flow cannot reach the product verification standard. The invention designs a novel process for preparing multi-donor liver cells, and the whole process is accurately controlled, so that all steps are coordinated, the large-scale and high-efficiency preparation of liver cells with high cell activity and purity based on a plurality of donors can be realized, the stability among batches is high, the prepared cells can be stored for a long time, the prepared cells still have high activity rate and activity after recovery, and CYP450 metabolic enzyme still has higher activity.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A method of preparing a multi-donor hepatocyte, comprising the steps of:

(1) Starting a perfusion system to perform constant-temperature perfusion of the liver, wherein the constant-temperature perfusion temperature is 42-44 ℃ of first perfusion liquid, the perfusion flow rate is 10-50 mL/min, and the time is 4-5 min;

(4) Mixing the cell suspension obtained in the step (3) with a culture medium, sieving cells, centrifuging, discarding the supernatant, collecting cells, and mixing the cells with a hepatocyte preservation solution to obtain a cell suspension;

(5) Centrifuging the cell suspension obtained in the step (4), discarding the supernatant, collecting cells, mixing with a culture medium, adding the mixture into a percoll solution, centrifuging, and collecting cell sediment at the bottom to obtain liver cells;

the method further comprises the steps of preheating and loading the perfusion liquid before the step (1), wherein the steps comprise the steps of placing the first perfusion liquid and the second perfusion liquid in a water bath with the temperature of 42-44 ℃ for at least 30min, and pouring the first perfusion liquid into a conduit of a perfusion system and circulating;

The first perfusion liquid in the step (1) contains sodium lactate, sodium chloride, potassium chloride, calcium chloride and sterile water;

the second perfusion liquid in the step (2) contains type II collagenase.

2. The method of preparing multi-donor hepatocytes of claim 1, wherein said medium of step (4) comprises Williams' E medium;

The hepatocyte preservation solution in step (4) contains a cell culture medium and additives including an electrolyte, a buffer, an oxygen radical scavenger, an impermeable substance adenosine and deferoxamine; the electrolyte includes metal ions and chloride ions; the oxygen radical scavenger comprises glutathione, allopurinol and alpha-lipoic acid; the impermeable substance comprises glucose, raffinose, mannitol, lactobionic acid, sucrose, fructose and dextran; the metal ions comprise any one or a combination of at least two of potassium ions, sodium ions, magnesium ions or calcium ions; the buffer comprises any one or a combination of at least two of dihydrogen phosphate, sulfate, bicarbonate or 4-hydroxyethyl piperazine ethane sulfonic acid.

3. The method of preparing multi-donor hepatocytes of claim 2, wherein said additives further comprise polyethylene glycol and/or antibiotics;

The hepatocyte preservation solution comprises, in terms of concentration: DMEM basal medium, 30-40 mM of potassium ion, 90-130 mM of sodium ion, 4-6 mM of magnesium ion, 0.15-0.25 mM of calcium ion, 15-22 mM of chloride ion, 14-20 mM of dihydric phosphate, 2-3 mM of sulfate, 2-3 mM of bicarbonate, 10-15 mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 2.5-3.5 mM of glutathione, 0.4-0.6 mM of allopurinol, 1.5-2.5 mM of alpha-lipoic acid, 2-3 mM of glucose, 12-18 mM of raffinose, 8-12 mM of mannitol, 120-140 mM of lactobionic acid, 8-12 mM of sucrose, 8-12 mM of fructose, 4-6 mM of glucan, 2.5-5 mM of adenosine, 0.8-1.2 mM of deferoxamine, 0.4-0.6 g/L penicillin 75-125 mM and 75-125 mM of streptomycin.

4. The method for preparing multi-donor liver cells according to claim 1, wherein the pore size of the cell sieve in step (4) is 30-40 μm.

5. The method of preparing multi-donor hepatocytes of claim 1, wherein the concentration of the percoll solution of step (5) is 25% -35%.

6. The method of preparing multi-donor hepatocytes of claim 1, wherein step (4) further comprises the step of cell counting the cell suspension, said cell counting method comprising trypan blue staining counting.

7. The method for preparing multi-donor liver cells according to claim 1, wherein the step (5) further comprises a step of screening the cell suspension obtained in the step (4), comprising selecting a cell suspension having a cell viability of 80% or more and a dead cell mass of less than 4.

8. A method of preparing multi-donor hepatocytes according to any of claims 1-7, comprising the steps of:

(1) Placing the first perfusion liquid and the second perfusion liquid in a water bath at 42-44 ℃ for at least 30min, and pouring the first perfusion liquid into a conduit of a perfusion system and circulating;

(2) Starting a perfusion system to perform constant-temperature liver perfusion, wherein the perfusion flow rate is 10-50 mL/min, and the operation is 4-5 min; switching the first perfusion liquid into the second perfusion liquid for constant-temperature perfusion at 42-44 ℃, wherein the perfusion flow rate is 5-25 mL/min, and the operation is 5-10 min;

(4) Mixing the cell suspension obtained in the step (3) with a culture medium, sieving with a 30-40 mu m cell sieve, centrifuging, discarding the supernatant, collecting cells, mixing the cells with a cell preservation solution to obtain a cell suspension, and performing trypan blue staining counting; the hepatocyte preservation solution comprises, in terms of concentration: DMEM basal medium, 30-40 mM of potassium ion, 90-130 mM of sodium ion, 4-6 mM of magnesium ion, 0.15-0.25 mM of calcium ion, 15-22 mM of chloride ion, 14-20 mM of dihydric phosphate, 2-3 mM of sulfate, 2-3 mM of bicarbonate, 10-15 mM of 4-hydroxyethyl piperazine ethane sulfonic acid, 2.5-3.5 mM of glutathione, 0.4-0.6 mM of allopurinol, 1.5-2.5 mM of alpha-lipoic acid, 2-3 mM of glucose, 12-18 mM of raffinose, 8-12 mM of mannitol, 120-140 mM of lactobionic acid, 8-12 mM of sucrose, 8-12 mM of fructose, 4-6 mM of glucan, 2.5-5 mM of adenosine, 0.8-1.2 mM of deferoxamine, 0.4-0.6 g/L of polyethylene glycol and 150-250 mM of penicillin-streptomycin;

(5) Taking the cell suspension obtained in the step (4), selecting the cell suspension with the activity ratio of more than or equal to 80% and the visual field dead cell mass of less than 4, centrifuging, discarding the supernatant, collecting cells, mixing with a culture medium, adding the cells into a percoll solution with the concentration of 25-35%, centrifuging, and collecting cell sediment at the bottom to obtain the liver cells.